Archive for the ‘Hematopoiesis’ Category

Blast crisis in myeloid leukemia and the activation of a microRNA-editing enzyme called ADAR1

Curator: Larry H. Bernstein, MD, FCAP


Fix to RNA-Editing Glitch May Defuse Blast Crisis

GEN News Highlights Jun 10, 2016   http://www.genengnews.com/gen-news-highlights/fix-to-rna-editing-glitch-may-defuse-blast-crisis/81252818/

The self-renewal of leukemia stem cells depends on the activation of a microRNA-editing enzyme called ADAR1. According to a new study, ADAR1 activation represents a unique therapeutic vulnerability in leukemia stem cells, which can give rise to blast crisis in chronic myeloid leukemia.     http://www.genengnews.com/Media/images/GENHighlight/thumb_Jun10_2016_ChronicMyeloidLeukemiaBloodCells6222419925.jpg

Few cancer mechanisms are as devastating as the generation of cancer stem cells, which arise in leukemia from white blood cell precursors. The mechanisms of this transition have been obscure, but the consequences are all too clear. Leukemia stem cells promote an aggressive, therapy-resistant form of disease called blast crisis.

Delving into the mechanisms by which leukemia stem cells are primed, a team of scientists at the University of California, San Diego (UCSD), uncovered a misfiring RNA-editing system. The main problem the scientists found was an enzyme called ADAR1 (adenosine deaminase acting on RNA1), which mediates post-transcriptional adenosine-to-inosine (A-to-I) RNA editing.

ADAR1 can edit the sequence of microRNAs (miRNAs), small pieces of genetic material. By swapping out just one miRNA building block for another, ADAR1 alters the carefully orchestrated system cells use to control which genes are turned on or off at which times.

ADAR1 is known to promote cancer progression and resistance to therapy. To study ADAR1, the UCSD team used human blast crisis chronic myeloid leukemia (CML) cells in the lab, and mice transplanted with these cells, to determine the enzyme’s role in governing leukemia stem cells.

The scientists, led by Catriona Jamieson, M.D., Ph.D., published their work June 9 in Cell Stem Cell, in an article entitled, “ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.” The article presented the first mechanistic link between pro-cancer inflammatory signals and RNA editing–driven reprogramming of precursor cells into leukemia stem cells.

The article describes how ADAR1-mediated A-to-I RNA editing is activated by Janus kinase 2 (JAK2) signaling and BCR-ABL1 signaling. Also, it indicated, in a model of blast crisis (BC) CML, that combined JAK2 and BCR-ABL1 inhibition prevents leukemia stem cell self-renewal commensurate with ADAR1 downregulation.

Essentially, the scientists were able to trace a series of molecular events: First, white blood cells with a leukemia-promoting gene mutation become more sensitive to signs of inflammation. That inflammatory response activates ADAR1. Then, hyper-ADAR1 editing slows down the miRNAs known as let-7. Ultimately, this activity increases cellular regeneration, or self-renewal, turning white blood cell precursors into leukemia stem cells.

“Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912A mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs,” wrote the author of the Cell Stem Cell article. “Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing andLIN28B upregulation.”

After learning how the ADAR1 system works, Dr. Jamieson’s team looked for a way to stop it. By inhibiting sensitivity to inflammation or inhibiting ADAR1 with a small-molecule tool compound, the researchers were able to counter ADAR1’s effect on leukemia stem cell self-renewal and restore let-7. Self-renewal of blast crisis CML cells was reduced by approximately 40% when treated with the small molecule called 8-Aza as compared to untreated cells.

“A small-molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis,” the study’s authors noted. “Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.”

“In this study, we showed that cancer stem cells co-opt a RNA editing system to clone themselves. What’s more, we found a method to dial it down,” said Dr. Catriona Jamieson. “Based on this research, we believe that detecting ADAR1 activity will be important for predicting cancer progression.

“In addition, inhibiting this enzyme represents a unique therapeutic vulnerability in cancer stem cells with active inflammatory signaling that may respond to pharmacologic inhibitors of inflammation sensitivity or selective ADAR1 inhibitors that are currently being developed.”


ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis

Maria Anna Zipeto, Angela C. Court, Anil Sadarangani, Nathaniel P. Delos Santos, Larisa Balaian, Hye-Jung Chun, Gabriel Pineda, Sheldon R. Morris, Cayla N. Mason, Ifat Geron, Christian Barrett, Daniel J. Goff, Russell Wall, Maurizio Pellecchia, Mark Minden, Kelly A. Frazer, Marco A. Marra, Leslie A. Crews, Qingfei Jiang, Catriona H.M. Jamieson
Published online: June 9, 2016
  • JAK2 signaling activates ADAR1-mediated A-to-I RNA editing
  • JAK2 and BCR-ABL1 signaling converge on ADAR1 activation through STAT5a
  • ADAR1-mediated microRNA editing impairs let-7 biogenesis and enhances LSC self-renewal
  • JAK2 and BCR-ABL1 inhibition reduces ADAR1 expression and prevents LSC self-renewal

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912A mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28Bupregulation. A small-molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.


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4014 Inflammatory Cytokine-Responsive ADAR1 Impairs Let-7 Biogenesis and Promotes Leukemia Stem Cell Generation

Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy
Program: Oral and Poster Abstracts
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Poster III
Monday, December 7, 2015, 6:00 PM-8:00 PM
Hall A, Level 2 (Orange County Convention Center)

Maria Anna Zipeto, Ph.D1*, Angela Court Recart2*, Nathaniel Delos Santos3*, Qingfei Jiang, PhD4*, Leslie A Crews, PhD3* and Catriona HM Jamieson, MD, PhD3

1Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA
2University of California San Diego, LA JOLLA, CA
3Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA
4University of California San Diego, La Jolla, CA

BackgroundIn advanced human malignancies, RNA sequencing (RNA-seq) has uncovered deregulation of adenosine deaminase acting on RNA (ADAR) editases that promote therapeutic resistance and leukemia stem cell (LSC) generation. Chronic myeloid leukemia (CML), an important paradigm for understanding LSC evolution, is initiated by BCR-ABL1 oncogene expression in hematopoietic stem cells (HSCs) but undergoes blast crisis (BC) transformation following aberrant self-renewal acquisition by myeloid progenitors harboring cytokine-responsive ADAR1 p150 overexpression. Emerging evidence suggests that adenosine to inosine editing at the level of primary (pri) or precursor (pre)-microRNA (miRNA), alters miRNA biogenesis and impairs biogenesis. However, relatively little is known about the role of inflammatory niche-driven ADAR1 miRNA editing in malignant reprogramming of progenitors into self-renewing LSCs.


Primary normal and CML progenitors were FACS-purified and RNA-Seq analysis as well as qRT-PCR validation were performed according to published methods (Jiang, 2013). MiRNAs were extracted from purified CD34+ cells derived from CP, BC CML and cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human JAK2, let-7a, wt-ADAR1 and mutant ADAR1, which lacks a functional deaminase domain.  Because STAT signaling triggers ADAR1 transcriptional activation and both BCR-ABL1 and JAK2 activate STAT5a, nanoproteomics analysis of STAT5a levels was performed.  Engrafted immunocompromised RAG2-/-γc-/- mice were treated with a JAK2 inhibitor, SAR302503, alone or in combination with a potent BCR-ABL1 TKI Dasatinib, for two weeks followed by FACS analysis of human progenitor engraftment in hematopoietic tissues and serial transplantation.


RNA-seq and qRT-PCR analysis in FACS purified BC CML progenitors revealed an over-representation of inflammatory pathway activation and higher levels of JAK2-dependent inflammatory cytokine receptors, when compared to normal and chronic phase (CP) progenitors. Moreover, RNA-seq and qRT-PCR analysis showed decreased levels of mature let-7 family of stem cell regulatory miRNA in BC compared to normal and CP progenitors. Lentiviral human JAK2 transduction of CD34+ progenitors led to an increase of ADAR1 transcript levels and to a reduction in let-7 family members. Interestingly, lentiviral human JAK2 transduction of normal progenitors enhanced ADAR1 activity, as revealed by RNA editing-specific qRT-PCR and RNA-seq analysis. Moreover, qRT-PCR analysis of CD34+ progenitors transduced with wt-ADAR1, but not mutant ADAR1 lacking functional deaminase activity, reduced let-7 miRNA levels. These data suggested that ADAR1 impairs let-7 family biogenesis in a RNA editing dependent manner. Interestingly, RNA-seq analysis confirmed higher frequency of A-to-I editing events in pri- and pre-let-7 family members in CD34+ BC compared to CP progenitors, as well as normal progenitors transduced with human JAK2 and ADAR1-wt, but not mutant ADAR1. Lentiviral ADAR1 overexpression enhanced CP CML progenitor self-renewal and decreased levels of some members of the let-7 family. In contrast, lentiviral transduction of human let-7a significantly reduced self-renewal of progenitors. In vivo treatments with Dasatinib in combination with a JAK2 inhibitor, significantly reduced self-renewal of BCR-ABL1 expressing BC progenitors in the bone marrow thereby prolonging survival of serially transplanted mice. Finally, a reduction in ADAR1 p150 transcripts was also noted following combination treatment only suggesting a role for ADAR1 in CSC propagation.


This is the first demonstration that intrinsic BCR-ABL oncogenic signaling and extrinsic cytokines signaling through JAK2 converge on activation of ADAR1 that drives LSC generation by impairing let-7 miRNA biogenesis. Targeted reversal of ADAR1-mediated miRNA editing may enhance eradication of inflammatory niche resident cancer stem cells in a broad array of malignancies, including JAK2-driven myeloproliferative neoplasms.

Disclosures: Jamieson: J&J: Research Funding ; GSK: Research Funding .

Interferon Receptor Signaling in Malignancy: a Network of Cellular Pathways Defining Biological Outcomes

Interferons (IFNs) are cytokines with important anti-proliferative activity and exhibit key roles in immune surveillance against malignancies. Early work initiated over 3 decades ago led to the discovery of IFN receptor activated Jak-Stat pathways and provided important insights into mechanisms for transcriptional activation of interferon stimulated genes (ISGs) that mediate IFN-biological responses. Since then, additional evidence has established critical roles for other receptor activated signaling pathways in the induction of IFN-activities. These include MAPK pathways, mTOR cascades and PKC pathways. In addition, specific microRNAs (miRNAs) appear to play a significant role in the regulation of IFN-signaling responses. This review focuses on the emerging evidence for a model in which IFNs share signaling elements and pathways with growth factors and tumorigenic signals, but engage them in a distinctive manner to mediate anti-proliferative and antiviral responses.

Because of their antineoplastic, antiviral, and immunomodulatory properties, recombinant interferons (IFNs) have been used extensively in the treatment of various diseases in humans (1). IFNs have clinical activity against several malignancies and are actively used in the treatment of solid tumors such as malignant melanoma and renal cell carcinoma; and hematological malignancies, such as myeloproliferative neoplasms (MPNs) (1). In addition, IFNs play prominent roles in the treatment of viral syndromes, such as hepatitis B and C (2). In contrast to their beneficial therapeutic properties, IFNs have been also implicated in the pathophysiology of certain diseases in humans. In many cases this involvement reflects abnormal activation of the endogenous IFN system, which has important roles in various physiological processes. Diseases in which dysregulation of the Type I IFN system has been implicated as a pathogenetic mechanism include autoimmune disorders such as systemic lupus erythematosous (3), Sjogren’s syndrome (3,4), dermatomyositis (5) and systemic sclerosis (3, 4). In addition, Type II IFN (IFNγ) overproduction has been implicated in bone marrow failure syndromes, such as aplastic anemia (6). There is also recent evidence for opposing actions of distinct IFN subtypes in the pathophysiology of certain diseases. For instance, a recent study demonstrated that there is an inverse association between IFNβ and IFNγ gene expression in human leprosy, consistent with opposing functions between Type I and II IFNs in the pathophysiology of this disease (7). Thus, differential targeting of components of the IFN-system, to either promote or block induction of IFN-responses depending on the disease context, may be useful in the therapeutic management of various human illnesses. The emerging evidence for the complex regulation of the IFN-system underscores the need for a detailed understanding of the mechanisms of IFN-signaling in order to target IFN-responses effectively and selectively.

It took over 35 years from the original discovery of IFNs in 1957 to the discovery of Jak-Stat pathways (8). The identification of the functions of Jaks and Stats dramatically advanced our understanding of the mechanisms of IFN-signaling and had a broad impact on the cytokine research field as a whole, as it led to the identification of similar pathways from other cytokine receptors (8). Subsequently, several other IFN receptor (IFNR)-regulated pathways were identified (9). As discussed below, in recent years there has been accumulating evidence that beyond Stats, non-Stat pathways play important and essential roles in IFN-signaling. This has led to an evolution of our understanding of the complexity associated with IFN receptor activation and how interacting signaling networks determine the relevant IFN response.

Interferons and their functions

The interferons are classified in 3 major categories, Type I (α, β, ω, ε, τ, κ, ν); Type II (γ) and Type III IFNs (λ1, λ2, λ3) (1, 9, 10). The largest IFN-gene family is the group of Type I IFNs. This family includes 14 IFNα genes, one of which is a pseudogene, resulting in the expression of 13 IFNα protein subtypes (1, 9). There are 3 distinct IFNRs that are specific for the 3 different IFN types. All Type I IFN subtypes bind to and activate the Type I IFNR, while Type II and III IFNs bind to and activate the Type II and III IFNRs, respectively (911). It should be noted that although all the different Type I IFNs bind to and activate the Type I IFNR, differences in binding to the receptor may account for specific responses and biological effects (9). For instance, a recent study provided evidence that direct binding of mouse IFNβ to the Ifnar1 subunit, in the absence of Ifnar2, regulates engagement of signals that control expression of genes specifically induced by IFNβ, but not IFNα (12). This recent discovery followed original observations from the 90s that revealed differential interactions between the different subunits of the Type I IFN receptor in response to IFNβ binding as compared to IFNα binding and partially explained observed differences in functional responses between different Type I IFNs (9).

A common property of all IFNs, independently of type and subtype, is the induction of antiviral effects in vitro and in vivo (1). Because of their potent antiviral properties, IFNs constitute an important element of the immune defense against viral infections. There is emerging information indicating that specificity of the antiviral response is cell type dependent and/or reflects specific tissue expression of certain IFNs. As an example, a recent comparative analysis of the involvement of the Type I IFN system as compared to the Type III IFN system in antiviral protection against rotavirus infection of intestinal epithelial cells demonstrated an almost exclusive requirement for IFNλ (Type III IFN) (13). The antiviral effects of IFNα have led to the introduction of this cytokine in the treatment of hepatitis C and B in humans (2) and different viral genotypes have been associated with response or failure to IFN-therapy (14).

Most importantly, IFNs exhibit important antineoplastic effects, reflecting both direct antiproliferative responses mediated by IFNRs expressed on malignant cells, as well as indirect immunomodulatory effects (15). IFNα and its pegylated form (peg IFNα) have been widely used in the treatment of several neoplastic diseases, such as hairy cell leukemia (HCL), chronic myeloid leukemia (CML), cutaneous T cell lymphoma (CTCL), renal cell carcinoma (RCC), malignant melanoma, and myeloproliferative neoplasms (MPNs) (1, 16). Although the emergence of new targeted therapies and more effective agents have minimized the use of IFNs in the treatment of diseases like HCL and CML, IFNs are still used extensively in the treatment of melanoma, CTCL and MPNs (1, 16, 17). Notably, recent studies have provided evidence for long lasting molecular responses in patients with polycythemia vera (PV), essential thrombocytosis (ET) and myelofibrosis (MF) who were treated with IFNα (16). Beyond their inhibitory properties on malignant hematopoietic progenitors, IFNs are potent regulators of normal hematopoiesis (9) and contribute to the regulation of normal homeostasis in the human bone marrow (18). Related to its effects in the central nervous system, IFNβ has clinical activity in multiple sclerosis (MS) and has been used extensively for the treatment of patients with MS (19). The immunoregulatory properties of Type I IFNs include key roles in the control of innate and adaptive immune responses, as well as positive and negative effects on the activation of the inflammasome (15). Dysregulation of the Type I IFN response is seen in certain autoimmune diseases, such as Aicardi-Goutières syndrome (20). In fact, self-amplifying Type I IFN-production is a key pathophysiological mechanism in autoimmune syndromes (21). There is also emerging evidence that IFNλ may contribute to the IFN signature in autoimmune diseases (3).

Jak-Stat pathways

Jak kinases and DNA binding Stat-complexes

Tyrosine kinases of the Janus family (Jaks) are associated in unique combinations with different IFNRs and their functions are essential for IFN-inducible biological responses. Stats are transcriptional activators whose activation depends on tyrosine phosphorylation by Jaks (8, 9). In the case of the Type I IFN receptor, Tyk2 and Jak1 are constitutively associated with the IFNAR1 and IFNAR2 subunits, respectively (8, 9) (Fig. 1). For the Type II IFN receptor, Jak1 and Jak2 are associated with the IFNGR1 and IFNGR2 receptor subunits, respectively (8, 9) (Fig. 1). Finally, in the case of the Type III IFNR, Jak1 and Tyk2 are constitutively associated with the IFN-λR1 and IL-10R2 receptor chains, respectively (10) (Fig. 1). Upon engagement of the different IFNRs by the corresponding ligands, the kinase domains of the associated Jaks are activated and phosphorylate tyrosine residues in the intracellular domains of the receptor subunits that serve as recruitmenst sites for specific Stat proteins. Subsequently, the Jaks phosphorylate Stat proteins that form unique complexes and translocate to the nucleus where they bind to specific sequences in the promoters of ISGs to initiate transcription. A major Stat complex in IFN-signaling is the interferon stimulated gene factor 3 (ISGF3) complex. This IFN-inducible complex is composed or Stat1, Stat2 and IRF9 and regulates transcription by binding to IFN stimulated response elements (ISRE) in the promoters of a large group of IFN stimulated genes (ISGs) (8, 9). ISGF3 complexes are induced during engagement of the Type I and III IFN receptors, but not in response to activation of Type II IFN receptors (810) (Table 1). Beyond ISGF3, several other Stat-complexes involving different Stat homodimers or heterodimers are activated by IFNs and bind to IFNγ-activated (GAS) sequences in the promoters of groups of ISGs (8, 9). Such GAS binding complexes are induced by all different IFNs (I, II and III), although there is variability in the engagement and utilization of different Stats by the different IFN-receptors (Table 1). It should also be noted that engagement of certain Stats, such as Stat4 and Stat6, is cell type-specific and may be relevant for tissue specific functions (9). The significance of different Stat binding complexes in the induction of Type I and II IFN responses was in part addressed in a study in which Stat1 cooperative DNA binding was disrupted by generating knock-in mice expressing cooperativity-deficient STAT1 (22). As expected, Type II IFN-induced gene transcription and antibacterial responses were essentially lost in these mice, but Type I IFN-dependent recruitment of Stat1 to ISRE elements and antiviral responses were not affected (22), demonstrating the existence of important differences in Stat1 cooperative DNA binding between Type I and II IFN signaling.

Type I, II, III interferon receptors subunits, associated kinases of the Janus family, and effector Stat-pathways. Note: Stat:Stat reflects multiple potential Stat:Stat compexes, as outlined in Table 2.

Table 1

Different Stat-DNA binding complexes induced by Type I, II and III IFNs.

Serine phosphorylation of Stats

The nuclear translocation of Stat-proteins occurs after their activation, following phosphorylation on specific sites by Jak kinases (8, 9). It is well established that phosphorylation on tyrosine 701 is required for activation of Stat1 and phosphorylation on tyrosine 705 is required for activation of Stat3 (8, 9). Beyond tyrosine phosphorylation, phosphorylation on serine 727 in the Stat1 and Stat3 transactivation domains is required for full and optimal transcriptional activation of ISGs (8, 9). There is evidence that serine phosphorylation occurs after the phosphorylation of Stat1 on tyrosine 701 and that translocation to the nucleus and recruitment to the chromatin are essential in order for Stat1 to undergo serine 727 phosphorylation (23). Several IFN-dependent serine kinases for Stat1 have been described, raising the possibility that this phosphorylation occurs in a cell type specific manner. After the original demonstration that protein kinase C (PKC) delta (PKCδ) is a serine kinase for Stat1 and is required for optimal transcriptional activation in response to IFNα (24), extensive work has confirmed the role of this PKC isoform in the regulation of serine 727 phosphorylation in Stat1 and has been extended to different cellular systems (2529) (Table 2). In the Type II IFN system five different serine kinases for the transactivation domain (TAD) of Stat1/phosphorylation on serine 727 have been demonstrated in different cell systems.  …..

Serine phosphorylation of Stats

The nuclear translocation of Stat-proteins occurs after their activation, following phosphorylation on specific sites by Jak kinases (8, 9). It is well established that phosphorylation on tyrosine 701 is required for activation of Stat1 and phosphorylation on tyrosine 705 is required for activation of Stat3 (8, 9). Beyond tyrosine phosphorylation, phosphorylation on serine 727 in the Stat1 and Stat3 transactivation domains is required for full and optimal transcriptional activation of ISGs (8, 9). There is evidence that serine phosphorylation occurs after the phosphorylation of Stat1 on tyrosine 701 and that translocation to the nucleus and recruitment to the chromatin are essential in order for Stat1 to undergo serine 727 phosphorylation (23). Several IFN-dependent serine kinases for Stat1 have been described, raising the possibility that this phosphorylation occurs in a cell type specific manner. After the original demonstration that protein kinase C (PKC) delta (PKCδ) is a serine kinase for Stat1 and is required for optimal transcriptional activation in response to IFNα (24), extensive work has confirmed the role of this PKC isoform in the regulation of serine 727 phosphorylation in Stat1 and has been extended to different cellular systems (2529) (Table 2). In the Type II IFN system five different serine kinases for the transactivation domain (TAD) of Stat1/phosphorylation on serine 727 have been demonstrated in different cell systems. ….

Protein tyrosine phosphatases with regulatory effects on Jak-Stat pathways in IFN-signaling.

MicroRNAs (miRs) and the IFN response

IFN-inducible JAK-STAT, MAPK and mTOR signaling cascades are also regulated potentially by microRNAs (miRs). miRs are important regulators of post-transcriptional events, leading to inhibition of mRNA translation or mRNA degradation (105). In recent years it has become apparent that the direct regulation of STAT activity by mIRs has profound effects on consequent gene expression, specifically in the context of cytokine-inducible events (106). Pertinent for this review of IFN-inducible STAT activation, miR-145, miR-146A and miR-221/222 target STAT1 and miR-221/222 target STAT2 (106). Numerous studies describe different miRs that target STAT3: mIR-17, miR-17-5p, mIR-17-3p, mIR-18a, miR-19b, mIR-92-1, miR-20b, Let-7a, miR-106a, miR-106-25, miR-106a-362 and miR-125b (106) (Fig. 4). mIR-132, miR-212 and miR-200a have been implicated in negatively regulating STAT4 expression in human NK cells (107) and miR-222 has been shown to regulate STAT5 expression (108). In addition, JAK-STAT signaling is affected by miR targeting of suppressors of cytokine signaling (SOCS) proteins. miR-122 and miR-155 targeting of SOCS1 releases the inhibition of STAT1 (and STAT5a/b) (109111), and mIR-19a regulation of SOCS1 and SOCS3 effectively prolongs activation of both STAT1 and STAT3 (112). There is also evidence that miR-155 targets the inositol phosphatase SHIP1, effectively prolonging/inducing IFN-γ expression (113). Much of the evidence associated with miRs prolonging JAK-STAT activation relates to cancer studies, where tumor-secreted miRs promote cell migration and angiogenesis by prolonging JAK-STAT activation (114). miR-145 targeting of SOCS7 affects nuclear translocation of STAT3 and has been associated with enhanced IFNβ production (115). Beyond inhibition of SOCS proteins, miRs may influence the expression of other inhibitory factors associated with JAK-STAT signaling, and miR-301a and miR-18a have been shown to inhibit PIAS3, a negative regulator of STAT3 activation (116). There is also the potential for STATS to directly regulate miR gene expression. STAT5 suppresses expression of miR15/16 (117) and there is evidence that there are potential STAT3 binding sites in the promoters of about 200 miRs (118). Viewed altogether, there is compelling evidence for miR-STAT interactions, yet few studies have considered the contributions of miRs to IFN-inducible JAK-STAT signaling.

Targeting and regulation of various proteins known to be involved in IFN-signaling by different miRNAs.  ….

Evolution of our understanding of IFN-signals and future perspectives

A substantial amount of knowledge has accumulated since the original discovery of the Jak-Stat pathway in the early 90s. It is now clear that several key signaling cascades are essential for the induction of Type I, II and III IFN-responses. The original view that IFN-signals can be transmitted from the cell surface to the nucleus in two simple steps involving tyrosine phosphorylation of Stat proteins (8) now appears somewhat simplistic, as it has been established that modifications of Jak-Stat signals by other pathways and/or simultaneous engagement of other essential complementary cellular cascades is essential for induction of ISG transcriptional activation, mRNA translation, protein expression and subsequent induction of IFN-responses. Such pathways include PKC and MAP kinase pathways and mTORC1 and mTORC2-dpendent signaling cascades.

Over the next decade our understanding of the mechanisms by which IFN-signals are induced will likely continue to evolve, with the anticipated outcome that it will be possible exploit this new knowledge for translational-therapeutic purposes. For instance, selective targeting of kinase-elements of the IFN-pathway with kinase inhibitors may be useful in the treatment of autoimmune diseases where dysregulated/excessive Type I IFN production contributes to the pathophysiology of disease. On the other hand, efforts to promote the induction of specific IFN-signals, may lead to novel, less toxic, therapeutic interventions for a variety of viral infectious diseases and neoplastic disorders.

Exploring the RNA World in Hematopoietic Cells Through the Lens of RNA-Binding Proteins

The discovery of microRNAs has renewed interest in post-transcriptional modes of regulation, fueling an emerging view of a rich RNA world within our cells that deserves further exploration. Much work has gone into elucidating genetic regulatory networks that orchestrate gene expression programs and direct cell fate decisions in the hematopoietic system. However, the focus has been to elucidate signaling pathways and transcriptional programs. To bring us one step closer to reverse engineering the molecular logic of cellular differentiation, it will be necessary to map post-transcriptional circuits as well and integrate them in the context of existing network models. In this regard, RNA-binding proteins (RBPs) may rival transcription factors as important regulators of cell fates and represent a tractable opportunity to connect the RNA world to the proteome. ChIP-seq has greatly facilitated genome-wide localization of DNA-binding proteins, helping us to understand genomic regulation at a systems level. Similarly, technological advances such as CLIP-seq allow transcriptome-wide mapping of RBP binding sites, aiding us to unravel post-transcriptional networks. Here, we review RBP-mediated post-transcriptional regulation, paying special attention to findings relevant to the immune system. As a prime example, we highlight the RBP Lin28B, which acts as a heterochronic switch between fetal and adult lymphopoiesis.

The basis of cellular differentiation and function can be represented as integrated circuits that are genetically programmed. Identification of the master regulators within these complex circuits that can switch on or off a genetic program will enable us to reprogram cells to suit biomedical needs. A remarkable example was the discovery by Takahashi and Yamanaka (1) that somatic cells could be reprogrammed into induced pluripotent stem (iPS) cells via the ectopic expression of four key transcription factors. Interestingly, a specific set of microRNAs (miRNAs) could also mediate this reprogramming (2, 3), revealing a powerful layer of post-transcriptional regulation that is able to override a pre-existing transcriptional program (4). Similarly, miR-9 and miR-124 were sufficient to mediate transdifferentiation of human fibroblasts into neurons (5). Accordingly, we are enamored by the RNA world and pay special attention in our investigations to regulatory non-coding RNAs (ncRNAs), particularly miRNAs and long non-coding RNAs (lncRNAs) and how they integrate with known genetic regulatory networks (Fig. 1). With the exception of certain ribozymes, regulatory RNAs generally do not work alone. Instead, they are physically organized as RNA-protein (RNP) complexes. Operationally, RNA-binding proteins (RBPs) and their interactome work in concert as post-transcriptional networks, or RNA regulons, in response to developmental and environmental cues (6). Inspired by this concept and other pioneering studies in the worm, we recently demonstrated that a single RBP Lin28 was sufficient to reprogram adult hematopoietic progenitors to adopt fetal-like properties (7). We discuss these and related findings, which begin to disentangle the complex functions of RBPs in the context of recent advances in post-transcriptional regulation, starting with the discovery of miRNAs.

Fig. 1

Updated model of gene regulation that integrates RBPs and ncRNAs

The Lin28/let-7 circuit: from worm development to lymphopoiesis

Inspiration from the worm

Working in C. elegans, Ambros and Horvitz (8) identified a set of genes that control developmental timing, a category that they termed heterochronic genes. Heterochrony is a term coined by evolutionary biologists and popularized by the worm community to denote events that either positively or negatively regulate developmental timing in multicellular organisms. The discovery of two heterochronic genes, lin-4 and lin-28, which encode a miRNA and RBP respectively, is particularly relevant to this review. The lineage (lin) mutants were previously identified and named because they displayed abnormalities in cell lineage differentiation. Furthermore, some of them were considered heterochronic, as adult mutants harbored immature characteristics (retarded phenotype) or, conversely, larval mutants displayed adult characteristics (precocious phenotype). It was not until 1993 that lin-4 was characterized molecularly, because contrary to popular expectations, the gene did not encode a protein but instead a small RNA now appreciated as the first miRNA to be discovered (9). The lin-4 miRNA acts in part by inhibiting the expression of the LIN-14 transcription factor through imperfect basepairing to sites in the 3′ untranslated region (UTR) of lin-14 mRNA (9, 10). However, it was not apparent initially whether lin-4 or lin-14 is evolutionarily conserved, potentially relegating these findings to be relevant only to the worm. Interestingly, Lin28, a gene conserved in mammals, was later identified to be a direct target of the lin-4 miRNA (11). Lin28 loss-of-function resulted in a precocious phenotype, whereas gain-of-function resulted in a retarded phenotype; thus, Lin28 acts as a heterochronic switch during C. elegans larval development (11).

The possibility that lin-4 may be an oddity of the worm was dissolved with the discovery of the second miRNA, again in C. elegans, let-7 (12). Unlike lin-4, the evolutionary conservation of let-7 from sea urchin to human was quickly appreciated (13). Importantly, expression analysis showed that let-7 expression is temporally regulated from molluscs to vertebrates in all three major clades of bilaterian animals, implying that its role as a developmental timekeeper is conserved (14). This established miRNAs as a field unto its own that has progressed rapidly with the identification of Drosha, Dgcr8, Dicer, and Argonaute (Ago) RBPs as core components of the miRNA pathway (15). Orthologs of lin-4were eventually found in mammals (mir-125a, -b-1, and -b-2) (16) along with hundreds of novel miRNAs from numerous organisms (17). We now recognize that miRNAs, in complex with the RBP Ago, frequently bind their cognate targets via imperfect complementarity to evolutionarily conserved sequences in 3′ UTRs (1820) and mediate post-transcriptional repression (21).


One diverse group of RBPs appreciated to be important in the immune system, even before the discovery of miRNAs, is distinguished by their ability to bind to AU-rich elements (AREs) often found in 3′ UTRs of genes involved in inflammation, growth, and survival. Such RBPs are known as ARE-BPs and have been implicated in mRNA decay, alternative splicing, translation, as well as both alleviating and enhancing miRNA-mediated mRNA repression (104107). Genetic inactivation of several ARE-BPs have been linked to aberrant cytokine expression due to impaired ARE-mediated decay (5, 108111) (Table 1). In addition, deficiency of HuR and AUF1 has uncovered a pro-survival role for both in lymphocytes (112, 113), while ectopic expression of Tis11b (ZFP36L1) negatively regulates erythropoiesis by down-regulating Stat5b mRNA stability (114). The KH-type splicing regulatory protein (KSRP) originally identified as an alternative-splicing factor is a multi-functional RBP. It has been shown to associate with both Drosha and Dicer complexes to positively regulate the biogenesis of a subset of miRNAs including mir-155 and let-7 (73, 108, 115120). In addition, KSRP, like many other ARE-BPs, mediate selective decay of mRNAs by recruitment of exosome complexes to mRNA targets (121) and constitutes a prime example of a multi-functional RBP.


Biological processes involved in the development and function of the immune system require programmed changes in protein production and constitute prime candidates for post-transcriptional regulation. While the ENCODE project initially aimed to identify all functional elements in the human DNA sequence, recent discoveries centered around miRNAs and multi-tasking RBPs, such as Lin28, have highlighted the need for a similar systematic effort in mapping post-transcriptional functional elements within the transcriptome. Integration of genomic, transcriptomic, and proteomic data remains a daunting but necessary task to achieve understanding of the full impact of genetic programs and the enigmatic roles of regulatory RNAs. Mastering the science of (re)programming cell fates promises to unleash the potential of stem cells for Regenerative Medicine.

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Immune System in Perspective

Curator: Larry H. Bernstein, MD, FCAP


How regulatory T cells work
Vignali DAA, Collison LW & Workman CJ
Nature Reviews Immunology 8, 523-532 (July 2008) |   doi:10.1038/nri2343

Regulatory T (TReg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting antitumour immunity. Given that TReg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we describe the basic mechanisms used by TReg cells to mediate suppression and discuss whether one or many of these mechanisms are likely to be crucial for TReg-cell function. In addition, we propose the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate TReg-cell function.

How regulatory T cells work.

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Basic mechanisms used by Treg cells

This schematic depicts the various regulatory T (Treg)-cell mechanisms arranged into four groups centred around four basic modes of action. ‘Inhibitory cytokines’ include interleukin-10 (IL-10), interleukin-35 (IL-35) and transforming growth factor-β (TGF-β). ‘Cytolysis’ includes granzyme-A- and granzyme-B-dependent and perforin-dependent killing mechanisms. ‘Metabolic disruption’ includes high affinity IL-2 receptor α (CD25)-dependent cytokine-deprivation-mediated apoptosis, cyclic AMP (cAMP)-mediated inhibition, and CD39- and/or CD73-generated, adenosine–purinergic adenosine receptor (A2A)-mediated immunosuppression. ‘Targeting dendritic cells’ includes mechanisms that modulate DC maturation and/or function such as lymphocyte activation gene-3 (LAG3; also known as CD223)–MHC-class-II-mediated suppression of DC maturation, and cytotoxic T lymphocyte antigen-4 (CTLA4)–CD80/CD86-mediated induction of indoleamine 2,3-dioxygenase (IDO), which is an immunosuppressive molecule, by DCs.

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Model for how effector T cells might boost Treg-cell function

This occurs in three stages. (a) Initial regulatory T (Treg)-cell activation induces production of regulatory factors such as interleukin-35 (IL-35). (b) Treg cells ‘sense’ the presence of recently activated effector T cells through a receptor–ligand interaction (cell surface or soluble). (c) This in turn boosts or potentiates Treg-cell function resulting in the enhanced production of regulatory mediators, such as IL-35, and perhaps the induction of new mediators.


Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity. Given that Treg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we discuss the basic mechanisms used by Treg cells to mediate suppression, and discuss whether one or many of these mechanisms are likely to be crucial for Tregcell function. In addition, we present the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate Treg-cell function.

Several sophisticated regulatory mechanisms are used to maintain immune homeostasis, prevent autoimmunity and moderate inflammation induced by pathogens and environmental insults. Chief amongst these are regulatory T (Treg) cells that are now widely regarded as the primary mediators of peripheral tolerance. Although Treg cells play a pivotal role in preventing autoimmune diseases, such as type 1 diabetes1,2, and limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease (IBD)3,4, they also block beneficial responses by preventing sterilizing immunity to certain pathogens5,6 and limiting anti-tumour immunity7. A seminal advance in the analysis of Treg cells came with the identification of a key transcription factor, forkhead box P3 (FOXP3), that is required for their development, maintenance and function8,9. Mice and patients that lack FOXP3 develop a profound autoimmune-like lymphoproliferative disease that graphically emphasizes the importance of Treg cells in maintaining peripheral tolerance10-12 (BOX 1). Although FOXP3 has been proposed as the master regulator of Treg cells that controls the expression of multiple genes that mediate their regulatory activity13,14, this has been recently challenged raising the possibility that other transcriptional events may operate upstream of and/or concurrently with FOXP3 to mediate Treg-cell development15.

While Foxp3 has proven to be an invaluable marker for murine Treg cells, its role in human Treg cells is less straightforward (see BOX 2 for a discussion of Treg-cell markers). Humans that lack FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), a severe autoimmune disease that presents early in infancy. Although FOXP3 appears to be required for human Treg-cell development and function, expression of FOXP3 alone is clearly not sufficient as a significant percentage of human activated T cells express FOXP3 and yet do not possess regulatory activity16-20. Furthermore, induction of FOXP3 in human T cells by transforming growth factor-β (TGFβ) does not confer a regulatory phenotype, in contrast to their murine counterparts20. Consequently, FOXP3 is not a good marker for human Treg cells (BOX 2). Whether this distinction is due to intrinsic differences between mouse and human FOXP3 and/or a requirement for an additional cofactor/ transcription factor is an important question that needs to be resolved.

Significant progress has been made over the last few years in delineating the molecules and mechanisms that Treg cells use to mediate suppression21,22. In this Review, we outline our current understanding of the mechanisms used by Treg cells to mediate suppression, and the challenges that lie ahead in defining their mode of action. We also discuss whether Treg cells are likely to depend on one, a few or many of these mechanisms. In addition, we propose that effector T cells may have a significant role in boosting and/or modulating Treg-cell function. Unless stated, we focus here primarily on the mechanisms that are used by thymus-derived natural CD4+CD25+ FOXP3+ Treg cells.

Basic mechanisms of Treg-cell function Defining the mechanisms of Treg-cell function is clearly of crucial importance. Not only would this provide insight into the control processes of peripheral tolerance but it would probably provide a number of potentially important therapeutic targets. Although this quest has been ongoing since interest in Treg cells was reignited in 199523, there has been significant progress in the last few years. From a functional perspective, the various potential suppression mechanisms of Treg cells can be grouped into four basic ‘modes of action’: suppression by inhibitory cytokines, suppression by cytolysis, suppression by metabolic disruption, and suppression by modulation of dendritic-cell (DC) maturation or function (FIG. 1).

Suppression by inhibitory cytokines Inhibitory cytokines, such as interleukin-10 (IL-10) and TGFβ, have been the focus of considerable attention as a mechanism of Treg-cell-mediated suppression. There has also been significant interest in their ability to generate induced (also known as adaptive) Treg-cell populations, either naturally in vivo or experimentally as a potential therapeutic modality (BOX 3). Although the general importance of IL-10 and TGFβ as suppressive mediators is undisputed, their contribution to the function of thymus-derived, natural Treg cells is still a matter of debate24. This is partly due to the general perception that Treg cells function in a contactdependent manner25,26. Indeed, in vitro studies using neutralizing antibodies or T cells that are unable to produce or respond to IL-10 and TGFβ suggested that these cytokines may not be essential for Treg-cell function25-28. However, this contrasts with data from in vivo studies29,30.

In allergy and asthma models, evidence suggests that both natural and antigen-specific Treg cells control disease in a manner that is, in part, dependent on IL-1029 and in some reports dependent on both IL-10 and TGFβ 31. Adoptive transfer of allergen-specific Treg cells induced significant IL-10 production by CD4+ effector T cells in the lung following allergen challenge and this Treg-cell-mediated control of disease was reversed by treatment with an IL-10- receptor-specific antibody32. However, suppression of allergic inflammation and airway hyper-reactivity, and increased production of IL-10 still occurred following transfer of IL-10- deficient Treg cells, suggesting that Treg cells can suppress the Th2-driven response to allergens in vivo through an IL-10-dependent mechanism, but that the production of IL-10 by Treg cells themselves is not required for the suppression observed. This contrasts with a recent study suggesting that the Treg-cell-specific ablation of IL-10 expression resulted in increased lung allergic inflammation and hyperreactivity33.

This scenario might occur in other disease models. For instance, the effects of IL-10 can only be partially attributed to Treg-cell-derived IL-10 in the immune response to hepatitis B virus34 and in the allograft tolerance response elicited by splenocytes exposed to non-inherited maternal antigens35. Recently, it was also shown that IL-10 is crucial for the control of various infections in which Treg cells have been reported to be involved including Mycobacterium tuberculosis36, Toxoplasma gondii37, Leishmania major38, and Trichinella spiralis39. However, Treg cells were not the source of IL-10 in all of these infection models.

By contrast, several studies have shown that IL-10 production by Treg cells is essential for the prevention of colitis in mouse models of IBD40. Moreover, it appears that the tumour microenvironment promotes the generation of FOXP3+ Treg cells that mediate IL-10- dependent, cell-contact independent, suppression41. Similarly, in UV-radiation-induced carcinogenesis, IL-10 production by Treg cells appears to be important for blocking anti-tumour immunity42. IL-10 produced by Treg cells also appears to be crucial for IL-10-mediated tolerance in a model of hepatitis induced by concanavalin A43 and tolerance to bacterial and viral superantigens44. In addition, recent papers suggest new roles for Treg-cell-derived IL-10 in the induction of feto-maternal tolerance45 and B-cell-enhanced recovery from experimental autoimmune encephalomyelitis46. Collectively, the picture that appears to be emerging is that the relative importance of Treg-cell-derived IL-10 is very dependent on the target organism or disease and on the experimental system. Furthermore, the Treg-cell-specific deletion of IL-10 did not result in the development of spontaneous systemic autoimmunity, but did result in enhanced pathology in the colon of older mice and in the lungs of mice with induced airway hypersensitivity, suggesting that the function of Treg-cell-derived IL-10 may be restricted to controlling inflammatory responses induced by pathogens or environmental insults33.

While some early in vitro studies using neutralizing antibodies to TGFβ or Treg cells lacking TGFβ 25,47 indicated that TGFβ was not required for natural Treg-cell function, other studies, both in vitro and in vivo suggested a critical role for Treg-cell surface bound TGFβ 48,49. Therefore, the importance of TGFβ for natural Treg-cell function has also been a controversial topic. Indeed, there has been considerably more focus recently on the importance of TGFβ in the development of induced Treg cells and perhaps in Treg-cell maintenance in general (BOX 3). However, there are studies that suggest that TGFβ produced by Treg cells may directly participate in effector T-cell suppression. For instance, effector T cells that are resistant to TGFβ-mediated suppression cannot be controlled by Treg cells in an IBD model50. In addition, TGFβ produced by Treg cells has been found to be important in the control of the host immune response to M. tuberculosis36, suppression of allergic responses31 and prevention of colitis in an IBD model51. Interestingly, TGFβ produced by Treg cells has also been implicated in limiting anti-tumour immunity in head and neck squamous-cell carcinoma52 and in follicular lymphoma53 by rendering T cells unresponsive to the tumour. TGFβ also appears to limit the anti-tumour activity of cytokine-induced killer cells54.

Membrane-tethered TGFβ can also mediate suppression by Treg cells in a cell-cell contactdependent manner48. Treg cells can control islet infiltration of CD8+ T cells and delay the progress of diabetes through membrane-tethered TGFβ 49. However, experiments using mice deficient in TGFβ-receptor (TGFβR) signalling in effector T cells or using TGFβ or TGFβR blocking reagents failed to show that membrane-tethered TGFβ is required for natural Treg cell development or function47. More recently, however, interest in membrane-tethered TGFβ has re-surfaced with the description of a previously unappreciated role for it in the tumour microenvironment. TGFβ associated with tumour exosome membranes appears to enhance the suppressive function of Treg cells and skew T cells away from their effector functions and towards regulatory functions55. Furthermore, ovalbumin-induced airway inflammation can be attenuated by heme oxygenase-1 through membrane-tethered TGFβ and IL-10 secretion by Treg cells56, a process that activates the Notch1–HES1 (hairy and enhancer of split 1) axis in target cells57. Thus, in light of the most current data, it now appears that soluble and/or membrane-tethered TGFβ may have a previously unappreciated role in natural Treg-cell function.

Recently, a new inhibitory cytokine, IL-35, has been described that is preferentially expressed by Treg cells and is required for their maximal suppressive activity58. IL-35 is a novel member of the IL-12 heterodimeric cytokine family and is formed by the pairing of Epstein–Barr virus induced gene 3 (Ebi3), which normally pairs with p28 to form IL-27, and p35 (also known as  Il12a), which normally pairs with p40 to form IL-12. Both Ebi3 and Il12a are preferentially expressed by murine Foxp3+ Treg cells58,59, but not resting or active effector T cells, and are significantly upregulated in actively suppressing Treg cells58. As predicted for a heterodimeric cytokine, both Ebi3−/− and Il12a−/− Treg cells had significantly reduced regulatory activity in vitro and failed to control homeostatic proliferation and cure IBD in vivo. This precise phenocopy suggested that IL-35 is required for the maximal suppressive activity of Treg cells. Importantly IL-35 was not only required but sufficient, as ectopic expression of IL-35 conferred regulatory activity on naive T cells and recombinant IL-35 suppressed T cell proliferation in vitro58. Although IL-35 is an exciting addition to the Treg-cell portfolio, there is clearly much that remains to be defined about this cytokine and its contribution to Treg-cell function. For instance, it remains to be determined if IL-35 suppresses the development and/or function of other cell types such as DCs and macrophages.

It is now clear that three inhibitory cytokines, IL-10, IL-35 and TGFβ, are key mediators of Treg-cell function. Although they are all inhibitory, the extent to which they are utilized in distinct pathogenic/homeostatic settings differs suggesting a non-overlapping function, which needs further refinement.


How many mechanisms do Treg cells need? Although efforts to define the suppressive mechanisms used by Treg cells continue, an important question looms large. Is it likely that all these molecules and mechanisms will be crucial for Treg-cell function? There are three broad possibilities.

One, a single, overriding suppressive mechanism is required by all Treg cells Until the entire mechanistic panoply of Treg cells is defined, one cannot completely rule out this possibility. However, this possibility would seem unlikely as none of the molecules and/ or mechanisms that have been defined to date, when blocked or deleted, result in the complete absence of regulatory activity — a consequence that one might predict would result in a ‘Scurfy-like’ phenotype (BOX 1). So, although Treg cells that lack a single molecule, for instance IL-10, IL-35 or granzyme B, exhibit significantly reduced suppressor function, a scurfy phenotype does not ensue. Given that none of the current Treg-cell mechanisms can exclusively claim this distinction, it seems unlikely that any ‘unknown’ molecules or mechanisms could do so either.

Two, multiple, non-redundant mechanisms are required for maximal Treg-cell function In the studies conducted to date, Treg cells that lack various suppressive molecules have been shown to be functionally defective. This favours a scenario where there are multiple mechanisms that can be used by Treg cells but they are non-redundant, with each molecule contributing to the mechanistic whole. At present, this possibility would seem plausible. Indeed, this is supported by the recent analysis of mice possessing a Treg-cell-specific ablation of IL-10 expression, in which enhanced pathology was observed following environmental insult33. One would predict that at some point we should be able to generate knockout mice that lack a particular set of genes which results in a complete loss of Treg-cell activity. For this to be truly non-redundant, this list would probably be restricted and small (2–4 genes).

Three, multiple, redundant mechanisms are required for maximal Treg-cell function With the plethora of regulatory mechanisms described to date and the possibility of more yet to be identified, it is conceivable that there are multiple mechanisms that function redundantly. Such a redundant system would help to mitigate against effector T-cell escape from regulatory control. Also, given the very small size of the Treg-cell population, a sizable arsenal may be required at the height of an effector T-cell attack. Of course, it is possible that a semi-redundant scenario exists.

These possibilities have been discussed from the perspective of there being a single homogeneous Treg-cell population. However, as for helper T cell subsets it remains possible that a few or even many different Treg-cell subsets exist24. Each of these may rely on one or multiple regulatory mechanisms. Several recent studies have provided support for both phenotypic and functional heterogeneity amongst Treg cells. For instance, it has recently been shown that a small sub-population of Treg cells express the chemokine receptor CCR6, which is associated with T cells possessing an effector-memory phenotype102. CCR6+ Treg cells appeared to accumulate in the central nervous systems of mice with experimental autoimmune encephalomyelitis (EAE) suggesting that they may have a prevalent role in controlling responses in inflamed tissues. Heterogeneous expression of HLA-DR has also been suggested to mark different subpopulations of functionally distinct human Treg cells103. Indeed, HLADR positive Treg cells were found to be more suppressive than their DR negative counterparts. One might speculate that their enhanced inhibitory activity is due to DR-mediated ligation of the inhibitory molecule LAG3 expressed by activated effector T cells95,96.

So, if multiple suppressor mechanisms exist, how might these be integrated and used productively by Treg cells in vivo? We would propose the following possible models21. First, a ‘hierarchical’ model in which Treg cells possess many mechanisms that could be used but only one or two that are really crucial and consistently important in a variety of regulatory settings. Second, a ‘contextual’ model where different mechanisms become more or less important depending on the background or context in which the Treg cells reside and the type of target cell that they have to repress. For example, some cell types may be inhibited primarily by cytokines, whereas others are most effectively suppressed through lysis by Treg cells. Alternatively, different mechanisms may be more effective in different tissue compartments or in different disease settings. This notion is supported by the recent analysis of mice in which IL-10 expression was specifically ablated in Treg cells33. Whereas Treg-cell-derived IL-10 was not required for the systemic control of autoimmunity, it did seem to be required from the control of inflammatory events at mucosal interfaces such as the lungs and colon. As a clear picture of the available Treg-cell weaponry emerges, an important challenge will be to determine their relative importance and contribution to Treg-cell function in different disease models.

A hypothesis: effector T cells potentiate Treg-cell function? Most cellular interactions within the immune system are bidirectional, with molecular signals moving in both directions even though the interaction has broader unidirectional intentions (for example, CD4+ T-cell help). However, to date the general perception is that Treg cells suppress and effector T cells capitulate. We hypothesize that this is in fact an incomplete picture and that effector T cells have a very active role in their own functional demise. Three recent observations support this view. First, we have recently examined the molecular signature of activated Treg cells in the presence and absence of effector T cells and were surprised to find that it was strikingly different, with hundreds of genes differentially modulated as a consequence of the presence of effector T cells (C.J.W. and D.A.A.V., unpublished observations). Second, we have shown that Ebi3 and Il12a mRNA are markedly upregulated in Treg cells that were co-cultured with effector T cells, supporting the idea that effector T cells may provide signals which boost IL-35 production in trans58. Third, we found that Treg cells were able to mediate suppression of effector T cells across a permeable membrane when placed in direct contact with effector T cells in the upper chamber of a Transwell™ plate (L.W.C. and D.A.A.V., unpublished observations). Interestingly, this suppression was IL-35 dependent, as Ebi3−/− Treg cells were unable to mediate this ‘long-distance’ suppression. Collectively, these data suggest that it is the ‘induction’, rather than the ‘function’, of Treg-cell suppression that is contact-dependent and that effector T cells have an active role in potentiating Treg-cellmediated suppression. Therefore, we hypothesize that receptor–ligand interactions between the co-cultured CD4+ effector T cells and Treg cells initiate a signalling pathway that leads to enhanced IL-35 secretion and regulatory activity (FIG. 2). While the molecule that mediates this enhanced Treg-cell suppression is unknown, it is possible that IL-2 may serve this function104. Given the contrasting genetic profiles of activated Treg cells in the presence and absence of effector T cells, it seems possible that this interaction may boost the expression of other regulatory proteins. It may well be that effector T cells unwittingly perform the ultimate act of altruism.

Concluding remarks Although significant progress has been made over the last few years in defining the mechanisms that Treg cells use to mediate their suppressive function, there is clearly much that remains to be elucidated and many questions persist. First, are there more undiscovered mechanisms and/ or molecules that mediate Treg-cell suppression? What is clear is that the transcriptional landscape of Treg cells is very different from naive or activated effector T cells. There are literally thousands of genes that are upregulated (or downregulated) in Treg cells compared with effector T cells. Although it seems unlikely that all or many of these will be crucial for Treg-cell function, it is quite possible that a few undiscovered genes might be important. It should be noted that although we are discussing mechanisms here, it is clear that some of these molecules may perform key Treg-cell functions, such as Treg-cell homing and homeostasis, which are likely to indirectly influence their suppressive capacity in vivo but don’t directly contribute to their inhibitory activity. It is also possible that some of these unknown molecules may represent more specific markers for the characterization and isolation of Treg cells, a particularly important issue for the analysis and use of human Treg cells (BOX 2).

Second, which mechanisms are most important? An important but potentially complex challenge will be to determine if a few mechanisms are important in many Treg-cell settings or whether different mechanisms are required in different cellular scenarios. At present it is difficult to assess this objectively as these mechanisms have predominantly been elucidated in different labs using distinct experimental systems and thus none have really been compared in side-by-side experiments. Furthermore, only recently have conditional mutant mice been examined that have a regulatory component specifically deleted in Treg cells33.

It almost goes without saying that although defining the Treg-cell mode of action is of great academic importance, it is also essential in order to develop effective approaches for the clinical manipulation of Treg cells. Given the capacity of Treg cells to control inflammation and autoimmunity, and their implication in blocking effective anti-tumour immunity and preventing sterilizing immunity, it seems probable that a clear understanding of how Treg cells work will present definitive opportunities for therapeutic intervention.

Box 1 Scurfy mice: misplaced mechanistic expectations?

Mice that carry a spontaneous loss-of-function mutation (known as Scurfy mice) or a deletion of Foxp3 develop a fatal autoimmune-like disease with hyperresponsive CD4+ T cells9,12. More recently Foxp3:diptheria toxin receptor (DTR) knockin mice have allowed for the selective depletion of Treg cells following DT treatment105. These mice have been invaluable for dissecting the role of Foxp3 in Treg-cell function. Given the profound phenotype in these mice, there is a general expectation that genetic disruption of any key Treg-cell inhibitory molecule or mechanism would probably result in a Scurfy-like phenotype. Of course, it is also possible that deletion of a key Treg-cell gene may be more synonymous with DT-mediated Treg-cell depletion where Foxp3 may still serve to prevent expression of proinflammatory cytokines105. Nonetheless, this has lead to the notion that if mutant mice don’t have a Scurfy-like or a Treg-cell-depleted phenotype, then the disrupted gene probably isn’t important for Treg-cell function. This may not necessarily be correct. Indeed, it is possible that no mouse lacking a Treg-cell inhibitory effector molecule will ever be generated that develops a profound, spontaneous autoimmune disease21. It should be noted that mutant mice that are Helicobacter spp. and/or Citrobacter rodentium positive may have an exacerbated phenotype, as several studies have shown that opportunistic enteric bacteria can significant exacerbate gut pathology4. Ultimately, the occurrence of disease in knockout mice will depend on whether Treg cells rely on a single or multiple suppressive mechanisms. Given the number of genes induced or modulated by FOXP3, it is probable that a programme of intrinsic and extrinsic regulation is induced that involves multiple proteins9,13. Therefore, it would not be surprising if deletion of a single molecule does not provoke the profound Scurfy-like phenotype seen in mice that lack Foxp3.

Box 2. Treg-cell markers

Identifying discriminatory cell surface markers for the characterization and isolation of Treg cells has always been a critical goal. Although excellent markers exist for murine Treg cells, this goal has remained elusive for human Treg cells. Traditionally, murine and human Treg cells have been characterized as CD4+CD25+ (also known as interleukin-2 receptor α (IL-2Rα)). Indeed, murine Treg cells can be effectively isolated based on staining for CD4+CD25+CD45RBlow expression. However, the purity of isolated human Treg cells has always been an issue because T cells up-regulate CD25 upon activation106. Indeed, during the influenza or allergy season a substantial proportion of human CD4+ T cells can express CD25. Although the identification of forkhead box P3 (Foxp3) as a key regulator of Treg-cell development and function has facilitated their identification in the mouse8, many activated (non-regulatory) human T cells express FOXP3, precluding it as a useful marker for human Treg cells16-20. Consequently, the search for Treg-cell-specific cellsurface markers, particularly in humans, has continued in earnest with a growing number of candidates proposed (reviewed by Zhao and colleagues107). For instance, it was shown that the expression of CD127 (also known as IL-7R) is down-regulated on Treg cells and that this could be used to increase the purity of human Treg-cell isolation. Indeed, there is a 90% correlation between CD4+CD25+CD127low T cells and FOXP3 expression108, 109. In addition, it was recently found that Treg cells expressed a higher level of folate receptor 4 (FR4) compared with activated effector T cells110. It is also important to recognize that Treg cells, like their T helper cell counterparts, may be heterogeneous and thus a collection of cell surface markers could facilitate their isolation and functional characterization. Indeed, such heterogeneity has recently been described based on differential expression of HLA-DR or CCR6102,103. However, the general use of both markers remains to be fully established so it is quite probable that the search for better Treg-cell markers will continue for some time.

Box 3 Induced or adaptive Treg cells: development and mode of action

Naturally occurring FOXP3+CD4+CD25+ Treg cells develop in the thymus and display a diverse T-cell receptor (TCR) repertoire that is specific for self-antigens111,112. However, Treg cells can also be ‘induced’, ‘adapted’ or ‘converted’ from effector T cells during inflammatory processes in peripheral tissues, or experimentally generated as a possible therapeutic29,113,114. For instance, T regulatory 1 cells (Tr1) and T helper 3 cells (Th3) can be generated experimentally by, and mediate their suppressive activity through interleukin-10 (IL-10) and transforming growth factor-β (TGFβ), respectively114,115. Typically, these regulatory populations do not express FOXP3. In vivo, it has recently been suggested that stimulation of mouse effector T cells by CD103+ dendritic cells (DCs) in the presence of TGFβ and retinoic acid induces the generation of Foxp3+ T cells in the gutassociated lymphoid tissue (GALT)116-121. Furthermore, Treg cells can be preferentially induced in the periphery by exposure to αVβ8-integrin-expressing DCs122 or suppressor of cytokine signalling 3 (Socs3) −/− DCs123. Interestingly, independent of its role in generating induced Treg cells, TGFβ may also have an important role in helping to maintain Foxp3 expression in natural Treg cells124, a process that can be blocked by IL-4 or interferon-γ (IFNγ) 125. In contrast to mouse T cells, FOXP3 induction by TCR stimulation in the presence of TGFβ in human T cells does not confer a regulatory phenotype20. The mechanism of action of adaptive Treg cells may not necessarily be restricted to suppressive cytokines. Indeed, human adaptive Treg cells (CD4+CD45RA+ T cells stimulated with CD3- and CD46-specific antibodies) have also been shown to express granzyme B and killing target cells in a perforin-dependent manner126. Treg cells often have a restricted specificity for particular cell types, tumours or foreign antigens127. Therefore, induced Treg cells may be ideally suited to respond to infectious agents. This may also be of particular importance in the GALT and in the tumour microenvironment where TGFβ drives the conversion of induced Treg cells118,128. A significant challenge in deciphering data from in vivo experiments is to assess the contribution of natural Treg cells versus induced Treg cells, and to determine whether inhibitory molecules, such as IL-10 or TGFβ, were derived from the former or the latter (or elsewhere).



Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers.

Keisuke Kataoka, Yuichi Shiraishi, Yohei Takeda, Seiji Sakata, et al.
Nature may 23,2016; http://dx.doi.org:/10.1038/nature18294

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development1, 2, 3, 4, 5, 6. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma6, 7, 8, 9, 10. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3′ region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3′-untranslated region (UTR). Disruption of the Pd-l1 3′-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3′-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.


Viruses are a dominant driver of protein adaptation in mammals.

David Enard, Le Cai, Carina Gwennap and Dmitri A Petrov.
eLife May 16, 2016; 5:e12469. http://dx.doi.org/10.7554/eLife.12469

Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes.


Purdue scientists use adaptors to advance CAR-T therapy

by Oliver Worsley | May 25, 2016


Chimeric antigen receptor (CAR) T cells, developed in the 1990s, are a genetically engineered type of T cell that can target a specific cancer. Now, scientists at Purdue University say they’ve made improvements in this strategy–overcoming the several limitations of traditional CAR-T therapy.

Purdue professor of chemistry Philip Low and his team presented their findings at the American Association for Cancer Research meeting in New Orleans last month.

T cells are a type of immune cell that recognizes and clears the body of invading cells or pathogens, like cancer. They are fine-tuned by the immune system in order to specifically target and kill these foreign invaders–but cancer cells may respond by jumping these safety barriers.

CAR-T therapy was therefore proposed and has been recently used for cancer treatment. It has been hailed for its promising remission rates after early stage clinical trials for acute lymphoblastic leukemia.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said in a university news release. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

They did this by teaming up with Endocyte ($ECYT) scientist Haiyan Chu and designing CAR T cells that require activation by a small molecule adaptor before proceeding. In this way, they can carefully control the amount of active CAR T cells in the circulation.

So far, they have only tried the novel therapy in animal models, but when they tested it in mice they observed antitumor activity only when both the CAR T cells and the correct adaptor molecules were present.

Low believes it will allow clinicians to target multiple cancer subtypes at once. “Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said in the release. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

– here’s the release

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Read More: CAR-T   Cancer


Purdue research may expand engineered T-cell cancer treatment



A graphic depicting the activation and inactivation of CAR T cells through a small molecule adaptor is shown. Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery, and graduate student Yong Gu Lee led a team that designed new engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The system has the potential to control the engineered cells to overcome existing limitations in CAR T-cell therapy. CREDIT Purdue University image courtesy of Yong Gu Lee

Purdue University researchers may have figured out a way to call off a cancer cell assassin that sometimes goes rogue and assign it a larger tumor-specific “hit list.”

T cells are the immune system’s natural defense against cancer and other harmful entities in the human body. However, the cells must be activated and taught by the immune system to recognize cancer cells in order to seek out and destroy them. Unfortunately, many types of cancer manage to thwart this process.


In the 1990s scientists found a way to genetically engineer T cells to recognize a specific cancer. These engineered T cells, called chimeric antigen receptor, or CAR, T cells, have been recently used as treatment for cancer, said Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery who led the work.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

Low and Purdue graduate student Yong Gu Lee collaborated with Endocyte Inc. scientist Haiyan Chu to design genetically engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The technology has been tested in animal models but no human trials have been performed. A poster presentation describing the work was presented Tuesday (April 19, 2016) at the American Association for Cancer Research annual meeting in New Orleans.

“While the traditional CAR T cells could remain and replicate in the human body for many years, the adaptors we have created are expected to be excreted fairly quickly,” Lee said. “By controlling the level of adaptors in the system, we can control the numbers and potencies of active CAR T cells. Those that aren’t stimulated by an adaptor molecule are blind and do not recognize or target any cells. Eventually, if they remain inactive for a while, they should die and be eliminated from the body.”

A study in mice showed the anti-tumor activity was induced only when both the engineered CAR T cell and the correct adaptor molecules were present.

The system also offers the potential to treat multiple cancer subtypes at once, Low said.

“Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

The adaptor molecule serves as a bridge between the CAR T-cell and the cancer cell. It is made with a yellow dye called fluorescein isothiocyanate on one end, to which the engineered CAR T cells have been designed to bind, and a cancer-targeting molecule on the other.

Low’s research has focused on the design and synthesis of technologies for targeted delivery of therapeutic and imaging agents to treat cancer, inflammatory and autoimmune diseases, and infectious diseases.

He has developed molecules that target folate-receptors and prostate-specific membrane antigen on the surfaces of cancer cells. Approximately 85 percent of ovarian cancers; 80 percent of endometrial and lung cancers; and 50 percent of breast, kidney and colon cancers express folate receptors on their cellular surfaces. Prostate-specific membrane antigen receptors are found on nearly 90 percent of all prostate cancers. Other tumor-specific ligands developed by Low’s lab can target each of the other major human cancers, he said.

Each CAR T cell has thousands of receptors on its surface to which an adaptor molecule can bind. One CAR T cell could have a variety of adaptor molecules bound to its surface and the cancer cell it targets will depend on which of those adaptors first encounters a targeted cancer cell. Once the CAR T cell binds to a cancer cell, it begins the process of destroying it. When that process is complete, the CAR T cell is released and can bind to a new cancer cell, he said.

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

In addition to Low, Chu and Lee, members of the research group include Purdue postdoctoral research associates at the time of the study Srinivasarao Tenneti and Ananda Kumar Kanduluru.

Drug discovery is one of the priorities within Purdue Moves, an initiative designed to broaden the university’s global impact and enhance educational opportunities for its students. All of the moves fall into four broad categories: science, technology, engineering and math (STEM) leadership; world-changing research; transformative education; and affordability and accessibility.

The Purdue University Center for Drug Discovery supports more than 100 faculty in six colleges with research focused on several major disease categories: cancer; diabetes, obesity and cardiovascular; immune and infectious disease; and neurological disorders and trauma.

The center and drug discovery initiative builds upon Purdue’s strengths along all points of the drug discovery pipeline, including 14 core units to provide shared resources for analysis, screening, synthesis and testing of potential therapeutic compounds.

With more than 44 Purdue-developed compounds at various stages of preclinical development, and 16 in human clinical trials, Purdue is among the most productive universities in the world of drug discovery.

The center also is aligned with the university’s recently announced $250 million investment in the life sciences.

Endocyte Inc., a Purdue Research Park-based company that develops receptor-targeted therapeutics for the treatment of cancer and autoimmune diseases, funded the study, holds exclusive rights to the technology and assisted Purdue researchers in the development of the technology. Low is a founder and chief science officer of Endocyte Inc. and serves on the Endocyte board of directors.

AACR press release: http://www.aacr.org/Newsroom/Pages/News-Release-Detail.aspx?ItemID=874#.VxZFs2N8V0c

Endocyte press release: http://investor.endocyte.com/releasedetail.cfm?ReleaseID=965753


A Universal Remedy for CAR T Cell Limitations

Yong Gu Lee, Haiyan Chu, Srinivasarao Tenneti, Ananda Kumar Kanduluru, Philip S. Low

Chimeric antigen receptor (CAR) T cells show significant potential for treating cancer due to their tumor-specific activation and ability to focus their killing activity on cells that express a tumor antigen. Unfortunately, this promising therapeutic technology is still limited by: (1) an inability to control the rate of cytokine release and tumor lysis; (2) the absence of an “off switch” that can terminate cytotoxic activity when tumor eradication is complete; (3) a failure to eliminate tumor cells that do not express the targeted antigen; and (4) a requirement to generate a different CAR T cell for each unique tumor antigen. In order to address these limitations, we have exploited a low molecular weight bi-specific adaptor molecule that must bridge between the CAR T cell and its targeted tumor cell by simultaneously binding to the chimeric antigen receptor on the CAR T cell and the unique antigen on the tumor. Using this bispecific adaptor, one can control CAR T cell cytotoxicity by adjusting the concentration and rate of administration of the adaptor. Because the half life of the adapter is <20 minutes in vivo, termination of CAR T cell killing can be accomplished by cessation of adapter administration. Moreover, when heterogeneous tumors containing cells that express orthogonal antigens must be treated, the same CAR T cell can be targeted to multiple antigens by attachment of the same CAR ligand to the appropriate selection of tumor-specific ligands. Finally, when the targeted tumor antigen is also expressed at low levels on normal cells, tumor specificity can be achieved by adjusting the affinity of the tumor-specific ligand to enable CAR T cell engagement only when a highly multivalent interaction is possible. To experimentally demonstrate the aforementioned benefits of using low molecular weight bispecific adaptors, CAR T cells were constructed by fusing an anti-fluorescein isothiocyanate (FITC) scFv to a CD3 zeta chain containing the intracellular domain of CD137 (i.e. CAR4-1BBZ T cells). Then, to enable their tumor-specific cytotoxicity, a bispecific adaptor molecule comprised of fluorescein linked to a small organic ligand with high affinity and specificity for a tumor-specific antigen (FITC-SMC) was synthesized. For these studies, the tumor-specific ligands were: i) folate for recognition of the folate receptor that is over-expressed on ~1/3 of human cancers, ii) DUPA for binding to prostate specific membrane antigen that is over-expressed on prostate cancers, and iii) NK-1R ligand that is over-expressed on neuroendocrine tumors. The ability of the same clone of CAR4-1BBZ T cells to eliminate tumors expressing each of the above antigens was then demonstrated by administration of the desired FITC-SMC to mice injected with the CAR4-1BBZ T cells. Our data show that anti-tumor activity: i) is only induced when both CAR4-1BBZ T cells and the correct antigen-specific FITC-SMC are present, ii) anti-tumor activity and toxicity can be sensitively controlled by adjusting the dosing of FITC-SMC, and iii) treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired FITC-SMCs. Taken together, these data show that many of the limitations of CAR T cell technology can be addressed by use of a bi-specific adaptor molecule to mediate tumor cell recognition and killing.



CTLA-4 found in dendritic cells suggests New cancer treatment possibilities

Matthew Halpert, et al. Dendritic Cell Secreted CTLA-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Stem Cells and Development, 2016; http://dx.doi.org:/10.1089/scd.2016.0009

Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the “general” leading T cells “soldiers” to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.

  Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Halpert MM1, Konduri V1, Liang D1, Chen Y1, Wing JB2, Paust S3,4, Levitt JM1,5, Decker WK1,6.  Stem Cells Dev. 2016 May 15;25(10):774-87. doi: 10.1089/scd.2016.0009. Epub 2016 May 2.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.


Non-invasive strategy to guide personalized cancer immunotherapy

Cancer immunotherapy is the rising hope to offer ultimate solutions for cancer. Neoantigens, derived from products of mutated genes in tumor cells, are found to be closely related to the efficacy of cancer immunotherapies. A non-invasive approach to identify unique, patient-specific neoantigens has been advanced by Dr. Steven Rosenberg’s group. A recent article published in Nature Medicine reported that a small population of circulating CD8+PD-1+ tumor-reactive T lymphocytes can be used to identify neoantigens, in addition to tumor-infiltrating T cells. The study paves the way for designing personalized cancer immunotherapy with a novel non-invasive approach.

Gros, A. et al.
Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.
Nat. Med. (2016)   http://dx. doi.org:/10.1038/nm.4501

Detection of lymphocytes that target tumor-specific mutant neoantigens-derived from products encoded by mutated genes in the tumor-is mostly limited to tumor-resident lymphocytes, but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(-), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8+ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8+ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8+lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8+ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8+PD-1+ compared with CD8+PD-1 TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8+ and the CD8+PD-1+ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8+ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

Cancer immunotherapy has experienced major progress in the last decade. Adoptive transfer of ex vivo–expanded tumor-infiltrating lymphocytes (TILs) can cause substantial regression of metastatic melanoma (1, 2). Blockade of the interaction of cytotoxic T lymphocyte antigen 4 (CTLA-4; also known as CD152) or programmed cell death 1 receptor (PD-1; also known as CD279) with their ligands using blocking antibodies alone or in combination have been shown to unleash an otherwise-ineffective immune response against melanoma (37), renal cell carcinoma (3), and non–small cell lung cancer (3). The antitumor responses observed in these clinical trials support the presence of naturally occurring tumor-reactive CD8+ T cells and their immunotherapeutic potential. In the particular case of TIL therapy, persistence of transferred tumor-specific T cell clones is associated with tumor regression (8). Moreover, retrospective clinical studies have shown an association of autologous tumor recognition by TILs and clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. However, the identification of the diverse repertoire of tumor-reactive cells limits the ability to study these cells, enhance clinical efficacy, and extend this therapy to other malignancies.

Melanoma TILs represent a heterogeneous population that can target a variety of antigens, including melanocyte differentiation antigens, cancer germline antigens, self-antigens overexpressed by the tumor, and mutated tumor neoantigens (11). The latter appear to be of critical importance for the antitumor responses observed after transfer of TILs, given the substantial regression of metastatic melanoma in up to 72% of patients in phase 2 clinical trials, in the absence of any autoimmune side effects in the great majority of patients (2). This contrasts with the modest antitumor activity but high prevalence of severe autoimmune manifestations observed after transfer of peripheral blood gene-engineered T cells expressing TCRs targeting shared melanocyte differentiation antigens MART1 and gp100 (12,13). Furthermore, T cells targeting mutated neoepitopes are not subject to negative selection in the thymus and may constitute the predominant naturally occurring tumor-reactive population in cancer patients. In support of this notion, a recent study reported the frequent detection and dominance of T cell populations targeting mutated epitopes in melanoma-derived TILs (14). Conversely, T cells targeting shared melanocyte differentiation antigens and cancer germline antigens in bulk melanoma TILs were represented at a strikingly low frequency (15). These findings have shifted our interest from the more accessible and commonly studied T cells targeting melanocyte differentiation antigens to T cells targeting unique patient-specific mutations. However, the often rare availability of autologous tumor cell lines necessary to study these reactivities, and the hurdles associated with the identification of the unique mutations targeted, have thus far hindered immunobiological studies of these T cell populations in the tumor.

Naturally occurring tumor-reactive cells are exposed to their antigen at the tumor site. Thus, the immunobiological characterization of T cells infiltrating tumors represents a unique opportunity to study their function and to identify the patient-specific repertoire of tumor-reactive cells. TCR stimulation triggers simultaneous upregulation of both costimulatory and coinhibitory receptors, which can either promote or inhibit T cell activation and function. Expression of the inhibitory receptors PD-1, CTLA-4, lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) is regulated in response to activation and throughout differentiation (16, 17). Chronic antigen stimulation has been shown to induce coexpression of inhibitory receptors and is associated with T cell hyporesponsiveness, termed exhaustion (18). Exhaustion in response to persistent exposure to antigen was first delineated in a murine model of chronic lymphocytic choriomeningitis virus (19), but has been observed in multiple human chronic viral infections (2022) as well as in tumor-reactive MART1-specific TILs (23, 24), and has provided the rationale for restoring immune function using immune checkpoint blockade. Conversely, 4-1BB (also known as CD137) is a costimulatory member of the TNF receptor family that has emerged as an important mediator of survival and proliferation, particularly in CD8+ T cells (2527). 4-1BB is transiently expressed upon TCR stimulation, and its expression has been used to enrich for antigen-specific T cells in response to acute antigen stimulation (28). However, expression of this marker has not been extensively explored in CD8+ lymphocytes infiltrating human tumors. In addition to changes in the expression of cosignaling receptors on the surface of T cells, antigen-specific stimulation typically results in clonal expansion. TCR sequence immunoprofiling can be used to monitor T cell responses to a given immune challenge even without a priori knowledge of the specific epitope targeted, through determination of the abundance of specific clonotypes (29, 30). However, there is limited knowledge regarding the TCR repertoire and the frequency of tumor-reactive clonotypes infiltrating human tumors.

We hypothesized that the assessment of unique phenotypic traits expressed by CD8+ TILs and TCR β chain (TCRβ; encoded by TRB) clonotypic immunoprofiling of lymphocytes infiltrating the tumor could provide a powerful platform to study antitumor T cell responses and evaluated their usefulness in identifying the diverse repertoire of tumor-reactive cells. Despite the accepted negative regulatory role of PD-1 in T cells, our findings establish that expression of PD-1 on CD8+ melanoma TILs accurately identifies the repertoire of clonally expanded tumor-reactive, mutation-specific lymphocytes and suggest that cells derived from this population play a critical role in tumor regression after TIL administration.

PD-1 was initially described to be expressed on a T cell hybridoma undergoing cell death (37). Its negative effect on T cell responses was first delineated in PD-1 knockout mice (38, 39). Since then, PD-1 expression and coexpression of other inhibitory receptors such as CTLA-4, TIM-3, BTLA, CD160, LAG-3, and 2B4 have become a hallmark of chronically stimulated T cells during chronic infection or in the tumor microenvironment. This altered phenotype, and the interaction of these receptors with their corresponding ligands on target cells, is associated with impaired proliferation and effector function frequently referred to as exhaustion (18, 24, 40). Expression of PD-1 in patients with chronic viral infections correlates with disease progression (22, 41). Additionally, CD8+ lymphocytes targeting melanoma differentiation antigens in the tumor express PD-1, CTLA-4, TIM-3, and LAG-3 and exhibit impaired IFN-γ and IL-2 secretion (23, 24), supporting a negative regulatory role of PD-1 and inhibitory receptors in naturally occurring T cell responses to cancer and providing a rationale for the treatment of cancer with immune checkpoint inhibitors.

In the present study, we found that expression of PD-1 on CD8+ melanoma TILs captured the diverse repertoire of clonally expanded tumor-reactive lymphocytes. TCRβ sequencing revealed that tumor-reactive and mutation-specific clonotypes were highly expanded in the CD8+ population and preferentially expanded in the PD-1+ population. This is consistent with the TCR stimulation-driven expression of this receptor on T cells (42). The inhibitory receptors TIM-3 and LAG-3 and the costimulatory receptor 4-1BB were also expressed on CD8+PD-1+ TILs and could also be used to enrich for tumor-reactive cells. PD-1 was consistently expressed at a higher frequency and was found to be more comprehensive at identifying the diverse repertoire of tumor-reactive cells infiltrating melanoma tumors, although the less frequent PD-1/TIM-3+ and PD-1/LAG-3+ subpopulations could also represent tumor-reactive cells (Supplemental Figure 4 and Supplemental Table 6). Additionally, previous studies from our laboratory showing coexpression of PD-1 and CTLA-4 (23), and our preliminary data supporting coexpression of PD-1 and ICOS (Supplemental Figure 5), suggest that other receptors may also be used to distinguish tumor-reactive cells. Our present results further support immunotherapeutic intervention using immune checkpoint blockade using PD-1, TIM-3, and LAG-3 blocking antibodies or 4-1BB agonistic antibody to restore the function of tumor-reactive lymphocytes, which is currently being actively pursued in the clinic (3, 4, 6, 7, 43). The potential cooperative mechanisms of inhibition of these receptors when engaged with their ligands (44, 45) suggests that the combined targeting of different inhibitory receptors can further enhance antitumor efficacy, as already shown with the combination of anti–PD-1 and anti–CTLA-4 (5). Our present results demonstrate that PD-1 identifies the clonally expanded CD8+ tumor-reactive population and suggest that expression of PD-1 on CD8+TILs could function as a potential predictive biomarker of antitumor efficacy using immune checkpoint inhibitors.

Naturally occurring tumor-reactive cells play a pivotal role in mediating antitumor responses after TIL transfer. Currently, expansion of TILs for patient treatment involves nonspecific growth of TILs from tumor fragments in IL-2, and the diversity and frequency of antitumor T cells present in the final T cell product used for treatment remains largely uncharacterized. Prospective clinical studies have reported that in vitro recognition of autologous tumor by TILs is associated with a higher probability of clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. This is consistent with the idea that both tumor-reactive and non–tumor-reactive cells may compete for cytokines in vivo, especially in the absence of vaccination. However, the isolation of the patient-specific repertoire of tumor-reactive cells is not possible with current technologies (14, 28, 4650). Our findings established that expression of PD-1, TIM-3, LAG-3, and 4-1BB in CD8+ TILs can be used to enrich for tumor-reactive cells, regardless of the specific antigen targeted. One potential concern with isolating T cells expressing inhibitory receptors for therapy is that these cells may be exhausted or functionally impaired (23, 24, 44, 51, 52). However, we found that PD-1+, TIM-3+, and LAG-3+ CD8+ cells expanded in IL-2 were capable of secreting IFN-γ and lyse tumor in vitro. This supports the notion that immune dysfunction associated with coexpression of inhibitory receptors on CD8+ TILs can be reversed (21, 41, 51, 53), and may enable the reproducible enrichment of tumor-reactive cells for patient treatment. Notably, in a preliminary experiment (n= 8 nonresponders; 14 responders), there was no association between the frequency of expression of any of the markers studied in the CD8+ TILs in the fresh tumor and the clinical response to TILs derived from these tumor samples. However, the fresh tumors included in this study belonged to patients treated in several TIL protocols over the course of 10 years, and TILs were generated from these tumors using different methods, which makes these data difficult to interpret. In addition, the frequency of cells initially expressing PD-1 in the tumor may not reflect the frequency of the PD-1 derived cells in the infusion bag. For example, a low frequency of PD-1+ cells may be highly enriched during the process of TIL culture as a result of the presence of tumor cells. Although in vivo antitumor activity of tumor-isolated TILs based on PD-1 expression requires testing in a clinical trial, the observation that the overwhelming majority of tumor-reactive cells were derived from cells expressing PD-1 suggests that cells expressing PD-1 and inhibitory receptors in the tumor play a critical role in tumor regression after TIL administration.

The functional implications of selecting PD-1–, LAG-3–, TIM-3–, or 4-1BB–expressing T cells to enrich for tumor-reactive cells for patient treatment remain unclear. Although previous studies have reported differential expression of PD-1, LAG-3, and TIM-3 throughout differentiation (17), or preferential expression of TIM-3 in IFN-γ–secreting cells (54), our preliminary results have failed to show consistent phenotypic or functional differences between PD-1+, LAG-3+, TIM-3+, and 4-1BB+ selected TILs, including cytokine secretion, proliferation, and susceptibility to apoptosis (data not shown). We found that PD-1 expression was almost completely lost in the PD-1+ derived populations upon in vitro culture in IL-2. Conversely, TIM-3 and LAG-3 expression increased in the TIM-3 and LAG-3 populations after expansion. Overall, there were no differences in the expression of PD-1, TIM-3, or LAG-3 between any the populations after expansion. Thus, in agreement with previous reports (55, 56), we conclude that expansion in IL-2 alters the expression of these markers and compromises the potential use of inhibitory receptors to select for tumor-reactive cells after in vitro expansion. Recent work in animal models suggests that chronic antigen stimulation (5759) or a tolerizing microenvironment (60) may lead to permanent epigenetic changes in T cells, raising the possibility that the restoration of function observed in previously exhausted or tolerized cells in presence of cytokines may only be transient. These results have not yet been corroborated in human tumor-specific cells. However, given that the overwhelming majority of tumor-reactive cells appear to derive from cells expressing PD-1 in the tumor, studying permanent versus transient reversion of exhaustion may have important implications for adoptive cell transfer of TILs.

Tumor-reactive cells can also be found infiltrating other tumor malignancies, such as renal cell carcinoma (61) or ovarian (62), cervical (63), or gastrointestinal tract cancers (64), albeit at lower frequencies. Our findings provide alternatives to enrich and study tumor-reactive CD8+ TILs through selection of cells expressing the cell surface receptors PD-1, LAG-3, TIM-3, and 4-1BB, a hypothesis that we are actively investigating. Additionally, our present findings showed that the frequency of a specific clonotype in the CD8+ and PD-1+ populations can be used to predict its ability to recognize tumor and isolate tumor-specific TCRs, thus providing means to overcome potential irreversible functional impairments of TILs (52).

2 reports with opposing results have generated controversy regarding which may be the optimal marker for the identification of the tumor-reactive repertoire, PD-1 or 4-1BB. In one report studying PD-1 expression in the tumor, the authors showed promising although inconsistent ability to enrich for shared melanoma-reactive cells (55). In a more recent article studying the role of 4-1BB in fresh ovarian TILs, Ye et al. concluded that expression of 4-1BB, but not PD-1, on lymphocytes defines the population of tumor-reactive cells in the tumor (65). The results of Ye et al. appear to contradict our present findings, showing that expression of PD-1 rather than 4-1BB more comprehensively identifies the repertoire of tumor-reactive cells in the tumor. However, these inconsistencies can be explained by different experimental approaches undertaken to study the immunobiology of TILs. First, Ye et al. found that expression of 4-1BB in fresh ovarian TILs and tumor-associated lymphocytes was low, and thus exposed the tumor to IL-7 and IL-15 (65). In the 1 patient sample in which the authors enriched for tumor-reactive cells from fresh ovarian TILs or tumor-associated lymphocytes exposed to IL-7 and IL-15, expression of 4-1BB was dependent on in vitro activation, but no longer represented the natural expression of 4-1BB in the fresh tumor. Second, with the exception of the 1 experiment described above, the enrichment experiments reported were carried out with melanoma or ovarian TIL lines expanded in IL-2 and cocultured with tumor cell lines in vitro. It is well known that IL-2 can change the activation status and also the expression of inhibitory receptors on T cells (data not shown and ref. 56). Thus, the experiment comparing expression of PD-1 and 4-1BB performed by Ye et al. (65) addressed the significance of these receptors after in vitro coculture of a highly activated melanoma TIL line with a tumor cell line, rather than the role of PD-1 and 4-1BB expression in CD8+ lymphocytes in the fresh tumor. Finally, both Inozume et al. and Ye et al. used matched HLA-A2 cell lines to assess tumor reactivity (55, 65). However, the use of HLA-matched tumor cell lines does not enable the assessment of reactivities against unique mutations that are present only in the autologous tumor cell line. In our current study, we used fresh melanoma tumors for all our experiments, and these were rested in the absence of cytokines to preserve the phenotype of TILs. Moreover, we used autologous tumor cell lines to assess tumor recognition. We believe that our experimental approach overcomes the limitations described above, enabling us to conclude that tumor-reactive cells can be detected in both the PD-1+/4-1BB+ and PD-1+/4-1BB CD8+ TIL populations.

In summary, expression of PD-1 in CD8+ TILs in the fresh tumor identified and selected for the diverse patient-specific repertoire of tumor-reactive cells, including mutation-specific cells. In addition, analysis of the CD8+ TIL TCRβ repertoire in 2 melanomas showed that the frequency of a specific TCRβ clonotype in the CD8+ and PD-1+ populations could be used to predict its ability to recognize the autologous tumor. The use of inhibitory receptors and the frequency of individual TCRs to prospectively identify and select the diverse repertoire of tumor-reactive cells holds promise for the personalized treatment of cancer with T cell therapies, but may also facilitate the dissection and understanding of the immune response in human cancer patients.

Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?

Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.

Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:

  • LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
  • TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
  • VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice

In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets

  • OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
  • 4-1BB – Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
  • Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.

Sharma P and Allison JP.
Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential.
Cell. April 2015;161:205-214


Targeting single immune-checkpoint proteins has proven to be clinically effective at treating specific tumor types; can targeting two different proteins synergize effects?

Despite the success of targeting immune-checkpoint proteins, such as CTLA-4, PD-1, LAG-3, TIM-3 among others, percentages of patient response vary and rarely exceed 50%. It is highly tempting to speculate a strategy of dual-targeting of these checkpoint proteins. A recent presentation at the Keystone Symposium for Tumor Immunology: Multidisciplinary Science Driving Combination Therapy detailed findings of dual-targeting two immune-checkpoint proteins in mouse tumor models. Their key findings are summarized below:

  • Dual-targeting PD-1 and LAG-3 demonstrates superior efficacy over blocking either target alone
  • In addition to previous reported data on superior dual-targeting efficacy against fibrosarcoma (Sa1N) and colorectal adenosarcoma (MC38) tumor types1, anti-tumor activity against myeloma (SC J558L) and B-cell lymphoma (A20) hematological tumor types were also reported to be effacious.2

These exciting pre-clinical findings may result in further exploration of dual-targeting antibodies in the clinic, either as combination of existing antibody therapies, or as a new bi-specific antibody therapeutic.

Camelid single domain antibodies are a novel bi-specific antibody platform that may be used to develop a new generation of dual-targeting antibodies against multiple immune-checkpoint proteins.

1Woo SR et al.
Immune Inhibitory Molecules Lag-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape.
Cancer Res. Feb 2012. 15(4):917-927.

2Lewis KE et al.
Dual Targeting of PD-1 and LAG-3 demonstrates Superior Efficacy to Blocking Either PD-1 or LAG-3 Alone in Pre-Clinical Solid and Hematological Tumor Models.
Abstract J7 2033. Keystone Symposia: Tumor Immunology: Multidisciplinary Science Driving Combination Therapy. February 8-13, 2015. Banff, Alberta, Canada.


New insight behind the success of fighting cancer by targeting immune checkpoint proteins

Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.

In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.

In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.

Gubin MM. et al.
Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.
Nature. Nov 2014. 515:577-584



Myeloid-derived-suppressor cells as regulators of the immune system
Dmitry I. Gabrilovich and Srinivas Nagaraj  Nat Rev Immunol. 2009 March ; 9(3): 162–174. http://dx.doi.org:/10.1038/nri2506

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expands during cancer, inflammation and infection, and that has a remarkable ability to suppress T-cell responses. These cells constitute a unique component of the immune system that regulates immune responses in healthy individuals and in the context of various diseases. In this Review, we discuss the origin, mechanisms of expansion and suppressive functions of MDSCs, as well as the potential to target these cells for therapeutic benefit.

The first observations of suppressive myeloid cells were described more than 20 years ago in patients with cancer1-3. However, the functional importance of these cells in the immune system has only recently been appreciated due to accumulating evidence that has demonstrated their contribution to the negative regulation of immune responses during cancer and other diseases. It is now becoming increasingly clear that this activity is contained within a population known as myeloid-derived suppressor cells (MDSCs). Features common to all MDSCs are their myeloid origin, immature state and a remarkable ability to suppress T-cell responses (Box 1). In addition to their suppressive effects on adaptive immune responses, MDSCs have also been reported to regulate innate immune responses by modulating the cytokine production of macrophages4. Non-immunological functions of MDSC have also been described, such as the promotion of tumour angiogenesis, tumour-cell invasion and metastasis. However, as a discussion of these aspects of MDSC biology is beyond the scope of this article, the reader is referred to another recent Review on this topic5.

MDSCs represent an intrinsic part of the myeloid-cell lineage and are a heterogeneous population that is comprised of myeloid-cell progenitors and precursors of myeloid cells. In healthy individuals, immature myeloid cells (IMCs) generated in bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). In pathological conditions such as cancer, various infectious diseases, sepsis, trauma, bone marrow transplantation or some autoimmune disorders, a partial block in the differentiation of IMCs into mature myeloid cells results in an expansion of this population. Importantly, the activation of these cells in a pathological context results in the upregulated expression of immune suppressive factors such as arginase (encoded by ARG1) and inducible nitric oxide synthase (iNOS; also known as NOS2) and an increase in the production of NO (nitric oxide) and reactive oxygen species (ROS). Together, this results in the expansion of an IMC population that has immune suppressive activity; these cells are now collectively known as MDSCs. In this

Origin and subsets of MDSCs It is important to note that MDSCs that are expanded in pathological conditions (see later) are not a defined subset of myeloid cells but rather a heterogeneous population of activated IMCs that have been prevented from fully differentiating into mature cells. MDSCs lack the expression of cell-surface markers that are specific for monocytes, macrophages or DCs and are comprised of a mixture of myeloid cells with granulocytic and monocytic morphology6. Early studies showed that 1–5% of MDSCs are able to form myeloid-cell colonies7-9 and that about one third of this population can differentiate into mature macrophages and DCs in the presence of appropriate cytokines in vitro and in vivo7-9. In mice, MDSCs are characterized by the co-expression of the myeloid lineage differentiation antigen Gr1 (also known as Ly6G) and CD11b (also known as αM-integrin)10. Normal bone marrow contains 20–30% of cells with this phenotype, but these cells make up only a small proportion (2–4%) of spleen cells and are absent from the lymph nodes in mice (Fig. 1). In humans, MDSCs are most commonly defined as CD14-CD11b+ cells or, more narrowly, as cells that express the common myeloid marker CD33 but lack the expression of markers of mature myeloid and lymphoid cells and the MHC-class-II molecule HLA-DR11, 12. MDSCs have also been identified within a CD15+ population in human peripheral blood13. In healthy individuals, immature myeloid cells with described above phenotype comprise ∼0.5% of peripheral blood mononuclear cells.
Recently, the morphological heterogeneity of these cells has been defined more precisely in part based on their expression of Gr1. Notably, Gr1-specific antibodies bind to both Ly6G and Ly6C,  which are encoded by separate genes. However, these epitopes are recognized by different antibodies specific for each individual epitopes: anti-Ly6C and anti-Ly6G. Granulocytic MDSCs have a CD11b+Ly6G+Ly6Clow phenotype, whereas MDSCs with monocytic morphology are CD11b+Ly6G-Ly6Chigh 6,14. Importantly, evidence indicates that these two subpopulations may have different functions in cancer and infectious and autoimmune diseases15-17. During the analysis of ten different experimental tumour models, we found that both of these subsets of MDSCs were expanded. In most cases, however, the expansion of the granulocytic MDSC population was much greater than that of the monocytic subset6 and, interestingly, the two subpopulations used different mechanisms to suppress Tcell function (see later). In addition, the ability to differentiate into mature DCs and macrophages in vitro has been shown to be restricted to monocytic MDSCs6.
In recent years, several other surface molecules have been used to identify additional subsets of suppressive MDSCs, including CD80 (also known as B7.1)18, CD115 (the macrophage colony-stimulating factor receptor)19, 20 and CD124 (the IL-4 receptor α-chain)20. In our own studies, we observed that many MDSCs in tumour-bearing mice co-express CD115 and CD1246; however, direct comparison of MDSCs from tumour-bearing mice and Gr1+CD11b+ cells from naive mice showed that they expressed similar levels of CD115 and CD124. In addition, sorted CD115+ or CD124+ MDSCs from EL-4 tumour-bearing mice had the same ability to suppress T-cell proliferation on a per cell basis as did CD115- or CD124-MDSCs. This suggests that, although these molecules are associated with MDSCs, they might not be involved in the immunosuppressive function of these cells in all tumour models.

Overall, current data suggest that MDSCs are not a defined subset of cells but rather a group of phenotypically heterogeneous myeloid cells that have common biological activity.

MDSCs in pathological conditions MDSCs were first characterized in tumour-bearing mice or in patients with cancer. Inoculation of mice with transplantable tumour cells, or the spontaneous development of tumours in transgenic mice with tissue-restricted oncogene expression, results in a marked systemic expansion of these cells (Fig. 1 and Table 1). In addition, up to a tenfold increase in MDSC numbers was detected in the blood of patients with different types of cancer11, 12, 21, 22. In many mouse tumour models, as many as 20–40% of nucleated splenocytes are represented by MDSCs (in contrast to the 2-4% seen in normal mice). In addition, these cells are found in tumour tissues and in the lymph nodes of tumour-bearing mice.
Although initial observations and most of the current information regarding the role of MDSCs in immune responses has come from studies in the cancer field, accumulating evidence has shown that MDSCs also regulate immune responses in bacterial and parasitic infections, acute and chronic inflammation, traumatic stress, surgical sepsis and transplantation. A systemic expansion of both the granulocytic and monocytic subset of MDSCs was observed in mice primed with Mycobacterium tuberculosis as part of complete Freund’s adjuvant (CFA). Acute Trypanosoma cruzi infection, which induces T-cell activation and increased production of interferon-γ (IFNγ), also leads to the expansion of MDSCs23, 24. A similar expansion of MDSCs has been reported during acute toxoplasmosis25, polymicrobial sepsis26, acute infection with Listeria monocytogenes or chronic infection with Leishmania major27 and infection with helminths28,29, 30, Candida albicans31 or Porphyromonas gingivalis32.

MDSC expansion is also associated with autoimmunity and inflammation. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, an increase in CD11b+Ly6ChiLy6G− MDSCs was observed in the spleen and blood and these cells were found to enter the central nervous system during the inflammatory phase of the disease16. A significant increase in the number of MDSCs was also detected in experimental autoimmune uveoretinitis, an animal model of human intraocular inflammatory disease33, in the skin and spleens of mice that were repeatedly treated with a contact sensitizer to induce an inflammatory response34 and in inflammatory bowel diseases35. MDSCs were also found to infiltrate the spleen and suppress T-cell function in a model of traumatic stress36. Finally, a significant transient increase in MDSC numbers was also demonstrated in normal mice following immunization with different antigens such as ovalbumin or peptide together with CFA, a recombinant vaccinia virus expressing interleukin-2 (IL-2) or staphylococcal enterotoxin A 8, 37, 38. Therefore, current information clearly indicates that the expansion of an immunosuppressive MDSC population is frequently observed in many pathological conditions.

Expansion and activation of MDSCs Studies have demonstrated that the MDSC population is influenced by several different factors (Table 1), which can be divided into two main groups. The first group includes factors that are produced mainly by tumour cells and promote the expansion of MDSC through stimulation of myelopoiesis and inhibiting of the differentiation of mature myeloid cells. The second group of factors is produced mainly by activated T cells and tumour stroma, and is involved in directly activating MDSCs.
Mechanisms of MDSC expansion—Factors that induce MDSC expansion can include cyclooxygenase-2 (COX2), prostaglandins 39-41, stem-cell factor (SCF)39, macrophage colony-stimulating factor (M-CSF), IL-642, granulocyte/macrophage colony-stimulating factor (GM-CSF)41 and vascular endothelial growth factor (VEGF) 43 (Table 1). The signalling pathways in MDSCs that are triggered by most of these factors converge on Janus kinase (JAK) protein family members and signal transducer and activator of transcription 3 (STAT3) (Fig. 2), which are signalling molecules that are involved in cell survival, proliferation, differentiation and apoptosis44. STAT3 is arguably the main transcription factor that regulates the expansion of MDSCs. MDSCs from tumour-bearing mice have markedly increased levels of phosphorylated STAT3 compared with IMCs from naive mice45. Exposure of haematopoietic progenitor cells to tumour-cell-conditioned medium resulted in the activation of JAK2 and STAT3 and was associated with an expansion of MDSCs in vitro, whereas inhibition of STAT3 expression in haematopoietic progenitor cells abrogated the effect of tumour-derived factors on MDSC expansion46. Ablation of STAT3 expression in conditional knockout mice or selective STAT3 inhibitors markedly reduced the expansion of MDSCs and increased T-cell responses in tumour-bearing mice45, 47. STAT3 activation is associated with increased survival and proliferation of myeloid progenitor cells, probably through upregulated expression of STAT3 target genes including B-cell lymphoma XL, (BCL-XL), cyclin D1, MYC and survivin. So, abnormal and persistent activation of STAT3 in myeloid progenitors prevents their differentiation into mature myeloid cells and thereby promotes MDSC expansion.

Recent findings suggest that STAT3 also regulates MDSC expansion through inducing the expression of S100A8 and S100A9 proteins. In addition, it has been shown that MDSCs also express receptors for these proteins on their cell surface. S100A8 and S100A9 belong to the family of S100 calcium-binding proteins that have been reported to have an important role in inflammation48. STAT3-dependent upregulation of S100A8 and S100A9 expression by myeloid progenitor cells prevented their differentiation and resulted in the expansion of MDSCs in the spleens of tumor-bearing and naive S100A9-transgenic mice. By contrast, MDSCs did not expand in the peripheral blood and spleens of mice deficient for S100A9 following challenge with tumour cells or CFA49. In a different study, S100A8 and S100A9 proteins were shown to promote MDSC migration to the tumour site through binding to carboxylated N-glycan receptors expressed on the surface of these cells 50. Blocking the binding of S100A8 and S100A9 to their receptors on MDSCs in vivo with a carboxylated glycan-specific antibody reduced MDSC levels in the blood and secondary lymphoid organs of tumour-bearing mice50. In human colon tumour tissue, and in a mouse model of colon cancer, myeloid progenitor cells expressing S100A8 and S100A9 have been shown to infiltrate regions of dysplasia and adenoma. Furthermore, administration of a carboxylated glycan-specific monoclonal antibody (mAbGB3.1) was found to markedly reduced chronic inflammation and tumorigenesis51. Although the mechanisms involved require further study, these studies suggest that S100A9 and/or S100A8 proteins have a crucial role in regulating MDSC expansion, and may provide a link between inflammation and immune suppression in cancer.

Mechanisms of MDSC activation—Recently, it has become clear that the suppressive activity of MDSCs requires not only factors that promote their expansion but those that induce their activation. The expression of these factors, which are produced mainly by activated T cells and tumour stromal cells, is induced by different bacterial or viral products or as a result of tumour cell death 26. These factors, which include IFNγ, ligands for Toll-like receptors (TLRs), IL-13, IL-4 and transforming growth factor-β (TGFβ), activate several different signalling pathways in MDSCs that involve STAT6, STAT1, and nuclear factor-κB (NF-κB) (Fig. 2).

Blockade of IFNγ, which is produced by activated T cells, abolishes MDSC-mediated T-cell suppression17, 52. STAT1 is the major transcription factor activated by IFNγ-mediated signalling and, in the tumour microenvironment, the upregulation of ARG1 and iNOS expression in MDSCs involved a STAT1-dependent mechanism. Indeed, MDSCs from Stat1-/- mice failed to up regulate ARG1 and iNOS expression and therefore did not inhibit Tcell responses53. Consistent with other findings, IFNγ produced by activated T cells and by MDSCs triggered iNOS expression and synergized with IL-4Rα and ARG1 pathways that have been implicated in the suppressive function of MDSCs20.
An important role for the signalling pathway that involves IL-4 receptor α-chain (IL-4Rα) and STAT6 (which is activated by the binding of either IL-4 or IL-13 to IL-4Rα) in MDSC activation has been demonstrated in several studies. It has been shown that ARG1 expression is induced by culturing freshly isolated MDSCs or cloned MDSC lines with IL-454. In addition, IL-4 and IL-13 upregulate arginase activity, which increases the suppressive function of MDSCs55. In line with these observations, other experiments have shown that STAT6 deficiency prevents signalling downstream of the IL-4Rα and thereby blocks the production of ARG1 by MDSCs56. In addition, the IL-4Rα–STAT6 pathway was also found to be involved in IL-13-induced TGFβ1 production by MDSCs in mice with sarcoma, which resulted in decreased tumour immunosurveillance57. This could be regulated by neutralizing both TGFβ and IL-1357. However, in breast tumor model IL-4Rα knockout mice retain high levels of MDSC after surgery56. In a different study that evaluated the separate role of TGFβ (not involving study of IL-4Rα) TGFβ-specific blocking antibody failed to reverse T-cell anergy in B-cell lymphoma in vitro58. It is possible that, the IL4Rα–STAT6 pathway might not be involved in promoting tumour immunosuppression in all tumour models.

TLRs have a central role in the activation of innate immune responses. Polymicrobial sepsis induced by the ligation and puncture of the caecum, which releases microbial products into the peritoneum and systemic circulation, was shown to result in an expansion of the MDSC population in the spleen that was dependent on the TLR adaptor molecule myeloid differentiation primary-response gene 88 (MyD88)26. However, wild-type mice and mice lacking a functional TLR4 protein had comparable expansion of the MDSC during polymicrobial sepsis, which suggests that signalling through TLR4 is not required for MDSC expansion and that MyD88-dependent signalling pathways that are triggered by other TLRs probably contribute to the expansion of MDSCs in sepsis26. This indicates that the activation of MDSCs is a fundamental outcome of the host innate immune response to pathogens that express TLR ligands.

It is important to note that an increase in the production and/or recruitment of IMCs in the context of acute infectious diseases or following vaccination does not necessarily represent an expansion of an immunosuppressive MDSC population. It is likely that under pathological conditions, the expansion of a suppressive MDSC population is regulated by two different groups of factors that have partially overlapping activity: those that induce MDSC expansion and those that induce their activation (which leads to increased levels of ROS, arginase, and/ or NO). This two-tiered system may allow for flexibility in the regulation of these cells under physiological and pathological conditions.
Mechanisms of MDSC suppressive activity Most studies have shown that the immunosuppressive functions of MDSCs require direct cell– cell contact, which suggests that they act either through cell-surface receptors and/or through the release of short-lived soluble mediators. The following sections describe the several mechanisms that have been implicated in MDSC-mediated suppression of T-cell function.

Arginase and iNOS—Historically, the suppressive activity of MDSCs has been associated with the metabolism of L-arginine. L-arginine serves as a substrate for two enzymes: iNOS, which generates NO, and arginase, which converts L-arginine into urea and L-ornithine. MDSCs express high levels of both arginase and iNOS, and a direct role for both of these enzymes in the inhibition of T-cell function is well established; this has been reviewed recently59, 60. Recent data suggest that there is a close correlation between the availability of arginine and the regulation of T-cell proliferation11, 61. The increased activity of arginase in MDSCs leads to enhanced L-arginine catabolism, which depletes this non-essential amino acid from the microenvironment. The shortage of L-arginine inhibits T-cell proliferation through several different mechanisms, including decreasing their CD3ζ expression62 and preventing their upregulation of the expression of the cell cycle regulators cyclin D3 and cyclin-dependent kinase 4 (CDK4)63. NO suppresses T-cell function through a variety of different mechanisms that involve the inhibition of JAK3 and STAT5 in T cells64, the inhibition of MHC class II expression 65 and the induction of T-cell apoptosis66.

ROS—Another important factor that contributes to the suppressive activity of MDSCs is ROS. Increased production of ROS has emerged as one of the main characteristics of MDSCs in both tumour-bearing mice and patients with cancer6, 10, 13, 53, 67-70. Inhibition of ROS production by MDSCs isolated from mice and patients with cancer completely abrogated the suppressive effect of these cells in vitro10, 13, 67. Interestingly, ligation of integrins expressed on the surface of MDSCs was shown to contribute to increased ROS production following the interaction of MDSCs with T cells10. In addition, several known tumour-derived factors, such as TGFβ, IL-10, IL-6, IL-3, platelet-derived growth factor (PDGF) and GM-CSF, can induce the production of ROS by MDSCs (for review see Ref 71).

The involvement of ROS and NO in mechanisms of MDSC suppression are not restricted to neoplastic conditions, as inflammation and microbial products are also known to induce the development of a MDSC population that produces ROS and NO following interactions with activated T cells15. Similar findings were observed in models of EAE16 and acute Toxoplasmosis infection 16. In addition, it has been observed that MDSCs mediated their suppressive function through IFNγ-dependent NO production in an experimental model of Trypanosoma cruzi infection23.

Peroxynitrite—More recently, it has emerged that peroxynitrite (ONOO-) is a crucial mediator of MDSC-mediated suppression of T-cell function. Peroxynitrite is a product of a chemical reaction between NO and superoxide anoion (O2-) and is one of the most powerful oxidants produced in the body. It induces the nitration and nitrosylation of the amino acids cystine, methionine, tryptophan and tyrosine72. Increased levels of peroxynitrite are present at sites of MDSC and inflammatory-cell accumulation, including sites of ongoing immune reactions. In addition, high levels of peroxynitrite are associated with tumour progression in many types of cancer72, 73,74-78, which has been linked with T-cell unresponsiveness. Bronte and colleagues reported that human prostate adenocarcinomas were infiltrated by terminallydifferentiated CD8+ T cells that were in an unresponsive state. High levels of nitrotyrosine were present in the T cells, which suggested the production of peroxynitrites in the tumour environment. Inhibiting the activity of arginase and iNOS, which are expressed in malignant but not in normal prostate tissue and are key enzymes of L-arginine metabolism,, led to decreased tyrosine nitration and restoration of T-cell responsiveness to tumour antigens79. In addition, we have demonstrated that peroxynitrite production by MDSCs during direct contact with T cells results in nitration of the T-cell receptor (TCR) and CD8 molecules, which alters the specific peptide binding of the T cells and renders them unresponsive to antigen-specific stimulation. However, the T cells maintained their responsiveness to nonspecific stimuli80. This phenomenon of MDSC induced antigen-specific T-cell unresponsiveness was also observed in vivo in tumour-bearing mice53.

Subset-specific suppressive mechanisms?—Recent findings indicate that different subsets of MDSC might use different mechanisms by which to suppress T-cell proliferation. As described earlier, two main subsets of MDSCs have been identified: a granulocytic subset and a monocytic subset. The granulocytic subset of MDSC was found to express high levels of ROS and low levels of NO, whereas the monocytic subset expressed low levels of ROS and high levels of NO and both subsets expressed ARG16 (Fig.3). Interestingly, both populations suppressed antigen-specific T-cell proliferation to an equal extent, despite their different mechanisms of action. Consistent with these observations, Movahedi et al. also reported two distinct MDSC subsets in tumour-bearing mice, one that consisted of mononuclear cells that resembled inflammatory monocytes and a second that consisted of polymorphonuclear cells that were similar to immature granulocytes. Again, both populations were found to suppress antigen-specific T-cell responses, although by using distinct effector molecules and signalling pathways. The suppressive activity of the granulocytic subset was ARG1-dependent, in contrast to the STAT1- and iNOS-dependent mechanism of the monocyte fraction17. Finally, the same trend was observed in Trypanosoma cruzii infection. In this case, monocytic MDSCs produced NO and strongly inhibited T-cell proliferation, and granulocytic MDSCs produced low levels of NO and did not inhibit T-cell proliferation, although they did produce superoxide15. The biological significance of such functional dichotomy of these two MDSC subsets remains to be elucidated.
Induction of TReg cells—Recently, the ability of MDSCs to promote the de novo development of FOXP3+ regulatory T (TReg) cells in vivo has been described18, 19. The induction of TReg cells by MDSCs was found to require the activation of tumour-specific Tcells and the presence of IFNγ and IL-10 but was independent of NO19. In mice bearing 1D8 ovarian tumours, the induction of TReg cells by MDSCs required the expression of cytotoxic lymphocyte antigen 4 (CTLA-4; also known as CD152) by MDSCs18. In a mouse model of lymphoma, MDSCs were shown to induce TReg-cell expansion through a mechanism that required arginase and the capture, processing and presentation of tumour-associated antigens by MDSCs, but not TGFβ58. By contrast, Movahedi et al. found that the percentage of TReg cells was invariably high throughout tumour growth and did not relate to the kinetics of expansion of the MDSC population, suggesting that MDSCs were not involved in TReg-cell expansion17. Furthermore, in a rat model of kidney allograft tolerance that was induced with a CD28-specific antibody, MDSCs that were co-expressing CD80 and CD86 were found to have a limited effect on the expansion of the TReg-cell population81. Although further work is required to resolve these discrepancies and to determine the physiological relevance of these studies, it seems possible that MDSCs are involved in TReg-cell differentiation through the production of cytokines or direct cell–cell interactions. Furthermore, MDSCs and TReg cells might be linked in a common immunoregulatory network (see later).
Tissue-specific effects on MDSCs A major unresolved question in this field is whether MDSCs mediate antigen-specific or nonspecific suppression of T-cell responses. Provided that MDSCs and T cells are in close proximity, the factors that mediate MDSC suppressive function (ROS, arginase and NO) can inhibit T-cell proliferation regardless of the antigen specificity of the T cells. Indeed, numerous in vitro studies have demonstrated the antigen nonspecific nature of MDSC-mediated suppression of T cells82 83. However, whether the situation is the same in vivo is not clear, and evidence suggests that MDSC-mediated immunosuppression in peripheral lymphoid organs is mainly antigen-specific. The idea that MDSC-mediated T-cell suppression occurs in an antigen-specific manner is based on findings that antigen-specific interactions between antigen-presenting cells and T cells result in much more stable and more prolonged cell–cell contact than nonspecific interactions82, 84, 85. Such stable contacts are necessary for MDSCderived ROS and peroxynitrite to mediate effects on the molecules on the surface of T cells that render the T cells unresponsive to specific antigen. It should be noted that such modification of cell-surface molecules does not lead to T-cell death nor prevent nonspecific T-cell activation. Other evidence that supports the idea that MDSCs mediate antigen-specific suppression is the finding that that MDSCs can take up soluble antigens, including tumourassociated antigens, and process and present them to T cells17 80; blockade of MDSC–T-cell interactions with a MHC-class-I-specific antibody abrogated MDSC-mediated inhibition of T cell responses in vitro86. The MHC-class-I-restricted nature of MDSC-mediated CD8+ T-cell suppression has also been demonstrated in vivo in tumor models53 and in the model of inflammatory bowel disease 35. This is consistent with the recent observation that large numbers of tumour-induced MDSCs did not inhibit CD8+ T-cell responses specific for unrelated antigens in a model of sporadic cancer87. Notably, it is currently unclear whether similar antigen-specific mechanisms of MDSC-mediated suppression operate on CD4+ T cells, as published studies have only assessed the effects of MDSCs on CD8+ T cells. Addressing this question is complicated by the fact that only a small proportion of MDSCs in many tumour models expresses MHC class II molecules.

The theory that MDSCs suppress T-cell responses in an antigen-specific manner helps to explain the finding that T cells in the peripheral lymphoid organs of tumour-bearing mice and in the peripheral blood of cancer patients can still respond to stimuli other than tumourassociated antigens, including viruses, lectins, co-stimulatory molecules, IL-2 and CD3- and CD28-specific antibodies21, 80, 88-90. Furthermore, even patients with advanced stage cancer do not have systemic immunodeficiency except in cases in which the patient has received high doses of chemotherapy or is at a terminal stage of the disease.

Evidence suggets that the nature of MDSC-mediated suppression at the tumour site is quite different to that which occurs in the periphery. MDSCs actively migrate into the tumour site10, where they upregulate the expression of ARG1 and iNOS, downregulate the production of ROS and/or rapidly differentiate into tumour-associated macrophages (TAMs) 52. The levels of NO and arginase produced by tumour-associated MDSCs and TAMs are much higher than those of MDSCs found in peripheral lymphoid organs of the same animals. In addition, TAMs produce several cytokines (reviewed in REFs91, 92) that suppress T-cell responses in a nonspecific manner (Fig. 4). The mechanisms by which MDSC functions are regulated within the tumour microenvironment, and how they differ from those that operate at peripheral sites, remain unclear. It is possible that tumour stroma, hypoxia and/or the acidophilic environment have a role.
Therapeutic targeting of MDSCs The recognition that immune suppression has a crucial role in promoting tumour progression and contributes to the frequent failure of cancer vaccines to elicit an immune response has resulted in a paradigm shift with respect to approaches for cancer immunotherapy. Indeed, it has become increasingly clear that successful cancer immunotherapy will be possible only with a strategy that involves the elimination of suppressive factors from the body. As MDSCs are one of the main immunosuppressive factors in cancer and other pathological conditions, several different therapeutic strategies that target these cells are currently being explored (Table 2). Although the studies described below were carried out in tumor-bearing hosts, it is likely that the same strategies will be useful in other pathological conditions in which inhibition or elimination of MDSCs is a therapeutic aim.

Promoting myeloid-cell differentiation—One of the most promising approaches by which to target MDSCs for therapy is to promote their differentiation into mature myeloid cells that do not have suppressive abilities. Vitamin A has been identified as a compound that can mediate this effect: vitamin A metabolites such as retinoic acid have been found to stimulate the differentiation of myeloid progenitors into DCs and macrophages 86, 93. Mice that are deficient in vitamin A94 or that have been treated with a pan-retinoic-acid-receptor antagonist95, show an expansion of MDSCs in the bone marrow and spleen. Conversely, therapeutic concentrations of all-trans retinoic acid (ATRA) results in substantial decrease in the presence of MDSCs in cancer patients and tumour-bearing mice. ATRA induced MDSCs to differentiate into DCs and macrophages in vitro and in vivo 12, 86, 96. It is probable that ATRA preferentially induces the differentiation of the monocytic subset of MDSCs, whereas it causes apoptosis of the granulocytic subset. The main mechanism of ATRA-mediated differentiation involved an upregulation of glutathione synthesis and a reduction in ROS levels in MDSCs 97. Decreasing the number of MDSCs in tumour-bearing mice resulted in increased tumour-specific T-cell responses, and the combination of ATRA and two different types of cancer vaccine prolonged the anti-tumour effect of the vaccine treatment in two different tumour models 96. Moreover, administration of ATRA to patients with metastatic renal cell carcinoma resulted in a substantial decrease in the number of MDSCs in the peripheral blood and improved antigen-specific response of T cells 21. Further studies will lead to identification of other agents that have a similar effect. So far, evidence suggests that Vitamin D3 may be another agent with the potential to decrease MDSC numbers in patients with cancer, as it is also known to promote myeloid-cell differentiation98.

Inhibition of MDSC expansion—Because MDSC expansion is known to be regulated by tumour-derived factors (Table 1), several studies have focused on neutralizing the effects of these factors. Recently, SCF has been implicated in causing MDSC expansion in tumourbearing mice39. Inhibition of SCF-mediated signalling by blocking its interaction with its receptor, c-kit, decreased MDSC expansion and tumor angiogenesis39. VEGF, another tumourderived factor that is involved in promoting MDSC expansion, might also be a useful target by which to manipulate MDSC. However, in a clinical trial of 15 patients with refractory solid tumours, treatment with VEGF–trap (a fusion protein that binds all forms of VEGF-A and placental growth factor) showed no effect on MDSC numbers and did not result in increased T-cell responses99. By contrast, treatment of patients with metastatic renal cell cancer with a VEGF-specific blocking antibody (known as avastin) resulted in a decrease in the size of a CD11b+VEGFR1+ population of MDSCs in the peripheral blood 100. However, whether avastatin treatment resulted in an improvement in antitumour responses in these patients has not been determined. Finally, inhibition of matrix metalloproteinase 9 function in tumorbearing mice decreased the number of MDSCs in the spleen and tumour tissues and resulted in a significant delay in the growth of spontaneous NeuT tumours in transgenic BALB/c mice101. However, the mechanism responsible for this outcome remains to be elucidated.

Inhibition of MDSC function—Another approach by which to inhibit MDSCs is to block the signalling pathways that regulate the production of suppressive factors by these cells. One potential target by which this might be achieved is COX2. COX2 is required for the production of prostaglandin E2, which in 3LL tumour cells61 and mammary carcinoma40 has been shown to induce the upregulation of ARG1 expression by MDSCs, thereby inducing their suppressive function. Accordingly, COX2 inhibitors were found to downregulate the expression of ARG1 by MDSCs, which improved antitumour T-cell responses and enhanced the therapeutic efficacy of immunotherapy102, 103. Similarly, phosphodiesterase-5 inhibitors such as sildenafil were found to downregulate the expression of arginase and iNOS expression by MDSCs, thereby inhibiting their suppressive function in growing tumours104. This resulted in the induction of a measurable anti-tumour immune response and a marked delay of tumour progression in several mouse models 104.
ROS inhibitors have also been shown to be effective for decreasing MDSC-mediated immune suppression in tumour-bearing mice. The coupling of a NO-releasing moiety to a conventional non-steroidal anti-inflammatory drug has proven to be an efficient means by which to inhibit the production of ROS. One such drug, nitroaspirin, was found to limit the activity of ARG1 and iNOS in spleen MDSCs105. In combination with vaccination with endogenous retroviral gp70 antigen, nitroaspirin inhibited MDSCs function and increased the number and function of tumour-antigen-specific T cells105.

Elimination of MDSCs—MDSCs can be directly eliminated in pathological settings by using some chemotherapeutic drugs. Administration of one such drug, gemcitabine, to mice that were bearing large tumours resulted in a dramatic reduction in the number of MDSCs in the spleen and resulted in a marked improvement in the anti-tumour response induced by immunotherapy106, 107. This effect was specific to MDSCs, as a significant decrease in the number of T or B cells was not observed in these animals. Furthermore, in a study of 17 patients with early-stage breast cancer that were treated with doxorubicin–cyclophosphamide chemotherapy, a decrease in the level of MDSCs in the peripheral blood was observed22.

Evidence suggests that there is a broad range of methods that will be effective for targeting of the number and/or function of MDSCs in vivo. These strategies will undoubtedly help to further investigate the biology of these cells as well as expedite clinical applications to treat cancer and other pathological conditions.

MDSCs as regulatory myeloid cells? The wealth of information that has accumulated in recent years regarding the biology of MDSCs suggests that these cells might have evolved as a regulatory component of the immune system. These cells are absent under physiological conditions, as IMCs in naive mice are an intrinsic part of normal haematopoiesis that are not immunosuppressive in an unactivated state. In conditions of acute stress, infection or immunization, there is a transient expansion of this IMC population, which then quickly differentiates into mature myeloid cells. This transient IMC population can mediate the suppressive functions that are characteristic of MDSCs but, because the acute conditions are short-lived, the suppressive functions of this transient population have a minimal impact on the overall immune response. However, these cells probably function as important ‘gatekeepers’ that prevent pathological immune-mediated damage.

The role of the MDSC population in settings of chronic infections and cancer is very different. In these pathological conditions, the prolonged and marked expansion of IMCs and their subsequent activation leads to the expansion of a large population of MDSCs with immunosuppressive abilities. MDSCs accumulate in peripheral lymphoid organs and migrate to tumour sites, where they contribute to immunosuppression. Furthermore, some evidence suggests that MDSCs can also induce expansion of regulatory T cells. Future studies will reveal whether MDSCs can be considered part of a natural immune regulatory network.

Concluding remarks The field of MDSC research has more outstanding questions than answers. The roles of specific MDSC subsets in mediating T-cell suppression, and the molecular mechanisms responsible for inhibition of myeloid-cell differentiation, need to be elucidated. The issue of whether Tcell suppression occurs in an antigen-specific manner remains to be clarified, as do the mechanisms that cause MDSC migration to peripheral lymphoid organs. Some of the main priorities in this field should include a better characterization of human MDSCs and a clear understanding of whether targeting these cells in patients with various pathological conditions will be of clinical significance. Conversely, adoptive cellular therapy with MDSCs may be an attractive opportunity by which to inhibit immune responses in the setting of autoimmune disease or transplantation. The challenge for these approaches will be to devise methods by which to generate these cells ex vivo in clinical-grade conditions such that they are suitable for administration to patients. If the past 5–6 years are an indication of the potential for progress in this area, it is safe to estimate that there will soon be significantly more discoveries that further our understanding about the biology and clinical utility of MDSCs.

Box 1. Definition of myeloid-derived suppressor cells (MDSCs)

• a heterogeneous population of cells of myeloid origin that consist of myeloid progenitors and immature macrophages, immature granulocytes and immature dendritic cells

• present in activated state that is characterized by the increased production of reactive oxygen and nitrogen species, and of arginase

• potent suppressors of various T-cell functions • in mice, their phenotype is CD11b+Gr1+, although functionally distinct subsets within this population have been identified (see main text)

• in humans, their phenotype is Lin-HLA-DR-CD33+ or CD11b+CD14-CD33+.

Human cells do not express a marker homologous to mouse Gr1. MDSC have also been identified within a CD15+ population in human peripheral blood.

• in the steady state, immature myeloid cells lack suppressive activity and are present in the bone marrow, but not in secondary lymphoid organs

• accumulation of MDSCs in lymphoid organs and in tumours in response to various growth factors and cytokines is associated with various pathological conditions (most notably cancer)

• in tumour tissues, MDSCs can be differentiated from tumour-associated macrophages (TAMs) by their high expression of Gr1 (not expressed by TAMs) by their low expression of F4/80 (expressed by TAMs), by the fact that a large proportion of MDSCs have a granulocytic morphology and based the upregulated expression of both arginase and inducible nitric oxide synthase by MDSCs but not TAMs.


1. Young MRI, Newby M, Wepsic TH. Hematopoiesis and suppressor bone marrow cells in mice bearing large metastatic Lewis lung carcinoma tumors. Cancer Res 1987;47:100–106. [PubMed: 2947676]
2. Buessow SC, Paul RD, Lopez DM. Influence of mammary tumor progression on phenotype and function of spleen and in situ lymphocytes in mice. J Natl Cancer Inst 1984;73:249–255. [PubMed: 6610791]
3. Seung L, Rowley D, Dubeym P, Schreiber H. Synergy between T-cell immunity and inhibition of paracrine stimulation causes tumor rejection. Proc Natl Acad Sci U S A 1995;92:6254–6258. [PubMed: 7603979]
4. Sinha P, Clements VK, Bunt SK, Albelda SM, Ostrand-Rosenberg S. Crosstalk between myeloidderived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. J Immunol 2007;179:977–983. [PubMed: 17617589]
5. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008;8:618–631. [PubMed: 18633355]
6. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumorbearing mice. J Immunol 2008;181:5791–5802. [PubMed: 18832739] Together with reference # 17 this paper described functional differences between subsets of MDSC.
7. Bronte V, et al. Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells. Blood 2000;96:3838. [PubMed: 11090068]
8. Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in cancer: The role of reactive oxygen species. J Leukoc Biol 2003;74:186–196. [PubMed: 12885935]
9. Li Q, Pan PY, Gu P, Xu D, Chen SH. Role of immature myeloid Gr-1+ cells in the development of antitumor immunity. Cancer Res 2004;64:1130–1139. [PubMed: 14871848] …..



Aurelian Udristioiu commented on your update
“The proto-oncogenic transcription factor Myc is known to promote transcription of genes for the cell cycle as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role to induce the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor HIF1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions. “

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Biology, Physiology and Pathophysiology of Heat Shock Proteins

Curation: Larry H. Bernstein, MD, FCAP



Heat Shock Proteins (HSP)

  1. Exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes

Hsp70 chaperones: Cellular functions and molecular mechanism

M. P. MayerB. Bukau
Cell and Molec Life Sci  Mar 2005; 62:670  http://dx.doi.org:/10.1007/s00018-004-4464-6

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [17]. Hsp70s have thus housekeeping functions in the cell in which they are built-in components of folding and signal transduction pathways, and quality control functions in which they proofread the structure of proteins and repair misfolded conformers. All of these activities appear to be based on the property of Hsp70 to interact with hydrophobic peptide segments of proteins in an ATP-controlled fashion. The broad spectrum of cellular functions of Hsp70 proteins is achieved through

  • the amplification and diversification of hsp70genes in evolution, which has generated specialized Hsp70 chaperones,
  • co-chaperones which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and
  • cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines.

Protein folding processes assisted by Hsp70

The role of Hsp70s in the folding of non-native proteins can be divided into three related activities: prevention of aggregation, promotion of folding to the native state, and solubilization and refolding of aggregated proteins. In the cellular milieu, Hsp70s exert these activities in the quality control of misfolded proteins and the co- and posttranslational folding of newly synthesized proteins. Mechanistically related but less understood is the role of Hsp70s in the disassembly of protein complexes such as clathrin coats, viral capsids and the nucleoprotein complex, which initiates the replication of bacteriophage λ DNA. A more complex folding situation exists for the Hsp70-dependent control of regulatory proteins since several steps in the folding and activation process of these substrates are assisted by multiple chaperones.

Hsp70 proteins together with their co-chaperones of the J-domain protein (JDP) family prevent the aggregation of non-native proteins through association with hydrophobic patches of substrate molecules, which shields them from intermolecular interactions (‘holder’ activity). Some JDPs such as Escherichia coli DnaJ and Saccharomyces cerevisiae Ydj1 can prevent aggregation by themselves through ATP-independent transient and rapid association with the substrates. Only members of the Hsp70 family with general chaperone functions have such general holder activity.

Hsp70 chaperone systems assist non-native folding intermediates to fold to the native state (‘folder’ activity). The mechanism by which Hsp70-chaperones assist the folding of non-native substrates is still unclear. Hsp70-dependent protein folding in vitro occurs typically on the time scale of minutes or longer. Substrates cycle between chaperone-bound and free states until the ensemble of molecules has reached the native state. There are at least two alternative modes of action. In the first mechanism Hsp70s play a rather passive role. Through repetitive substrate binding and release cycles they keep the free concentration of the substrate sufficiently low to prevent aggregation, while allowing free molecules to fold to the native state (‘kinetic partitioning’). In the second mechanism, the binding and release cycles induce local unfolding in the substrate, e.g. the untangling of a misfolded β-sheet, which helps to overcome kinetic barriers for folding to the native state (‘local unfolding’) [8–11]. The energy of ATP may be used to induce such conformational changes or alternatively to drive the ATPase cycle in the right direction.

Hsp70 in cellular physiology and pathophysiology

Two Hsp70 functions are especially interesting, de novo folding of nascent polypeptides and interaction with signal transduction proteins, and therefore some aspects of these functions shall be discussed below in more detail. Hsp70 chaperones were estimated to assist the de novo folding of 10–20% of all bacterial proteins whereby the dependence on Hsp70 for efficient folding correlated with the size of the protein [12]. Since the average protein size in eukaryotic cells is increased (52 kDa in humans) as compared to bacteria (35 kDa in E. coli) [25], it is to be expected that an even larger percentage of eukaryotic proteins will be in need of Hsp70 during de novo folding. This reliance on Hsp70 chaperones increases even more under stress conditions. Interestingly, mutated proteins [for example mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxid dismutase (SOD) 1] seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein [2629]. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations [30], a function demonstrated earlier for Hsp90 [3132]. Such mutations are only uncovered when the overall need for Hsp70 action exceeds the chaperone capacity of the Hsp70 proteins, for example during stress conditions [30], at certain stages in development or during aging, when the magnitude of stress-induced increase in Hsp70 levels declines [3334]. Alternatively, the mutant protein can be targeted by Hsp70 and its co-chaperones to degradation as shown e.g. for CFTRΔF508 and some of the SOD1 mutant proteins [35,36]. Deleterious mutant proteins may then only accumulate when Hsp70 proteins are overwhelmed by other, stress-denatured proteins. Both mechanisms may contribute to pathological processes such as oncogenesis (mutant p53) and neurodegenerative diseases, including amyotrophic, lateral sclerosis (SOD1 mutations), Parkinsonism (α-synuclein mutations), Huntington’s chorea (huntingtin with polyglutamin expansions) and spinocerebellar ataxias (proteins with polyglutamin expansions).

De novo folding is not necessarily accelerated by Hsp70 chaperones. In some cases folding is delayed for different reasons. First, folding of certain proteins can only proceed productively after synthesis of the polypeptide is completed as shown, e.g. for the reovirus lollipop-shaped protein sigma 1 [37]. Second, proteins destined for posttranslational insertion into organellar membranes are prevented from aggregation and transported to the translocation pore [38]. Third, in the case of the caspase-activated DNase (CAD), the active protein is dangerous for the cell and therefore can only complete folding in the presence of its specific inhibitor (ICAD). Hsp70 binds CAD cotranslationally and mediates folding only to an intermediate state. Folding is completed after addition of ICAD, which is assembled into a complex with CAD in an Hsp70-dependent manner [39]. Similar folding pathways may exist also for other potentially dangerous proteins.

As mentioned above Hsp70 interacts with key regulators of many signal transduction pathways controlling cell homeostasis, proliferation, differentiation and cell death. The interaction of Hsp70 with these regulatory proteins continues in activation cycles that also involve Hsp90 and a number of co-chaperones. The regulatory proteins, called clients, are thereby kept in an inactive state from which they are rapidly activated by the appropriate signals. Hsp70 and Hsp90 thus repress regulators in the absence of the upstream signal and guarantee full activation after the signal transduction pathway is switched on [6]. Hsp70 can be titrated away from these clients by other misfolded proteins that may arise from internal or external stresses. Consequently, through Hsp70 disturbances of the cellular system induced by environmental, developmental or pathological processes act on these signal transduction pathways.

In this way stress response and apoptosis are linked to each other. Hsp70 inhibits apoptosis acting on the caspase-dependent pathway at several steps both upstream and downstream of caspase activation and on the caspase-independent pathway. Overproduction of Hsp70 leads to increased resistance against apoptosis-inducing agents such as tumor necrosis factor-α(TNFα), staurosporin and doxorubicin, while downregulation of Hsp70 levels by antisense technology leads to increased sensitivity towards these agents [1840]. This observation relates to many pathological processes, such as oncogenesis, neurodegeneration and senescence. In many tumor cells increased Hsp70 levels are observed and correlate with increased malignancy and resistance to therapy. Downregulation of the Hsp70 levels in cancer cells induce differentiation and cell death [41]. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s corea and spinocerebellar ataxias are characterized by excessive apoptosis. In several different model systems overexpression of Hsp70 or one of its co-chaperones could overcome the neurodegenerative symptoms induced by expression of a disease-related gene (huntingtin, α-synuclein or ataxin) [20,42]. Senescence in cell culture as well as aging in vivo is correlated with a continuous decline in the ability to mount a stress response [3443]. Age-related symptoms and diseases reflect this decreased ability to cope with cellular stresses. Interestingly, centenarians seem to be an exception to the rule, as they show a significant induction of Hsp70 production after heat shock challenge [44].

ATPase domain and ATPase cycle

Substrate binding

The coupling mechanism: nucleotide-controlled opening and closing of the substrate binding cavity

The targeting activity of co-chaperones

J-domain proteins

Bag proteins

Hip, Hop and CHIP


The Hsp70 protein family and their co-chaperones constitute a complex network of folding machines which is utilized by cells in many ways. Despite considerable progress in the elucidation of the mechanistic basis of these folding machines, important aspects remain to be solved. With respect to the Hsp70 proteins it is still unclear whether their activity to assist protein folding relies on the ability to induce conformational changes in the bound substrates, how the coupling mechanism allows ATP to control substrate binding and to what extent sequence variations within the family translate into variations of the mechanism. With respect to the action of co-chaperones we lack a molecular understanding of the coupling function of JDPs and of how co-chaperones target their Hsp70 partner proteins to substrates. Furthermore, it can be expected that more cellular processes will be discovered that depend on the chaperone activity of Hsp70 chaperones.


  1. The biochemistry and ultrastructure of molecular chaperones

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Laurence H. Pearl and Chrisostomos Prodromou
Ann Rev of Biochem July 2006;75:271-294

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90’s diverse “clientele.” Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.


  1. Properties of heat shock proteins (HSPs) and heat shock factor (HSF)

Heat shock factors: integrators of cell stress, development and lifespan

Malin Åkerfelt,*‡ Richard I. Morimoto,§ and Lea Sistonen*‡
Nat Rev Mol Cell Biol. 2010 Aug; 11(8): 545–555.  doi:  10.1038/nrm2938

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

In the early 1960s, Ritossa made the seminal discovery of temperature-induced puffs in polytene chromosomes of Drosophila melanogaster larvae salivary glands1. A decade later, it was shown that the puffing pattern corresponded to a robust activation of genes encoding the heat shock proteins (HSPs), which function as molecular chaperones2. The heat shock response is a highly conserved mechanism in all organisms from yeast to humans that is induced by extreme proteotoxic insults such as heat, oxidative stress, heavy metals, toxins and bacterial infections. The conservation among different eukaryotes suggests that the heat shock response is essential for survival in a stressful environment.

The heat shock response is mediated at the transcriptional level by cis-acting sequences called heat shock elements (HSEs; BOX 1) that are present in multiple copies upstream of the HSP genes3. The first evidence for a specific transcriptional regulator, the heat shock factor (HSF) that can bind to the HSEs and induce HSP gene expression, was obtained through DNA–protein interaction studies on nuclei isolated from D. melanogaster cells4,5. Subsequent studies showed that, in contrast to a single HSF in invertebrates, multiple HSFs are expressed in plants and vertebrates68. The mammalian HSF family consists of four members: HSF1,HSF2, HSF3 and HSF4. Distinct HSFs possess unique and overlapping functions (FIG. 1), exhibit tissue-specific patterns of expression and have multiple post-translational modifications (PTMs) and interacting protein partners7,9,10. Functional crosstalk between HSF family members and PTMs facilitates the fine-tuning of HSF-mediated gene regulation. The identification of many targets has further extended the impact of HSFs beyond the heat shock response. Here, we present the recent discoveries of novel target genes and physiological functions of HSFs, which have changed the view that HSFs act solely in the heat shock response. Based on the current knowledge of small-molecule activators and inhibitors of HSFs, we also highlight the potential for pharmacologic modulation of HSF-mediated gene regulation.

Box 1

The heat shock element


Heat shock factors (HSFs) act through a regulatory upstream promoter element, called the heat shock element (HSE). In the DNA-bound form of a HSF, each DNA-binding domain (DBD) recognizes the HSE in the major groove of the double helix6. The HSE was originally identified using S1 mapping of transcripts of the Drosophila melanogaster heat shock protein (HSP) genes3 (see the figure; part a). Residues –47 to –66 are necessary for heat inducibility. HSEs in HSP gene promoters are highly conserved and consist of inverted repeats of the pentameric sequence nGAAn132. The type of HSEs that can be found in the proximal promoter regions of HSP genes is composed of at least three contiguous inverted repeats: nTTCnnGAAnnTTCn132134. The promoters of HSF target genes can also contain more than one HSE, thereby allowing the simultaneous binding of multiple HSFs. The binding of an HSF to an HSE occurs in a cooperative manner, whereby binding of an HSF trimer facilitates binding of the next one135. More recently, Trinklein and colleagues used chromatin immunoprecipitation to enrich sequences bound by HSF1 in heat-shocked human cells to define the HSE consensus sequence. They confirmed the original finding of Xiao and Lis, who identified guanines as the most conserved nucleotides in HSEs87,133 (see the figure; part b). Moreover, in a pair of inverted repeats, a TTC triplet 5′ of a GAA triplet is separated by a pyrimidine–purine dinucleotide, whereas the two nucleotides separating a GAA triplet 5′ from a TTC triplet is unconstrained87. The discovery of novel HSF target genes that are not involved in the heat shock response has rendered it possible that there may be HSEs in many genes other than the HSP genes. Although there are variations in these HSEs, the spacing and position of the guanines are invariable7. Therefore, both the nucleotides and the exact spacing of the repeated units are considered as key determinants for recognition by HSFs and transcriptional activation. Part b of the figure is modified, with permission, from REF. 87 © (2004) The American Society for Cell Biology.

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f1.gif

The mammalian HSF machinery

HSFs as stress integrators

A hallmark of stressed cells and organisms is the increased synthesis of HSPs, which function as molecular chaperones to prevent protein misfolding and aggregation to maintain protein homeostasis, also called proteostasis11. The transcriptional activation of HSP genes is mediated by HSFs (FIG. 2a), of which HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance1215. HSF1 is constitutively expressed in most tissues and cell types16, where it is kept inactive in the absence of stress stimuli. Thus, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple PTMs, protein–protein interactions and subcellular localization. HSF1 also has an intrinsic stress-sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a homotrimer in vitro in response to thermal or oxidative stress1719.

Figure 2    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f2.gif

Members of the mammalian HSF family

Functional domains

HSFs, like other transcription factors, are composed of functional domains. These have been most thoroughly characterized for HSF1 and are schematically presented in FIG. 2b. The DNA-binding domain (DBD) is the best preserved domain in evolution and belongs to the family of winged helix-turn-helix DBDs2022. The DBD forms a compact globular structure, except for a flexible wing or loop that is located between β-strands 3 and 4 (REF. 6). This loop generates a protein– protein interface between adjacent subunits of the HSF trimer that enhances high-affinity binding to DNA by cooperativity between different HSFs23. The DBD can also mediate interactions with other factors to modulate the transactivating capacity of HSFs24. Consequently, the DBD is considered as the signature domain of HSFs for target-gene recognition.

The trimerization of HSFs is mediated by arrays of hydrophobic heptad repeats (HR-A and HR-B) that form a coiled coil, which is characteristic for many Leu zippers6,25 (FIG. 2b). The trimeric assembly is unusual, as Leu zippers typically facilitate the formation of homodimers or heterodimers. Suppression of spontaneous HSF trimerization is mediated by yet another hydrophobic repeat, HR-C2628. Human HSF4 lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity29. Positioned at the extreme carboxyl terminus of HSFs is the transactivation domain, which is shared among all HSFs6except for yeast Hsf, which has transactivation domains in both the amino and C termini, and HSF4A, which completely lacks a transactivation domain2931. In HSF1, the transactivation domain is composed of two modules — AD1 and AD2, which are rich in hydrophobic and acidic residues (FIG. 3a) — that together ensures a rapid and prolonged response to stress32,33. The transactivation domain was originally proposed to provide stress inducibility to HSF1 (REFS 34,35), but it soon became evident that an intact regulatory domain, located between the HR-A and HR-B and the transactivation domain, is essential for the responsiveness to stress stimuli32,33,36,37. Because several amino acids that are known targets for different PTMs reside in the regulatory domain33,3842, the structure and function of this domain are under intensive investigation.

Figure 3    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f3.gif

HSF1 undergoes multiple PTMs on activation

Regulation of the HSF1 activation–attenuation cycle

The conversion of the inactive monomeric HSF1 to high-affinity DNA-binding trimers is the initial step in the multistep activation process and is a common feature of all eukaryotic HSFs43,44 (FIG. 3b). There is compelling evidence for HSF1 interacting with multiple HSPs at different phases of its activation cycle. For example, monomeric HSF1 interacts weakly with HSP90 and, on stress, HSF1 dissociates from the complex, allowing HSF1 trimerization45,46 (FIG. 3b). Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex of HSP90, co-chaperone p23 (also known as PTGES3) and immunophilin FK506-binding protein 5 (FKBP52; also known as FKBP4)4651. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation on heat shock52. Activated HSF1 trimers also interact with HSP70 and the co-chaperone HSP40 (also known as DNAJB1), but instead of suppressing the DNA-binding activity of HSF1, this interaction inhibits its transactivation capacity5254. Although the inhibitory mechanism is still unknown, the negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides.

A ribonucleoprotein complex containing eukaryotic elongation factor 1A (eEF1A) and a non-coding RNA, heat shock RNA-1 (HSR-1), has been reported to possess a thermosensing capacity. According to the proposed model, HSR-1 undergoes a conformational change in response to heat stress and together with eEF1A facilitates trimerization of HSF1 (REF. 55). How this activation mode relates to the other regulatory mechanisms associated with HSFs remains to be elucidated.

Throughout the activation–attenuation cycle, HSF1 undergoes extensive PTMs, including acetylation, phosphorylation and sumoylation (FIG. 3). HSF1 is also a phosphoprotein under non-stress conditions, and the results from mass spectrometry (MS) analyses combined with phosphopeptide mapping experiments indicate that at least 12 Ser residues are phosphorylated41,5659. Among these sites, stress-inducible phosphorylation of Ser230 and Ser326 in the regulatory domain contributes to the transactivation function of HSF1 (REFS 38,41). Phosphorylation-mediated sumoylation on a single Lys residue in the regulatory domain occurs rapidly and transiently on exposure to heat shock; Ser303 needs to be phosphorylated before a small ubiquitin-related modifier (SUMO) can be conjugated to Lys298 (REF. 39). The extended consensus sequence ΨKxExxSP has been named the phosphorylation-dependent sumoylation motif (PDSM; FIG. 3)40. The PDSM was initially discovered in HSF1 and subsequently found in many other proteins, especially transcriptional regulators such as HSF4, GATA1, myocyte-specific enhancer factor 2A (MEF2A) and SP3, which are substrates for both SUMO conjugation and Pro-directed kinases40,6062.

Recently, Mohideen and colleagues showed that a conserved basic patch on the surface of the SUMO-conjugating enzyme ubiquitin carrier protein 9 (UBC9; also known as UBE2I) discriminates between the phosphorylated and non-phosphorylated PDSM of HSF1 (REF. 63). Future studies will be directed at elucidating the molecular mechanisms for dynamic phosphorylation and UBC9-dependent SUMO conjugation in response to stress stimuli and establishing the roles of kinases, phosphatases and desumoylating enzymes in the heat shock response. The kinetics of phosphorylation-dependent sumoylation of HSF1 correlates inversely with the severity of heat stress, and, as the transactivation capacity of HSF1 is impaired by sumoylation and this PTM is removed when maximal HSF1 activity is required40, sumoylation could modulate HSF1 activity under moderate stress conditions. The mechanisms by which SUMO modification represses the transactivating capacity of HSF1, and the functional relationship of this PTM with other modifications that HSF1 is subjected to, will be investigated with endogenous substrate proteins.

Phosphorylation and sumoylation of HSF1 occur rapidly on heat shock, whereas the kinetics of acetylation are delayed and coincide with the attenuation phase of the HSF1 activation cycle. Stress-inducible acetylation of HSF1 is regulated by the balance of acetylation by p300–CBP (CREB-binding protein) and deacetylation by the NAD+-dependent sirtuin, SIRT1. Increased expression and activity of SIRT1 enhances and prolongs the DNA-binding activity of HSF1 at the human HSP70.1promoter, whereas downregulation of SIRT1 enhances the acetylation of HSF1 and the attenuation of DNA-binding without affecting the formation of HSF1 trimers42. This finding led to the discovery of a novel regulatory mechanism of HSF1 activity, whereby SIRT1 maintains HSF1 in a state that is competent for DNA binding by counteracting acetylation (FIG. 3). In the light of current knowledge, the attenuation phase of the HSF1 cycle is regulated by a dual mechanism: a dependency on the levels of HSPs that feed back directly by weak interactions with HSF1, and a parallel step that involves the SIRT1-dependent control of the DNA-binding activity of HSF1. Because SIRT1 has been implicated in caloric restriction and ageing, the age-dependent loss of SIRT1 and impaired HSF1 activity correlate with an impairment of the heat shock response and proteostasis in senescent cells, connecting the heat shock response to nutrition and ageing (see below).

HSF dynamics on the HSP70 promoter

For decades, the binding of HSF to the HSP70.1 gene has served as a model system for inducible transcription in eukaryotes. In D. melanogaster, HSF is constitutively nuclear and low levels of HSF are associated with the HSP70promoter before heat shock6466. The uninduced HSP70 promoter is primed for transcription by a transcriptionally engaged paused RNA polymerase II (RNAP II)67,68. RNAP II pausing is greatly enhanced by nucleosome formation in vitro, implying that chromatin remodelling is crucial for the release of paused RNAP II69. It has been proposed that distinct hydrophobic residues in the transactivation domain of human HSF1 can stimulate RNAP II release and directly interact withBRG1, the ATPase subunit of the chromatin remodelling complex SWI/SNF70,71. Upon heat shock, RNAP II is released from its paused state, leading to the synthesis of a full-length transcript. Rapid disruption of nucleosomes occurs across the entire HSP70 gene, at a rate that is faster than RNAP II-mediated transcription72. The nucleosome displacement occurs simultaneously with HSF recruitment to the promoter in D. melanogaster. Downregulation of HSF abrogates the loss of nucleosomes, indicating that HSF provides a signal for chromatin rearrangement, which is required for HSP70 nucleosome displacement. Within seconds of heat shock, the amount of HSF at the promoter increases drastically and HSF translocates from the nucleoplasm to several native loci, including HSP genes. Interestingly, the levels of HSF occupying the HSP70 promoter reach saturation soon after just one minute65,73.

HSF recruits the co-activating mediator complex to the heat shock loci, which acts as a bridge to transmit activating signals from transcription factors to the basal transcription machinery. The mediator complex is recruited by a direct interaction with HSF: the transactivation domain of D. melanogaster HSF binds to TRAP80(also known as MED17), a subunit of the mediator complex74. HSF probably has other macromolecular contacts with the preinitiation complex as it binds to TATA-binding protein (TBP) and the general transcription factor TFIIB in vitro75,76. In contrast to the rapid recruitment and elongation of RNAP II on heat shock, activated HSF exchanges very slowly at the HSP70 promoter. HSF stays stably bound to DNA in vivo and no turnover or disassembly of transcription activator is required for successive rounds of HSP70 transcription65,68.

Functional interplay between HSFs

Although HSF1 is the principal regulator of the heat shock response, HSF2 also binds to the promoters of HSP genes. In light of our current knowledge, HSF2 strictly depends on HSF1 for its stress-related functions as it is recruited to HSP gene promoters only in the presence of HSF1 and this cooperation requires an intact HSF1 DBD77. Nevertheless, HSF2 modulates, both positively and negatively, the HSF1-mediated inducible expression of HSP genes, indicating that HSF2 can actively participate in the transcriptional regulation of the heat shock response. Coincident with the stress-induced transcription of HSP genes, HSF1 and HSF2 colocalize and accumulate rapidly on stress into nuclear stress bodies (NSBs; BOX 2), where they bind to a subclass of satellite III repeats, predominantly in the human chromosome 9q12 (REFS 7880). Consequently, large and stable non-coding satellite III transcripts are synthesized in an HSF1-dependent manner in NSBs81,82. The function of these transcripts and their relationship with other HSF1 targets, and the heat shock response in general, remain to be elucidated.


Box 2

Nuclear stress bodies  


The cell nucleus is highly compartmentalized and dynamic. Many nuclear factors are diffusely distributed throughout the nucleoplasm, but they can also accumulate in distinct subnuclear compartments, such as nucleoli, speckles, Cajal bodies and promyelocytic leukaemia (PML) bodies136. Nuclear stress bodies (NSBs) are different from any other known nuclear bodies137,138. Although NSBs were initially thought to contain aggregates of denatured proteins and be markers of heat-shocked cells, their formation can be elicited by various stresses, such as heavy metals and proteasome inhibitors137. NSBs are large structures, 0.3–3 μm in diameter, and are usually located close to the nucleoli or nuclear envelope137,138. NSBs consist of two populations: small, brightly stained bodies and large, clustered and ring-like structures137.

NSBs appear transiently and are the main site of heat shock factor 1 (HSF1) and HSF2 accumulation in stressed human cells80. HSF1 and HSF2 form a physically interacting complex and colocalize into small and barely detectable NSBs after only five minutes of heat shock, but the intensity and size of NSBs increase after hours of continuous heat shock. HSF1 and HSF2 colocalize in HeLa cells that have been exposed to heat shock for one hour at 42°C (see the figure; confocal microscopy image with HSF1–green fluorescent protein in green and endogenous HSF2 in red). NSBs form on specific chromosomal loci, mainly on q12 of human chromosome 9, where HSFs bind to a subclass of satellite III repeats78,79,83. Stress-inducible and HSF1-dependent transcription of satellite III repeats has been shown to produce non-coding RNA molecules, called satellite III transcripts81,82. The 9q12 locus consists of pericentromeric heterochromatin, and the satellite III repeats provide scaffolds for docking components, such as splicing factors and other RNA-processing proteins139143.

HSF2 also modulates the heat shock response through the formation of heterotrimers with HSF1 in the NSBs when bound to the satellite III repeats83 (FIG. 4). Studies on the functional significance of heterotrimerization indicate that HSF1 depletion prevents localization of HSF2 to NSBs and abolishes the stress-induced synthesis of satellite III transcripts. By contrast, increased expression of HSF2 leads to its own activation and the localization of both HSF1 and HSF2 to NSBs, where transcription is spontaneously induced in the absence of stress stimuli. These results suggest that HSF2 can incorporate HSF1 into a transcriptionally competent heterotrimer83. It is possible that the amounts of HSF2 available for heterotrimerization with HSF1 influence stress-inducible transcription, and that HSF1–HSF2 heterotrimers regulate transcription in a temporal manner. During the acute phase of heat shock, HSF1 is activated and HSF1–HSF2 heterotrimers are formed, whereas upon prolonged exposures to heat stress the levels of HSF2 are diminished, thereby limiting heterotrimerization83. Intriguingly, in specific developmental processes such as corticogenesis and spermatogenesis, the expression of HSF2 increases spatiotemporarily, leading to its spontaneous activation. Therefore, it has been proposed that HSF-mediated transactivation can be modulated by the levels of HSF2 to provide a switch that integrates the responses to stress and developmental stimuli83 (FIG. 4). Functional relationships between different HSFs are emerging, and the synergy of DNA-binding activities among HSF family members offers an efficient way to control gene expression in a cell- and stimulus-specific manner to orchestrate the differential upstream signalling and target-gene networks.

Figure 4   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f4.gif


Interactions between different HSFs provide distinct functional modes in transcriptional regulation

A new member of the mammalian HSF family, mouse HSF3, was recently identified10. Avian HSF3 was shown to be activated at higher temperatures and with different kinetics than HSF1 (REF. 84), whereas in mice, heat shock induces the nuclear translocation of HSF3 and activation of stress-responsive genes other than HSP genes10. Future experiments will determine whether HSF3 is capable of interacting with other HSFs, potentially through heterocomplex formation. HSF4 has not been implicated in the heat shock response, but it competes with HSF1 for common target genes in mouse lens epithelial cells85, which will be discussed below. It is important to elucidate whether the formation of homotrimers or hetero trimers between different family members is a common theme in HSF-mediated transcriptional regulation.


HSFs as developmental regulators

Evidence is accumulating that HSFs are highly versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physioogical functions, especially during development. The initial observations using deletion experiments of the D. melanogaster Hsf gene revealed defective oogenesis and larvae development86. These effects were not caused by obvious changes in HSP gene expression patterns, which is consistent with the subsequent studies showing that basal expression of HSP genes during mouse embryogenesis is not affected by the lack of HSF1 (REF. 13). These results are further supported by genome-wide gene expression studies revealing that numerous genes, not classified as HSP genes or molecular chaperones, are under HSF1-dependent control87,88.

Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility13. Fertilized oocytes do not develop past the zygotic stage when HSF1-deficient female mice are mated with wild-type male mice, indicating that HSF1 is a maternal factor that is essential for early post-fertilization development89. Recently, it was shown that HSF1 is abundantly expressed in maturing oocytes, where it regulates specifically Hsp90α transcription90. The HSF1-deficient oocytes are devoid of HSP90α and exhibit a blockage of meiotic maturation, including delayed G2–M transition or germinal vesicle breakdown and defective asymmetrical division90. Moreover, intra-ovarian HSF1-depleted oocytes contain dysfunctional mitochondria and are sensitive to oxidative stress, leading to reduced survival91. The complex phenotype of Hsf1-knockout mice also demonstrates the involvement of HSF1 in placenta formation, placode development and the immune system15,85,92,93, further strengthening the evidence for a protective function of HSF1 in development and survival.

Both HSF1 and HSF2 are key regulators in the developing brain and in maintaining proteostasis in the central nervous system. Disruption of Hsf1 results in enlarged ventricles, accompanied by astrogliosis, neurodegeneration, progressive myelin loss and accumulation of ubiquitylated proteins in specific regions of the postnatal brain under non-stressed conditions94,95. The expression of HSP25 (also known as HSPB1) and α-crystallin B chain (CRYAB), which are known to protect cells against stress-induced protein damage and cell death, is dramatically decreased in brains lacking HSF1 (REF. 13). In contrast to HSF1, HSF2 is already at peak levels during early brain development in mice and is predominantly expressed in the proliferative neuronal progenitors of the ventricular zone and post-mitotic neurons of the cortical plate9699. HSF2-deficient mice have enlarged ventricles and defects in cortical lamination owing to abnormal neuronal migration9799. Incorrect positioning of superficial neurons during cortex formation in HSF2-deficient embryos is caused by decreased expression of the cyclin-dependent kinase 5 (CDK5) activator p35, which is a crucial regulator of the cortical migration signalling pathway100,101. The p35 gene was identified as the first direct target of HSF2 in cortex development99. As correct cortical migration requires the coordination of multiple signalling molecules, it is likely that HSF2, either directly or indirectly, also regulates other components of the same pathway.


Cooperativity of HSFs in development

In adult mice, HSF2 is most abundantly expressed in certain cell types of testes, specifically pachytene spermatocytes and round spermatids102. The cell-specific expression of HSF2 in testes is regulated by a microRNA, miR-18, that directly binds to the 3′ untranslated region (UTR) of HSF2 (J.K. Björk, A. Sandqvist, A.N. Elsing, N. Kotaja and L.S., unpublished observations). Targeting of HSF2 in spermatogenesis reveals the first physiological role for miR-18, which belongs to the oncomir-1 cluster associated mainly with tumour progression103. In accordance with the expression pattern during the maturation of male germ cells, HSF2-null male mice display several abnormal features in spermatogenesis, ranging from smaller testis size and increased apoptosis at the pachytene stage to a reduced amount of sperm and abnormal sperm head shape97,98,104. A genome-wide search for HSF2 target promoters in mouse testis revealed the occupancy of HSF2 on the sex chromosomal multi-copy genes spermiogenesis specific transcript on the Y 2 (Ssty2), Sycp3-like Y-linked (Sly) and Sycp3-like X-linked (Slx), which are important for sperm quality104. Compared with the Hsf2-knockout phenotype, disruption of both Hsf1 and Hsf2 results in a more pronounced phenotype, including larger vacuolar structures, more widely spread apoptosis and a complete lack of mature spermatozoa and male sterility105. The hypo thesis that the activities of HSF1 and HSF2 are intertwined and essential for spermatogenesis is further supported by our results that HSF1 and HSF2 synergistically regulate the sex chromosomal multi-copy genes in post-meiotic round spermatids (M.Å., A. Vihervaara, E.S. Christians, E. Henriksson and L.S., unpublished observations). Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are currently the only known transcriptional regulators during post-meiotic repression. These results, together with the earlier findings that HSF2 can also form heterotrimers with HSF1 in testes83, strongly suggest that HSF1 and HSF2 act in a heterocomplex and fine-tune transcription of their common target genes during the maturation of male germ cells.

HSF1 and HSF4 are required for the maintenance of sensory organs, especially when the organs are exposed to environmental stimuli for the first time after birth85,88. During the early postnatal period, Hsf1-knockout mice display severe atrophy of the olfactory epithelium, increased accumulation of mucus and death of olfactory sensory neurons88. Although lens development in HSF4-deficient mouse embryos is normal, severe abnormalities, including inclusion-like structures in lens fibre cells, appear soon after birth and the mice develop cataracts85,106,107. Intriguingly, inherited severe cataracts occurring in Chinese and Danish families have been associated with a mutation in the DBD of HSF4 (REF. 108). In addition to the established target genes, Hsp25Hsp70 and Hsp90, several new targets for HSF1 and HSF4, such as crystallin γF (Crygf), fibroblast growth factor 7 (Fgf7) and leukaemia inhibitory factor (Lif) have been found to be crucial for sensory organs85,88. Furthermore, binding of either HSF1 or HSF4 to the Fgf7 promoter shows opposite effects on gene expression, suggesting competitive functions between the two family members85. In addition to the proximal promoters, HSF1, HSF2 and HSF4 bind to other genomic regions (that is, introns and distal parts of protein-coding genes in mouse lens), and there is also evidence for either synergistic interplay or competition between distinct HSFs occupying the target-gene promoters109. It is possible that the different HSFs are able to compensate for each other to some extent. Thus, the identification of novel functions and target genes for HSFs has been a considerable step forward in understanding their regulatory mechanisms in development.


HSFs and lifespan

The lifespan of an organism is directly linked to the health of its tissues, which is a consequence of the stability of the proteome and functionality of its molecular machineries. During its lifetime, an organism constantly encounters environmental and physiological stress and requires an efficient surveillance of protein quality to prevent the accumulation of protein damage and the disruption of proteostasis. Proteotoxic insults contribute to cellular ageing, and numerous pathophysiological conditions, associated with impaired protein quality control, increase prominently with age11. From studies on the molecular basis of ageing, in which a wide range of different model systems and experimental strategies have been used, the insulin and insulin-like growth factor 1 receptor (IGF1R) signalling pathway, which involves the phosphoinositide 3-kinase (PI3K) and AKT kinases and the Forkhead box protein O (FOXO) transcription factors (such as DAF-16 in Caenorhabditis elegans), has emerged as a key process. The downregulation of HSF reduces the lifespan and accelerates the formation of protein aggregates in C. elegans carrying mutations in different components of the IGF1R-mediated pathway. Conversely, inhibition of IGF1R signalling results in HSF activation and promotes longevity by maintaining proteostasis110,111. These results have prompted many laboratories that use other model organisms to investigate the functional relationship between HSFs and the IGF1R signalling pathway.

The impact of HSFs on the lifespan of whole organisms is further emphasized by a recent study, in which proteome stability was examined during C. elegansageing112. The age-dependent misfolding and downregulation of distinct metastable proteins, which display temperature-sensitive missense mutations, was examined in different tissues. Widespread failure in proteostasis occurred rapidly at an early stage of adulthood, coinciding with the severely impaired heat shock response and unfolded protein response112. The age-dependent collapse of proteostasis could be restored by overexpression of HSF and DAF-16, strengthening the evidence for the unique roles of these stress-responsive transcription factors to prevent global instability of the proteome.

Limited food intake or caloric restriction is another process that is associated with an enhancement of lifespan. In addition to promoting longevity, caloric restriction slows down the progression of age-related diseases such as cancer, cardiovascular diseases and metabolic disorders, stimulates metabolic and motor activities, and increases resistance to environmental stress stimuli113. To this end, the dynamic regulation of HSF1 by the NAD+-dependent protein deacetylase SIRT1, a mammalian orthologue of the yeast transcriptional regulator Sir2, which is activated by caloric restriction and stress, is of particular interest. Indeed, SIRT1 directly deacetylates HSF1 and keeps it in a state that is competent for DNA binding. During ageing, the DNA-binding activity of HSF1 and the amount of SIRT1 are reduced. Consequently, a decrease in SIRT1 levels was shown to inhibit HSF1 DNA-binding activity in a cell-based model of ageing and senescence42. Furthermore, an age-related decrease in the HSF1 DNA-binding activity is reversed in cells exposed to caloric restriction114. These results indicate that HSF1 and SIRT1 function together to protect cells from stress insults, thereby promoting survival and extending lifespan. Impaired proteostasis during ageing may at least partly reflect the compromised HSF1 activity due to lowered SIRT1 expression.


Impact of HSFs in disease

The heat shock response is thought to be initiated by the presence of misfolded and damaged proteins, and is thus a cell-autonomous response. When exposed to heat, cells in culture, unicellular organisms, and cells in a multicellular organism can all trigger a heat shock response autonomously115117. However, it has been proposed that multicellular organisms sense stress differently to isolated cells. For example, the stress response is not properly induced even if damaged proteins are accumulated in neurodegenerative diseases like Huntington’s disease and Parkinson’s disease, suggesting that there is an additional control of the heat shock response at the organismal level118. Uncoordinated activation of the heat shock response in cells in a multicellular organism could cause severe disturbances of interactions between cells and tissues. In C. elegans, a pair of thermosensory neurons called AFDs, which sense and respond to temperature, regulate the heat shock response in somatic tissues by controlling HSF activity119,120. Moreover, the heat shock response in C. elegans is influenced by the metabolic state of the organism and is reduced under conditions that are unfavourable for growth and reproduction121. Neuronal control may therefore allow organisms to coordinate the stress response of individual cells with the varying metabolic requirements in different tissues and developmental stages. These observations are probably relevant to diseases of protein misfolding that are highly tissue-specific despite the often ubiquitous expression of damaged proteins and the heat shock response.

Elevated levels of HSF1 have been detected in several types of human cancer, such as breast cancer and prostate cancer122,123. Mice deficient in HSF1 exhibit a lower incidence of tumours and increased survival than their wild-type counterparts in a classical chemical skin carcinogenesis model and in a genetic model expressing an oncogenic mutation of p53. Similar results have been obtained in human cancer cells lines, in which HSF1 was depleted using an RNA interference strategy124. HSF1 expression is likely to be crucial for non-oncogene addiction and the stress phenotype of cancer cells, which are attributes given to many cancer cells owing to their high intrinsic level of proteotoxic and oxidative stress, frequent spontaneous DNA damage and aneuploidy125. Each of these features may disrupt proteostasis, raising the need for efficient chaperone and proteasome activities. Accordingly, HSF1 would be essential for the survival of cancer cells that experience constant stress and develop non-oncogene addiction.


HSFs as therapeutic targets

Given the unique role of HSF1 in stress biology and proteostasis, enhanced activity of this principal regulator during development and early adulthood is important for the stability of the proteome and the health of the cell. However, HSF1 is a potent modifier of tumorigenesis and, therefore, a potential target for cancer therapeutics125. In addition to modulating the expression of HSF1, the various PTMs of HSF1 that regulate its activity should be considered from a clinical perspective. As many human, age-related pathologies are associated with stress and misfolded proteins, several HSF-based therapeutic strategies have been proposed126. In many academic and industrial laboratories, small molecule regulators of HSF1 are actively being searched for (see Supplementary information S1 (table)). For example, celastrol, which has antioxidant properties and is a natural compound derived from the Celastreace family of plants, activates HSF1 and induces HSP expression with similar kinetics to heat shock, and could therefore be a potential candidate molecule for treating neurodegenerative diseases127,128. In a yeast-based screen, a small-molecule activator of human HSF1 was found and named HSF1A129. HSF1A, which is structurally distinct from the other known activators, activates HSF1 and enhances chaperone expression, thereby counteracting protein misfolding and cell death in polyQ-expressing neuronal precursor cells129. Triptolide, also from the Celastreace family of plants, is a potent inhibitor of the transactivating capacity of HSF1 and has been shown to have beneficial effects in treatments of pancreatic cancer xenografts130,131. These examples of small-molecule regulators of HSF1 are promising candidates for drug discovery and development. However, the existence of multiple mammalian HSFs and their functional interplay should also be taken into consideration when planning future HSF-targeted therapies.


Concluding remarks and future perspectives

HSFs were originally identified as specific heat shock-inducible transcriptional regulators of HSP genes, but now there is unambiguous evidence for a wide variety of HSF target genes that extends beyond the molecular chaperones. The known functions governed by HSFs span from the heat shock response to development, metabolism, lifespan and disease, thereby integrating pathways that were earlier strictly divided into either cellular stress responses or normal physiology.

Although the extensive efforts from many laboratories focusing on HSF biology have provided a richness of understanding of the complex regulatory mechanisms of the HSF family of transcription factors, several key questions remain. For example, what are the initial molecular events (that is, what is the ‘thermometer’) leading to the multistep activation of HSFs? The chromatin-based interaction between HSFs and the basic transcription machinery needs further investigation before the exact interaction partners at the chromatin level can be established. The activation and attenuation mechanisms of HSFs require additional mechanistic insights, and the roles of the multiple signal transduction pathways involved in post-translational regulation of HSFs are only now being discovered and are clearly more complex than anticipated. Although still lacking sufficient evidence, the PTMs probably serve as rheostats to allow distinct forms of HSF-mediated regulation in different tissues during development. Further emphasis should therefore be placed on understanding the PTMs of HSFs during development, ageing and different protein folding diseases. Likewise, the subcellular distribution of HSF molecules, including the mechanism by which HSFs shuttle between the cytoplasm and the nucleus, remains enigmatic, as do the movements of HSF molecules in different nuclear compartments such as NSBs.

Most studies on the impact of HSFs in lifespan and disease have been conducted with model organisms such as D. melanogaster and C. elegans, which express a single HSF. The existence of multiple members of the HSF family in mammals warrants further investigation of their specific and overlapping functions, including their extended repertoire of target genes. The existence of multiple HSFs in higher eukaryotes with different expression patterns suggests that they may have functions that are triggered by distinct stimuli, leading to activation of specific target genes. The impact of the HSF family in the adaptation to diverse biological environments is still poorly understood, and future studies are likely to broaden the prevailing view of HSFs being solely stress-inducible factors. To this end, the crosstalk between distinct HSFs that has only recently been uncovered raises obvious questions about the stoichiometry between the components in different complexes residing in different cellular compartments, and the mechanisms by which the factors interact with each other. Interaction between distinct HSF family members could generate new opportunities in designing therapeutics for protein-folding diseases, metabolic disorders and cancer.


  1. Role in the etiology of cancer

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Dan Tang,1 Md Abdul Khaleque,2 Ellen L. Jones,1 Jimmy R. Theriault,2 Cheng Li,3 Wing Hung Wong,3 Mary Ann Stevenson,2 and Stuart K. Calderwood1,2,4
Cell Stress Chaperones. 2005 Mar; 10(1): 46–58. doi:  10.1379/CSC-44R.1

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.

Heat shock proteins (HSPs) were first discovered as a cohort of proteins that is induced en masse by heat shock and other chemical and physical stresses in a wide range of species (Lindquist and Craig 1988Georgopolis and Welch 1993). The HSPs (Table 1) have been subsequently characterized as molecular chaperones, proteins that have in common the property of modifying the structures and interactions of other proteins (Lindquist and Craig 1988Beckmann et al 1990;Gething and Sambrook 1992Georgopolis and Welch 1993Netzer and Hartl 1998). Molecular chaperone function dictates that the HSP often interact in a stoichiometric, one-on-one manner with their substrates, necessitating high intracellular concentrations of the proteins (Lindquist and Craig 1988Georgopolis and Welch 1993). As molecules that shift the balance from denatured, aggregated protein conformation toward ordered, functional conformation, HSPs are particularly in demand when the protein structure is disrupted by heat shock, oxidative stress, or other protein-damaging events (Lindquist and Craig 1988;Gething and Sambrook 1992Georgopolis and Welch 1993). The HSP27, HSP40,HSP70, and HSP110 genes have therefore evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation (Voellmy 1994). HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress (Hickey and Weber 1982Landry et al 1982Li and Werb 1982Subjeck et al 1982Henics et al 1999) (Zhao et al 2002). Heat shock factor (HSF) proteins have been shown to interact with the promoters of many HSP genes and ensure prompt transcriptional activation in stress and equally precipitous switch off after recovery (Sorger and Pelham 1988Wu 1995). The hsf gene family includes HSF1 (hsf1), the molecular coordinator of the heat shock response, as well as 2 less well-characterized genes, hsf2 and hsf4(Rabindran et al 1991Schuetz et al 1991) (Nakai et al 1997). In addition to the class of HSPs induced by heat, cells also contain a large number of constitutively expressed HSP homologs, which are also listed in Table 1. The constitutive HSPs are found in a variety of multiprotein complexes containing both HSPs and cofactors (Buchner 1999). These include HSP10-HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with regulatory proteins within the cell (Netzer and Hartl 1998). HSP90 plays a particularly versatile role in cell regulation, forming complexes with a large number of cellular kinases, transcription factors, and other molecules (Buchner 1999Grammatikakis et al 2002).


Table 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1074571/bin/i1466-1268-10-1-46-t01.jpg


Heat shock protein family genes studied by microchip array analysis

Many tumor types contain high concentrations of HSP of the HSP28, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca et al 1993Yano et al 1999Cornford et al 2000Strik et al 2000Ricaniadis et al 2001Ciocca and Vargas-Roig 2002). We have concentrated here on HSP gene expression in prostate carcinoma. The progression of prostatic epithelial cells to the fully malignant, metastatic phenotype is a complex process and involves the expression of oncogenes as well as escape from androgen-dependent growth and survival (Cornford et al 2000). There is a molecular link between HSP expression and tumor progression in prostate cancer in that HSP56, HSP70, and HSP90 regulate the function of the androgen receptor (AR) (Froesch et al 1998Grossmann et al 2001). Escape from AR dependence during tumorigenesis may involve altered HSP-AR interactions (Grossmann et al 2001). The role of HSPs in tumor development may also be related to their function in the development of tolerance to stress (Li and Hahn 1981). Thermotolerance is induced in cells preconditioned by mild stress coordinately with the expression of high HSP levels (Landry et al 1982Li and Werb 1982Subjeck et al 1982). Elevated HSP expression appears to be a factor in tumor pathogenesis, and, among other mechanisms, this may involve the ability of individual HSPs to block the pathways of apoptosis and permit malignant cells to arise despite the triggering of apoptotic signals during transformation (Volloch and Sherman 1999). De novo HSP expression may also afford protection of cancer cells from treatments such as chemotherapy and hyperthermia by thwarting the proapoptotic influence of these modalities (Gabai et al 1998Hansen et al 1999Blagosklonny 2001Asea et al 2001Van Molle et al 2002). The mechanisms underlying HSP induction in tumor cells are not known but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment, which would be expected to lead to cellular stress.

Here, we have examined expression of hsf and HSP genes in immortalized normal human prostate epithelial cells and a range of prostate carcinoma cells obtained from human tumors at the mRNA and protein levels. Our aim was to determine whether hsf-HSP expression profiles are conserved in cells that express varying degrees of malignancy, under resting conditions and after heat and ionizing radiation. In addition, we have compared HSP expression profiles of a metastatic human prostate carcinoma cell line growing either in monolayer culture or as a tumor xenograft in nude mice. These studies were prompted by findings in our laboratory that prostate carcinoma cells are considerably more sensitive to heat-induced apoptosis in vivo growing as tumors compared with similar cells growing in tissue culture in vitro. Our studies show that, although the hsf-HSP expression profiles are similar in normal and malignant prostate-derived cells at the mRNA level, expression at the protein level was very different. HSF1 and HSP protein expression was highest in the 3 aggressively metastatic prostate cancer cell lines (PC-3, DU-145, and CA-HPV-10). Although the gene expression patterns of constitutive HSP differ enormously in PC-3 cells in vitro and in xenografts in vivo, stress induction of HSP genes is not markedly altered by exposure to the tumor microenvironment, indicating the hierarchical rank of the stress response that permits it to override other forms of regulation. ……

The experiments described here are largely supportive of the notion that HSP gene expression and HSF activity and expression are increased in more advanced stages of cancer (Fig 4). The most striking finding in the study was the elevation of HSF1 and HSP levels in aggressively malignant prostate carcinoma cell lines (Fig 4). It is significant that these changes in HSF and HSP levels would not have been predicted from microarray studies of HSF (Fig 3) and HSP (Fig 1) mRNA levels. The increased HSF levels observed in the metastatic prostate carcinoma cell lines in particular appear to be due to altered regulation of either mRNA translation or protein turnover (or both) (Figs 3 and ​and4).4). Although we do not at this stage know the mechanisms involved, 1 candidate could be differential activity of the proteosome in the metastatic cell lines: both HSF1 and HSF2 are targets for proteosomal degradation (Mathew et al 1998). Despite these differences in HSP expression between cells of varying degrees of malignancy under growth conditions, stress caused a major shift in HSP gene expression and activation of HSP40-1, HSP70-1A, HSP70-1B, HSP70-6 (HSP70B), DNA-J2–like, and HSP105 in all cells (Fig 2). Even in LnCap cells with minimal HSF1 and HSF2 expression, heat-inducible HSP70 protein expression was observed (Fig 4). Interestingly, we observed minimal induction of the HSP70B gene in LnCap cells: because the HSP70B promoter is known to be almost exclusively induced by stress through the HSE in its promoter, the findings may suggest that a mechanism for HSP70 induction alternative to HSF1 activation may be operative in LnCap cells (Schiller et al 1988). Increased HSP expression in cancer patients has been shown to signal a poor response to treatment by a number of modalities, suggesting that HSP expression is involved with development of resistance to treatment in addition to being involved in the mechanisms of malignant progression (Ciocca et al 1993Cornford et al 2000Yamamoto et al 2001Ciocca and Vargas-Roig 2002;Mese et al 2002). In addition, subpopulations of LnCap-derived cells, selected for enhanced capacity to metastasize, have been shown to express elevated levels of HSF1, HSP70, and HSP27 compared with nonselected controls (Hoang et al 2000). This may be highly significant because our studies indicate minimal levels of HSF1 and HSP in the poorly metastatic parent LnCap cells (Figs 1 and ​and4).4). Previous studies have also indicated that elevated HSP70 expression occurs at an early stage in cellular immortalization from embryonic stem cells (Ravagnan et al 2001). We had to use immortalized prostatic epithelial cells for our normal controls and may have missed a very early change in HSP expression during the immortalization process.

As indicated by the kinetic studies (Figs 5–7), HSPs are activated at a number of regulatory levels by stress in addition to transcriptional activation, and these may include stress-induced mRNA stabilization, differential translation, and protein stabilization (Hickey and Weber 1982Zhao et al 2002). HSF1 activity and HSP expression appear to be subject to differential regulation by a number of pathways at normal temperatures but are largely independent of such regulation when exposed to heat shock, which overrides constitutive regulation and permits prompt induction of this emergency response.

Growth of PC-3 cells in vivo as tumor xenografts was accompanied by a marked decrease in constitutive HSP expression (Figs 8 and ​and11).11). Decreased HSP expression was part of a global switch in gene expression that accompanies the switch of PC-3 cells from growth as monolayers in tissue culture to growth as tumors in vivo (D. Tang and S.K. Calderwood, in preparation). Many reports indicate changes in a wide range of cellular properties as cells grow as tumors, and these properties may reflect the remodeling of gene expression patterns. These changes may reflect adaptation to the chemical nature of the tumor microenvironment and the alterations in cell-cell interaction in growth as a tumor in vivo. Our studies also indicate the remarkable sturdiness of the heat shock response that remains intact in the PC-3 cells growing in vivo despite the global rearrangements in other gene expressions mentioned above (Figs 10 and ​and1111).

The elevation in HSF1 and HSP levels in cancer shown in our studies and in those of others and its association with a poor prognosis and inferior response to therapy suggests the strategy of targeting HSP in cancer therapy. Treatment with HSP70 antisense oligonucleotides, for instance, can cause tumor cell apoptosis on its own and can synergize with heat shock in cell killing (Jones et al 2004). Indeed, it has been shown that antagonizing heat-inducible HSP expression with quercitin, a bioflavonoid drug that inhibits HSF1 activation, or by using antisense oligonucleotides directed against HSP70 mRNA further sensitizes PC-3 cells to heat-induced apoptosis in vitro and leads to tumor regression in vivo (Asea et al 2001Lepchammer et al 2002Jones et al 2004) (A. Asea et al, personal communication). The strategy of targeting HSP expression or function in cancer cells may thus be indicated. Such a strategy might prove particularly effective because constitutive HSP expression is reduced in tumors, and this might be related to increased killing of PC-3 tumor cells by heat (Fig 12).


  1. Molecular chaperones in aging

Aging and molecular chaperones

Csaba So˝ti*, Pe´ter Csermely
Exper Geront 2003; 38:1037–1040

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Molecular chaperones Chaperones are ubiquitous, highly conserved proteins (Hartl, 1996), either assisting in the folding of newly synthesized or damaged proteins in an ATP-dependent active process or working in an ATP-independent passive mode sequestering damaged proteins for future refolding or digestion. Environmental stress leads to proteotoxic damage. Damaged, misfolded proteins bind to chaperones, and liberate the heat shock factor (HSF) from its chaperone complexes. HSF is activated and transcription of chaperone genes takes place (Morimoto, 2002). Most chaperones, therefore, are also called stress or (after the archetype of experimental stress) heat shock proteins (Hsp-s).

Aging proteins—proteins of aging organisms During the life-span of a stable protein, various posttranslational modifications occur including backbone and side chain oxidation, glycation, etc. In aging organisms, the disturbed cellular homeostasis leads to an increased rate of protein modification: in an 80-year old human, half of all proteins may become oxidized (Stadtman and Berlett, 1998). Susceptibility to various proteotoxic damages is mainly increased due to dysfunction of mitochondrial oxidation of starving yeast cells (Aguilaniu et al., 2001). In prokaryotes, translational errors result in folding defects and subsequent protein oxidation (Dukan et al., 2000), which predominantly takes place in growth arrested cells (Ballesteros et al., 2001). Additionally, damaged signalling networks loose their original stringency, and irregular protein phosphorylation occurs (e.g.: the Parkinson disease-related a-synuclein also becomes phosphorylated, leading to misfolding and aggregation; Neumann et al., 2002).

Aging protein degradation Irreversibly damaged proteins are recognized by chaperones, and targeted for degradation. Proteasome level and function decreases with aging, and some oxidized, aggregated proteins exert a direct inhibition on proteasome activity. Chaperones also aid in lysosomal degradation. The proteolytic changes are comprehensively reviewed by Szweda et al. (2002). Due to the degradation defects, damaged proteins accumulate in the cells of aged organisms, and by aggregation may cause a variety of protein folding diseases (reviewed by So˝ti and Csermely, 2002a).

Aging chaperones I: defects in chaperone induction Damaged proteins compete with the HSF in binding to the Hsp90-based cytosolic chaperone complex, which may contribute to the generally observed constitutively elevated chaperone levels in aged organisms (Zou et al., 1998; So˝ti and Csermely, 2002b). On the contrary, the majority of the reports showed that stress-induced synthesis of chaperones is impaired in aged animals. While HSF activation does not change, DNA binding activity may be reduced during aging (Heydari et al., 2000). A number of signaling events use an overlapping network of chaperones not only to establish the activation-competent state of different transcription factors (e.g. steroid receptors), but also as important elements in the attenuation of respective responses. HSF transcriptional activity is also negatively influenced by higher levels of chaperones (Morimoto, 2002). Differential changes of these proteins in various organisms and tissues may lead to different extents of (dys)regulation. More importantly, the cross-talk between different signalling pathways through a shared pool of chaperones may have severe consequences during aging when the cellular conformational homeostasis is deranged (see below).

Aging chaperones II: defects in chaperone function   Direct studies on chaperone function in aged organisms are largely restricted to a-crystallin having a decreased activity in aged human lenses (Cherian and Abraham, 1995; Cherian-Shaw et al., 1999). In a recent study, an initial test of passive chaperone function of whole cytosols was assessed showing a decreased chaperone capacity in aged rats compared to those of young counterparts (Nardai et al., 2002). What can be the mechanism behind these deleterious changes in chaperone function? Chaperones may also be prone to oxidative damage, as GroEL is preferentially oxidized in growth-arrested E. coli (Dukan and Nystro¨m, 1999). Macario and Conway de Macario (2002) raised the idea of ‘sick chaperones’ in aged organisms in a recent review. Indeed, chaperones are interacting with a plethora of other proteins (Csermely, 2001a), which requires rather extensive binding surfaces. These exposed areas may make chaperones a preferential target for proteotoxic damage: chaperones may behave as ‘suicide proteins’ during aging, sacrificing themselves instead of ‘normal’ proteins. The high abundance of chaperones (which may constitute more than 5% of cellular proteins), and their increased constitutive expression in aged organisms makes them a good candidate for this ‘altruistic courtesy.’ It may be especially true for mitochondrial Hsp60, the role of which would deserve extensive studies.

Aging chaperones III: defects in capacity, the chaperone overload Another possible reason of decreased chaperone function is chaperone overload (Csermely, 2001b). In aging organisms, the balance between misfolded proteins and available free chaperones is grossly disturbed: increased protein damage, protein degradation defects increase the amount of misfolded proteins, while chaperone damage, inadequate synthesis of molecular chaperones and irreparable folding defects (due to posttranslational changes) decrease the amount of available free chaperones. Chaperone overload occurs, where the need for chaperones may greatly exceed the available chaperone capacity (Fig. 1). Under these conditions, the competition for available chaperones becomes fierce and the abundance of damaged proteins may disrupt the folding assistance to other chaperone targets, such as: (1) newly synthesized proteins; (2) ‘constantly damaged’ (mutant) proteins; and (3) constituents of the cytoarchitecture (Csermely, 2001a). This may cause defects in signal transduction, protein transport, immune recognition, cellular organization as well as the appearance of previously buffered, hidden mutations in the phenotype of the cell (Csermely, 2001b). Chaperone overload may significantly decrease the robustness of cellular networks, as well as shift their function towards a more stochastic behavior. As a result of this, aging cells become more disorganized, their adaptation is impaired.

Fig. 1. Chaperone overload: a shift in the balance between misfolded proteins and available free chaperones in aging organisms. The accumulation of chaperone substrates along with an impaired chaperone function may exhaust the folding assistance to specific chaperone targets and leads to deterioration in vital processes. Chaperone overload may significantly decrease the robustness of cellular networks, and compromise the adaptative responses. See text for details.

Senescent cells and chaperones The involvement of chaperones in aging at the cellular level is recently reviewed (So˝ti et al., 2003). Non-dividingsenescent-peripheral cells tend to have increased chaperone levels (Verbeke et al., 2001), and cannot preserve the induction of several chaperones (Liu et al., 1989), similarly to cells from aged animals. Activation and binding of HSF to the heat shock element is decreased in aged cells (Choi et al., 1990). Interestingly, cellular senescence seems to unmask a proteasomal activity leading to the degradation of HSF (Bonelli et al., 2001). Chaperone induction per se seems to counteract senescence. Repeated mild heat shock (a kind of hormesis) has been reported to delay fibroblast aging (Verbeke et al., 2001), though it does not seem to extend replicative lifespan. A major chaperone, Hsp90 is required for the correct function of telomerase, an important enzyme to extend the life-span of cells (Holt et al., 1999). Mortalin (mtHsp70/Grp75), a member of the Hsp70 family, produces opposing phenotypic effects related to its localization. In normal cells, it is pancytoplasmically distributed, and its expression causes senescence. Its upregulation and perinuclear distribution, however, is connected to transformation, probably via p53 inactivation. Mortalin also induces life-span extension in human fibroblasts or in C. elegans harboring extra copies of the orthologous gene (Kaul et al., 2002).

Aging organisms and chaperones: age-related diseases Unbalanced chaperone requirement and chaperone capacity in aged organisms helps the accumulation of aggregated proteins, which often cause folding diseases, mostly of the nervous system, due to the very limited proliferation potential of neurons. Over expression of chaperones often delays the onset or diminishes the symptoms of the disease (So˝ti and Csermely, 2002b). Other aging diseases, such as atherosclerosis and cancer are also related to chaperone action. Here space limitation precludes a detailed description of these rapidly developing fields, however, numerous recent reviews were published on these subjects, where the interested readers may find a good summary and several hints for further readings (Ferreira and Carlos, 2002; Neckers, 2002; Sarto et al., 2000; Wick and Xu, 1999).


Chaperones and Longevity

Increased chaperone induction leads to increased longevity (Tatar et al., 1997). Moreover, a close correlation exists between stress resistance and longevity in several long-lived C. elegans and Drosophila mutants (Lithgow and Kirkwood, 1996). As the other side of the same coin, damaged HSF has been found as an important gene to cause accelerated aging in C. elegans (Garigan et al., 2002). Caloric restriction, the only effective experimental manipulation known to retard aging in rodents and primates (Ramsey et al., 2000), restores age-impaired chaperone induction, while reversing the age-induced changes in constitutive Hsp levels (see So˝ti and Csermely, 2002a,b). These examples confirm the hypothesis that a better adaptation capacity to various stresses greatly increases the chances to reach longevity. 10. Conclusions and perspectives Aging can be defined as a multicausal process leading to a gradual decay of self-defensive mechanisms, and an exponential accumulation of damage at the molecular, cellular and organismal level. The protein oxidation, damage, misfolding and aggregation together with the simultaneously impaired function and induction of chaperones in aged organisms disturb the balance between chaperone requirement and availability. There are several important aspects for future investigation of this field: † the measurement of active chaperone function (i.e. chaperone-assisted refolding of damaged proteins) in cellular extracts does not have a well-established method yet; † we have no methods to measure free chaperone levels; † among the consequences of chaperone overload, changes in signal transduction, protein transport, immune recognition and cellular organization have not been systematically measured and/or related to the protein folding homeostasis of aging organisms and cells.


  1. Extracellular HSPs in inflammation and immunity

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1

Amir Kol,* Andrew H. Lichtman,† Robert W. Finberg,‡ Peter Libby,*† and Evelyn A. Kurt-Jones2‡
J  Immunol 2000; 164: 13–17.  https://www.researchgate.net/profile/Robert_Finberg/publication/12696457_Cutting_Edge_Heat_Shock_Protein_(HSP)_60_Activates_the_Innate_Immune_Response_CD14_Is_an_Essential_Receptor_for_HSP60_Activation_of_Mononuclear_Cells/links/53ee00460cf23733e80b21c0.pdf

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

There is increasing interest in the role of nontraditional mediators of inflammation in atherosclerosis (1). Recent studies from our laboratory have shown that chlamydial and human heat shock protein 60 (HSP60)3 colocalize in human atheroma (2), and either HSP60 induces adhesion molecule and cytokine production by human vascular cells and macrophages, in a pattern similar to that induced by Escherichia coli LPS (3, 4). These results suggested that HSP60 and LPS might share similar signaling mechanisms. CD14 is the major high-affinity receptor for bacterial LPS on the cell membrane of mononuclear cells and macrophages (5, 6). In addition to LPS, CD14 functions as a signaling receptor for other microbial products, including peptidoglycan from Gram-positive bacteria and mycobacterial lipoarabinomann (7, 8). CD14 is considered a pattern recognition receptor for microbial Ags and, with Toll-like receptor (TLR) proteins, an important mediator of innate immune responses to infection (9–14). We have examined the role of CD14 in the response of human monocytes and macrophages to HSP60.  …..

HSP may play a central role in the innate immune response to microbial infections. Because both microbes and stressed or injured host cells produce abundant HSP (36), and dying cells likely release these proteins, it is conceivable that HSP furnish signals that inform the innate immune system of the presence of infection and cell damage. The findings reported here, that human HSP60 induces IL-6 production by mononuclear cells and macrophages via the CD14, supports this hypothesis, suggesting that human HSP60 may act together with LPS or other microbial products to provoke innate immune responses.

Inflammation and immunity can contribute to the pathogenesis and complications of atherosclerosis (37). Moreover, the search for novel risk factors for atherosclerosis has revived the concept that microbial products might substantially contribute to the inflammatory reaction in the atheromatous vessel wall (38, 39). We have shown that chlamydial HSP60 colocalizes with human HSP60 in the macrophages of human atheroma (2). Therefore, bacterial and human HSP60, released from dying or injured cells during atherogenesis (40) or myocardial injury (41), may further promote local inflammation and possibly activate the innate immune system. Previous reports that immunization with mycobacterial HSP65 enhances atheroma formation in rabbits (42), have suggested an important role for HSPs in atherogenesis, particularly because the high degree of homology between HSPs of the same m.w. among different species might stimulate autoimmunity (43).

In conclusion, our findings, that CD14 mediates cellular activation induced by human HSP60 provide further insight into the molecular mechanisms by which HSP may activate the innate immune system and participate in atherogenesis and other inflammatory disorders.

DAMPs, PAMPs and alarmins: all we need to know about danger

Marco E. Bianchi1
J. Leukoc. Biol. 81: 1–5; 2007.   http://aerozon.ru/documents/publications/37_Bianche.pdf

Multicellular animals detect pathogens via a set of receptors that recognize pathogen associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term “alarmin” is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage associated molecular patterns (DAMPs).

Multicellular animals must distinguish whether their cells are alive or dead and detect when microorganisms intrude, and have evolved surveillance/defense/repair mechanisms to this end. How these mechanisms are activated and orchestrated is still incompletely understood, and I will argue that that these themes define a unitary field of investigation, of both basic and medical interest.

A complete system for the detection, containment, and repair of damage caused to cells in the organism requires warning signals, cells to respond to them via receptors and signaling pathways, and outputs in the form of physiological responses. Classically, a subset of this system has been recognized and studied in a coherent form: pathogen-associated molecular patterns (PAMPs) are a diverse set of microbial molecules which share a number of different recognizable biochemical features (entire molecules or, more often, part of molecules or polymeric assemblages) that alert the organism to intruding pathogens [1]. Such exogenous PAMPs are recognized by cells of the innate and acquired immunity system, primarily through toll-like receptors (TLRs), which activate several signaling pathways, among which NF-kB is the most distinctive. As a result, some cells are activated to destroy the pathogen and/or pathogen-infected cells, and an immunological response is triggered in order to produce and select specific T cell receptors and antibodies that are best suited to recognize the pathogen on a future occasion. Most of the responses triggered by PAMPs fall into the general categories of inflammation and immunity.

However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Tissues can be ripped, squashed, or wounded by mechanical forces, like falling rocks or simply the impact of one’s own body hitting the ground. Animals can be wounded by predators. In addition, tissues can be damaged by excessive heat (burns), cold, chemical insults (strong acids or bases, or a number of different cytotoxic poisons), radiation, or the withdrawal of oxygen and/or nutrients. Finally, humans can also be damaged by specially designed drugs, such as chemotherapeutics, that are meant to kill their tumor cells with preference over their healthy cells. Very likely, we would not be here to discuss these issues if evolution had not incorporated in our genetic program ways to deal with these damages, which are not caused by pathogens but are nonetheless real and common enough. Tellingly, inflammation is also activated by these types of insults. A frequently quoted reason for the similarity of the responses evoked by pathogens and trauma is that pathogens can easily breach wounds, and infection often follows trauma; thus, it is generally effective to respond to trauma as if pathogens were present. In my opinion, an additional reason is that pathogens and trauma both cause tissue and cell damage and thus trigger similar responses.

None of these considerations is new; however, a new awareness of the close relationship between trauma- and pathogenevoked responses emerged from the EMBO Workshop on Innate Danger Signals and HMGB1, which was held in February 2006 in Milano (Italy); many of the findings presented at the meeting are published in this issue of the Journal of Leukocyte Biology. At the end of the meeting, Joost Oppenheim proposed the term “alarmin” to differentiate the endogenous molecules that signal tissue and cell damage. Together, alarmins and PAMPs therefore constitute the larger family of damage-associated molecular patterns, or DAMPs.

Extranuclear expression of HMGB1 has been involved in a number of pathogenic conditions: sepsis [44], arthritis [45, 46], atherosclerosis [10], systemic lupus erythematosus (SLE) [47], cancer [48] and hepatitis [49, this issue]. Uric acid has been known to be the aethiologic agent for gout since the 19th century. S100s may be involved in arthritis [31, this issue] and psoriasis [50]. However, although it is clear that excessive alarmin expression might lead to acute and chronic diseases, the molecular mechanisms underlying these effects are still largely unexplored.

The short list of alarmins presented above is certainly both provisional and incomplete and serves only as an introduction to the alarmin concept and to the papers published in this issue of JLB. Other molecules may be added to the list, including cathelicidins, defensins and eosinophil-derived neurotoxin (EDN) [51], galectins [52], thymosins [53], nucleolin [54], and annexins [55; and 56, this issue]; more will emerge with time. Eventually, the concept will have to be revised and adjusted to the growing information. Indeed, I have previously argued that any misplaced protein in the cell can signal damage [57], and Polly Matzinger has proposed that any hydrophobic surface (“Hyppo”, or Hydrophobic protein part) might act as a DAMP [58]. As most concepts in biology, the alarmin category serves for our understanding and does not correspond to a blueprint or a plan in the construction of organisms. Biology proceeds via evolution, and evolution is a tinkerer or bricoleur, finding new functions for old molecules. In this, the reuse of cellular components as signals for alerting cells to respond to damage and danger, is a prime example.


  1. Role of heat shock and the heat shock response in immunity and cancer


Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

Stuart K. Calderwood,1,* Ayesha Murshid,1 and Jianlin Gong1
Front Immunol. 2012; 3: 75.  doi:  10.3389/fimmu.2012.00075

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock protein, vaccine, inflammation, antigen presentation

Heat shock proteins (HSP) were first discovered as a group of polypeptides whose level of expression increases to dominate the cellular proteome after stress (Lindquist and Craig, 1988). These increases in HSPs synthesis correlate with a marked resistance to potentially toxic stresses such as heat shock (Li and Werb,1982). The finding that such proteins have extracellular immune functions suggested that, as highly abundant intracellular proteins they could be prime candidates as danger signals to the immune response (Srivastava and Amato,2001). There are several human HSP gene families with known immune significance and their classification is reviewed in Kampinga et al. (2009). These include the HSPA (Hsp70) family, which includes the HPA1A and HSPA1B genes encoding the two major stress-inducible Hsp70s, that together are often referred to as Hsp72. When referring to Hsp70 in this chapter, we generally refer to the products of these two genes. The Hsp70 family also includes two other members with immune function – HSPA8 and HSPA5 genes, whose protein products are known as Hsc70 the major constitutive Hsp70 family member and Grp78, a key ER-resident protein. In addition two more Hsp70 related genes have immune significance and these include HSPH2 (Hsp110) and HSPH4 the ER-resident class H protein Grp170. The Hsp90 family also has major functions in tumor immunity and these include HSPC2 and HSPC3, which encode the major cytoplasmic proteins Hsp90a and Hsp90b, and HSPC4 that encodes ER chaperone Grp94. In addition, the product of the HSPD1 gene, the mitochondrial chaperone Hsp60 has some immunological functions. Mice have been shown to encode orthologs of each of these genes (Kampinga et al., 2009).

It has been suggested that many of the HSPs have the property of damage associated molecular patterns (DAMPs), inducers of sterile inflammation and innate immunity (Kono and Rock, 2008). The additional discovery that intracellular HSPs function as molecular chaperones and can bind to a wide spectrum of intracellular polypeptides further indicated that they could play a broad role in the immune response and might mediate both innate immunity due to their status as DAMPs and adaptive immunity by chaperoning antigens.

Heat shock proteins are currently employed as vaccines in cancer immunotherapy (Tamura et al., 1997; Murshid et al., 2011a). The rationale behind the approach is that if HSPs can be extracted from tumor tissue bound to the polypeptides which they chaperone during normal metabolism, they may retain antigenic peptides specific to the tumor (Noessner et al., 2002; Srivastava, 2002; Wang et al., 2003; Enomoto et al., 2006; Gong et al., 2010). Indeed, vaccines based on Hsp70, Hsp90, Grp94, Hsp110, and Grp170 polypeptide complexes have been used successfully to immunize mice to a range of tumor types and Hsp70 and Grp94 vaccines have undergone recent clinical trials (rev: Murshid et al., 2011a). These effects of the HSP vaccines on tumor immunity appear to be mediated largely to the associated, co-isolated tumor polypeptides, although in the case of Grp94 this question is still controversial and tumor regression was observed in mice treated with the chaperone devoid of its peptide binding domain (Udono and Srivastava, 1993; Srivastava, 2002; Nicchitta, 2003; Chandawarkar et al., 2004; Nicchitta et al.,2004). Use of such HSP vaccines is potentially a powerful approach to tumor immunotherapy as the majority of the antigenic repertoire of most individual tumor cells is unknown (Srivastava and Old, 1988; Srivastava, 1996). Individual cancer cells are likely to take a lone path in accumulating a spectrum of random mutations. Although some mutations are functional, permitting cells to become transformed and to progress into a highly malignant state, many such changes are likely to be passenger mutations not required to drive tumor growth (Srivastava and Old, 1988; Srivastava, 1996). Some of these individual mutant sequences will be novel antigenic epitopes and together with the few known shared tumor antigens comprise an “antigenic fingerprint” for each individual tumor (Srivastava,1996). Accumulation of mutations in cancer appears to be related to, and may drive the increases in HSPs observed in many tumors (Kamal et al., 2003; Whitesell and Lindquist, 2005; Trepel et al., 2010). As the mutant conformations of tumor proteins are “locked in” due to the covalent nature of the alterations, cancer cells appear to be under permanent proteotoxic stress and rich in HSP expression (Ciocca and Calderwood, 2005). For tumor immunology these conditions may offer a therapeutic opportunity as individual HSPs, whose expression is expanded in cancer will chaperone a cross-section of the “antigenic fingerprint” of the individual tumors (Murshid et al., 2011a). This approach was first utilized by Srivastava (20002006) and led to the development of immunotherapy using HSP–peptide complexes.

In addition to using HSP–peptide complexes extracted from tumors, in cases where tumor antigens are known, these can be directly loaded onto purified or recombinant HSPs and the complex used as a vaccine. This procedure has been carried out successfully in the case of the “large HSPs,” Hsp110 and Grp170 (Manjili et al., 20022003). A variant of this approach employs the molecular engineering of tumor antigens in order to produce molecular chaperone-fusion genes which encode products in which the HSP is fused covalently to the antigen. The fusion proteins are then employed as vaccines. This approach was pioneered by Young et al. who showed that a fusion between mycobacterial Hsp70 and ovalbumin could induced cytotoxic lymphocytes (CTL) in mice with the capacity to kill Ova-expressing cancer cells (Suzue et al., 1997). The vaccines could be used effectively without adjuvant and adjuvant properties were ascribed to the molecular chaperone component of the fusion protein. Subsequent studies have confirmed the utility of the approach in targeting common tumor antigens such as the melanoma antigen Mage3 (Wang et al., 2009).

HSPs and Immunosurveillance in Cancer

The question next arises as to the role of endogenous HSPs, with or without bound antigens in immunosurveillance of cancer cells. Although the immune system can recognize tumor antigens and generate a CTL response, most cancers evade immune cell killing by a range of strategies (van der Bruggen et al., 1991; Pardoll,2003). These include the down-regulation of surface MHC class I molecules by individual tumor cells and release of immunosuppressive IL-10 by tumors (Moller and Hammerling, 1992; Chouaib et al., 2002). Tumors in vivo also appear to attract a range of hematopoietic cells with immunosuppressive action including regulatory CD4+CD25+FoxP3+ T cells (Treg), M2 macrophages, myeloid-derived suppressor cells (MDSC) and some classes of natural killer cells (Pekarek et al.,1995; Terabe et al., 2005; Mantovani et al., 2008; Marigo et al., 2008). The tumor milieu also contain a small fraction of cells of mesenchymal origin identified by surface fibroblast activation protein-a (FAP cells) that suppress antitumor immune responses (Kraman et al., 2010). Endogenous tumor HSPs may also participate in immune suppression. Although the majority of the HSPs function as intracellular molecular chaperones, a fraction of these proteins can be released from cells even under unstressed conditions and may participate in immune functions (rev: Murshid and Calderwood, 2012). Intracellular Hsp70 can be actively secreted from tumor cells in either free form or packaged into lipid-bounded structures called exosomes (Mambula and Calderwood, 2006b; Chalmin et al., 2010). In addition Hsp70 and Hsp90 can also be found associated with the surfaces of tumor cells where they can function as molecular chaperones or as recognition structures for immune cells (Sidera et al., 2008; Qin et al., 2010; Multhoff and Hightower, 2011). As Hsp70 was shown in a number of earlier studies to be pro-inflammatory due to its interaction with pattern recognition receptors such as Toll-like receptors 2 and 4 (TLR2 and TLR4), these findings might suggest, as mentioned above, that Hsp70 released by tumors could be pro-inflammatory and possess the properties of DAMPs (Asea et al., 20002002; Vabulas et al., 2002). However, subsequent studies indicated that a portion of the TLR4 activation detected in the earlier reports, involving exposure of monocytes, macrophages, or dendritic cells (DC) to HSPs in vitro may be due to trace contamination with bacterial pathogen associated molecular patterns (PAMPs), potent TLR activators (Tsan and Gao,2004). In spite of these drawbacks, an overwhelming amount of evidence now seems to indicate the interaction of Hsp70 and other HSPs with TLRs (particularly TLR4) in vivo – in a wide range of physiological and pathological conditions, leading to acute inflammation in many conditions (Chase et al., 2007; Wheeler et al., 2009; see Appendix for a full list of references). Thus both TLR2 and TLR4 appear to be important components of inflammatory responses to Hsp70 under many pathophysiological conditions. In cancer therapy it has been shown that autoimmunity can be triggered in mice through necrotic killing of melanocytes engineered to overexpress Hsp70; such treatment led to the concomitant immune destruction of B16 melanoma tumors that share patterns of antigen expression with the killed melanocytes (Sanchez-Perez et al., 2006). Hsp70 appears to play an adjuvant role in this form of therapy through its interaction with TLR4 and induction of the cytokine TNF-a (Sanchez-Perez et al., 2006). However, despite these findings it has also been shown that depletion of Hsp70 in cancer cells can, in the absence of other treatments lead to tumor regression by inducing antitumor immunity (Rerole et al., 2011). This effect appears to be due to the secretion by cancer cells of immunosuppressive exosomes containing Hsp70 that activate MDSC and lead to local immunosuppression (Chalmin et al., 2010). Under normal circumstances therefore, release of endogenous Hsp70 into the extracellular microenvironment may be a component of the tumor defenses against immunosurveillance. Extracellular Hsp60 has also been shown be immunomodulatory and can increase levels of FoxP3 Treg in vitro and suppress T cell-mediated immunity (de Kleer et al., 2010; Aalberse et al., 2011).

The pro-inflammatory properties of extracellular HSPs may be more evident underin vivo situations particularly in the context of tissue damage (Sanchez-Perez et al.,2006). For instance when elevated temperatures were used to boost Hsp70 release from Lewis Lung carcinoma cells in vivo, antitumor immunity was activated along with release of chemokines CCL2, CCL5, and CCL10, in a TLR4-dependent manner, leading to attraction of DC and T cells into the tumor (Chen et al., 2009). Thus under resting conditions, the tumor milieu appears to be a specialized immunosuppressive environment, rich in inhibitory cells such as Treg, MDSC, and M2 macrophages and inaccessible to “exhausted” CD8+ T cells that often fail to penetrate the tumor microcirculation. However, under inflammatory conditions involving necrotic cell killing of tumor cells, extracellular HSPs may be able to amplify the anticancer immune response, intracellular HSPs may be released to further increase such a response and CTL may triggered to penetrate the tumor milieu, inducing antigen-specific cancer cell killing (Evans et al., 2001; Mambula and Calderwood, 2006a; Sanchez-Perez et al., 2006; Chen et al., 2009).


HSP-Based Anticancer Vaccines

It is apparent that a number of HSP types, conjugated to peptide complexes (HSP.PC) from cancer cells form effective bases for immunotherapy approaches with unique properties, as mentioned above (Calderwood et al., 2008; Murshid et al., 2011a). The immunogenicity of most HSP.PC appears to involve the ability of the HSPs to sample the tumor “antigenic fingerprint,” deliver the antigens to antigen presenting cells (APC) such as DC and stimulate activation of CTL (Tamura et al., 1997; Singh-Jasuja et al., 2000b; Wang et al., 2003; Murshid et al.,2010). A number of studies show that HSPs can chaperone tumor antigens and deliver them to the appropriate destination – MHC class I molecules on the DC surface (Singh-Jasuja et al., 2000a,b; Srivastava and Amato, 2001; Delneste et al.,2002; Enomoto et al., 2006; Gong et al., 2009). In addition, Hsp70 has been shown to chaperone viral antigenic peptides and increase cross priming of human CTL under ex vivo conditions (Tischer et al., 2011). However, it is still far from clear how the process of HSP-mediated cross priming unfolds. For instance, the CD8+ expressing DC subpopulation in lymph nodes is regarded as the primary cross-presenting APC (Heath and Carbone, 2009). It is not however currently known whether the CD8+ DC subset or other peripheral or lymph-node resident, DC interact with HSP vaccines to induce cross presentation. HSPs appear to be able to enter APC, such as mouse bone marrow derived DC (BMDC) and human DC in a receptor-mediated manner (Basu et al., 2001; Delneste et al., 2002; Gong et al.,2009; Murshid et al., 2010). However, no unique endocytosing HSP receptor has emerged and HSP–antigen complexes appear instead to be taken up by proteins with “scavenger” function such as LOX-1, SRECI, and CD91 that can each take up a wide range of extracellular ligands (Basu et al., 2001; Delneste et al., 2002; Theriault et al., 2006; Murshid et al., 2010). A pathway for Hsp90–peptide (Hsp90.PC) uptake has been characterized in mouse BMDC by scavenger receptor SRECI (Murshid et al., 2010). SRECI is able to mediate the whole process of Hsp90.PC endocytosis, trafficking through the cytoplasm to the sites of antigen processing and presentation of antigens to CD8+ T lymphocytes on MHC class I molecules (Murshid et al., 2010). This process is known as antigen cross presentation (Kurts et al., 2010). It is not currently clear what the relative contribution to antigen cross presentation of the various HSP receptors might be under in vivo conditions. It may be that each receptor class contributes to an individual aspect of CTL activation by HSP peptide complexes although a definitive understanding may await studies in mice deficient in each receptor class.


HSPs and CTL Programming

It is evident that that HSPs can mediate antigen cross presentation and activate CD8+ T lymphocytes. However, presentation of tumor antigens by DC is not sufficient for CTL programming and, in the absence of co-stimulatory molecules and innate immunity, the “helpless” CD8+ cells will cease to proliferate abundantly and will most likely undergo apoptosis (Schurich et al., 2009; Kurts et al., 2010). One mechanism for enhancing CTL programming involves activation of the TLR pathways that lead to synthesis of co-stimulatory molecules (Rudd et al.,2009; Yamamoto and Takeda, 2010). The co-stimulatory molecules, including CD80 and CD86 then become expressed on the DC cell surface and amplify the signals induced by binding of the T cell receptor on CD8+ T cells to MHC class I peptide complexes on the presenting DC (Parra et al., 1995; Rudd et al., 2009). This process is important in pathogen infection in which microbially derived antigens are encountered in the presence of inflammatory PAMPs that can activate innate immune transcriptional networks. Originally it had been thought that HSPs could provide analogous stimulation through their suspected activity as DAMPs and their inbuilt ability to trigger innate immunity through TLR2 and TLR4 on DC (Asea et al., 20002002; Vabulas et al., 2002). (The potential role of HSPs as DAMPs has been the subject of a recent review: van Eden et al., 2012). Subsequent studies on the capacity of HSPs to bind TLRs do not indicate avid binding of Hsp70 to either TLR2 or TLR4 when expressed in cells deficient in HSP receptors in vitro (Theriault et al., 2006). In vivo however, TLR signaling is essential for Hsp70 vaccine-induced tumor cell killing. Studies of tumor-bearing mice treated with an Hsp70 vaccine in vivo indicated that vaccine function is depleted by knockout of the TLR signaling intermediate Myd88 and completely abrogated by double knockout of TLR2 and TLR4 (Gong et al., 2009). These findings were somewhat complicated by the fact that TLR4 is involved in upstream regulation of the expression of Hsp70 receptor SRECI, but do strongly implicate a role for these receptors in amplifying immune signaling by Hsp70 vaccines and Hsp70-based immunotherapy (Sanchez-Perez et al., 2006; Gong et al., 2009). It is still not clear to what degree HSPs are capable of providing a sturdy DC maturing signal through TLR2/TLR4. The potency of HSP anticancer vaccines could potentially be improved by addition of PAMPs such as CpG DNA shown to activate TLR9, or double stranded RNA that can activate TLR3 (Murshid et al., 2011a). As mentioned, one contradictory factor in the earlier studies was that, although TLR2 and TLR4 are required for a sturdy Hsp70 vaccine-mediated immune response, direct binding of Hsp70 to these receptors was not observed (Theriault et al., 2006; Gong et al., 2009; Murshid et al., 2012). A rationale for these findings might be that HSPs can activate TLR signaling indirectly through primary binding to established HSP receptors such as LOX-1 and SRECI which secondarily recruit and activate the TLRs (Murshid et al., 2011b). Both of these scavenger receptors bind to TLR2 upon stimulation and activate TLR2-based signaling (Jeannin et al., 2005; A. Murshid and SK Calderwood, in preparation). In addition, we have found that Hsp90–SRECI complexes move to the lipid raft compartment of the cell, an environment highly enriched in TLR2 and TLR4 (Triantafilou et al., 2002; Murshid et al., 2010).


Heat shock protein–peptide complexes extracted from tumor cells interact with endocytosing receptors (HSP-R) such as SRECI or signaling receptors (TLR) such as TLR4 on DC. SREC1 mediates uptake and intracellular processing of antigens and the presentation of resulting peptides on surface MHC class I and MHC class II proteins. MHC class II receptor–peptide complexes then bind to T cell receptors on CD4+ cells. One consequence of binding is interaction of CD40 ligand on the MHC class II cell with CD40 on the DC leading to the licensing interaction that results in enhanced expression of co-stimulatory proteins on the DC cell surface. The licensed DC may then interact with CD8+ T cells through T cell interaction with MHC class I peptide complexes. This effect will be enhanced by simultaneous interaction of CD80 or Cd86 co-stimulatory complexes on the DC with CD28 on the CD8+ cells, leading to effective CD8+ CTL that can lyse tumor cells. T cell programming can also be amplified by signals emanating from activated TLR that can boost levels of CD80 and CD86 as well as inflammatory cytokines (not shown).


Hsp70, Cell Damage, and Inflammation

The question of whether Hsp70 acts as DAMP and could by itself induce an inflammatory response in cancer patients in vivo is still open. However, some recent studies by Vile et al. using a gene therapy approach may shed some light on the inflammatory role of Hsp70 in tumor therapy. In this approach, as mentioned above, normal murine tissues were engineered to express high Hsp70 levels then subjected to treatments that lead to necrotic killing. The aim was to stimulate an autoimmune response that could lead to bystander immune killing of tumor cells that share the antigenic repertoire as the killed normal cells (Sanchez-Perez et al.,2006). In the initial studies, normal melanocytes were preloaded with Hsp70 plasmids and then necrotic cell death was triggered (Daniels et al., 2004). This treatment led to T cell-mediated immune killing of syngeneic B16 melanoma cells transplanted at a distant site in the mouse, presumably in response to antigens shared by the killed normal melanocytes and melanoma cell (Daniels et al., 2004). This effect only occurred when melanocytes were induced to undergo necrosis and Hsp70 levels were elevated, indicating a role for high levels of Hsp70 in the tumor specific immune response. Interestingly, these conditions did not lead to a prolonged autoimmune response, an effect mediated by the induction of a delayed Treg response (Srivastava, 2003; Daniels et al., 2004). It is notable that some early studies of chaperone-based tumor vaccines in animal models demonstrated a primary CTL response to tumors in response to treatment followed by delayed activation of a Treg reaction, and that chaperone levels must be carefully titrated for effective induction of tumor immunity (Udono and Srivastava, 1993; Liu et al.,2009). The role of Hsp70 in autoimmune rejection of tumors was also investigated in prostate cancer (Kottke et al., 2007). Ablation of normal prostate cells by necrotic killing with fusogenic viruses in the absence of Hsp70 elevation led to the induction of the cytokines IL-10 and TGF-b in the mouse prostate and a Treg response. However, when Hsp70 levels were elevated in these cells, IL-10, TGF-b, and IL-6 were induced simultaneously, the IL-6 component leading to further induction of IL-17, a profound Th17 response and tumor rejection (Kottke et al.,2007). Thus elevated levels of Hsp70, presumably released from cells undergoing necrosis can influence the local cytokine patterns and lead to an inflammatory statein vivo. Interestingly, these results seem to be tissue specific as inflammatory killing of pancreatic cells even in the presence of elevated Hsp70 did not provoke IL-6 release, a Th17 response or tumor rejection and the Treg response dominated under these conditions (Kottke et al., 2009). Thus the role of Hsp70 in tissue inflammation and tumor rejection seems to require elevated concentrations of extracellular chaperones, significant levels of necrotic cell killing, and tissue specific cytokine release.


  • Earlier studies investigating HSP vaccines considered such structures to be the “Swiss penknives” of immunology able to deliver antigens directly to APC and confer a maturing signal that could render DC able to effectively program CTL (Srivastava and Amato, 2001; Noessner et al., 2002). It is well established now that Hsp70, Hsp90, Hsp110, and GRP170 can chaperone tumor antigens and activate antigen cross presentation (Murshid et al., 2011a). In addition, HSPs were thought to be DAMPs with ability to strongly activate TLR signaling and innate immunity (Asea et al., 2000). However, although there is compelling evidence to indicate that Hsp70, for instance can interact with TLR4 under a number of pathological situations (see Appendix, Sanchez-Perez et al., 2006), it remains unclear whether free Hsp70 binds directly to the Toll-like receptor and induces innate immunity in the absence of other treatments in vitro(Tsan and Gao, 2004).
  • Elevated levels of extracellular HSPs appear to have the capacity to amplify the effects of inflammatory signals emanating from necrotic cells in vivoin a TLR4-dependent manner (Daniels et al., 2004; Sanchez-Perez et al., 2006; Kottke et al., 2007). In the presence of cell injury and death, elevated levels of Hsp70 appear to increase the production of inflammatory signals that involve cytokines such as IL-6 and IL-17 and lead to a specific T cell-mediated immune response to tumor cells sharing antigens with the dying cells (Kottke et al., 2007). The mechanisms involved in these processes are not clear although one possibility is that HSPs can induce the engulfment of necrotic cells. Hsp70 has been shown to increase bystander engulfment of a variety of structures (Wang et al., 2006a,b). In addition, tumor cells treated with elevated temperatures release inflammatory chemokines in an Hsp70 and TLR4-dependent mechanisms and this effect may be significant in CTL programming and tumor cell killing (Chen et al., 2009). Our studies indicate that CTL induction by Hsp70 vaccines in vivo has an absolute requirement for TLR2 and TLR4 suggesting that at least in vivo HSPs can trigger innate immunity through TLR signaling (Gong et al., 2009).
  • HSPs appear also to be able to direct antigen presentation through the class II pathway in DC and may stimulate T helper cells (Gong et al., 2009). It may thus be possible that HSPs participate in DC licensing and reinforce CTL programming during exposure to HSP vaccines. Future studies will address these questions.
  • A further interesting consideration is whether HSPs released from untreated tumor cells enhance or depress tumor immunity. One initial study shows that Hsp70 released from tumor cells in exosomes can strongly decrease tumor immunity through effects on MDSC (Chalmin et al., 2010). Further studies will be required to make a definitive statement on these questions.


  1. Protein aggregation disorders and HSP expression

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Christopher J. Cummings1,5, Michael A. Mancini3, Barbara Antalffy4, Donald B. DeFranco7, Harry T. Orr8 & Huda Y. Zoghbi1,2,6
Nature Genetics 19, 148 – 154 (1998) http://dx.doi.org:/10.1038/502

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease

Andreas Wyttenbach, Jenny Carmichael, Jina Swartz, Robert A. Furlong, Yolanda Narain, Julia Rankin, and David C. Rubinsztein*

Huntington’s disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGypolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2yHSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2yHSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2yHSDJ in protein folding.


  1. Hsp70 in blood cell differentiation.


Apoptosis Versus Cell Differentiation -Role of Heat Shock Proteins HSP90, HSP70 and HSP27

David Lanneau, Aurelie de Thonel, Sebastien Maurel, Celine Didelot, and Carmen Garrido
Prion. 2007 Jan-Mar; 1(1): 53–60.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs

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Stress or heat shock proteins (HSPs) were first discovered in 19621 as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.2 Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.3 Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α’s functions.4 HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.7 HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.8 This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.9

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.10 Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.11 One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.12Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases15 (Fig. 1).

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/figure/F1/

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and 

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.16 It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.


HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.2,1921 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.2224 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).


HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.25

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.26 A direct interaction between Akt and HSP90 was reported later.27 Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.26 Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.30 When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.27 Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.26 Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.31 HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.32 Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.33 The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p5334 and the transcription factors Her2 and Hif1α.35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.37

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,23 suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.38

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.


HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.39Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.40 In cancer cells, depletion of HSP70 results in spontaneous apoptosis.41

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation44 in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.45

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.46

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.47However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.48

HSP70 also prevents cell death in conditions in which caspase activation does not occur.49 Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.52 AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.53 HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation54 but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.55 During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.56

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).57 Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-150 and AIF binding,51 it is dispensable for JNK60 or GATA-161binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.61


HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.6466 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.65 In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.67

HSP27 can also interfere with caspase activation upstream of the mitochondria.66This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin68preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release66 (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,69 to decrease reactive oxygen species cell content,19 and to neutralize the toxic effects of oxidized proteins.70 These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.71

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.72 HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.73 In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).74 Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival75 while that of p27kip1facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.76

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.77 Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.78 In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary73,79 and it is its phosphorylated form that protects against neurotoxicity.80


HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.81 In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.82 In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.83

In leukemic cells HSP27 has been described as a pre-differentiation marker84because its induction occurs early during differentiation.8588 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes89 and for C2C12 muscle cells.90 This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G0/G1).19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells’ formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.91 HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.61 HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.18

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate92 and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.93 This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,94 RIP32 or Rb.95 Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.96 In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.97 In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.98 In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.4

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.99 In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G1 block. These G1 arrested cells do not differentiate but instead die by apoptosis.100

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.101Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.102 In breast cancer cells, 17-AAG-induced G1 block is accompanied by differentiation followed by apoptosis.103 The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.104

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.105 Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.16,17Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.61 Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.106It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.18 Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.


Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells’ aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells’ tumorigenicty and their depletion can induce a spontaneous regression of the tumors.24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.108 However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.

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HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death inC. elegans

A new pathway for non-apoptotic cell death

The results presented here allow us to construct a model for the initiation and execution of LCD in C. elegans (Figure 7). The logic of the LCD pathway may be similar to that of developmental apoptotic pathways. In C. elegans and Drosophila, where the control of specific cell deaths has been primarily examined, cell lineage or fate determinants control the expression of specific transcription factors that then impinge on proteins regulating caspase activation (Fuchs and Steller, 2011). Likewise, LCD is initiated by redundant determinants that require a transcription factor to activate protein degradation genes.

Figure 7.


Figure 7. Model for linker cell death.

Green, upstream regulators. Orange, HSF-1. Purple, proteolytic components.    DOI: http://dx.doi.org/10.7554/eLife.12821.016


Our data suggest that three partially redundant signals control LCD initiation. The antagonistic Wnt pathways we describe may provide positional information to the linker cell, as the relevant ligands are expressed only near the region where the linker cell dies. The LIN-29 pathway, which controls timing decisions during the L4-adult molt, may ensure that LCD takes place only at the right time. Finally, while the TIR-1/SEK-1 pathway could act constitutively in the linker cell, it may also respond to specific cues from neighboring cells. Indeed, MAPK pathways are often induced by extracellular ligands. We propose that these three pathways, together, trigger activation of HSF-1. Our data support a model in which HSF-1 is present in two forms, HSF-1LC, promoting LCD, and HSF-1HS, protecting cells from stresses, including heat shock. We postulate that the redundant LCD initiation pathways tip the balance in favor of HSF-1LC, allowing this activity to bind to promoters and induce transcription of key LCD effectors, including LET-70/UBE2D2 and other components of the ubiquitin proteasome system (UPS), functioning through E3 ligase complexes consisting of CUL-3, RBX-1, BTBD-2, and SIAH-1.

Importantly, the molecular identification of LCD components and their interactions opens the door to testing the impact of this cell death pathway on vertebrate development. For example, monitoring UBE2D2 expression during development could reveal upregulation in dying cells. Likewise, genetic lesions in pathway components we identified may lead to a block in cell death. Double mutants in apoptotic and LCD genes would allow testing of the combined contributions of these processes.

The proteasome and LCD

As is the case with caspase proteases that mediate apoptosis (Pop and Salvesen, 2009), how the UPS induces LCD is not clear, and remains an exciting area of future work. That loss of BTBD-2, a specific E3 ligase component, causes extensive linker cell survival suggests that a limited set of targets may be required for LCD. Previous work demonstrated that BTBD2, the vertebrate homolog of BTBD-2, interacts with topoisomerase I (Khurana et al., 2010; Xu et al., 2002), raising the possibility that this enzyme may be a relevant target, although other targets may exist.

The UPS has been implicated in a number of cell death processes in which it appears to play a general role in cell dismantling, most notably, perhaps, in intersegmental muscle remodeling during metamorphosis in moths (Haas et al., 1995). However, other studies suggest that the UPS can have specific regulatory functions, as with caspase inhibition by IAP E3 ligases (Ditzel et al., 2008).

During Drosophila sperm development, caspase activity is induced by the UPS to promote sperm individualization, a process that resembles cytoplasm-specific activation of apoptosis (Arama et al., 2007). While C. elegans caspases are dispensible for LCD, it remains possible that they participate in linker cell dismantling or serve as a backup in case the LCD program fails.

Finally, the proteasome contains catalytic domains with target cleavage specificity reminiscent of caspases; however, inactivation of the caspase-like sites does not, alone, result in overt cellular defects (Britton et al., 2009), suggesting that this activity may be needed to degrade only specific substrates. Although the proteasome generally promotes proteolysis to short peptides, site-specific cleavage of proteins by the proteasome has been described (Chen et al., 1999). It is intriguing to speculate, therefore, that caspases and the proteasome may have common, and specific, targets in apoptosis and LCD.

A pro-death developmental function for HSF-1

Our discovery that C. elegans heat-shock factor, HSF-1, promotes cell death is surprising. Heat-shock factors are thought to be protective proteins, orchestrating the response to protein misfolding induced by a variety of stressors, including elevated temperature. Although a role for HSF1 has been proposed in promoting apoptosis of mouse spermatocytes following elevated temperatures (Nakai et al., 2000), it is not clear whether this function is physiological. In this context, HSF1 induces expression of the gene Tdag51 (Hayashida et al., 2006). Both pro- and anti-apoptotic activities have been attributed to Tdag51 (Toyoshima et al., 2004), and which is activated in sperm is not clear. Recently, pathological roles for HSF1 in cancer have been detailed (e.g. Mendillo et al., 2012), but in these capacities HSF1 still supports cell survival.

Developmental functions for HSF1 have been suggested in which HSF1 appears to act through transcriptional targets different from those of the heat-shock response (Jedlicka et al., 1997), although target identity remains obscure. Here, we have shown that HSF-1 has at least partially non-overlapping sets of stress-induced and developmental targets. Indeed, typical stress targets of HSF-1, such as the small heat-shock gene hsp-16.49 as well as genes encoding larger chaperones, likehsp-1, are not expressed during LCD, whereas let-70, a direct transcriptional target for LCD, is not induced by heat shock. Interestingly, the yeast let-70 homologs ubc4 and ubc5 are induced by heat shock (Seufert and Jentsch, 1990), supporting a conserved connection between HSF and UBE2D2-family proteins. However, the distinction between developmental and stress functions is clearly absent in this single-celled organism, raising the possibility that this separation of function may be a metazoan innovation.

What distinguishes the stress-related and developmental forms of HSF-1? One possibility is that whereas the stress response appears to be mediated by HSF-1 trimerization, HSF-1 monomers or dimers might promote LCD roles. Although this model would nicely account for the differential activities in stress responses and LCD of the HSF-1(R145A) transgenic protein, which would be predicted to favor inactivation of a larger proportion of higher order HSF-1 complexes, the identification of conserved tripartite HSEs in the let-70 and rpn-3 regulatory regions argues against this possibility. Alternatively, selective post-translational modification of HSF-1 could account for these differences. In mammals, HSF1 undergoes a variety of modifications including phosphorylation, acetylation, ubiquitination, and sumoylation (Xu et al., 2012), which, depending on the site and modification, stimulate or repress HSF1 activity. In this context, it is of note that p38/MAPK-mediated phosphorylation of HSF1 represses its stress-related activity (Chu et al., 1996), and the LCD regulator SEK-1 encodes a MAPKK. However, no single MAPK has been identified that promotes LCD (E.S.B., M.J.K. unpublished results), suggesting that other mechanisms may be at play.

Our finding that POP-1/TCF does not play a significant role in LCD raises the possibility that Wnt signaling exerts direct control over HSF-1 through interactions with β-catenin. However, we have not been able to demonstrate physical interactions between these proteins to date (M.J.K, unpublished results).

Finally, a recent paper (Labbadia and Morimoto, 2015) demonstrated that in young adult C. elegans, around the time of LCD, global binding of HSF-1 to its stress-induced targets is reduced through changes in chromatin modification. Remarkably, we showed that chromatin regulators play a key role in let-70 induction and LCD (J.A.M., M.J.K and S.S., manuscript in preparation), suggesting, perhaps, that differences in HSF-1 access to different loci may play a role in distinguishing its two functions.

LCD and neurodegeneration

Previous studies from our lab raised the possibility that LCD may be related to degenerative processes that promote vertebrate neuronal death. Nuclear crenellation is evident in dying linker cells and in degenerating cells in polyQ disease (Abraham et al., 2007) and the TIR-1/Sarm adapter protein promotes LCD in C. elegans as well as degeneration of distal axonal segments following axotomy in Drosophila and vertebrates (Osterloh et al., 2012). The studies we present here, implicating the UPS and heat-shock factor in LCD, also support a connection with neurodegeneration. Indeed, protein aggregates found in cells of patients with polyQ diseases are heavily ubiquitylated (Kalchman et al., 1996). Chaperones also colocalize with protein aggregates in brain slices from SCA patients, and HSF1 has been shown to alleviate polyQ aggregation and cellular demise in both polyQ-overexpressing flies and in neuronal precursor cells (Neef et al., 2010). While the failure of proteostatic mechanisms in neurodegenerative diseases is generally thought to be a secondary event in their pathogenesis, it is possible that this failure reflects the involvement of a LCD-like process, in which attempts to engage protective measures instead result in activation of a specific cell death program.

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Conduction, graphene, elements and light

Larry H. Bernstein, MD, FCAP, Curator



New 2D material could upstage graphene   Mar 25, 2016

Can function as a conductor or semiconductor, is extremely stable, and uses light, inexpensive earth-abundant elements
The atoms in the new structure are arranged in a hexagonal pattern as in graphene, but that is where the similarity ends. The three elements forming the new material all have different sizes; the bonds connecting the atoms are also different. As a result, the sides of the hexagons formed by these atoms are unequal, unlike in graphene. (credit: Madhu Menon)

A new one-atom-thick flat material made up of silicon, boron, and nitrogen can function as a conductor or semiconductor (unlike graphene) and could upstage graphene and advance digital technology, say scientists at the University of Kentucky, Daimler in Germany, and the Institute for Electronic Structure and Laser (IESL) in Greece.

Reported in Physical Review B, Rapid Communications, the new Si2BN material was discovered in theory (not yet made in the lab). It uses light, inexpensive earth-abundant elements and is extremely stable, a property many other graphene alternatives lack, says University of Kentucky Center for Computational Sciences physicist Madhu Menon, PhD.

Limitations of other 2D semiconducting materials

A search for new 2D semiconducting materials has led researchers to a new class of three-layer materials called transition-metal dichalcogenides (TMDCs). TMDCs are mostly semiconductors and can be made into digital processors with greater efficiency than anything possible with silicon. However, these are much bulkier than graphene and made of materials that are not necessarily earth-abundant and inexpensive.

Other graphene-like materials have been proposed but lack the strengths of the new material. Silicene, for example, does not have a flat surface and eventually forms a 3D surface. Other materials are highly unstable, some only for a few hours at most.

The new Si2BN material is metallic, but by attaching other elements on top of the silicon atoms, its band gap can be changed (from conductor to semiconductor, for example) — a key advantage over graphene for electronics applications and solar-energy conversion.

The presence of silicon also suggests possible seamless integration with current silicon-based technology, allowing the industry to slowly move away from silicon, rather than precipitously, notes Menon.


Abstract of Prediction of a new graphenelike Si2BN solid

While the possibility to create a single-atom-thick two-dimensional layer from any material remains, only a few such structures have been obtained other than graphene and a monolayer of boron nitride. Here, based upon ab initiotheoretical simulations, we propose a new stable graphenelike single-atomic-layer Si2BN structure that has all of its atoms with sp2 bonding with no out-of-plane buckling. The structure is found to be metallic with a finite density of states at the Fermi level. This structure can be rolled into nanotubes in a manner similar to graphene. Combining first- and second-row elements in the Periodic Table to form a one-atom-thick material that is also flat opens up the possibility for studying new physics beyond graphene. The presence of Si will make the surface more reactive and therefore a promising candidate for hydrogen storage.


Nano-enhanced textiles clean themselves with light

Catalytic uses for industrial-scale chemical processes in agrochemicals, pharmaceuticals, and natural products also seen
Close-up of nanostructures grown on cotton textiles. Image magnified 150,000 times. (credit: RMIT University)

Researchers at at RMIT University in Australia have developed a cheap, efficient way to grow special copper- and silver-based nanostructures on textiles that can degrade organic matter when exposed to light.

Don’t throw out your washing machine yet, but the work paves the way toward nano-enhanced textiles that can spontaneously clean themselves of stains and grime simply by being put under a light or worn out in the sun.

The nanostructures absorb visible light (via localized surface plasmon resonance — collective electron-charge oscillations in metallic nanoparticles that are excited by light), generating high-energy (“hot”) electrons that cause the nanostructures to act as catalysts for chemical reactions that degrade organic matter.

Steps involved in fabricating copper- and silver-based cotton fabrics: 1. Sensitize the fabric with tin. 2. Form palladium seeds that act as nucleation (clustering) sites. 3. Grow metallic copper and silver nanoparticles on the surface of the cotton fabric. (credit: Samuel R. Anderson et al./Advanced Materials Interfaces)

The challenge for researchers has been to bring the concept out of the lab by working out how to build these nanostructures on an industrial scale and permanently attach them to textiles. The RMIT team’s novel approach was to grow the nanostructures directly onto the textiles by dipping them into specific solutions, resulting in development of stable nanostructures within 30 minutes.

When exposed to light, it took less than six minutes for some of the nano-enhanced textiles to spontaneously clean themselves.

The research was described in the journal Advanced Materials Interfaces.

Scaling up to industrial levels

Rajesh Ramanathan, a RMIT postdoctoral fellow and co-senior author, said the process also had a variety of applications for catalysis-based industries such as agrochemicals, pharmaceuticals, and natural productsand could be easily scaled up to industrial levels. “The advantage of textiles is they already have a 3D structure, so they are great at absorbing light, which in turn speeds up the process of degrading organic matter,” he said.

Cotton textile fabric with copper-based nanostructures. The image is magnified 200 times. (credit: RMIT University)

“Our next step will be to test our nano-enhanced textiles with organic compounds that could be more relevant to consumers, to see how quickly they can handle common stains like tomato sauce or wine,” Ramanathan said.

“There’s more work to do to before we can start throwing out our washing machines, but this advance lays a strong foundation for the future development of fully self-cleaning textiles.”

Abstract of Robust Nanostructured Silver and Copper Fabrics with Localized Surface Plasmon Resonance Property for Effective Visible Light Induced Reductive Catalysis

Inspired by high porosity, absorbency, wettability, and hierarchical ordering on the micrometer and nanometer scale of cotton fabrics, a facile strategy is developed to coat visible light active metal nanostructures of copper and silver on cotton fabric substrates. The fabrication of nanostructured Ag and Cu onto interwoven threads of a cotton fabric by electroless deposition creates metal nanostructures that show a localized surface plasmon resonance (LSPR) effect. The micro/nanoscale hierarchical ordering of the cotton fabrics allows access to catalytically active sites to participate in heterogeneous catalysis with high efficiency. The ability of metals to absorb visible light through LSPR further enhances the catalytic reaction rates under photoexcitation conditions. Understanding the modes of electron transfer during visible light illumination in Ag@Cotton and Cu@Cotton through electrochemical measurements provides mechanistic evidence on the influence of light in promoting electron transfer during heterogeneous catalysis for the first time. The outcomes presented in this work will be helpful in designing new multifunctional fabrics with the ability to absorb visible light and thereby enhance light-activated catalytic processes.


New type of molecular tag makes MRI 10,000 times more sensitive

Could detect biochemical processes in opaque tissue without requiring PET radiation or CT x-rays

Duke scientists have discovered a new class of inexpensive, long-lived molecular tags that enhance MRI signals by 10,000 times. To activate the tags, the researchers mix them with a newly developed catalyst (center) and a special form of hydrogen (gray), converting them into long-lived magnetic resonance “lightbulbs” that might be used to track disease metabolism in real time. (credit: Thomas Theis, Duke University)

Duke University researchers have discovered a new form of MRI that’s 10,000 times more sensitive and could record actual biochemical reactions, such as those involved in cancer and heart disease, and in real time.

Let’s review how MRI (magnetic resonance imaging) works: MRI takes advantage of a property called spin, which makes the nuclei in hydrogen atoms act like tiny magnets. By generating a strong magnetic field (such as 3 Tesla) and a series of radio-frequency waves, MRI induces these hydrogen magnets in atoms to broadcast their locations. Since most of the hydrogen atoms in the body are bound up in water, the technique is used in clinical settings to create detailed images of soft tissues like organs (such as the brain), blood vessels, and tumors inside the body.

MRI’s ability to track chemical transformations in the body has been limited by the low sensitivity of the technique. That makes it impossible to detect small numbers of molecules (without using unattainably more massive magnetic fields).

So to take MRI a giant step further in sensitivity, the Duke researchers created a new class of molecular “tags” that can track disease metabolism in real time, and can last for more than an hour, using a technique called hyperpolarization.* These tags are biocompatible and inexpensive to produce, allowing for using existing MRI machines.

“This represents a completely new class of molecules that doesn’t look anything at all like what people thought could be made into MRI tags,” said Warren S. Warren, James B. Duke Professor and Chair of Physics at Duke, and senior author on the study. “We envision it could provide a whole new way to use MRI to learn about the biochemistry of disease.”

Sensitive tissue detection without radiation

The new molecular tags open up a new world for medicine and research by making it possible to detect what’s happening in optically opaque tissue instead of requiring expensive positron emission tomography (PET), which uses a radioactive tracer chemical to look at organs in the body and only works for (typically) about 20 minutes, or CT x-rays, according to the researchers.

This research was reported in the March 25 issue of Science Advances. It was supported by the National Science Foundation, the National Institutes of Health, the Department of Defense Congressionally Directed Medical Research Programs Breast Cancer grant, the Pratt School of Engineering Research Innovation Seed Fund, the Burroughs Wellcome Fellowship, and the Donors of the American Chemical Society Petroleum Research Fund.

* For the past decade, researchers have been developing methods to “hyperpolarize” biologically important molecules. “Hyperpolarization gives them 10,000 times more signal than they would normally have if they had just been magnetized in an ordinary magnetic field,” Warren said. But while promising, Warren says these hyperpolarization techniques face two fundamental problems: incredibly expensive equipment — around 3 million dollars for one machine — and most of these molecular “lightbulbs” burn out in a matter of seconds.

“It’s hard to take an image with an agent that is only visible for seconds, and there are a lot of biological processes you could never hope to see,” said Warren. “We wanted to try to figure out what molecules could give extremely long-lived signals so that you could look at slower processes.”

So the researchers synthesized a series of molecules containing diazarines — a chemical structure composed of two nitrogen atoms bound together in a ring. Diazirines were a promising target for screening because their geometry traps hyperpolarization in a “hidden state” where it cannot relax quickly. Using a simple and inexpensive approach to hyperpolarization called SABRE-SHEATH, in which the molecular tags are mixed with a spin-polarized form of hydrogen and a catalyst, the researchers were able to rapidly hyperpolarize one of the diazirine-containing molecules, greatly enhancing its magnetic resonance signals for over an hour.

The scientists believe their SABRE-SHEATH catalyst could be used to hyperpolarize a wide variety of chemical structures at a fraction of the cost of other methods.

Abstract of Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal 15N2-diazirine molecular tags

Abstract of Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal 15N2-diazirine molecular tags

Conventional magnetic resonance (MR) faces serious sensitivity limitations, which can be overcome by hyperpolarization methods, but the most common method (dynamic nuclear polarization) is complex and expensive, and applications are limited by short spin lifetimes (typically seconds) of biologically relevant molecules. We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize 15N2 magnetization and long-lived 15N2singlet spin order, with signal decay time constants of 5.8 and 23 min, respectively. We find >10,000-fold enhancements generating detectable nuclear MR signals that last for more than an hour. 15N2-diazirines represent a class of particularly promising and versatile molecular tags, and can be incorporated into a wide range of biomolecules without significantly altering molecular function.


[Seems like they have a great idea, now all they need to do is confirm very specific uses or types of cancers/diseases or other processes they can track or target. Will be interesting to see if they can do more than just see things, maybe they can use this to target and destroy bad things in the body also. Keep up the good work….. this sounds like a game changer.]


Scientists time-reverse developed stem cells to make them ‘embryonic’ again

May help avoid ethically controversial use of human embryos for research and support other research goals
Researchers have reversed “primed” (developed) “epiblast” stem cells (top) from early mouse embryos using the drug MM-401, causing the treated cells (bottom) to revert to the original form of the stem cells. (credit: University of Michigan)

University of Michigan Medical School researchers have discovered a way to convert mouse stem cells (taken from an embryo) that have  become “primed” (reached the stage where they can  differentiate, or develop into every specialized cell in the body) to a “naïve” (unspecialized) state by simply adding a drug.

This breakthrough has the potential to one day allow researchers to avoid the ethically controversial use of human embryos left over from infertility treatments. To achieve this breakthrough, the researchers treated the primedembryonic stem cells (“EpiSC”) with a drug called MM-401* (a leukemia drug) for a short period of time.

Embryonic stem cells are able to develop into any type of cell, except those of the placenta (credit: Mike Jones/CC)


* The drug, MM-401, specifically targets epigenetic chemical markers on histones, the protein “spools” that DNA coils around to create structures called chromatin. These epigenetic changes signal the cell’s DNA-reading machinery and tell it where to start uncoiling the chromatin in order to read it.

A gene called Mll1 is responsible for the addition of these epigenetic changes, which are like small chemical tags called methyl groups. Mll1 plays a key role in the uncontrolled explosion of white blood cells in leukemia, which is why researchers developed the drug MM-401 to interfere with this process. But Mll1 also plays a role in cell development and the formation of blood cells and other cells in later-stage embryos.

Stem cells do not turn on the Mll1 gene until they are more developed. The MM-401 drug blocks Mll1’s normal activity in developing cells so the epigenetic chemical markers are missing. These cells are then unable to continue to develop into different types of specialized cells but are still able to revert to healthy naive pluripotent stem cells.

Abstract of MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency

The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.

Abstract of Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.



How to kill bacteria in seconds using gold nanoparticles and light

March 24, 2016


zapping bacteria ft Could treat bacterial infections without using antibiotics, which could help reduce the risk of spreading antibiotics resistance

Researchers at the University of Houston have developed a new technique for killing bacteria in 5 to 25 seconds using highly porous gold nanodisks and light, according to a study published today in Optical Materials Express. The method could one day help hospitals treat some common infections without using antibiotics

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Metformin and vitamin B12 deficiency?

Larry H. Bernstein, MD, FCAP, Curator



Years of taking popular diabetes drug tied to risk of B12 deficiency


Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study


Metformin linked to vitamin B12 deficiency

David Holmes   Nature Reviews Endocrinology(2016)    http://dx.doi.org:/10.1038/nrendo.2016.39

Secondary analysis of data from the Diabetes Prevention Program Outcomes Study (DPPOS), one of the largest and longest studies of metformin treatment in patients at high risk of developing type 2 diabetes mellitus, shows that long-term use of metformin is associated with vitamin B12deficiency.

Aroda, V. R. et al. Long-term metformin use and vitamin B12 deficiency in the Diabetes Prevention Program Outcomes Study. J. Clin. Endocrinol. Metab. http://dx.doi.org/10.1210/jc.2015-3754 (2016)


Long-term Follow-up of Diabetes Prevention Program Shows Continued Reduction in Diabetes Development


San Francisco, California
June 16, 2014

Treatments used to decrease the development of type 2 diabetes continue to be effective an average of 15 years later, according to the latest findings of the Diabetes Prevention Program Outcomes Study, a landmark study funded by the National Institutes of Health (NIH).

The results, presented at the American Diabetes Association’s 74th Scientific Sessions®, come more than a decade after the Diabetes Prevention Program, or DPP, reported its original findings. In 2001, after an average of three years of study, the DPP announced that the study’s two interventions, a lifestyle program designed to reduce weight and increase activity levels and the diabetes medicinemetformin, decreased the development of type 2 diabetes in a diverse group of people, all of whom were at high risk for the disease, by 58 and 31 percent, respectively, compared with a group taking placebo.

The Diabetes Prevention Program Outcomes Study, or DPPOS, was conducted as an extension of the DPP to determine the longer-term effects of the two interventions, including further reduction in diabetes development and whether delaying diabetes would reduce the development of the diabetes complications that can lead to blindness, kidney failure, amputations and heart disease. Funded largely by the NIH’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the new findings show that the lifestyle intervention and metformin treatment have beneficial effects, even years later, but did not reduce microvascular complications.

Delaying Type 2 Diabetes

Participants in the study who were originally assigned to the lifestyle intervention and metformin during DPP continued to have lower rates of type 2 diabetes development than those assigned to placebo, with 27 percent and 17 percent reductions, respectively, after 15 years.

“What we’re finding is that we can prevent or delay the onset of type 2 diabetes, a chronic disease, through lifestyle intervention or with metformin, over a very long period of time,” said David M. Nathan, MD, Chairman of the DPP/DPPOS and Professor of Medicine at Harvard Medical School. “After the initial randomized treatment phase in DPP, all participants were offered lifestyle intervention and the rates of diabetes development fell in the metformin and former placebo groups, leading to a reduction in the treatment group differences over time.  However, the lifestyle intervention and metformin are still quite effective at delaying, if not preventing, type 2 diabetes,” Dr. Nathan said. Currently, an estimated 79 million American adults are at high-risk for developing type 2 diabetes.

Microvascular Complications
The DPPOS investigators followed participants for an additional 12 years after the end of the DPP to determine both the extent of diabetes prevention over time and whether the study treatments would also decrease the small vessel -or microvascular- complications, such as eye, nerve and kidney disease. These long-term results did not demonstrate significant differences among the lifestyle intervention, metformin or placebo groups on the microvascular complications, reported Kieren Mather, MD, Professor of Medicine at Indiana University School of Medicine and a study investigator.

“However, regardless of type of initial treatment, participants who didn’t develop diabetes had a 28 percent lower occurrence of the microvascular complications than those participants who did develop diabetes. These findings show that intervening in the prediabetes phase is important in reducing early stage complications,” Dr. Mather noted. The absence of differences in microvascular complications among the intervention groups may be explained by the small differences in average glucose levels among the groups at this stage of follow-up.

Risk for Cardiovascular Disease

The DPP population was relatively young and healthy at the beginning of the study, and few participants had experienced any severe cardiovascular events, such as heart attack or stroke, 15 years later. The relatively small number of events meant that the DPPOS researchers could not test the effects of interventions on cardiovascular disease. However, the research team did examine whether the study interventions, or a delay in the onset of type 2 diabetes, improved cardiovascular risk factors.

“We found that cardiovascular risk factors, such as hypertension, are generally improved by the lifestyle intervention and somewhat less by metformin,” said Ronald Goldberg, MD, Professor of Medicine at the University of Miami and one of the DPPOS investigators. “We know that people with type 2 diabetes are at much higher risk for heart disease and stroke than those who do not have diabetes, so a delay in risk factor development or improvement in risk factors may prove to be beneficial.”

Long-term Results with Metformin

The DPP/DPPOS is the largest and longest duration study to examine the effects of metformin, an inexpensive, well-known and generally safe diabetes medicine, in people who have not been diagnosed with diabetes. For DPPOS participants, metformin treatment was associated with a modest degree of long-term weight loss. “Other than a small increase in vitamin B-12 deficiency, which is a recognized consequence of metformin therapy, it has been extremely safe and well-tolerated over the 15 years of our study,” said Jill Crandall, MD, Professor of Medicine at Albert Einstein College of Medicine and a DPPOS investigator. “Further study will help show whether metformin has beneficial effects on heart disease and cancer, which are both increased in people with type 2 diabetes.”

Looking to the Future

In addition to the current findings, the DPPOS includes a uniquely valuable population that can help researchers understand the clinical course of type 2 diabetes.  Since the participants did not have diabetes at the beginning of the DPP, for those who have developed diabetes, the data show precisely when they developed the disease, which is rare in previous studies. “The DPP and DPPOS have given us an incredible wealth of information by following a very diverse group of people with regard to race and age as they have progressed from prediabetes to diabetes,” said Judith Fradkin, MD, Director of the NIDDK Division of Diabetes, Endocrinology and Metabolic Diseases. “The study provides us with an opportunity to make crucial discoveries about the clinical course of type 2 diabetes.”

Dr. Fradkin noted that the study population held promise for further analyses because researchers would now be able to examine how developing diabetes at different periods of life may cause the disease to progress differently. “We can look at whether diabetes behaves differently if you develop it before the age of 50 or after the age of 60,” she said. “Thanks to the large and diverse population of DPPOS that has remained very loyal to the study, we will be able to see how and when complications first develop and understand how to intervene most effectively.”

She added that NIDDK had invited the researchers to submit an application for a grant to follow the study population for an additional 10 years.

The Diabetes Prevention Program Outcomes Study was funded under NIH grant U01DK048489 by the NIDDK; National Institute on Aging; National Cancer Institute; National Heart, Lung, and Blood Institute; National Eye Institute; National Center on Minority Health and Health Disparities; and the Office of the NIH Director; Eunice Kennedy Shriver National Institute of Child Health and Human Development; Office of Research on Women’s Health; and Office of Dietary Supplements, all part of the NIH, as well as the Indian Health Service, Centers for Disease Control and Prevention and American Diabetes Association. Funding in the form of supplies was provided by Merck Sante, Merck KGaA and LifeScan.

The American Diabetes Association is leading the fight to Stop Diabetes® and its deadly consequences and fighting for those affected by diabetes. The Association funds research to prevent, cure and manage diabetes; delivers services to hundreds of communities; provides objective and credible information; and gives voice to those denied their rights because of diabetes. Founded in 1940, our mission is to prevent and cure diabetes and to improve the lives of all people affected by diabetes. For more information please call the American Diabetes Association at 1-800-DIABETES (1-800-342-2383) or visit http://www.diabetes.org. Information from both these sources is available in English and Spanish.

Association of Biochemical B12Deficiency With Metformin Therapy and Vitamin B12Supplements  

The National Health and Nutrition Examination Survey, 1999–2006

Lael ReinstatlerYan Ping QiRebecca S. WilliamsonJoshua V. Garn, and Godfrey P. Oakley Jr.
Diabetes Care February 2012 vol. 35 no. 2 327-333 

OBJECTIVE To describe the prevalence of biochemical B12deficiency in adults with type 2 diabetes taking metformin compared with those not taking metformin and those without diabetes, and explore whether this relationship is modified by vitamin B12supplements.

RESEARCH DESIGN AND METHODS Analysis of data on U.S. adults ≥50 years of age with (n = 1,621) or without type 2 diabetes (n = 6,867) from the National Health and Nutrition Examination Survey (NHANES), 1999–2006. Type 2 diabetes was defined as clinical diagnosis after age 30 without initiation of insulin therapy within 1 year. Those with diabetes were classified according to their current metformin use. Biochemical B12 deficiency was defined as serum B12concentrations ≤148 pmol/L and borderline deficiency was defined as >148 to ≤221 pmol/L.

RESULTS Biochemical B12 deficiency was present in 5.8% of those with diabetes using metformin compared with 2.4% of those not using metformin (P = 0.0026) and 3.3% of those without diabetes (P = 0.0002). Among those with diabetes, metformin use was associated with biochemical B12 deficiency (adjusted odds ratio 2.92; 95% CI 1.26–6.78). Consumption of any supplement containing B12 was not associated with a reduction in the prevalence of biochemical B12deficiency among those with diabetes, whereas consumption of any supplement containing B12 was associated with a two-thirds reduction among those without diabetes.

CONCLUSIONS Metformin therapy is associated with a higher prevalence of biochemical B12 deficiency. The amount of B12recommended by the Institute of Medicine (IOM) (2.4 μg/day) and the amount available in general multivitamins (6 μg) may not be enough to correct this deficiency among those with diabetes.

It is well known that the risks of both type 2 diabetes and B12deficiency increase with age (1,2). Recent national data estimate a 21.2% prevalence of diagnosed diabetes among adults ≥65 years of age and a 6 and 20% prevalence of biochemical B12 deficiency (serum B12<148 pmol/L) and borderline deficiency (serum B12 ≥148–221 pmol/L) among adults ≥60 years of age (3,4).

The diabetes drug metformin has been reported to cause a decrease in serum B12 concentrations. In the first efficacy trial, DeFronzo and Goodman (5) demonstrated that although metformin offers superior control of glycosylated hemoglobin levels and fasting plasma glucose levels compared with glyburide, serum B12 concentrations were lowered by 22% compared with placebo, and 29% compared with glyburide therapy after 29 weeks of treatment. A recent, randomized control trial designed to examine the temporal relationship between metformin and serum B12 found a 19% reduction in serum B12 levels compared with placebo after 4 years (6). Several other randomized control trials and cross-sectional surveys reported reductions in B12ranging from 9 to 52% (716). Although classical B12 deficiency presents with clinical symptoms such as anemia, peripheral neuropathy, depression, and cognitive impairment, these symptoms are usually absent in those with biochemical B12 deficiency (17).

Several researchers have made recommendations to screen those with type 2 diabetes on metformin for serum B12 levels (6,7,1416,1821). However, no formal recommendations have been provided by the medical community or the U.S. Prevention Services Task Force. High-dose B12 injection therapy has been successfully used to correct the metformin-induced decline in serum B12 (15,21,22). The use of B12supplements among those with type 2 diabetes on metformin in a nationally representative sample and their potentially protective effect against biochemical B12 deficiency has not been reported. It is therefore the aim of the current study to use the nationally representative National Health and Nutrition Examination Survey (NHANES) population to determine the prevalence of biochemical B12deficiency among those with type 2 diabetes ≥50 years of age taking metformin compared with those with type 2 diabetes not taking metformin and those without diabetes, and to explore how these relationships are modified by B12 supplement consumption.

Design overview

NHANES is a nationally representative sample of the noninstitutionalized U.S. population with targeted oversampling of U.S. adults ≥60 years of age, African Americans, and Hispanics. Details of these surveys have been described elsewhere (23). All participants gave written informed consent, and the survey protocol was approved by a human subjects review board.

Setting and participants

Our study included adults ≥50 years of age from NHANES 1999–2006. Participants with positive HIV antibody test results, high creatinine levels (>1.7 mg/dL for men and >1.5 mg/dL for women), and prescription B12 injections were excluded from the analysis. Participants who reported having prediabetes or borderline diabetes (n = 226) were removed because they could not be definitively grouped as having or not having type 2 diabetes. We also excluded pregnant women, those with type 1 diabetes, and those without diabetes taking metformin. Based on clinical aspects described by the American Diabetes Association and previous work in NHANES, those who were diagnosed before the age of 30 and began insulin therapy within 1 year of diagnosis were classified as having type 1 diabetes (24,25). Type 2 diabetes status in adults was dichotomized as yes/no. Participants who reported receiving a physician’s diagnosis after age 30 (excluding gestational diabetes) and did not initiate insulin therapy within 1 year of diagnosis were classified as having type 2 diabetes.

Outcomes and follow-up

The primary outcome was biochemical B12 deficiency determined by serum B12 concentrations. Serum B12 levels were quantified using the Quantaphase II folate/vitamin B12 radioassay kit from Bio-Rad Laboratories (Hercules, CA). We defined biochemical B12 deficiency as serum levels ≤148 pmol/L, borderline deficiency as serum B12 >148 to ≤221 pmol/L, and normal as >221 pmol/L (26).

The main exposure of interest was metformin use. Using data collected in the prescription medicine questionnaire, those with type 2 diabetes were classified as currently using metformin therapy (alone or in combination therapy) versus those not currently using metformin. Length of metformin therapy was used to assess the relationship between duration of metformin therapy and biochemical B12 deficiency. In the final analysis, two control groups were used to allow the comparison of those with type 2 diabetes taking metformin with those with type 2 diabetes not taking metformin and those without diabetes.

To determine whether the association between metformin and biochemical B12 deficiency is modified by supplemental B12 intake, data from the dietary supplement questionnaire were used. Information regarding the dose and frequency was used to calculate average daily supplemental B12 intake. We categorized supplemental B12 intake as 0 μg (no B12 containing supplement), >0–6 μg, >6–25 μg, and >25 μg. The lower intake group, >0–6 μg, includes 6 μg, the amount of vitamin B12 typically found in over-the-counter multivitamins, and 2.4 μg, the daily amount the IOM recommends for all adults ≥50 years of age to consume through supplements or fortified food (1). The next group, >6–25 μg, includes 25 μg, the amount available in many multivitamins marketed toward senior adults. The highest group contains the amount found in high-dose B-vitamin supplements.


In the final analysis, there were 575 U.S. adults ≥50 years of age with type 2 diabetes using metformin, 1,046 with type 2 diabetes not using metformin, and 6,867 without diabetes. The demographic and biological characteristics of the groups are shown in Table 1. Among metformin users, mean age was 63.4 ± 0.5 years, 50.3% were male, 66.7% were non-Hispanic white, and 40.7% used a supplement containing B12. The median duration of metformin use was 5 years. Compared with those with type 2 diabetes not taking metformin, metformin users were younger (P < 0.0001), reported a lower prevalence of insulin use (P < 0.001), and had a shorter duration of diabetes (P = 0.0207). Compared with those without diabetes, metformin users had a higher proportion of nonwhite racial groups (P< 0.0001), a higher proportion of obesity (P < 0.0001), a lower prevalence of macrocytosis (P = 0.0017), a lower prevalence of supplemental folic acid use (P = 0.0069), a lower prevalence of supplemental vitamin B12 use (P = 0.0180), and a lower prevalence of calcium supplement use (P = 0.0002). There was a twofold difference in the prevalence of anemia among those with type 2 diabetes versus those without, and no difference between the groups with diabetes.    

Association of Biochemical B12Deficiency With Metformin Therapy and Vitamin B12Supplements

Demographic and biological characteristics of U.S. adults ≥50 years of age: NHANES 1999–2006

Table 1
The geometric mean serum B12 concentration among those with type 2 diabetes taking metformin was 317.5 pmol/L. This was significantly lower than the geometric mean concentration in those with type 2 diabetes not taking metformin (386.7 pmol/L; P = 0.0116) and those without diabetes (350.8 pmol/L; P = 0.0011). As seen in Fig. 1, the weighted prevalence of biochemical B12 deficiency adjusted for age, race, and sex was 5.8% for those with type 2 diabetes taking metformin, 2.2% for those with type 2 diabetes not taking metformin (P = 0.0002), and 3.3% for those without diabetes (P = 0.0026). Among the three aforementioned groups, borderline deficiency was present in 16.2, 5.5, and 8.8%, respectively (P < 0.0001). Applying the Fleiss formula for calculating attributable risk from cross-sectional data (27), among all of the cases of biochemical B12 deficiency, 3.5% of the cases were attributable to metformin use; and among those with diabetes, 41% of the deficient cases were attributable to metformin use. When the prevalence of biochemical B12 deficiency among those with diabetes taking metformin was analyzed by duration of metformin therapy, there was no notable increase in the prevalence of biochemical B12 deficiency as the duration of metformin use increased. The prevalence of biochemical B12 deficiency was 4.1% among those taking metformin <1 year, 6.3% among those taking metformin ≥1–3 years, 4.1% among those taking metformin >3–10 years, and 8.1% among those taking metformin >10 years (P = 0.3219 for <1 year vs. >10 years). Similarly, there was no clear increase in the prevalence of borderline deficiency as the duration of metformin use increased (15.9% among those taking metformin >10 years vs. 11.4% among those taking metformin <1 year; P = 0.4365).
Figure 1
Weighted prevalence of biochemical B12 deficiency and borderline deficiency adjusted for age, race, and sex in U.S. adults ≥50 years of age: NHANES 1999–2006. Black bars are those with type 2 diabetes on metformin, gray bars are those with type 2 diabetes not on metformin, and the white bars are those without diabetes. *P = 0.0002 vs. type 2 diabetes on metformin. †P < 0.0001 vs. type 2 diabetes on metformin. ‡P = 0.0026 vs. type 2 diabetes on metformin.
Table 2 presents a stratified analysis of the weighted prevalence of biochemical B12 deficiency and borderline deficiency by B12supplement use. For those without diabetes, B12 supplement use was associated with an ∼66.7% lower prevalence of both biochemical B12deficiency (4.8 vs. 1.6%; P < 0.0001) and borderline deficiency (16.6 vs. 5.5%; P < 0.0001). A decrease in the prevalence of biochemical B12deficiency was seen at all levels of supplemental B12 intake compared with nonusers of supplements. Among those with type 2 diabetes taking metformin, supplement use was not associated with a decrease in the prevalence of either biochemical B12 deficiency (5.6 vs. 5.3%; P= 0.9137) or borderline deficiency (15.5 vs. 8.8%; P = 0.0826). Among the metformin users who also used supplements, those who consumed >0–6 μg of B12 had a prevalence of biochemical B12 deficiency of 14.1%. However, consumption of a supplement containing >6 μg of B12 was associated with a prevalence of biochemical B12 deficiency of 1.8% (P = 0.0273 for linear trend). Similar trends were seen in the association of supplemental B12 intake and the prevalence of borderline deficiency. For those with type 2 diabetes not taking metformin, supplement use was also not associated with a decrease in the prevalence of biochemical B12 deficiency (2.1 vs. 2.0%; P = 0.9568) but was associated with a 54% reduction in the prevalence of borderline deficiency (7.8 vs. 3.4%; P = 0.0057 for linear trend).
Table 2
Comparison of average daily B12 supplement intake by weighted prevalence of biochemical B12 deficiency (serum B12 ≤148 pmol/L) and borderline deficiency (serum B12 >148 to ≤221 pmol/L) among U.S. adults ≥50 years of age: NHANES 1999–2006.
Table 3 demonstrates the association of various risk factors with biochemical B12 deficiency. Metformin therapy was associated with biochemical B12 deficiency (odds ratio [OR] 2.89; 95% CI 1.33–6.28) and borderline deficiency (OR 2.32; 95% CI 1.31–4.12) in a crude model (results not shown). After adjusting for age, BMI, and insulin and supplement use, metformin maintained a significant association with biochemical B12 deficiency (OR 2.92; 95% CI 1.28–6.66) and borderline deficiency (OR 2.16; 95% CI 1.22–3.85). Similar to Table 2, B12 supplements were protective against borderline (OR 0.43; 95% CI 0.23–0.81), but not biochemical, B12 deficiency (OR 0.76; 95% CI 0.34–1.70) among those with type 2 diabetes. Among those without diabetes, B12 supplement use was ∼70% protective against biochemical B12 deficiency (OR 0.26; 95% CI 0.17–0.38) and borderline deficiency (OR 0.27; 95% CI 0.21–0.35).
Table 3
Polytomous logistic regression for potential risk factors of biochemical B12 deficiency and borderline deficiency among U.S. adults ≥50 years of age: NHANES 1999–2006, OR (95% CI)

The IOM has highlighted the detection and diagnosis of B12 deficiency as a high-priority topic for research (1). Our results suggest several findings that add to the complexity and importance of B12 research and its relation to diabetes, and offer new insight into the benefits of B12 supplements. Our data confirm the relationship between metformin and reduced serum B12 levels beyond the background prevalence of biochemical B12 deficiency. Our data demonstrate that an intake of >0–6 μg of B12, which includes the dose most commonly found in over-the-counter multivitamins, was associated with a two-thirds reduction of biochemical B12 deficiency and borderline deficiency among adults without diabetes. This relationship has been previously reported with NHANES and Framingham population data (4,29). In contrast, we did not find that >0–6 μg of B12 was associated with a decrease in the prevalence of biochemical B12 deficiency or borderline deficiency among adults with type 2 diabetes taking metformin. This observation suggests that metformin reduces serum B12 by a mechanism that is additive to or different from the mechanism in older adults. It is also possible that metformin may exacerbate the deficiency among older adults with low serum B12. Our sample size was too small to determine which amount >6 μg was associated with maximum protection, but we did find a dose-response trend.

We were surprised to find that those with type 2 diabetes not using metformin had the lowest prevalence of biochemical B12 deficiency. It is possible that these individuals may seek medical care more frequently than the general population and therefore are being treated for their biochemical B12 deficiency. Or perhaps, because this population had a longer duration of diabetes and a higher proportion of insulin users compared with metformin users, they have been switched from metformin to other diabetic treatments due to low serum B12 concentrations or uncontrolled glucose levels and these new treatments may increase serum B12 concentrations. Despite the observed effects of metformin on serum B12 levels, it remains unclear whether or not this reduction is a public health concern. With lifetime risks of diabetes estimated to be one in three and with metformin being a first-line intervention, it is important to increase our understanding of the effects of oral vitamin B12 on metformin-associated biochemical deficiency (20,21).

The strengths of this study include its nationally representative, population-based sample, its detailed information on supplement usage, and its relevant biochemical markers. This is the first study to use a nationally representative sample to examine the association between serum B12 concentration, diabetes status, and metformin use as well as examine how this relationship may be modified by vitamin B12 supplementation. The data available regarding supplement usage provided specific information regarding dose and frequency. This aspect of NHANES allowed us to observe the dose-response relationship in Table 2 and to compare it within our three study groups.

This study is also subject to limitations. First, NHANES is a cross-sectional survey and it cannot assess time as a factor, and therefore the results are associations and not causal relationships. A second limitation arises in our definition of biochemical B12 deficiency. There is no general consensus on how to define normal versus low serum B12levels. Some researchers include the functional biomarker methylmalonic acid (MMA) in the definition, but this has yet to be agreed upon (3034). Recently, an NHANES roundtable discussion suggested that definitions of biochemical B12 deficiency should incorporate one biomarker (serum B12 or holotranscobalamin) and one functional biomarker (MMA or total homocysteine) to address problems with sensitivity and specificity of the individual biomarkers. However, they also cited a need for more research on how the biomarkers are related in the general population to prevent misclassification (34). MMA was only measured for six of our survey years; one-third of participants in our final analysis were missing serum MMA levels. Moreover, it has recently been reported that MMA values are significantly greater among the elderly with diabetes as compared with the elderly without diabetes even when controlling for serum B12 concentrations and age, suggesting that having diabetes may independently increase the levels of MMA (35). This unique property of MMA in elderly adults with diabetes makes it unsuitable as part of a definition of biochemical B12 deficiency in our specific population groups. Our study may also be subject to misclassification bias. NHANES does not differentiate between diabetes types 1 and 2 in the surveys; our definition may not capture adults with type 2 diabetes exclusively. Additionally, we used responses to the question “Have you received a physician’s diagnosis of diabetes” to categorize participants as having or not having diabetes. Therefore, we failed to capture undiagnosed diabetes. Finally, we could only assess current metformin use. We cannot determine if nonmetformin users have ever used metformin or if they were not using it at the time of the survey.

Our data demonstrate several important conclusions. First, there is a clear association between metformin and biochemical B12 deficiency among adults with type 2 diabetes. This analysis shows that 6 μg of B12 offered in most multivitamins is associated with two-thirds reduction in biochemical B12 deficiency in the general population, and that this same dose is not associated with protection against biochemical B12 deficiency among those with type 2 diabetes taking metformin. Our results have public health and clinical implications by suggesting that neither 2.4 μg, the current IOM recommendation for daily B12 intake, nor 6 μg, the amount found in most multivitamins, is sufficient for those with type 2 diabetes taking metformin.

This analysis suggests a need for further research. One research design would be to identify those with biochemical B12 deficiency and randomize them to receive various doses of supplemental B12chronically and then evaluate any improvement in serum B12concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B12 deficiency and how they are affected by B12supplements at various doses. Given that a significant proportion of the population ≥50 years of age have biochemical B12 deficiency and that those with diabetes taking metformin have an even higher proportion of biochemical B12 deficiency, we suggest that support for further research is a reasonable priority.


One research design would be to identify those with biochemical B12 deficiency and randomize them to receive various doses of supplemental B12chronically and then evaluate any improvement in serum B12concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B12 deficiency and how they are affected by B12supplements at various doses.
This is of considerable interest.  As far as I can see, there is insufficient data presented to discern all of the variables entangled.  In a study of 8000 hemograms several years ago, it was of some interest that there were a large percentage of patients who were over age 75 years having a MCV of 94 – 100, not considered indicative of macrocytic anemia.  It would have been interesting to explore that set of the data further.
UPDATED 3/17/2020
 2019 May 7;11(5). pii: E1020. doi: 10.3390/nu11051020.

Monitoring Vitamin B12 in Women Treated with Metformin for Primary Prevention of Breast Cancer and Age-Related Chronic Diseases.


Metformin (MET) is currently being used in several trials for cancer prevention or treatment in non-diabetics. However, long-term MET use in diabetics is associated with lower serum levels of total vitamin B12. In a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET, whose participants were characterized by different components of metabolic syndrome, we tested the effect of MET on serum levels of B12, holo transcobalamin II (holo-TC-II), and methylmalonic acid (MMA). The study was conducted on 165 women receiving MET or placebo for three years. Results of the study indicate a significant overall reduction in both serum total B12 and holo-TC-II levels according with MET-treatment. In particular, in the MET group 26 of 81 patients and 10 of the 84 placebo-treated subjects had B12 below the normal threshold (<221 pmol/L) at the end of the study. Considering jointly all B12, Holo-TC-II, and MMA, 13 of the 165 subjects (10 MET and 3 placebo-treated) had at least two deficits in the biochemical parameters at the end of the study, without reporting clinical signs. Although our results do not affect whether women remain in the trial, B12 monitoring for MET-treated individuals should be implemented.


Metformin (MET) is the first-line treatment for type-2 diabetes and has been used for decades to treat this chronic condition [1]. Given its favorable effects on glycemic control, weight patterns, insulin requirements, and cardiovascular outcomes, MET has been recently proposed in addition to lifestyle interventions to reduce metabolic syndrome (MS) and age-related chronic diseases [2]. Observational studies have also suggested that diabetic patients treated with MET had a significantly lower risk of developing cancer or lower cancer mortality than those untreated or treated with other drugs [3,4]. For this reason, a number of clinical trials are in progress in different solid cancers.
One of the limitations in implementing long-term use of MET to prevent chronic conditions in healthy subjects relates to its potential lowering effect on vitamin B12 (B12). The aim of the present study was to assess the effect of three years of MET treatment in a randomized, controlled trial considering both B12 levels and biomarkers of its metabolism and biological effectiveness.
Cobalamin, also known as B12, is a water-soluble, cobalt-containing vitamin. All forms of B12 are converted intracellularly into adenosyl-Cbl and methylcobalamin—the biologically active forms at the cellular level [5]. Vitamin B12 is a vital cofactor of two enzymes: methionine synthase and L-methyl-malonyl-coenzyme. A mutase in intracellular enzymatic reactions related to DNA synthesis, as well as in amino and fatty acid metabolism. Vitamin B12, under the catalysis of the enzyme l-methyl-malonyl-CoA mutase, synthesizes succinyl-CoA from methylmalonyl-CoA in the mitochondria. Deficiency of B12, thus results in elevated methylmalonic acid (MMA) levels.
Dietary B12 is normally bound to proteins. Food-bound B12 is released in the stomach under the effect of gastric acid and pepsin. The free vitamin is then bound to an R-binder, a glycoprotein in gastric fluid and saliva that protects B12 from the highly acidic stomach environment. Pancreatic proteases degrade R-binder in the duodenum and liberate B12; finally, the free vitamin is then bound by the intrinsic factor (IF)—a glycosylated protein secreted by gastric parietal cells—forming an IF-B12 complex [6]. The IF resists proteolysis and serves as a carrier for B12 to the terminal ileum where the IF-B12 complex undergoes receptor (cubilin)-mediated endocytosis [7]. The vitamin then appears in circulation bound to holo-transcobalamin-I (holo-TC-I), holo-transcobalamin-II (holo-TC-II), and holo-transcobalamin-III (holo-TC-III). It is estimated that 20–30% of the total circulating B12 is bound to holo-TC-II and only this form is available to the cells [7]. Holo-TC-I binds 70–80% of circulating B12, preventing the loss of the free unneeded portion [6]. Vitamin B12 is stored mainly in the liver and kidneys.
Many mechanisms have been proposed to explain how MET interferes with the absorption of B12: diminished absorption due to changes in bacterial flora, interference with intestinal absorption of the IF–B12 complex (and)/or alterations in IF levels. The most widely accepted current mechanism suggests that MET antagonizes the calcium cation and interferes with the calcium-dependent IF–B12 complex binding to the ileal cubilin receptor [8,9]. The recognition and treatment of B12 deficiency is important because it is a cause of bone marrow failure, macrocytic anemia, and irreversible neuropathy [10].
In general, previous studies on diabetics have observed a reduction in serum levels of B12 after both short- and long-term MET treatment [1]. A recent review on observational studies showed significantly lower levels of B12 and an increased risk of borderline or frank B12 deficiency in patients on MET than not on MET [1]. The meta-analysis of four trials (only one double-blind) found a significant overall mean B12 reducing effect of MET after six weeks to three months of use [1]. A secondary analysis (13 years after randomization) of the Diabetes Prevention Program Outcomes Study, which randomized over 3000 persons at high risk for type 2 diabetes to MET or placebo, showed a 13% increase in the risk of B12 deficiency per year of total MET use [3]. In this study, B12 levels were measured from samples obtained in years 1 and 9. Stored serum samples from other time points, including baseline, were not available, and potentially informative red blood cell indices that might have demonstrated the macrocytic anemia, typical of B12 deficiency, were not recorded [3]. The HOME (Hyperinsulinaemia: the Outcome of its Metabolic Effects) study, a large randomized controlled trial investigating the long-term effects of MET versus placebo in patients with type 2 diabetes treated with insulin, showed that the addition of MET improved glycemic control, reduced insulin requirements, prevented weight gain but lowered serum B12 over time, and raised serum homocysteine, suggesting tissue B12 deficiency [4]. A recent analysis of 277 diabetics from the same trial showed that serum levels of MMA, the specific biomarker for tissue B12 deficiency [5], were significantly higher in people treated with MET than those receiving placebo after four years (on average) [4].
The risk of MET-associated B12 deficiency may be higher in older individuals and those with poor dietary habits. Prospective studies have found negative associations between obesity and B12 in numerous ethnicities [11,12]. An energy-dense but micronutrient-insufficient diet consumed by individuals who are overweight or obese might explain this [12]. Furthermore, obesity is associated with low-grade inflammation and these physiological changes have been shown to be associated, in several studies, with elevated C-reactive protein and homocysteine and with low concentrations of B12 and other vitamins [13,14].
As part of a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET for primary prevention of breast cancer and other chronic age-related diseases in healthy women with tracts of MS [15] we tested the effect of MET on serum levels of B12, holo-TC-II, and MMA.

Other articles of note on the Mediterranean Diet in this Online Open Access Scientific Journal Include

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Targeting hematopoietic stem cells

Larry H. Bernstein, MD, FCAP, Curator



New technology uncovered the stem cell niche in the bone marrow

HSCs, Stem cells, hematopoiesis

Hematopoietic stem cells (HSCs) are so rare that it’s difficult to comprehensively localize dividing and non-dividing HSCs. Thus, there has controversy about their specific location in the bone marrow. A recent Nature publication reported that the HSCs resides mainly in perisinusoidal niches through out the bone marrow and there are no spatially distinct niches for dividing and non-dividing blood-forming stem cells. This group of researchers at UT Southwestern Medical Center started the generation of a GFP knock-in for the gene Ctnnal1, a generic marker for HSCs in mice (α-catulinGFP mice) and confirmed that α-catulin-GFP+c-kit+ cells represent blood-forming HSCs by showing that α-catulin-GFP+c-kit+ cells gave long term multi-lineage reconstitution of irradiated mice. Using a tissue-clearing technique and deep confocal imaging, they were able to image thousands of α-catulin-GFP+c-kit+ cells and see their relation to other cells. This publication improved the understanding of the microenvironment of HSCs in the bone marrow, which would significantly improve the safety and effectiveness of bone marrow transplantation.

Melih Acar, etc. (October 2015) Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature


Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

AcarKS. KocherlakotaMM. MurphyJG. PeyerH OguroCN. InraC JaiyeolaZ ZhaoK Luby-Phelps & Sean J. Morrison
Nature526,126–130(01 October 2015)


Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial1. HSCs are rare and few can be found in thin tissue sections2, 3 or upon live imaging4, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as αcatulinGFP), we discover that αcatulinGFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of αcatulin−GFP+c-kit+ cells give long-term multilineage reconstitution of irradiated mice, indicating thatαcatulin−GFP+c-kit+ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of αcatulin–GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of αcatulin–GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr+) and Cxcl12high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67+) and non-dividing (Ki-67), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr+Cxcl12high cells throughout the bone marrow.


Figure 1: Deep imaging of αcatulin−GFP+ HSCs in digitally reconstructed bone marrow.close


Deep imaging of [agr]-catulin-GFP+ HSCs in digitally reconstructed bone marrow.

a, Only 0.021 ± 0.006% of αcatulinGFP/+ bone marrow cells were GFP+ (n = 14 mice in 11 independent experiments). b, Nearly allαcatulin−GFP+c-kit+ bone marrow cells were CD150+CD48 (n = 9 mice in 3 independent experiments;


Extended Data Figure 3: αcatulin−GFP expression among haematopoietic cells is highly restricted to HSCs.


[agr]-catulin-GFP expression among haematopoietic cells is highly restricted to HSCs.


a, The frequency of αcatulin−GFP+ bone marrow cells in negative control αcatulin+/+ (WT) mice and α-catulinGFP/+ mice (n = 14 mice per genotype in 11 independent experiments). In all cases in this figure, percentages refer to the frequency of each population as a percentage of WBM cells. b, αcatulin−GFP+c-kit+ cells from Fig. 1b are shown (blue dots) along with all other bone marrow cells in the same sample (red dots). c, CD150+CD48LSK HSCs express αcatulin−GFP but CD150CD48LSK MPPs do not (n = 17 mice in 12 independent experiments). A minority of the αcatulin−GFP+c-kit+ cells had high forward scatter, lacked reconstituting potential, and were gated out when isolating HSCs by flow cytometry and when identifying HSCs during imaging (see Extended Data Fig. 5for further explanation). d, Linc-kitlowSca-1lowCD127+CD135+ common lymphoid progenitors (CLPs), Linc-kit+Sca-1CD34+CD16/32 common myeloid progenitors (CMPs), Linc-kit+Sca-1CD34+CD16/32+ granulocyte-macrophage progenitors (GMPs), and Linc-kit+Sca-1CD34CD16/32 megakaryocyte-erythroid progenitors (MEPs) did not express αcatulin−GFP. αcatulinGFP/+ and control cell populations had similar levels of background GFP signals that accounted for fewer than 1% of the cells in each population (n = 9 mice per genotype in 2 independent experiments).


Extended Data Figure 7: HSC density is higher in the diaphysis as compared to the metaphysis.

HSC density is higher in the diaphysis as compared to the metaphysis.

a, Schematic of a femur showing the separation of epiphysis/metaphysis from diaphysis. We divided metaphysis from diaphysis at the point where the central sinus branched (see red line in panels a, f,and i). This is also the point at wh…



Extended Data Figure 9: Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.close

Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.

a, b, Schematic (a) and properties (b) of blood vessels in the bone marrow. Blood enters the marrow through arterioles that branch as they become smaller in diameter and approach the endosteum, where they connect to smaller diameter tra…

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Monitoring AML with “cell specific” blood test

Larry H. Bernstein, MD, FCAP, Curator



‘Liquid Biopsy’ Blood Test Replaces Painful Bone Marrow Biopsy for Leukemia

Mon, 01/11/2016  by BioFluidica, Inc.  http://www.mdtmag.com/news/2016/01/liquid-biopsy-blood-test-replaces-painful-bone-marrow-biopsy-leukemia


BioFluidica, Inc. has released the clinical data for minimal residual disease detection in Acute Myeloid Leukemia (AML) patients using circulating leukemic cells selected from blood. The data was published in the peer reviewed journal the Analyst (141 (2016) 640). AML is a rapidly developing leukemic disease with ~20,000 cases reported in 2015 with a 5-year survival rate of only 25%.

The goal of this study was to detect early stages of disease relapse following stem cell transplantation. Currently AML relapse is detected using bone marrow biopsy samples that are painful for the patient and using existing commercial tests, limits the frequency of testing and thus resulting in poor outcomes for AML patients. The paper describes that using BioFluidica’s analytical technology relapse could be detected nearly 2 months earlier than conventional tests. In addition, test frequency could be significantly increased using BioFluidica’s technology compared to tests requiring bone marrow biopsies.

Professor Steven A. Soper, the scientific founder of BioFluidica and co-author of the paper with Dr. Paul Armistead, a hematologist, both at the University of North Carolina states that “the use of a blood test compared to a bone marrow biopsy would be a tremendous advancement in diagnostic capability that can dramatically improve the survival rate of patients with AML.”

BioFluidica is developing innovative technologies for the isolation and analysis of rare, circulating biomarkers in the blood. The company’s first platform has the capacity to isolate circulating tumor cells, exosomes and cfDNA from the blood with unprecedented recovery and purity. The technology is based on patented microfluidics designs which has been clinically validated for 6 different cancer types including Colorectal, Pancreatic Ductal Adenocarcinoma, Ovarian, Breast, Multiple Myeloma and AML. Additionally, stroke detection and infectious disease identification have also obtained clinical validation using the BioFluidica test. The company was cofounded by Dr. Soper who is currently a Professor in Biomedical Engineering and Chemistry at the University of North Carolina at Chapel Hill (UNC-CH). He is also Director of a new center on the UNC-CH campus, Center for BioModular Multi-scale Systems for Precision Medicine, focused on developing new tools for the molecular analysis of circulating biomarkers.

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Larry H. Bernstein, MD, FCAP, Curator



Hematopoietic Stem Cells Use a Simple Heirarchy





These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?

In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.


Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).

Another paper in the journal Cell by Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.

One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.

Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.

 A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.
A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver.  Also, these HSCs seem to produce CMPs, which is more in line with the classical model.  Does the environmental difference or fetal liver and bone marrow make the difference?  In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.”  These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2).  What about fetal liver?  That’s not so clear – until now.

In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.

Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence.  What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.

When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.

These models give us a better view of the inner workings of HSC differentiation.  Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.


Distinct routes of lineage development reshape the human blood hierarchy across ontogeny

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult “two-tier” hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

Franziska Paul, et al.
Cell Dec 2015; Volume 163, Issue 7:1663–1677   http://dx.doi.org/10.1016/j.cell.2015.11.013
Figure thumbnail fx1
  • Transcriptionally primed single-cell subpopulations in early myeloid progenitors
  • Transcription factors and epigenetic landscapes that regulate myeloid priming
  • Mixed lineage states are not observed but appear when regulation is perturbed
  • New reference model for studying hematopoiesis at single-cell resolution



Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.



Fetal liver hematopoietic stem cell niches associate with portal vessels

Jalal A. Khan, et al.   Science  08 Jan 2016; 351(6269):176-180   http://dx.doi.org:/10.1126/science.aad0084
How HSCs populate the fetal liver

Hematopoietic stem cells (HSCs) undergo dramatic expansion in the fetal liver before migrating to their definitive site in the bone marrow. Khan et al. identify portal vessel–associated Nestin+NG2+ pericytes as critical HSC niche components (see the Perspective by Cabezas-Wallscheid and Trumpp). The portal vessel niche and HSCs expand according to fractal geometries, suggesting that niche cells—rather than factors expressed by the niche—drive HSC proliferation. After birth, arterial portal vessels transform into portal veins, and lose Nestin+NG2+pericytes. When this happens, the niche is lost and HSCs migrate away from the neonatal liver.

Science, this issue p. 176; see also p. 126


Whereas the cellular basis of the hematopoietic stem cell (HSC) niche in the bone marrow has been characterized, the nature of the fetal liver niche is not yet elucidated. We show that Nestin+NG2+ pericytes associate with portal vessels, forming a niche promoting HSC expansion. Nestin+NG2+ cells and HSCs scale during development with the fractal branching patterns of portal vessels, tributaries of the umbilical vein. After closure of the umbilical inlet at birth, portal vessels undergo a transition from Neuropilin-1+Ephrin-B2+ artery to EphB4+ vein phenotype, associated with a loss of periportal Nestin+NG2+ cells and emigration of HSCs away from portal vessels. These data support a model in which HSCs are titrated against a periportal vascular niche with a fractal-like organization enabled by placental circulation.



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Neutrophil Serine Proteases in Disease and Therapeutic Considerations

Larry H. Bernstein, MD, FCAP, Curator



SERPINB1 Regulates the activity of the neutrophil proteases elastase, cathepsin G, proteinase-3, chymase,
chymotrypsin, and kallikrein-3. Belongs to the serpin family. Ov-serpin subfamily. Note: This description may
include information from UniProtKB.
Chromosomal Location of Human Ortholog: 6p25
Cellular Component: extracellular space; membrane; cytoplasm
Molecular Function: serine-type endopeptidase inhibitor activity
Reference #:  P30740 (UniProtKB)
Alt. Names/Synonyms: anti-elastase; EI; ELANH2; ILEU; LEI; Leukocyte elastase inhibitor; M/NEI; MNEI; Monocyte/neutrophil elastase inhibitor; Peptidase inhibitor 2; PI-2; PI2; protease inhibitor 2 (anti-elastase), monocyte/neutrophil derived; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1; Serpin B1; serpin peptidase inhibitor, clade B (ovalbumin), member 1; SERPINB1
Gene Symbols: SERPINB1
Molecular weight: 42,742 Da


Alternative titles; symbols
HGNC Approved Gene Symbol: SERPINB1
Cloning and Expression
Monocyte/neutrophil elastase inhibitor (EI) is a protein of approximately 42,000 Mr with serpin-like functional properties.
Remold-O’Donnell et al. (1992) cloned EI cDNA and identified 3 EI mRNA species of 1.5, 1.9, and 2.6 kb in monocyte-like cells
and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI enzymatic activity. The cDNA open reading frame encoded
a 379-amino acid protein. Its sequence established EI as a member of the serpin superfamily. Sequence alignment indicated that
the reactive center P1 residue is cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making
EI a naturally occurring cys-serpin.



In the course of studying 4 closely linked genes encoding members of the ovalbumin family of serine proteinase inhibitors
(Ov-serpins) located on 18q21.3, Schneider et al. (1995) investigated the mapping of elastase inhibitor. They prepared PCR
primer sets of the gene, and by using the NIGMS monochromosomal somatic cell hybrid panel, showed that the EI gene maps
to chromosome 6.

By amplifying DNA of a somatic cell hybrid panel, Evans et al. (1995) unambiguously localized ELANH2 to chromosome 6.
With the use of a panel of radiation and somatic cell hybrids specific for chromosome 6, they refined the localization to
the short arm telomeric of D6S89, F13A (134570), and D6S202 at 6pter-p24.




Evans, E., Cooley, J., Remold-O’Donnell, E. Characterization and chromosomal localization of ELANH2, the gene encoding human
monocyte/neutrophil elastase inhibitor. Genomics 28: 235-240, 1995. [PubMed: 8530031related citations] [Full Text]
Remold-O’Donnell, E., Chin, J., Alberts, M. Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.
Proc. Nat. Acad. Sci. 89: 5635-5639, 1992. [PubMed: 1376927related citations][Full Text]
Schneider, S. S., Schick, C., Fish, K. E., Miller, E., Pena, J. C., Treter, S. D., Hui, S. M., Silverman, G. A. A serine proteinase inhibitor locus at
18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc. Nat. Acad. Sci. 92: 3147-3151, 1995.
[PubMed: 7724531,related citations] [Full Text]


Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Short name: Serpin_B1

Family relationships

  • Serpin family (IPR000215)
    • Leukocyte elastase inhibitor (serpin B1) (IPR015557)


Leukocyte elastase inhibitor is also known as serpin B1. Serpins (SERine Proteinase INhibitors) belong to MEROPS inhibitor family I4 (clan ID)
[PMID: 14705960].

Serpin B1 regulates the activity of neutrophil serine proteases such as elastase, cathepsin G and proteinase-3 and may play a regulatory role to
limit inflammatory damage due to proteases of cellular origin [PMID: 11747453]. It also functions as a potent intracellular inhibitor of granzyme
H [PMID: 23269243]. In mouse, four different homologues of human serpin B1 have been described [PMID: 12189154].


The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection

Dapeng Gong1,2, Charaf Benarafa1,2, Kevan L Hartshorn3 and Eileen Remold-O’Donnell1,2
J Immunol April 2009; 182(Meeting Abstract Supplement) 43.10

SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFN, TNF, IL-17, IFN, G-CSF) and chemokines (MIP-1, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection.


The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosainfection

Charaf Benarafa 1 , 2 Gregory P. Priebe 3 , 4 , and Eileen Remold-O’Donnell 1 , 2
JEM July 30, 2007; 204(8): 1901-1909   http://dx.doi.org:/10.1084/jem.20070494

Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1−/− mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein–D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance inserpinb1−/− mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.


Neutrophils are the first and most abundant phagocytes mobilized to clear pathogenic bacteria during acute lung infection. Prominent among their antimicrobial weapons, neutrophils carry high concentrations of a unique set of serine proteases in their granules, including neu trophil elastase (NE), cathepsin G (CG), and proteinase-3. These neutrophil serine proteases (NSPs) are required to kill phagocytosed bacteria and fungi (12). Indeed, neutrophils lacking NE fail to kill phagocytosed pathogens, and mice deficient for NE and/or CG have increased mortality after infection with pulmonary pathogens (34). However, NSPs in the lung airspace can have a detrimental effect in severe inflammatory lung disease through degradation of host defense and matrix proteins (57). Thus, understanding of the mechanisms that regulate NSP actions during lung infections associated with neutrophilia will help identify strategies to balance host defense and prevent infection-induced tissue injury.


SERPINB1, also known as monocyte NE inhibitor (8), is an ancestral serpin super-family protein and one of the most efficient inhibitors of NE, CG, and proteinase-3 (910). SERPINB1 is broadly expressed and is at particularly high levels in the cytoplasm of neutrophils (1112). SERPINB1 has been found complexed to neutro phil proteases in lung fluids of cystic fibrosis patients and in a baboon model of bronchopulmonary dysplasia (1314). Although these studies suggest a role for SERPINB1 in regulating NSP activity, it is unclear whether these complexes reflect an important physiological role for SERPINB1 in the lung air space.


To define the physiological importance of SERPINB1 in shaping the outcome of bacterial lung infection, we generated mice deficient for serpinb1 (serpinb1−/−) by targeted mutagenesis in embryonic stem (ES) cells (Fig. 1, A–C). Crossings of heterozygous mice produced WT (+/+), heterozygous (+/−), and KO (−/−) mice for serpinb1 at expected Mendelian ratios (25% +/+, 51% +/−, and 24% −/−; n = 225; Fig. 1 D), indicating no embryonic lethality. Bone marrow neutrophils of serpinb1−/− mice lacked expression of the protein, whereas heterozygous serpinb1+/− mice had reduced levels compared with WT mice (Fig. 1 E). Importantly, levels of the cognate neutrophil proteases NE and CG, measured as antigenic units, were not altered by deletion of serpinb1 (Fig. 1 F). When maintained in a specific pathogen-free environment, serpinb1−/− mice did not differ from WT littermates in growth, litter size, or life span (followed up to 12 mo), and no gross or histopathological defects were observed at necropsy in 8-wk-old mice.

6–8-wk-old animals were intranasally inoculated with the nonmucoid Pseudomonas aeruginosa strain PAO1. Using two infection doses (3 × 106 and 7 × 106 CFU/mouse),serpinb1−/− mice had a significantly lower survival probability and a shorter median survival time compared with WT mice (Fig. 2 A). Further groups of infected mice were used to evaluate bacterial clearance. At 6 h after infection, the bacteria were similarly restricted in mice of the two genotypes, suggesting that the serpinb1−/− mice have a normal initial response to infection. At 24 h, the median bacterial count in the lungs of serpinb1−/− mice was five logs higher than that of the WT mice (P < 0.001), and the infection had spread systemically in serpinb1−/− mice but not in WT mice, as shown by high median CFU counts in the spleen (Fig. 2 B). Histological examination at 24 h after infection revealed abundant neutrophil infiltration in the lungs of both WT and serpinb1−/− mice, and consistent with the bacteriological findings, numerous foci of bacterial colonies and large areas of alveolar exudates were found in serpinb1−/− mice only (Fig. 2 C). When challenged with the mucoid P. aeruginosa clinical strain PA M57-15 isolated from a cystic fibrosis patient, WT mice cleared >99.9% of the inoculum within 24 h, whereas serpinb1-deficient mice failed to clear the infection (Fig. 2 D). Thus, the NSP inhibitor serpinb1 is essential for maximal protection against pneumonia induced by mucoid and nonmucoid strains of P. aeruginosa.

Figure 2.

Serpinb1−/− mice fail to clear P. aeruginosalung infection. (A) Kaplan-Meier survival curves of WT (+/+) and serpinb1-deficient (−/−) mice intranasally inoculated with nonmucoid P. aeruginosa strain PAO1. Increased mortality of serpinb1−/− mice was statistically significant (P = 0.03 at 3 × 106CFU/mouse; P < 0.0001 at 7 × 106CFU/mouse). (B) CFUs per milligram of lung (left) and splenic (right) tissue determined 6 and 24 h after inoculation with 3 × 106 CFUP. aeruginosa PAO1 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Each symbol represents a value for an individual mouse. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test. Data below the limit of detection (dotted line) are plotted as 0.5 CFU × dilution factor. (C) Lung sections stained with hematoxylin and eosin show bacterial colonies (arrowheads) and alveolar exudate in lungs of serpinb1−/− mice 24 h after infection with P. aeruginosa PAO1. Bars, 50 μm. (D) Total CFUs in the lung and spleen 24 h after inoculation with 2 × 108 CFU of the mucoid P. aeruginosa strain PA M57-15 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test.

To verify specificity of the gene deletion, we tested whether delivering rSERPINB1 would correct the defective phenotype. Indeed, intranasal instillation of rSERPINB1 to serpinb1−/− mice at the time of inoculation significantly improved clearance of P. aeruginosa PAO1 from the lungs assessed at 24 h and reduced bacteremia compared with infectedserpinb1−/− mice that received PBS instead of the recombinant protein (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20070494/DC1). We have previously demonstrated that rSERPINB1 has no effect on the growth of P. aeruginosa in vitro (15) and does not induce bacterial aggrega tion (16). Also, rSERPINB1 mixed with PAO1 had no effect on adherence of the bacteria to human bronchial epithelial and corneal epithelial cell lines (unpublished data). Therefore, the improved bacterial clearance in treated serpinb1−/− mice is not related to a direct antibacterial role for rSERPINB1 but rather to reducing injury induced by excess neutrophil proteases. In addition, previous in vivo studies in WT rats showed that rSERPINB1 can protect against elastase-induced lung injury (17) and accelerate bacterial clearance two- to threefold in the Pseudomonas agar bead model (15).

Evidence of excess NSP action was examined in the lungs of infected serpinb1−/− mice by measuring surfactant protein–D (SP-D). SP-D, a multimeric collagenous C-type lectin produced by alveolar epithelial cells, is highly relevant as a host defense molecule, because it functions as an opsonin in microbial clearance (18) and acts on alveolar macrophages to regulate pro- and antiinflammatory cytokine production (19). SP-D is also relevant as an NSP target because it is degraded in vitro by trace levels of each of the NSPs (1620). SP-D levels in lung homogenates of WT and serpinb1−/− mice were similar 6 h after P. aeruginosa infection. At 24 h, SP-D levels were reduced in the lungs ofserpinb1−/− mice compared with WT mice, as indicated by immunoblots. A lower molecular mass band indicative of proteolytic degradation is also apparent (Fig. 3 A). Densitometry analysis of the 43-kD SP-D band relative to β-actin indicated that the reduction of SP-D level was statistically significant (+/+, 45 ± 6 [n = 8]; −/−, 10 ± 2 [n = 8]; P < 0.0001 according to the Student’s t test). Furthermore, rSERPINB1 treatment ofP. aeruginosa–infected serpinb1−/− mice partly prevented the degradation of SP-D in lung homogenates compared with nontreated mice (Fig. S1 B). As a further test of the impact of serpinb1 deletion on NSP activity, isolated neutrophils of serpinb1−/− mice were treated with LPS and FMLP and tested for their ability to cleave recombinant rat SP-D (rrSP-D) in vitro. The extent of rrSP-D cleavage by serpinb1−/− neutrophils was fourfold greater than by WT neutrophils, as determined by densitometry. The cleavage was specific for NSPs because it was abrogated by rSERPINB1 and diisopropyl fluorophosphate (Fig. 3 B). Collectively, these findings indicate a direct role for serpinb1 in regulating NSP activity released by neutrophils and in preserving SP-D, an important-host defense molecule.

Efficient clearance of P. aeruginosa infection requires an early cytokine and chemokine response coordinated by both resident alveolar macrophages and lung parenchymal cells (2122). The IL-8 homologue keratinocyte-derived chemokine (KC) and the cytokines TNF-α, IL-1β, and G-CSF were measured in cell-free bronchoalveolar (BAL) samples. Although the tested cytokines were undetectable in sham-infected mice of both genotypes (unpublished data), comparable induc tion of these cytokines was observed in BAL of WT and serpinb1−/− mice at 6 h after infection, demonstrating that there is no early defect in cytokine production in serpinb1−/− mice. At 24 h, levels of TNF-α, KC, and IL-1β were sustained or increased in serpinb1−/− mice and significantly higher than cytokine levels in WT mice. G-CSF levels at 24 h were elevated to a similar extent in BAL of WT and KO mice (Fig. 3 C). However, G-CSF levels were significantly higher in the serum of serpinb1−/− mice (WT, 336 ± 80 ng/ml; KO, 601 ± 13 ng/ml; n = 6 of each genotype; P < 0.01). In addition, serpinb1−/− mice that were treated at the time of infection with rSERPINB1 had cytokine levels in 24-h lung homogenates that were indistinguishable from those of infected WT mice (Fig. S1 C). The increased cytokine production in the lungs of infected serpinb1−/− mice may be caused by failed bacterial clearance but also by excess NSPs, which directly induce cytokine and neutrophil chemokine production in pulmonary parenchymal cells and alveolar macrophages (2324).

Neutrophil recruitment to the lungs was next examined as a pivotal event of the response to P. aeruginosa infection (25). Lung homogenates were assayed for the neutrophil-specific enzyme myeloperoxidase (MPO) to quantify marginating, interstitial, and alveolar neutrophils. Neutrophils in BAL fluid were directly counted as a measure of neutrophil accumulation in the alveolar and airway lumen. MPO in lung homo genates was undetectable in uninfected mice and was comparably increased in mice of both genotypes at 6 h, suggesting normal early serpinb1−/− neutrophil margination and migration into the interstitium. However, by 24 h after infection, MPO levels in lung homogenates remained high in WT mice but were significantly decreased in serpinb1−/− mice (Fig. 4 A). Importantly, the content of MPO per cell was the same for isolated neutrophils of WT andserpinb1−/− mice (+/+, 369 ± 33 mU/106 cells; −/−, 396 ± 27 mU/106 cells). The numbers of neutrophils in BAL were negligible in uninfected mice and were similarly increased in WT and serpinb1−/− mice at 6 h after infection. Neutrophil counts in BAL further increased at 24 h, but the mean BAL neutrophil numbers were significantly lower in serpinb1−/− mice compared with WT mice (Fig. 4 B). The evidence from the 6-h quantitation of MPO in homogenates and neutrophils in BAL strongly suggests that neutrophil recruitment is not defective in infected serpinb1−/− mice. Moreover, the high levels of cytokines and neutrophil chemoattractant KC in serpinb1−/− mice at 24 h (Fig. 3 C) also suggest that, potentially, more neutrophils should be recruited. Therefore, to examine neutrophil recruitment in serpinb1−/− mice, we used a noninfectious model in which neutrophils are mobilized to migrate to the lung after intranasal delivery of P. aeruginosa LPS. MPO levels in lung homogenate and neutrophil numbers in BAL were not statistically different in WT and serpinb1−/− mice 24 h after LPS instillation (Fig. 4, C and D). Furthermore, the number of circulating blood neutrophils and recruited peritoneal neutrophils after injection of sterile irritants glycogen and thioglycollate did not differ in WT and serpinb1−/− mice (unpublished data). Alveolar macrophage numbers were similar in uninfected mice of both genotypes (∼5 × 105 cells/mouse) and did not substantially change upon infection. Collectively, these findings show that neutrophil recruitment to the lungs in response to P. aeruginosa infection is not defective in serpinb1−/− mice, and therefore, the recovery of lower numbers of serpinb1−/− neutrophils at 24 h after infection suggests their decreased survival.

To examine the putative increased death of serpinb1−/− neutrophils in the lungs after P. aeruginosa infection, lung sections were analyzed by immunohistochemistry. Caspase-3–positive leukocytes were more relevant in the alveolar space of serpinb1−/− mice compared with WT mice at 24 h after infection, suggesting increased neutrophil apoptosis (Fig. 5 A). The positive cells were counted in 50 high power fields (hpf’s), and mean numbers of caspase-3–stained cells were increased in the lungs of serpinb1/− mice (1.8 ± 0.2 cells/hpf) compared with WT mice (0.4 ± 0.1 cells/hpf; P < 0.0001). To characterize neutrophils in the alveoli and airways, neutrophils in BAL were identified in flow cytometry by forward scatter (FSC) and side scatter and were stained with annexin V (AnV) and propidium iodide (PI). At 24 h after infection, the proportion of late apoptotic/necrotic neutrophils (AnV+PI+) was increased at the expense of viable neutrophils (AnVPI) in the BAL of serpinb1−/− mice compared with WT mice (Fig. 5 B). Neutrophil fragments in BAL were also identified in flow cytometry by low FSC (FSClow) within the neutrophil population defined by the neutrophil marker Gr-1. The number of neutrophil fragments (FSClow, Gr-1+) relative to intact neutrophils was increased two- to threefold at 24 h after infection for serpinb1−/− compared with WT mice (Fig. 5 C). Moreover, free MPO in BAL supernatants was increased in serpinb1−/− mice compared with WT mice at 24 h after infection, indicating increased PMN lysis or degranulation (Fig. 5 D).

Finally, we questioned whether the enhanced death of serpinb1−/− pulmonary neutrophils was a primary effect of gene deletion or a secondary effect caused by, for example, bacteria or components of inflammation. To address this, neutrophils were collected using the noninfectious LPS recruitment model and were cultured in vitro to allow for spontaneous cell death. After 24 h, the percentages of apoptotic and necrotic neutrophils evaluated by microscopy were increased in serpinb1−/− neutrophils compared with WT neutrophils (Fig. 6, A–C). A similar increase in apoptotic cells was observed using AnV/PI staining and measurements of hypodiploid DNA (unpublished data). Moreover, live cell numbers from serpinb1−/− mice remaining in culture after 24 h were significantly decreased compared with WT mice (Fig. 6 D). The in vitro findings indicate that enhanced death of pulmonary neutrophils of infected serpinb1−/− mice is at least in part a cell-autonomous defect likely mediated by unchecked NSP actions.


In this paper, we have demonstrated that serpinb1, an intracellular serpin family member, regulates the innate immune response and protects the host during lung bacterial infection. Serpinb1 is among the most potent inhibitors of NSPs and is carried at high levels within neutrophils. Serpinb1-deficient mice fail to clear P. aeruginosa PAO1 lung infection and succumb from systemic bacterial spreading. The defective immune function in serpinb1−/− mice stems at least in part from an increased rate of neutrophil necrosis, reducing the number of phagocytes and leading to increased NSP activity in the lungs with proteolysis of SP-D. In addition, serpinb1-deficient mice also have impaired clearance of the mucoid clinical strain PA M57-15. Interestingly, mucoid strains of P. aeruginosa are cleared with a very high efficiency from the lungs of WT and cystic fibrosis transmembrane conductance regulator–deficient mice (26). The phenotype of serpinb1−/− mice reproduces major pathologic features of human pulmonary diseases characterized by excessive inflammation, massive neutrophil recruitment to the air space, and destruction of cellular and molecular protective mechanisms. Importantly, serpinb1 deficiency may be helpful as an alternative or additional model of the inflammatory lung pathology of cystic fibrosis.

The present study documents a key protective role for serpinb1 in regulating NSP actions in the lung. This role has previously been attributed to the NSP inhibitors α1-antitrypsin and secretory leukocyte protease inhibitor, which are found in the airway and alveolar lining fluid (2728). However, patients with α1-antitrypsin deficiency do not present with pulmonary infection secondary to innate immune defects despite increased NSP activity that leads to reduced lung elasticity and emphysema. Moreover, there is so far no evidence that deficiency in secretory leukocyte protease inhibitor results in failure to clear pulmonary infection. Because synthesis and storage of NSPs in granules is an event that exclusively takes place in bone marrow promyelocytes (29), the regulation of NSPs in the lung relies entirely on NSP inhibitors. Thus, the extent of the innate immune defect inserpinb1−/− mice and the normalization of bacterial clearance with topical rSERPINB1 treatment indicate that serpinb1 is required to regulate NSP activity in the airway fluids and that, during acute lung infection associated with high neutrophilic recruitment, there is insufficient compensation by other NSP inhibitors. The devastating effects of NSPs when released in the lungs by degranulating and necrotic neutrophils are well documented in human pulmonary diseases (5630). Therefore, our findings clearly establish a physiological and nonredundant role for serpinb1 in regulating NSPs during pulmonary infection.

NSPs also cleave molecules involved in apoptotic cell clearance, including the surfactant protein SP-D and the phosphatidylserine receptor on macrophages (3132), thereby tipping the balance further toward a detrimental outcome. The increased numbers of leukocytes with active caspase-3 in the alveolar space of P. aeruginosa–infectedserpinb1−/− mice suggest that the removal of apoptotic cells may be inadequate during infection. SP-D has been shown to stimulate phagocytosis of P. aeruginosa by alveolar macrophages in vitro (33), and SP-D–deficient mice were found to have defective early (6-h) clearance of P. aeruginosa from the lung (34). Although the destruction of SP-D alone may not entirely account for the defective phenotype of serpinb1−/− mice, loss of SP-D likely diminishes bacterial clearance and removal of apop totic neutrophils.

Given that NSPs also mediate bacterial killing, why would NSP excess lead to a failed bacterial clearance? In the NE KO mice, the decreased killing activity of neutrophils is a direct consequence of the loss of the bactericidal activity of NE. The absence of an early bacterial clearance defect at 6 h after infection in serpinb1−/− mice suggests that there is initially normal bacterial killing. The current understanding is that the compartmentalization of the NSPs is crucial to the outcome of their actions: on the one hand, NSPs are protective when killing microbes within phagosomes, and on the other hand, extracellular NSPs destroy innate immune defense molecules such as lung collectins, immunoglobulins, and complement receptors. We have shown that the regulation of NSP activity is essential and that cytoplasmic serpinb1 provides this crucial shield. Neutrophils undergoing cell death gradually transition from apoptosis, characterized by a nonpermeable plasma membrane, to necrosis and lysis, where cellular and granule contents, including NSPs, are released. The increased pace of serpinb1−/− neutrophil cell death strongly suggests that unopposed NSPs may precipitate neutrophil demise and, therefore, reduce the neutrophil numbers leading to a late-onset innate immune defect. High levels of G-CSF, a prosurvival cytokine for neutrophils, also indicate that increased cell death is likely independent or downstream of G-CSF.

In conclusion, serpinb1 deficiency unleashes unbridled proteolytic activity during inflammation and thereby disables two critical components of the host response to bacterial infection, the neutrophil and the collectin SP-D. The phenotype of the infectedserpinb1-deficient mouse, characterized by a normal early antibacterial response that degenerates over time, highlights the delicate balance of protease–antiprotease systems that protect the host against its own defenses as well as invading microbes during infection-induced inflammation.



Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

K Kessenbrock,1 LFröhlich,2 M Sixt,3 …., A Belaaouaj,5 J Ring,6,7 M Ollert,6 R Fässler,3 and DE. Jenne1
J Clin Invest. 2008 Jul 1; 118(7): 2438–2447.   http://dx.doi.org:/10.1172/JCI34694

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.


Neutrophils belong to the body’s first line of cellular defense and respond quickly to tissue injury and invading microorganisms (1). In a variety of human diseases, like autoimmune disorders, infections, or hypersensitivity reactions, the underlying pathogenic mechanism is the formation of antigen-antibody complexes, so-called immune complexes (ICs), which trigger an inflammatory response by inducing the infiltration of neutrophils (2). The subsequent stimulation of neutrophils by C3b-opsonized ICs results in the generation of ROS and the release of intracellularly stored proteases leading to tissue damage and inflammation (3). It is therefore important to identify the mechanisms that control the activation of infiltrating neutrophils.

Neutrophils abundantly express a unique set of neutrophil serine proteases (NSPs), namely cathepsin G (CG), proteinase 3 (PR3; encoded by Prtn3), and neutrophil elastase (NE; encoded by Ela2), which are stored in the cytoplasmic, azurophilic granules. PR3 and NE are closely related enzymes, with overlapping and potentially redundant substrate specificities different from those of CG. All 3 NSPs are implicated in antimicrobial defense by degrading engulfed microorganisms inside the phagolysosomes of neutrophils (48). Among many other functions ascribed to these enzymes, PR3 and NE were also suggested to play a fundamental role in granulocyte development in the bone marrow (911).

While the vast majority of the enzymes is stored intracellularly, minor quantities of PR3 and NE are externalized early during neutrophil activation and remain bound to the cell surface, where they are protected against protease inhibitors (1213). These membrane presented proteases were suggested to act as path clearers for neutrophil migration by degrading components of the extracellular matrix (14). This notion has been addressed in a number of studies, which yielded conflicting results (1517). Thus, the role of PR3 and NE in leukocyte extravasation and interstitial migration still remains controversial.

Emerging data suggest that externalized NSPs can contribute to inflammatory processes in a more complex way than by simple proteolytic tissue degradation (18). For instance, recent observations using mice double-deficient for CG and NE indicate that pericellular CG enhances IC-mediated neutrophil activation and inflammation by modulating integrin clustering on the neutrophil cell surface (1920). Because to our knowledge no Prtn3–/– mice have previously been generated, the role of this NSP in inflammatory processes has not been deciphered. Moreover, NE-dependent functions that can be compensated by PR3 in Ela2–/–animals are still elusive.

One mechanism by which NSPs could upregulate the inflammatory response has recently been proposed. The ubiquitously expressed progranulin (PGRN) is a growth factor implicated in tissue regeneration, tumorigenesis, and inflammation (2123). PGRN was previously shown to directly inhibit adhesion-dependent neutrophil activation by suppressing the production of ROS and the release of neutrophil proteases in vitro (23). This antiinflammatory activity was degraded by NE-mediated proteolysis of PGRN to granulin (GRN) peptides (23). In contrast, GRN peptides may enhance inflammation (23) and have been detected in neutrophil-rich peritoneal exudates (24). In short, recent studies proposed PGRN as a regulator of the innate immune response, but the factors that control PGRN function are still poorly defined and its relevance to inflammation needs to be elucidated in vivo.

In the present study, we generated double-deficient Prtn3–/–Ela2–/– mice to investigate the role of these highly similar serine proteases in noninfectious neutrophilic inflammation. We established that PR3 and NE are required for acute inflammation in response to subcutaneous IC formation. The proteases were found to be directly involved in early neutrophil activation events, because isolated Prtn3–/–Ela2–/– neutrophils were poorly activated by ICs in vitro. These defects in Prtn3–/–Ela2–/– mice were accompanied by accumulation of PGRN. We demonstrated that PGRN represents a potent inflammation-suppressing factor that is cleaved by both PR3 and NE. Our data delineate what we believe to be a previously unknown proinflammatory role for PR3 and NE, which is accomplished via the local inactivation of antiinflammatory PGRN.


Generation of Prtn3–/–Ela2–/– mice.

To analyze the role of PR3 and NE in neutrophilic inflammation, we generated a Prtn3–/–Ela2–/– mouse line by targeted gene disruption in embryonic stem cells (see Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI34694DS1). Positive recombination of the Prtn3/Ela2locus was proven by Southern blotting of embryonic stem cell clones (Figure ​(Figure1A).1A). Prtn3–/–Ela2–/– mice showed no expression of mRNA for PR3 and NE in bone marrow cells, as assessed by RT-PCR (Figure ​(Figure1B).1B). The successful elimination of PR3 and NE was confirmed at the level of proteolytic activity in neutrophil lysates using a PR3/NE-specific chromogenic substrate (Supplemental Figure 3) as well as by casein zymography (Figure ​(Figure1C).1C). The substantially reduced casein degradation by heterozygous neutrophils indicates gene-dosage dependence of PR3/NE activities. Furthermore, PR3 and NE deficiency was proven by Western blotting using cell lysates from bone marrow–derived neutrophils, while other enzymes stored in azurophilic granula, such as CG and myeloperoxidase (MPO), were normally detected (Figure ​(Figure1D).1D). Crossing of heterozygous Prtn3+/–Ela2+/– mice resulted in regular offspring of WT, heterozygous, and homozygous genotype according to the Mendelian ratio. Despite the absence of 2 abundant serine proteases, and in contrast to expectations based on previous reports (911), we found unchanged neutrophil morphology (Figure ​(Figure1E)1E) and regular neutrophil populations in the peripheral blood of the mutant mice, the latter as assessed via flow cytometry to determine the differentiation markers CD11b and Gr-1 (Figure ​(Figure1F)1F) (2526). Moreover, Prtn3–/–Ela2–/– mice demonstrated normal percentages of the leukocyte subpopulations in the peripheral blood, as determined by the Diff-Quick staining protocol and by hemocytometric counting (Supplemental Figure 2, A and B). Hence, the proteases are not crucially involved in granulopoiesis, and ablating PR3 and NE in the germ line represents a valid approach to assess their biological significance in vivo.


Figure 1

Generation and characterization of Prtn3–/–Ela2–/– mice.

PR3 and NE are dispensable for neutrophil extravasation and interstitial migration.

To examine neutrophil infiltration into the perivascular tissue, we applied phorbol esters (croton oil) to the mouse ears. At 4 h after stimulation, we assessed the neutrophil distribution in relation to the extravascular basement membrane (EBM) by immunofluorescence microscopy of fixed whole-mount specimens (Figure ​(Figure2A).2A). We found that Prtn3–/–Ela2–/– neutrophils transmigrated into the interstitium without retention at the EBM (Figure ​(Figure2B),2B), resulting in quantitatively normal and widespread neutrophil influx compared with WT mice (Figure ​(Figure2C).2C). Moreover, we analyzed chemotactic migration of isolated neutrophils through a 3-dimensional collagen meshwork in vitro (Supplemental Video 1) and found unhampered chemotaxis toward a C5a gradient, based on the directionality (Figure ​(Figure2D)2D) and velocity (Figure ​(Figure2E)2E) of Prtn3–/–Ela2–/–neutrophils. These findings led us to conclude that PR3 and NE are not principally required for neutrophil extravasation or interstitial migration.


Figure 2

PR3 and NE are not principally required for neutrophil extravasation and interstitial migration.

Reduced inflammatory response to ICs in Prtn3–/–Ela2–/– mice.

The formation of ICs represents an important trigger of neutrophil-dependent inflammation in many human diseases (2). To determine the role of PR3 and NE in this context, we induced a classic model of subcutaneous IC-mediated inflammation, namely the reverse passive Arthus reaction (RPA) (27). At 4 h after RPA induction, we assessed the cellular inflammatory infiltrates by histology using H&E-stained skin sections (Figure ​(Figure3A).3A). Neutrophils, which were additionally identified by Gr-1 immunohistochemistry, made up the vast majority of all cellular infiltrates (Figure ​(Figure3A).3A). We found that neutrophil infiltration to the sites of IC formation was severely diminished in Prtn3–/–Ela2–/– mice. Indeed, histological quantification revealed significantly reduced neutrophil influx in Prtn3–/–Ela2–/– mice compared with WT mice, while Ela2–/– mice showed marginally reduced neutrophil counts (Figure ​(Figure3B).3B). These results indicate that PR3 and NE fulfill an important proinflammatory function during IC-mediated inflammation.


Figure 3

Impaired inflammatory response to locally formed ICs inPrtn3–/–Ela2–/– mice.

(A) Representative photomicrographs of inflamed skin sections 4 h after IC formation. Neutrophils were identified morphologically (polymorphic nucleus) in H&E stainings and by Gr-1 staining (red). The cellular infiltrates were located to the adipose tissue next to the panniculus carnosus muscle (asterisks) and were primarily composed of neutrophil granulocytes. Scale bars: 200 μm. (B) Neutrophil infiltrates in lesions from Prtn3–/–Ela2–/– mice were significantly diminished compared with Ela2–/– mice and WT mice. Neutrophil influx in Ela2–/–mice was slightly, but not significantly, diminished compared with WT mice. Results are mean ± SEM infiltrated neutrophils per HPF. *P < 0.05.

PR3 and NE enhance neutrophil activation by ICs in vitro.

PR3 and NE enhance neutrophil activation by ICs in vitro.

Because PR3 and NE were required for the inflammatory response to IC (Figure ​(Figure3),3), but not to phorbol esters (Figure ​(Figure2),2), we considered the enzymes as enhancers of the neutrophil response to IC. We therefore assessed the oxidative burst using dihydrorhodamine as a readout for cellular activation of isolated, TNF-α–primed neutrophils in the presence of ICs in vitro. While both WT and Prtn3–/–Ela2–/– neutrophils showed a similar, approximately 20-min lag phase before the oxidative burst commenced, the ROS production over time was markedly reduced, by 30%–40%, in the absence of PR3 and NE (Figure ​(Figure4A).4A). In contrast, oxidative burst triggered by 25 nM PMA was not hindered in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure4B),4B), which indicated no general defect in producing ROS. We also performed a titration series ranging from 0.1 to 50 nM PMA and found no reduction in oxidative burst activity in Prtn3–/–Ela2–/– neutrophils at any PMA concentration used (Supplemental Figure 4). These data are consistent with our in vivo experiments showing that neutrophil influx to ICs was impaired (Figure ​(Figure3),3), whereas the inflammatory response to phorbol esters was normal (Figure ​(Figure2,2, A–C), in Prtn3–/–Ela2–/– mice. To compare neutrophil priming in WT and Prtn3–/–Ela2–/–neutrophils, we analyzed cell surface expression of CD11b after 30 min of incubation at various concentrations of TNF-α and found no difference (Supplemental Figure 5). Moreover, we observed normal neutrophil adhesion to IC-coated surfaces (Supplemental Figure 6A) and unaltered phagocytosis of opsonized, fluorescently labeled E. coli bacteria (Supplemental Figure 6, B and C) in the absence of both proteases. We therefore hypothesized that PR3 and NE enhance early events of adhesion-dependent neutrophil activation after TNF-α priming and binding of ICs. It is important to note that Ela2–/– neutrophils were previously shown to react normally in the same setup (20). Regarding the highly similar cleavage specificities of both proteases, we suggested that PR3 and NE complemented each other during the process of neutrophil activation and inflammation.


Figure 4

Impaired oxidative burst and PGRN degradation by IC-activatedPrtn3–/–Ela2–/– neutrophils.

Oxidative burst as the readout for neutrophil activation by ICs was measured over time. (A) While no difference was observed during the initial 20-min lag phase of the oxidative burst, Prtn3–/–Ela2–/– neutrophils exhibited diminished ROS production over time compared with WT neutrophils. (B) Bypassing receptor-mediated activation using 25 nM PMA restored the diminished oxidative burst of Prtn3–/–Ela2–/–neutrophils. Results are presented as normalized fluorescence in AU (relative to maximum fluorescence produced by WT cells). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate. (C) Isolated mouse neutrophils were activated by ICs in vitro and tested for PGRN degradation by IB. In the cellular fraction, the PGRN (~80 kDa) signal was markedly increased in Prtn3–/–Ela2–/–cells compared with WT and Ela2–/– neutrophils. Intact PGRN was present in the supernatant (SN) of IC-activated Prtn3–/–Ela2–/–neutrophils only, not of WT or Ela2–/– cells. (D and E) Exogenous administration of 100 nM PGRN significantly reduced ROS production of neutrophils activated by ICs (D), but not when activated by PMA (E). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate.

Antiinflammatory PGRN is degraded by PR3 and NE during IC-mediated neutrophil activation.

PGRN inhibits neutrophil activation by ICs in vitro.

Both PR3 and NE process PGRN in vitro.

Figure 5

PR3 and NE are major PGRN processing enzymes of neutrophils.

PGRN inhibits IC-mediated inflammation in vivo.

Figure 6

PGRN is a potent inhibitor of IC-stimulated inflammation in vivo.

PR3 and NE cleave PGRN during inflammation in vivo.

Finally, we aimed to demonstrate defective PGRN degradation in Prtn3–/–Ela2–/– mice during neutrophilic inflammation in vivo. For practical reasons, we harvested infiltrated neutrophils from the inflamed peritoneum 4 h after casein injection and subjected the lysates of these cells to anti-PGRN Western blot. Intact, inhibitory PGRN was detected in Prtn3–/–Ela2–/– neutrophils, but not in WT cells (Figure ​(Figure6D).6D). These data prove that neutrophilic inflammation is accompanied by proteolytic removal of antiinflammatory PGRN and that the process of PGRN degradation is essentially impaired in vivo in the absence of PR3 and NE.


Chronic inflammatory and autoimmune diseases are often perpetuated by continuous neutrophil infiltration and activation. According to the current view, the role of NSPs in these diseases is mainly associated with proteolytic tissue degradation after their release from activated or dying neutrophils. However, recent observations suggest that NSPs such as CG may contribute to noninfectious diseases in a more complex manner, namely as specific regulators of inflammation (18). Here, we demonstrate that PR3 and NE cooperatively fulfilled an important proinflammatory role during neutrophilic inflammation. PR3 and NE directly enhanced neutrophil activation by degrading oxidative burst–suppressing PGRN. These findings support the use of specific serine protease inhibitors as antiinflammatory agents.

Much attention has been paid to the degradation of extracellular matrix components by NSPs. We therefore expected that ablation of both PR3 and NE would cause impaired neutrophil extravasation and interstitial migration. Surprisingly, we found that the proteases were principally dispensable for these processes:Prtn3–/–Ela2–/– neutrophils migrated normally through a dense, 3-dimensional collagen matrix in vitro and demonstrated regular extravasation in vivo when phorbol esters were applied (Figure ​(Figure2).2). This finding is in agreement with recent reports that neutrophils preferentially and readily cross the EBM through regions of low matrix density in the absence of NE (28).

Conversely, we observed that PR3 and NE were required for the inflammatory response to locally formed ICs (Figure ​(Figure3).3). Even isolated Prtn3–/–Ela2–/– neutrophils were challenged in performing oxidative burst after IC stimulation in vitro (Figure ​(Figure4A),4A), showing that the proteases directly enhanced the activation of neutrophils also in the absence of extracellular matrix. However, when receptor-mediated signal transduction was bypassed by means of PMA, neutrophils from Prtn3–/–Ela2–/– mice performed normal oxidative burst (Figure ​(Figure4B),4B), indicating that the function of the phagocyte oxidase (phox) complex was not altered in the absence of PR3 and NE. These findings substantiate what we believe to be a novel paradigm: that all 3 serine proteases of azurophilic granules (CG, PR3, and NE), after their release in response to IC encounter, potentiate a positive autocrine feedback on neutrophil activation.

In contrast to CG, the highly related proteases PR3 and NE cooperate in the effacement of antiinflammatory PGRN, leading to enhanced neutrophil activation. Previous studies already demonstrated that PGRN is a potent inhibitor of the adhesion-dependent oxidative burst of neutrophils in vitro, which can be degraded by NE (23). Here, we showed that PR3 and NE play an equally important role in the regulation of PGRN function. Ela2–/– neutrophils were sufficiently able to degrade PGRN. Only in the absence of both PR3 and NE was PGRN degradation substantially impaired, resulting in the accumulation of antiinflammatory PGRN during neutrophil activation in vitro (Figure ​(Figure4C)4C) and neutrophilic inflammation in vivo (Figure ​(Figure6D).6D). Moreover, we provided in vivo evidence for the crucial role of PGRN as an inflammation-suppressing mediator, because administration of recombinant PGRN potently inhibited the neutrophil influx to sites of IC formation (Figure ​(Figure6,6, A–C). Hence, the cooperative degradation of PGRN by PR3 and NE is a decisive step for the establishment of neutrophilic inflammation.

The molecular mechanism of PGRN function is not yet completely understood, but it seems to interfere with integrin (CD11b/CD18) outside-in signaling by blocking the function of pyk2 and thus dampens adhesion-related oxidative burst even when added after the initial lag phase of oxidase activation (23). PGRN is produced by neutrophils and stored in highly mobile secretory granules (29). It was recently shown that PGRN can bind to heparan-sulfated proteoglycans (30), which are abundant components of the EBM and various cell surfaces, including those of neutrophils. Also, PR3 and NE are known to interact with heparan sulfates on the outer membrane of neutrophils, where the enzymes appear to be protected against protease inhibitors (121331). These circumstantial observations support the notion that PGRN cleavage by PR3 and NE takes place at the pericellular microenvironment of the neutrophil cell surface.

Impaired outside-in signaling most likely reduced the oxidative burst in Prtn3–/–Ela2–/– neutrophils adhering to ICs. In support of this hypothesis, we excluded an altered response to TNF-α priming (Supplemental Figure 5) as well as reduced adhesion to immobilized ICs and defective endocytosis of serum-opsonized E. coli in Prtn3–/–Ela2–/– neutrophils (Supplemental Figure 6). MPO content and processing was also unchanged in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure1D);1D); hence, the previously discussed inhibitory effect of MPO on phox activity (3233) does not appear to be stronger in neutrophils lacking PR3 and NE. Because there was no difference in the lag phase of the oxidative burst, initial IC-triggered receptor activation was probably not affected by either PRGN or PR3/NE. Our concept is consistent with all these observations and takes into account that PGRN unfolds its suppressing effects in the second phase, when additional membrane receptors, endogenous PGRN, and some PR3/NE from highly mobile intracellular pools are translocated to the cell surface. The decline and cessation of ROS production suggested to us that outside-in signaling was not sustained and that active oxidase complexes were no longer replenished in the absence of PR3 and NE. Our present findings, however, do not allow us to exclude other potential mechanisms, such as accelerated disassembly of the active oxidase complex.



Proposed function of PR3 and NE in IC-mediated inflammation.

TNF-α–primed neutrophils extravasate from blood vessels, translocate PR3/NE to the cellular surface, and discharge PGRN to the pericellular environment (i). During transmigration of interstitial tissues (ii), neutrophil activation is initially suppressed by relatively high pericellular levels of antiinflammatory PGRN (green shading), which is also produced locally by keratinocytes and epithelial cells of the skin. Until IC depots are reached, neutrophil activation is inhibited by PGRN. Surface receptors (e.g., Mac-1) recognize ICs, which results in signal transduction (black dotted arrow) and activation of the phox. The molecular pathway of PGRN-mediated inhibition is not completely understood, but it may interfere with integrin signaling after IC encounter (green dotted line inside the cell). Adherence of neutrophils to ICs (iii) further increases pericellular PR3 and NE activity. PR3 and NE cooperatively degrade PGRN in the early stage of neutrophilic activation to facilitate optimal neutrophil activation (red shading), resulting in sustained integrin signaling (red arrow) and robust production of ROS by the phox system. Subsequently, neutrophils release ROS together with other proinflammatory mediators and chemotactic agents, thereby enhancing the recruitment of further neutrophils and establishing inflammation (iv). In the absence of PR3/NE, the switch from inflammation-suppressing (ii) to inflammation-enhancing (iii) conditions is substantially delayed, resulting in diminished inflammation in response to ICs (iv).


NSPs are strongly implicated as effector molecules in a large number of destructive diseases, such as emphysema or the autoimmune blistering skin disease bullous pemphigoid (143537). Normally, PR3/NE activity is tightly controlled by high plasma levels of α1-antitrypsin. This balance between proteases and protease inhibitors is disrupted in patients with genetic α1-antitrypsin deficiency, which represents a high risk factor for the development of emphysema and certain autoimmune disorders (38). The pathogenic effects of NSPs in these diseases have so far been associated with tissue destruction by the proteases after their release from dying neutrophils. Our findings showed that PR3 and NE were already involved in much earlier events of the inflammatory process, because the enzymes directly regulated cellular activation of infiltrating neutrophils by degrading inflammation-suppressing PGRN. This concept is further supported by previous studies showing increased inflammation in mice lacking serine protease inhibitors such as SERPINB1 or SLPI (3940). Blocking PR3/NE activity using specific inhibitors therefore represents a promising therapeutic strategy to treat chronic, noninfectious inflammation. Serine protease inhibitors as antiinflammatory agents can interfere with the disease process at 2 different stages, because they attenuate both early events of neutrophil activation and proteolytic tissue injury caused by released NSPs.





Editorial: Serine proteases, serpins, and neutropenia

David C. Dale

J Leuko Biol July 2011;  90(1): 3-4   http://dx.doi.org:/10.1189/jlb.1010592

Cyclic neutropenia and severe congenital neutropenia are autosomal-dominant diseases usually attributable to mutations in the gene for neutrophil elastase orELANE. Patients with these diseases are predisposed to recurrent and life-threatening infections [1]. Neutrophil elastase, the product of the ELANE gene, is a serine protease that is synthesized and packaged in the primary granules of neutrophils. These granules are formed at the promyelocytes stage of neutrophil development. Synthesis of mutant neutrophil elastase in promyelocytes triggers the unfolded protein response and a cascade of intracellular events, which culminates in death of neutrophil precursors through apoptosis [2]. This loss of cells causes the marrow abnormality often referred to as “maturation arrest” [34].

Neutrophil elastase is one of the serine proteases normally inhibited by serpinB1. In this issue of JLB, Benarafa and coauthors [5] present their intriguing studies of serpinB1 expression in human myeloid cells and their extensive investigations ofSERPINB1−/− mice. They observed that serpinB1 expression parallels protease expression. The peak of serpinB1 expression occurs in promyelocytes. Benarafa et al. [5] found that SERPINB1−/− mice have a deficiency of postmitotic neutrophils in the bone marrow. This change was accompanied by an increase in the plasma levels of G-CSF. The decreased supply of marrow neutrophils reduced the number of neutrophils that could be mobilized to an inflammatory site. Using colony-forming cell assays, they determined that the early myeloid progenitor pool was intact. Separate assays showed that maturing myeloid cells were being lost through accelerated apoptosis of maturing neutrophils in the marrow. The authors concluded that serpinB1 is required for maintenance of a healthy reserve of marrow neutrophils and a normal acute immune response [5].

This paper provides new and fascinating insights for understanding the mechanism for neutropenia. It also suggests opportunities to investigate potential therapies for patients with neutropenia and prompts several questions. As inhibition of the activity of intracellular serine proteases is the only known function of serpinB1, the findings reported by Benarafa et al. [5] suggest that uninhibited serine proteases perturbed neutrophil production severely. The SERPINB1−/− mice used in their work have accelerated apoptosis of myeloid cells, a finding suggesting that uninhibited serine proteases or mutant neutrophil elastase perturb myelopoiesis by similar mechanisms. It is now important to determine whether the defect in the SERPINB1−/− mice is, indeed, attributable to uninhibited activity of normal neutrophil elastase, other neutrophil proteases, or another mechanism. ″Double-knockout″ studies in mice deficient in neutrophil elastase and serpinB1 might provide an answer.

This report provides evidence regarding the intracellular mechanisms for the apoptosis of myeloid cells and indicates that other studies are ongoing. The key antiapoptotic proteins, Mcl-1, Bcl-XL, and A1/Bfl-I, are apparently not involved. A more precise understanding of the mechanisms of cell death is important for development of targeted therapies for neutropenia. It is also important to discover whether only cells of the neutrophil lineage are involved or whether monocytes are also affected. In cyclic and congenital neutropenia, patients failed to produce neutrophils, but they can produce monocytes; in fact, they overproduce monocytes and have significantly elevated blood monocyte counts. Neutropenia with monocytosis is probably attributable to differences in the expression of ELANE in the two lineages. Benarafa et al. [5] reported that human bone marrow monocytes contain substantially less serpinB1 than marrow neutrophils, suggesting that the expression of serpinB1 and the serine proteases are closely coordinated.

This report shows the importance of the marrow neutrophil reserves in the normal response to infections. Compared with humans, healthy mice are always neutropenic, but they have a bigger marrow neutrophil reserve, and their mature neutrophils in the marrow and blood look like human band neutrophils. These differences are well known, but they are critical for considering the clinical inferences that can be made from this report. For example, although theSERPINB1−/− mice were not neutropenic, human SERPINB1−/− might cause neutropenia because of physiological differences between the species. If some but not all mutations in SERPINB1 cause neutropenia, we might gain a better understanding about how serpinB1 normally inhibits the neutrophil’s serine proteases.

We do not know if some or all of the mutant neutrophil elastases can be inhibited by serpinB1. We do not know whether cyclic or congenital neutropenia are attributable to defects in this interaction. However, we do know that there are chemical inhibitors of neutrophil elastase that can abrogate apoptosis of myeloid cells in a cellular model for congenital neutropenia [6]. It would be interesting to see if these chemical inhibitors can replace the natural inhibitor and normalize neutrophil production in the SERPINB1−/− mice. This would provide evidence to support use of chemical protease inhibitors as a treatment for cyclic and congenital neutropenia.

Concerns with the use of G-CSF for the treatment of cyclic and congenital neutropenia are how and why some of these patients are at risk of developing leukemia. Are the SERPINB1−/− mice with a hyperproliferative marrow and high G-CSF levels also at risk of developing myeloid leukemia?

This is a very provocative paper, and much will be learned from further studies of the SERPINB1−/− mice.


SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Mathias Baumann1,2, Christine T. N. Pham3, and Charaf Benarafa1

Blood May 9, 2013; 121(19)   http://www.bloodjournal.org/content/121/19/3900

Key Points

  • Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G.
  • Granule permeabilization in neutrophils leads to cathepsin G–mediated death upstream and independent of apoptotic caspases.


Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1−/−) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia ofsB1−/− mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1−/− mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1−/− PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent L-leucyl-L-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.


Polymorphonuclear neutrophil (PMN) granulocytes are essential components of the innate immune response to infection. PMNs are relatively short-lived leukocytes that originate from hematopoietic stem cells in the bone marrow (BM) in a process called granulopoiesis. Granulopoiesis proceeds through a proliferative phase followed by a maturation phase. After maturation, the BM retains a large reserve of mature PMNs, which includes over 90% of the mature PMNs in the body while only a small proportion (1%-5%) is in the blood.1,2 Even in noninflammatory conditions, granulopoiesis is remarkable as >1011 PMNs are produced daily in an adult human, only to be disposed of, largely unused, a few hours later.3 There is evidence that the majority of PMNs produced never reach circulation and die within the BM.4 Congenital or acquired forms of neutropenia are associated with the highest risks of bacterial and fungal infection,5 indicating a strong evolutionary pressure to maintain granulopoiesis at high levels and sustain a large mobilizable pool of PMNs in the BM.

In steady state, PMNs die by apoptosis, a form of programmed cell death that allows for the safe disposal of aging PMNs and their potentially toxic cargo. Like in other cells, caspases participate in the initiation, amplification, and execution steps of apoptosis in PMNs.6,7 Interestingly, noncaspase cysteine proteases calpain and cathepsin D were reported to induce PMN apoptosis through activation of caspases.811 In addition, PMNs carry a unique set of serine proteases (neutrophil serine proteases [NSPs]) including elastase (NE), cathepsin G (CG), and proteinase-3 (PR3) stored active in primary granules. There is strong evidence for a role of NSPs in killing pathogens and inducing tissue injury when released extracellularly.1214 In contrast, the function of NSPs in PMN homeostasis and cell death remains elusive. In particular, no defects in granulopoiesis or PMN homeostasis have been reported in mice deficient in cathepsin G (CG−/−),15 neutrophil elastase (NE−/−),16,17 or dipeptidylpeptidase I (DPPI−/−), which lack active NSPs.18 We have recently shown that mice lacking the serine protease inhibitor serpinB1 (sB1−/−) have reduced PMN survival in the lungs following Pseudomonas infection and that these mice have a profound reduction in mature PMN numbers in the BM.19,20SerpinB1, also known as monocyte NE inhibitor, is expressed at high levels in the cytoplasm of PMNs and is one of the most potent inhibitors of NE, CG, and PR3.21,22 In this study, we tested the hypothesis that serpinB1 promotes PMN survival by inhibiting 1 or several NSPs, and we discovered a novel regulatory pathway in PMN homeostasis in vivo.



Figure 1

Defective PMN reserve in BM chimera depends on serpinB1 deficiency in the hematopoietic compartment. Flow cytometry analysis of major BM leukocyte subsets of lethally irradiated mice was performed 8 to 10 weeks after BM transfer. (A) Irradiated WT (CD45.1) mice were transferred with WT (●) or sB1−/− (○) BM cells. (B) Irradiated WT (●) andsB1−/− (○) mice both CD45.2 were transferred with WT (CD45.1) BM cells. Each circle represents leukocyte numbers for 1 mouse and horizontal line indicates the median. Median subsets numbers were compared by the Mann-Whitney test (*P < .05; ***P < .001).

CG regulates neutrophil numbers in the BM

Because serpinB1 is an efficient inhibitor of NE, CG, and PR3, we then examined PMN numbers in mice deficient in 1 or several NSPs in combination with serpinB1 deletion. As expected, sB1−/− mice had significantly reduced numbers and percentage of mature PMNs in the BM compared with WT and heterozygous sB1+/− mice. In addition, PMN numbers were normal in mice deficient in either DPPI, NE, or CG (Figure 2A). DPPI is not inhibited by serpinB1 but is required for the activation of all NSPs, and no NSP activity is detectable in DPPI−/− mice.18,23 PMN counts in DPPI−/−.sB1−/− BM were significantly higher than in sB1−/− BM, suggesting that 1 or several NSPs contribute to the PMN survival defect. To examine the role of NSPs in this process, we crossed several NSP-deficient strains with sB1−/− mice. We found that NE.CG.sB1−/− mice had normal PMN numbers indicating that these NSPs play a key role in the defective phenotype of sB1−/− PMNs (Figure 2A). Furthermore, CG.sB1−/− mice showed normal PMN numbers whereasNE.sB1−/− mice retained the BM neutropenia phenotype indicating that CG, but not NE, plays a significant role in the death of sB1−/− PMNs (Figure 2A). In addition, the double-deficient NE.sB1−/− mice had significantly lower BM myelocyte numbers than sB1−/− mice while the myelocyte numbers in singly deficient NE−/− and sB1−/− BM were normal (Figure 2B). These results suggest that NE may promote myeloid cell proliferation, an activity that is revealed only when serpinB1 is absent. This complex interaction between sB1 and NE requires further investigation. On the other hand, B-cell and monocyte numbers and relative percentage in the BM were largely similar in all genotypes (supplemental Figure 2). Total numbers of blood leukocytes, erythrocytes, and platelets were normal in mice deficient in NSPs and/or serpinB1 (supplemental Figure 3). PMN numbers in blood were normal insB1−/− mice in steady state and combined deficiency of NSPs did not significantly alter these numbers (Figure 2C). Taken together, our results indicate that serpinB1 likely sustains the survival of postmitotic PMNs through its interaction with CG.

Figure 2

PMN and myelocyte numbers in BM and blood of mice deficient in NSPs and serpinB1.


CG-mediated PMN death proceeds independent of caspase activity

Figure 4

sB1−/− PMN death mediated by CG does not require caspase activity


Granule membrane permeabilization induces CG-mediated death in PMNs

To test whether granule disruption contributes to the serpinB1-regulated CG-dependent cell death, BM cells were treated with the lysosomotropic agent LLME. LLME accumulates in lysosomes where the acyl transferase activity of DPPI generates hydrophobic (Leu-Leu)n-OMe polymers that induce lysosomal membrane permeabilization (LMP) and cytotoxicity in granule-bearing cells such as cytotoxic T lymphocytes, NK cells, and myeloid cells.29,30

Figure 5

LMP induces CG-mediated death in PMNs


G-CSF therapy increases sB1−/− PMN numbers via enhanced granulopoiesis

G-CSF therapy is an effective long-term treatment in many cases of severe congenital neutropenia and it is also used to prevent chemotherapy-induced febrile neutropenia by enhancing PMN production. In addition, G-CSF delays neutrophil apoptosis by differentially regulating proapoptotic and antiapoptotic factors.10 To test whether G-CSF could rescue sB1−/− PMN survival defect, WT and sB1−/− mice were treated with therapeutic doses of G-CSF or saline for 5 days and BM and blood PMNs were analyzed 24 hours after the last injection. Total counts of myelocytes and PMNs were significantly increased in the BM of treated mice compared with their respective untreated genotype controls (Figure 6A-B). The increase in myelocyte numbers was identical in G-CSF–treated WT and sB1−/− mice, indicating that G-CSF–induced granulopoiesis proceeds normally in sB1−/−myeloid progenitors (Figure 6B).

Figure 6

In vivo G-CSF therapy increases PMN numbers in BM of sB1−/− mice.


SerpinB1 is a member of the clade B serpins, a subfamily composed of leaderless proteins with nucleocytoplasmic localization. Clade B serpins are often expressed in cells that also carry target proteases, which led to the hypothesis that intracellular serpins protect against misdirected granule proteases and/or protect bystander cells from released proteases.31 We previously reported that deficiency in serpinB1 is associated with reduced PMN survival in the BM and at inflammatory sites.19,20 The evidence presented here demonstrates that the cytoprotective function of serpinB1 in PMNs is based on the inhibition of granule protease CG. Deficiency in CG was sufficient to rescue the defect of sB1−/− mice as illustrated by normal PMN counts in the BM of double knockout CG.sB1−/− mice. We also showed that the protease-serpin interaction occurred within PMNs. Indeed, WT PMNs had a greater survival over sB1−/− PMNs in mixed BM chimera, whereas the survival of CG.sB1−/− PMNs was similar to WT PMNs after BM transfer. SerpinB1 is an ancestral clade B serpin with a conserved specificity determining reactive center loop in all vertebrates.32 Furthermore, human and mouse serpinB1 have the same specificity for chymotrypsin-like and elastase-like serine proteases.21,22 Likewise, human and mouse CG have identical substrate specificities and the phenotype of CG−/− murine PMN can be rescued by human CG.33 Therefore, it is highly likely that the antagonistic functions of CG and serpinB1 in cellular homeostasis observed in mice can be extended to other species.

Extracellular CG was previously reported to promote detachment-induced apoptosis (anoikis) in human and mouse cardiomyocytes.34 This activity is mediated through the shedding and transactivation of epidermal growth factor receptor and downregulation of focal adhesion signaling.35,36 In our study, exogenous human CG also induced PMN death in vitro but these effects were not enhanced in sB1−/− PMNs and the neutropenia associated with serpinB1 deficiency was principally cell intrinsic. How intracellular CG induces PMN death remains to be fully investigated. However, our studies provide some indications on the potential pathways. Like other NSPs, the expression of CG is transcriptionally restricted to the promyelocyte stage during PMN development and NSPs are then stored in active form in primary azurophil granules.37 Because serpinB1 is equally efficient at inhibiting NE, CG, and PR3, it was surprising that deletion of CG alone was sufficient to achieve a complete reversal of the PMN survival defect in CG.sB1−/− mice. A possible explanation would be that CG gains access to targets more readily than other granule proteases. There is evidence that binding to serglycin proteoglycans differs between NE and CG resulting in altered sorting of NE but not CG into granules of serglycin-deficient PMNs.38 Different interactions with granule matrix may thus contribute to differential release of CG from the granules compared with other NSPs. However, because sB1−/− PMNs have similar levels of CG and NE as WT PMNs20 and because LLME-induced granule permeabilization likely releases all granule contents equally, we favor an alternative interpretation where CG specifically targets essential cellular components that are not cleaved by the other serpinB1-inhibitable granule proteases. Upon granule permeabilization, we found that CG can induce cell death upstream of caspases as well as independent of caspases. CG was previously shown to activate caspase-7 in vitro and it functions at neutral pH, which is consistent with a physiological role in the nucleocytoplasmic environment.39 Cell death induced by lysosomal/granule membrane permeabilization has previously been linked to cysteine cathepsins in other cell types. However, these proteases appear to depend on caspase activation to trigger apoptosis and they function poorly at neutral pH, questioning their potential role as regulators of cell death.40 In contrast, CG-mediated cell death is not completely blocked by caspase inhibition, which is a property reminiscent of granzymes in cytotoxic T cells.41 In fact, CG is phylogenetically most closely related to serine proteases granzyme B and H.42 Granzymes have numerous nuclear, mitochondrial, and cytoplasmic target proteins leading to cell death41 and we anticipate that this may also be the case for CG.


G-CSF therapy is successfully used to treat most congenital and acquired neutropenia through increased granulopoiesis, mobilization from the BM, and increased survival of PMNs. Prosurvival effects of G-CSF include the upregulation of antiapoptotic Bcl-2 family members, which act upstream of the mitochondria and the activation of effector caspases. In sB1−/− mice, G-CSF levels in serum are fourfold higher than in WT mice in steady state and this is accompanied by an upregulation of the antiapoptotic Bcl-2 family member Mcl-1 in sB1−/− PMNs.19 Here, G-CSF therapy significantly increased granulopoiesis in both WT and sB1−/− mice. However, the PMN numbers in treated sB1−/− BM and blood were significantly lower than those of treated WT mice, indicating only a partial rescue of the survival defect. This is consistent with our findings that CG-mediated death can proceed independent of caspases and can thus bypass antiapoptotic effects mediated by G-CSF.

CG has largely been studied in association with antimicrobial and inflammatory functions due to its presence in PMNs.1214,49 In this context, we have previously shown that serpinB1 contributes to prevent increased mortality and morbidity associated with production of inflammatory cytokines upon infection with Pseudomonas aeruginosa and influenza A virus.20,50 In this study, we demonstrate that serpinB1 inhibition of the primary granule protease CG in PMNs is essential for PMN survival and this ultimately regulates PMN numbers in vivo. Our findings also extend the roles of CG from antimicrobial and immunoregulatory functions to a novel role in inducing cell death.


Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

Brice KorkmazMarshall S. HorwitzDieter E. Jenne and Francis Gauthier
Pharma Rev Dec 2010; 62(4):726-759  http://dx.doi.org:/10.1124/pr.110.002733

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.


Human polymorphonuclear neutrophils represent 35 to 75% of the population of circulating leukocytes and are the most abundant type of white blood cell in mammals (Borregaard et al., 2005). They are classified as granulocytes because of their intracytoplasmic granule content and are characterized by a multilobular nucleus. Neutrophils develop from pluripotent stem cells in the bone marrow and are released into the bloodstream where they reach a concentration of 1.5 to 5 × 109 cells/liter. Their half-life in the circulation is only on the order of a few hours. They play an essential role in innate immune defense against invading pathogens and are among the primary mediators of inflammatory response. During the acute phase of inflammation, neutrophils are the first inflammatory cells to leave the vasculature, where they migrate toward sites of inflammation, following a gradient of inflammatory stimuli. They are responsible for short-term phagocytosis during the initial stages of infection (Borregaard and Cowland, 1997Hampton et al., 1998Segal, 2005). Neutrophils use complementary oxidative and nonoxidative pathways to defend the host against invading pathogens (Kobayashi et al., 2005).

The three serine proteases neutrophil elastase (NE1), proteinase 3 (PR3), and cathepsin G (CG) are major components of neutrophil azurophilic granules and participate in the nonoxidative pathway of intracellular and extracellular pathogen destruction. These neutrophil serine proteases (NSPs) act intracellularly within phagolysosomes to digest phagocytized microorganisms in combination with microbicidal peptides and the membrane-associated NADPH oxidase system, which produces reactive oxygen metabolites (Segal, 2005). An additional extracellular antimicrobial mechanism, neutrophil extracellular traps (NET), has been described that is made of a web-like structure of DNA secreted by activated neutrophils (Papayannopoulos and Zychlinsky, 2009) (Fig. 1). NETs are composed of chromatin bound to positively charged molecules, such as histones and NSPs, and serve as physical barriers that kill pathogens extracellularly, thus preventing further spreading. NET-associated NSPs participate in pathogen killing by degrading bacterial virulence factors extracellularly (Brinkmann et al., 2004;Papayannopoulos and Zychlinsky, 2009).


Fig. 1.

Polymorphonuclear neutrophil. Quiescent (A) and chemically activated (B) neutrophils purified from peripheral blood. C, PMA-activated neutrophils embedded within NET and neutrophil spreading on insoluble elastin.

In addition to their involvement in pathogen destruction and the regulation of proinflammatory processes, NSPs are also involved in a variety of inflammatory human conditions, including chronic lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, acute lung injury, and acute respiratory distress syndrome) (Lee and Downey, 2001Shapiro, 2002Moraes et al., 2003Owen, 2008b). In these disorders, accumulation and activation of neutrophils in the airways result in excessive secretion of active NSPs, thus causing lung matrix destruction and inflammation. NSPs are also involved in other human disorders as a consequence of gene mutations, altered cellular trafficking, or, for PR3, autoimmune disease. Mutations in the ELA2/ELANE gene encoding HNE are the cause of human cyclic neutropenia and severe congenital neutropenia (Horwitz et al., 19992007). Neutrophil membrane-bound proteinase 3 (mPR3) is the major target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA), which are associated with Wegener granulomatosis (Jenne et al., 1990). All three proteases are affected by mutation of the gene (CTSC) encoding dipeptidyl peptidase I (DPPI), which activates several granular hematopoietic serine proteases (Pham and Ley, 1999Adkison et al., 2002). Mutations of CTSC cause Papillon-Lefèvre syndrome and palmoplantar keratosis (Hart et al., 1999Toomes et al., 1999).


Fully processed mature HNE, PR3, and CG isolated from azurophilic granules contain, respectively, 218 (Bode et al., 1986Sinha et al., 1987), 222 (Campanelli et al., 1990b), and 235 (Salvesen et al., 1987Hof et al., 1996) residues. They are present in several isoforms depending on their carbohydrate content, with apparent mass of 29 to 33 kDa upon SDS-polyacrylamide gel electrophoresis (Twumasi and Liener, 1977Watorek et al., 1993). HNE and PR3 display two sites of N-glycosylation, whereas CG possesses only one. NSPs are stored mainly in neutrophil azurophilic granules, but HNE is also localized in the nuclear envelope, as revealed by immunostaining and electron microscopy (Clark et al., 1980;Benson et al., 2003), whereas PR3 is also found in secretory vesicles (Witko-Sarsat et al., 1999a). Upon neutrophil activation, granular HNE, PR3, and CG are secreted extracellularly, although some molecules nevertheless remain at the cell surface (Owen and Campbell, 1999Owen, 2008a). The mechanism through which NSPs are sorted from the trans-Golgi network to the granules has not been completely defined, even though an intracellular proteoglycan, serglycin, has been identified as playing a role in elastase sorting and packaging into azurophilic granules (Niemann et al., 2007). Unlike HNE and CG, PR3 is constitutively expressed on the membranes of freshly isolated neutrophils (Csernok et al., 1990Halbwachs-Mecarelli et al., 1995). Stimulation of neutrophils at inflammatory sites triggers intracytoplasmic granules to translocate to the phagosomes and plasma membrane, thereby liberating their contents. The first step of the translocation to the target membrane depends on cytoskeleton remodeling and microtubule assembly (Burgoyne and Morgan, 2003). This is followed by a second step of granule tethering and docking, which are dependent on the sequential intervention of SNARE proteins (Jog et al., 2007).


Exposure of neutrophils to cytokines (TNF-α), chemoattractants (platelet-activating factor, formyl-Met-Leu-Phe, or IL-8), or bacterial lipopolysaccharide leads to rapid granule translocation to the cell surface with secretion of HNE, PR3, and CG into the extracellular medium (Owen and Campbell, 1999). A fraction of secreted HNE, PR3, and CG is detected at the surface of activated neutrophils (Owen et al., 1995a1997Campbell et al., 2000). Resting purified neutrophils from peripheral blood express variable amounts of PR3 on their surface. A bimodal, apparently genetically determined, distribution has been observed with two populations of quiescent neutrophils that express or do not express the protease at their surface (Halbwachs-Mecarelli et al., 1995Schreiber et al., 2003). The percentage of mPR3-positive neutrophils ranges from 0 to 100% of the total neutrophil population within individuals. Furthermore, the percentage of mPR3-positive neutrophils remains stable over time and is not affected by neutrophil activation (Halbwachs-Mecarelli et al., 1995).

The mechanism through which HNE and CG are associated with the outer surface of the plasma membrane of neutrophils mainly involves electrostatic interactions with the sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans (Campbell and Owen, 2007). These two proteases are released from neutrophil cell surfaces by high concentrations of salt (Owen et al., 1995b1997;Korkmaz et al., 2005a) and after treatment with chondroitinase ABC and heparinase (Campbell and Owen, 2007). Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al., 1999aKorkmaz et al., 2009). Unlike HNE and CG, PR3 bears at its surface a hydrophobic patch formed by residues Phe166, Ile217, Trp218, Leu223, and Phe224 that is involved in membrane binding (Goldmann et al., 1999Hajjar et al., 2008) (Fig. 3B). Several membrane partners of PR3 have been identified, including CD16/FcγRIIIb (David et al., 2005Fridlich et al., 2006), phospholipid scramblase-1, a myristoylated membrane protein with translocase activity present in lipid rafts (Kantari et al., 2007), CD11b/CD18 (David et al., 2003), and human neutrophil antigen NB1/CD177 (von Vietinghoff et al., 2007Hu et al., 2009), a 58- to 64-kDa glycosyl-phosphatidylinositol anchored surface receptor belonging to the urokinase plasminogen activator receptor superfamily (Stroncek, 2007). NB1 shows a bimodal distribution that superimposes with that of PR3 on purified blood neutrophils (Bauer et al., 2007). Active, mature forms of PR3 but not pro-PR3 can bind to the surface of NB1-transfected human embryonic kidney 293 cells (von Vietinghoff et al., 2008) and Chinese hamster ovary cells (Korkmaz et al., 2008b). Interaction involves the hydrophobic patch of PR3 because specific amino acid substitutions disrupting this patch in the closely related gibbon PR3 prevent binding to NB1-transfected cells (Korkmaz et al., 2008b). Decreased interaction of pro-PR3 with NB1-transfected cells is explained by the topological changes affecting the activation domain containing the hydrophobic patch residues. Together, these results support the hydrophobic nature of PR3-membrane interaction.


Roles in Inflammatory Process Regulation

NSPs are abundantly secreted into the extracellular environment upon neutrophil activation at inflammatory sites. A fraction of the released proteases remain bound in an active form on the external surface of the plasma membrane so that both soluble and membrane-bound NSPs are able to proteolytically regulate the activities of a variety of chemokines, cytokines, growth factors, and cell surface receptors. Secreted proteases also activate lymphocytes and cleave apoptotic and adhesion molecules (Bank and Ansorge, 2001Pham, 2006Meyer-Hoffert, 2009). Thus, they retain pro- and anti-inflammatory activities, resulting in a modulation of the immune response at sites of inflammation.


Processing of Cytokines, Chemokines, and Growth Factors.

Processing and Activation of Cellular Receptors.

Induction of Apoptosis by Proteinase 3.

Physiological Inhibitors of Elastase, Proteinase 3, and Cathepsin G

During phagocytosis and neutrophil turnover, HNE, PR3, and CG are released into the extracellular space as active proteases. The proteolytic activity of HNE, PR3, and CG seems to be tightly regulated in the extracellular and pericellular space to avoid degradation of connective tissue proteins including elastin, collagen, and proteoglycans (Janoff, 1985). Protein inhibitors that belong to three main families, the serpins, the chelonianins, and the macroglobulins, ultimately control proteolytic activity of HNE, PR3, and CG activities. The individual contributions of these families depend on their tissue localization and that of their target proteases. The main characteristics of HNE, PR3, and CG physiological inhibitors are presented in Table 2.


Serine Protease Inhibitors

Serpins are the largest and most diverse family of protease inhibitors; more than 1000 members have been identified in human, plant, fungi, bacteria, archaea, and certain viruses (Silverman et al., 2001Mangan et al., 2008). They share a similar highly conserved tertiary structure and similar molecular weight of approximately 50 kDa. Human serpins belong to the first nine clades (A–I) of the 16 that have been described based on phylogenic relationships (Irving et al., 2000Silverman et al., 2001Mangan et al., 2008). For historical reasons, α1-protease inhibitor (α1-PI) was assigned to the first clade. Clade B, also known as the ov-serpin clan because of the similarity of its members to ovalbumin (a protein that belongs to the serpin family but lacks inhibitory activity), is the second largest clan in humans, with 15 members identified so far. Ov-serpin clan members are generally located in the cytoplasm and, to a lesser extent, on the cell surface and nucleus (Remold-O’Donnell, 1993).

Serpins play important regulatory functions in intracellular and extracellular proteolytic events, including blood coagulation, complement activation, fibrinolysis, cell migration, angiogenesis, and apoptosis (Potempa et al., 1994). Serpin dysfunction is known to contribute to diseases such as emphysema, thrombosis, angioedema, and cancer (Carrell and Lomas, 1997Lomas and Carrell, 2002). Most inhibitory serpins target trypsin-/chymotrypsin-like serine proteases, but some, termed “cross-class inhibitors,” have been shown to target cysteine proteases (Annand et al., 1999). The crystal structure of the prototype plasma inhibitor α1-PI revealed the archetype native serpin fold (Loebermann et al., 1984). All serpins typically have three β-sheets (termed A, B, and C) and eight or nine α-helices (hA–hI) arranged in a stressed configuration. The so-called reactive center loop (RCL) of inhibitory molecules determines specificity and forms the initial encounter complex with the target protease (Potempa et al., 1994Silverman et al., 2001). Serpins inhibit proteases by a suicide substrate inhibition mechanism. The protease initially recognizes the serpin as a potential substrate using residues of the reactive center loop and cleaves it between P1 and P1′ This cleavage allows insertion of the cleaved RCL into the β-sheet A of the serpin, dragging the protease with it and moving it over 71 Å to the distal end of the serpin to form a 1:1 stoichiometric covalent inhibitory complex (Huntington et al., 2000). Such cleavage generates a ∼4-kDa C-terminal fragment that remains noncovalently bound to the cleaved serpin. Displacement of the covalently attached active site serine residue from its catalytic partner histidine explains the loss of catalytic function in the covalent complex. The distortion of the catalytic site structure prevents the release of the protease from the complex, and the structural disorder induces its proteolytic inactivation (Huntington et al., 2000). Covalent complex formation between serpin and serine proteases triggers a number of conformational changes, particularly in the activation domain loops of the bound protease (Dementiev et al., 2006).


Pathophysiology of Elastase, Proteinase 3 and Cathepsin G in Human Diseases

In many instances, the initiation and propagation of lung damage is a consequence of an exaggerated inappropriate inflammatory response, which includes the release of proteases and leukocyte-derived cytotoxic products (Owen, 2008b;Roghanian and Sallenave, 2008). Inflammation is a physiological protective response to injury or infection consisting of endothelial activation, leukocyte recruitment and activation, vasodilation, and increased vascular permeability. Although designed to curtail tissue injury and facilitate repair, the inflammatory response sometimes results in further injury and organ dysfunction. Inflammatory chronic lung diseases, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and cystic fibrosis are syndromes of severe pulmonary dysfunction resulting from a massive inflammatory response and affecting millions of people worldwide. The histological hallmark of these chronic inflammatory lung diseases is the accumulation of neutrophils in the microvasculature of the lung. Neutrophils are crucial to the innate immune response, and their activation leads to the release of multiple cytotoxic products, including reactive oxygen species and proteases (serine, cysteine, and metalloproteases). The physiological balance between proteases and antiproteases is required for the maintenance of the lung’s connective tissue, and an imbalance in favor of proteases results in lung injury (Umeki et al., 1988Tetley, 1993). A number of studies in animal and cell culture models have demonstrated a contribution of HNE and related NSPs to the development of chronic inflammatory lung diseases. Available preclinical and clinical data suggest that inhibition of NSP in lung diseases suppresses or attenuates the contribution of NSP to pathogenesis (Chughtai and O’Riordan, 2004Voynow et al., 2008Quinn et al., 2010). HNE could also participate in fibrotic lung remodeling by playing a focused role in the conversion of latent transforming growth factor-β into its biologically active form (Chua and Laurent, 2006Lungarella et al., 2008).

Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides

ANCA-associated vasculitides encompasses a variety of diseases characterized by inflammation of blood vessels and by the presence of autoantibodies directed against neutrophil constituents. These autoantibodies are known as ANCAs (Kallenberg et al., 2006). In Wegener granulomatosis (WG), antibodies are mostly directed against PR3. WG is a relatively uncommon chronic inflammatory disorder first described in 1931 by Heinz Karl Ernst Klinger as a variant of polyarteritis nodosa (Klinger, 1931). In 1936, the German pathologist Friedrich Wegener described the disease as a distinct pathological entity (Wegener, 19361939). WG is characterized by necrotizing granulomatous inflammation and vasculitis of small vessels and can affect any organ (Fauci and Wolff, 1973Sarraf and Sneller, 2005). The most common sites of involvement are the upper and lower respiratory tract and the kidneys. WG affects approximately 1 in 20,000 people; it can occur in persons of any age but most often affects those aged 40 to 60 years (Walton, 1958Cotch et al., 1996). Approximately 90% of patients have cold or sinusitis symptoms that fail to respond to the usual therapeutic measures and that last considerably longer than the usual upper respiratory tract infection. Lung involvement occurs in approximately 85% of the patients. Other symptoms include nasal membrane ulcerations and crusting, saddle-nose deformity, inflammation of the ear with hearing problems, inflammation of the eye with sight problems, and cough (with or without hemoptysis).

Hereditary Neutropenias

Neutropenia is a hematological disorder characterized by an abnormally low number of neutrophils (Horwitz et al., 2007). The normal neutrophil count fluctuates across human populations and within individual patients in response to infection but typically lies in the range of 1.5 to 5 × 109 cells/liter. Neutropenia is categorized as severe when the cell count falls below 0.5 × 109 cells/liter. Hence, patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening. Common causes of neutropenia include cancer chemotherapy, drug reactions, autoimmune diseases, and hereditary disorders (Berliner et al., 2004Schwartzberg, 2006).

Papillon-Lefèvre Syndrome


New Strategies for Fighting Neutrophil Serine Protease-Related Human Diseases

Administration of therapeutic inhibitors to control unwanted proteolysis at inflammation sites has been tested as a therapy for a variety of inflammatory and infectious lung diseases (Chughtai and O’Riordan, 2004). Depending on the size and chemical nature of the inhibitors, they may be administered orally, intravenously, or by an aerosol route. Whatever the mode of administration, the access of therapeutic inhibitors to active proteases is often hampered by physicochemical constraints in the extravascular space and/or by the partitioning of proteases between soluble and solid phases.


Concluding Remarks

NSPs were first recognized as protein-degrading enzymes but have now proven to be multifunctional components participating in a variety of pathophysiological processes. Thus, they appear as potential therapeutic targets for drugs that inhibit their active site or impair activation from their precursor. Overall, the available preclinical and clinical data suggest that inhibition of NSPs using therapeutic inhibitors would suppress or attenuate deleterious effects of inflammatory diseases, including lung diseases. Depending on the size and chemical nature of inhibitors, those may be administered orally, intravenously, or by aerosolization. But the results obtained until now have not been fully convincing because of the poor knowledge of the biological function of each protease, their spatiotemporal regulation during the course of the disease, the physicochemical constraints associated with inhibitor administration, or the use of animal models in which NSP regulation and specificity differ from those in human. Two different and complementary approaches may help bypass these putative problems. One is to target active proteases by inhibitors at the inflammatory site in animal models in which lung anatomy and physiology are close to those in human to allow in vitro and in vivo assays of human-directed drugs/inhibitors. The other is to prevent neutrophil accumulation at inflammatory sites by impairing production of proteolytically active NSPs using an inhibitor of their maturation protease, DPPI. Preventing neutrophil accumulation at the inflammatory sites by therapeutic inhibition of DPPI represents an original and novel approach, the exploration of which has just started (Méthot et al., 2008). Thus pharmacological inactivation of DPPI in human neutrophils could well reduce membrane binding of PR3 and, as a consequence, neutrophil priming by pathogenic auto-antibodies in WG. In addition, it has been recognized that the intracellular level of NSPs depends on their correct intracellular trafficking. In the future, pharmacological targeting of molecules specifically involved in the correct intracellular trafficking of each NSP could possibly regulate their production and activity, a feature that could be exploited as a therapeutic strategy for inflammatory diseases.










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Understanding the Stem Cell Niche: A Webinar by The Scientist

Reporter: Stephen J. Williams, Ph.D.


The Scientist

nature stem cell

Schematic diagram showing some of the factors implicated in each process. Haematopoietic stem cells (HSCs) bound to the bone-marrow niche are mobilized in response to granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide, or after peripheral myeloablation following treatment with 5-fluorouracil (5-FU). After extravasation from the bone-marrow cords into the microvasculature, HSCs enter the circulation and are distributed to peripheral tissues such as the spleen or liver. HSCs locate close to endothelial cells in the splenic red pulp. They home to the bone-marrow cords through the circulation, a process that is controlled by a number of adhesion molecules such as very late antigen 4 (VLA4), VLA5, lymphocyte function-associated antigen 1 (LFA1) or selectins. After entering the bone marrow, HSCs specifically lodge in the niche, a process requiring membrane-bound stem-cell factor (SCF), CXC-chemokine ligand 12 (CXCL12), osteopontin (OPN), hyaluronic acid, and their corresponding receptors. CXCR4, CXC-chemokine receptor 4; E-selectin, endothelial-cell selectin; P-selectin, platelet selectin; PSGL1, P-selectin glycoprotein ligand 1.


Understanding the Stem Cell Niche

  This presentation will begin on Tuesday, December 01, 2015 at 02:30 PM Eastern Standard Time.

Free Webinar
Tuesday December 1, 2015
2:30 – 4:00 PM EST

Stem cells provide an attractive model to study human physiology and disease. However, technical challenges persist in the biological characterization and manipulation of stem cells in their native microenvironment. The Scientist brings together a panel of experts to discuss interactions between stem cells and external cues, and the role of the stem cell niche in development and disease. Topics to be covered include the molecular mechanisms of hematopoietic stem cell niche interactions and techniques for engineering 3-D stem-cell microenvironments. Following the presentations, attendees will have an opportunity to ask questions concerning their specific applications and receive answers in real-time.


Dr. Jon Hoggatt, Assistant Professor of Medicine, Cancer Center and Center for Transplantation Sciences, Harvard Medical School/Massachusetts General Hospital.

Dr. Todd McDevitt, Senior Investigator, Gladstone Institute of Cardiovascular Disease, Professor, Department of Bioengineering & Therapeutic Sciences, UCSF.


Understanding the Stem Cell Niche
Click Here To Watch The Video

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Notes from Webinar:

Hematopoetic stem cells good model since now we have liquid biopsies (as a result field has skyrocketed).

Two processes involved with stem cells finding their niche

  1. Homing; CXCR4-SDK1 dependent process into the bone marrow.
  2. Mobilization: stem cells moving from bone into blood (found that GMCSF main factor responsible for this process)

Dr. Raymond Schofield was one of the first to propose the existence of this stem cell niche (each progenitor will produce a unique factor {possibly a therapeutic target} for example leptin+ receptor target perivascular cells so one target is good for only a small subset of stem cells)

Therefore it may be possible or advantageous to target the whole stem cell milieu. One such possible target they are investigating is CD26 (dipeptyl peptidase). The diabetes drug Januvia is an inhibitor of CD26.

It was also noticed if inhibit the GMCSF receptor complex can inhibit the whole stem cell niche.

Prostoglandins and stem cell niche

  • Indomethacin blocks the mobilization step
  • Prostaglandin E increases homing
  • GMCSF and malaxocam (COX2 inhibitor) flattens osteoblast cells and may be a mechanism how inhibition of prostaglandin synthesis blocks mobilization
  • Found that the PGE4 receptor is ultimately responsible for the NSAID effect

The niche after G-CSF

Dr. Hoggat found that macrophages are supplying the factors that support the niche. He will be presenting the findings at 2015 Hematology conference. (See information about his conference presentation here).

From the 57th Annual American Society of Hematology Meeting (2015) please see Dr. Hoggat’s moderated section Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells: Hematopoietic Stem Cell Niche


Relevant articles from Dr. Hoggat

Anti-CD47 Therapy Is More Than a Dinner Bell October 19, 2015

Dr. Hoggatt looks at the therapeutic effects of blocking CD47 aside from alerting macrophages to devour tumor cells.

Hematopoietic Stem Cells Should Hold Their Breath August 12, 2015

Dr. Hoggatt and Hannah Rasmussen discuss new approaches to the use of hematopoietic stem cells considering observer effects that emerge due to our experimental systems for HSCs.

Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation. Hoggatt J, Singh P, Sampath J, Pelus LM. Blood. 2009 May 28;113(22):5444-55. doi: 10.1182/blood-2009-01-201335. Epub 2009 Mar 26.


Prostaglandin E2 enhances long-term repopulation but does not permanently alter inherent stem cell competitiveness. Hoggatt J, Mohammad KS, Singh P, Pelus LM. Blood. 2013 Oct 24;122(17):2997-3000. doi: 10.1182/blood-2013-07-515288. Epub 2013 Sep 18.


Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment. Speth JM, Hoggatt J, Singh P, Pelus LM. Blood. 2014 Jan 9;123(2):203-7. doi: 10.1182/blood-2013-07-516336. Epub 2013 Oct 28.


Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development from hematopoietic progenitor cells. Singh P, Hoggatt J, Hu P, Speth JM, Fukuda S, Breyer RM, Pelus LM. Blood. 2012 Feb 16;119(7):1671-82. doi: 10.1182/blood-2011-03-342428. Epub 2011 Nov 22.


Recovery from hematopoietic injury by modulating prostaglandin E(2) signaling post-irradiation. Hoggatt J, Singh P, Stilger KN, Plett PA, Sampson CH, Chua HL, Orschell CM, Pelus LM. Blood Cells Mol Dis. 2013 Mar;50(3):147-53. doi: 10.1016/j.bcmd.2012.11.006. Epub 2012 Nov 30.


Pulse exposure of haematopoietic grafts to prostaglandin E2 in vitro facilitates engraftment and recovery. Pelus LM, Hoggatt J, Singh P. Cell Prolif. 2011 Apr;44 Suppl 1:22-9. doi: 10.1111/j.1365-2184.2010.00726.x.


Pleiotropic effects of prostaglandin E2 in hematopoiesis; prostaglandin E2 and other eicosanoids regulate hematopoietic stem and progenitor cell function. Pelus LM, Hoggatt J. Prostaglandins Other Lipid Mediat. 2011 Nov;96(1-4):3-9. doi: 10.1016/j.prostaglandins.2011.06.004. Epub 2011 Jun 21. Review.


Differential stem- and progenitor-cell trafficking by prostaglandin E2. Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, Hu P, Poteat BA, Stilger KN, Ferraro F, Silberstein L, Wong FK, Farag SS, Czader M, Milne GL, Breyer RM, Serezani CH, Scadden DT, Guise TA, Srour EF, Pelus LM. Nature. 2013 Mar 21;495(7441):365-9. doi: 10.1038/nature11929. Epub 2013 Mar 13.


Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking.Hoggatt J, Pelus LM. Leukemia. 2010 Dec;24(12):1993-2002. doi: 10.1038/leu.2010.216. Epub 2010 Sep 30. Review.


Hematopoietic stem cell mobilization with agents other than G-CSF. Hoggatt J, Pelus LM. Methods Mol Biol. 2012;904:49-67. doi: 10.1007/978-1-61779-943-3_4.


Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment. Hoggatt J, Pelus LM. Stem Cell Res Ther. 2011 Mar 14;2(2):13. doi: 10.1186/scrt54. Review.


Engineering 3D Pluripotent Stem Cell Microenvironments by Todd McDevitt, Ph.D.

In recent years, it has finally been shown how to produce centrally derived (self assembling) organoids (microtissues).


How to specifically deliver specific morphogens in 3D organoids


  1. Microparticle (MP)-mediated delivery (can do in mouse and human): reduces the amount needed to be delivered



What are other effects of introduced MP in ES (embryonic stem cell) aggregates?

  1. a) physiocomechanical changes –mechanical effects of materials
  2. b) how changes in local presentation of factors affect bioavailbility and binding properties







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