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New Coronavirus Passive Vaccine Developed by Israeli Researchers
Reporter: Irina Robu, PhD
Researchers at Bar-Ilan University have identified short amino acid sequences that could help the development of a vaccine against COVID-19 virus. Of the 25 epitopes that were discovered to be 100% identical to SARS, seven are theoretically efficient vaccine candidates. Their research indicate that they could cover as much as 87% of the world population
Their study has identified a set of immunodominant epitopes from the SARS-CoV-2 proteome, which are capable of generating antibody and cell mediated immune responses. The epitopes, known as antigenic determinants, are the part of the antigen that binds to a specific antigen receptor on the surface of B cells or T cells and are able to provoke an immune response.
It is known that immune response occurs within an organism for the purpose of defending against foreign invaders such as viruses, bacteria, parasites and fungi. The immune responses that are based on specific immunodominant epitopes contain the generation of both antibody- and cell-mediated immunity against pathogens. Such immunity can facilitate fast and effective elimination of the pathogen. The end result is a passive vaccine capable of capable of activating both cellular and humoral immune responses in humans.
According to the team at Bar-Ilan University, the mapped coronavirus epitopes with those of the influenza virus. And they found that 85% of the sequence identity with experimentally detected epitopes of Severe Acute Respiratory Syndrome-related coronavirus (SARS-CoV).
Additional analysis indicated that the epitopes are non-allergic and non-toxic to humans and have very low risk for generating autoimmune responses. The team is looking to partner with companies to build vaccine constructs and test them in-vitro and on animal trials before starting any clinical trials.
When T cells receive the appropriate signals through the T cell receptor (TCR) complex and the costimulatory receptor CD28, a complex rearrangement of the cytoskeleton occurs that enables the formation of the immunological synapse, a specialized structure that forms between the antigen-presenting cell and the T cell. In this week’s issue, Roybal et al. used sophisticated imaging of live cells and computational image analysis to visualize the dynamic rearrangements of actin and various regulatory proteins in T cells activated through the TCR in the absence or presence of CD28 signaling. The regulatory proteins WAVE2 and cofilin were efficiently recruited to the immunological synapse only when both TCR and CD28 signaled. Fried et al. used a different fluorescence imaging approach, triple-color FRET (fluorescence resonance energy transfer), to visualize not the movement of proteins with the cell, but the interactions between the actin-regulatory proteins WASp and WIP in live cells. The WASp-WIP interaction is required for T cell activation. The triple-color FRET analysis revealed how changes in the interaction between WASp and WIP resulted in WASp functioning as both an on and off switch. Although most studies’ focus of T cell cytoskeletal dynamics is on the changes that occur at the immunological synapse, as González-Granado et al. showed, the nuclear cytoskeleton is also important for the immune response. In this study, live-cell imaging and immunofluorescence analysis revealed that nuclear lamin-A contributed to the polymerization of actin and, thus, immunological synapse formation. These studies highlight the insights that can be obtained about molecular dynamics from live-cell image analysis.
K. T. Roybal, T. E. Buck, X. Ruan, B. H. Cho, D. J. Clark, R. Ambler, H. M. Tunbridge, J. Zhang, P. Verkade, C. Wülfing, R. F. Murphy, Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics. Sci. Signal.9, rs3 (2016). [Abstract]
Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics
T cells must receive signals through the T cell receptor (TCR) and the costimulatory receptor CD28 to become fully activated. Critical to this process is the reorganization of plasma membrane actin at the immunological synapse, the interface between a T cell and an antigen-presenting cell. Roybal et al.imaged actin and fluorescently tagged actin regulatory proteins in T cells activated through the TCR in the absence or presence of CD28 signaling. Computational image processing to normalize differences in cell shape enabled tracking of the fluorescent proteins. The regulatory proteins WAVE2 and cofilin were efficiently recruited to the immunological synapse only when both TCR and CD28 signaled. Constitutive activation of either protein in TCR-stimulated T cells enabled normal actin reorganization even when CD28 signaling was blocked. This combination of imaging and computational analysis could be applied to other systems to determine the spatiotemporal dynamics of signaling molecules.
Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.
S. Fried, B. Reicher, M. H. Pauker, S. Eliyahu, O. Matalon, E. Noy, J. Chill, M. Barda-Saad, Triple-color FRET analysis reveals conformational changes in the WIP-WASp actin-regulating complex. Sci. Signal.7, ra60 (2014). [Abstract]
Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex
Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.
J. M. González-Granado, C. Silvestre-Roig, V. Rocha-Perugini, L. Trigueros-Motos, D. Cibrián, G. Morlino, M. Blanco-Berrocal, F. G. Osorio, J. M. P. Freije, C. López-Otín, F. Sánchez-Madrid, V. Andrés, Nuclear envelope Lamin-a couples actin dynamics with immunological synapse architecture and T cell activation.Sci. Signal.7, ra37 (2014). [Abstract]
Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation
In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal–regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A–dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response.
T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex at the site of the T cell receptors (TCRs). Srikanth et al. found that the lymphocyte-specific large guanosine triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human CD4+ T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T cell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium and kinase signaling.
More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.
T cell-mediated immune responses & signaling pathways activated by Toll-like Receptors
Curator: Larry H. Bernstein, MD, FCAP
Series E. 2; 6.10
Bruce A. Beutler, Jules A. Hoffmann, Ralph M. Steinman
Bruce Beutler, MD
Regental Professor; Raymond and Ellen Willie Distinguished Chair in Cancer Research, in Honor of Laverne and Raymond Willie, Sr.
Department Center for Genetics of Host Defense, Immunology
Bruce Beutler, MD, the son of the “father of red cell metabolic pathways”, Ernest Beutler, was born in Chicago and grew up in Los Angeles, where Beutler did his groundbreaking work on hemolytic red cell disorders at City of Hope Hospital. He was infected with an interest in medicine and research at an early age, somewhat analagous to a comparison with Roger and Arthur Kornberg. He attended University of California San Diego (UCSD) Medical School, where his interest in immunity was sparked by Abraham Braude, PhD, Professor Pathology and Medicine, and Chief of Microbiology.
Bruce Beutler, MD, discovered an important family of receptors that allow mammals to sense infections when they occur, triggering a powerful inflammatory response. For this work he received the 2011 Nobel Prize in Physiology or Medicine.
The period from 1950 to 1953 was critical to Abraham Braude’s career. During these years, Braude realized the importance of the microbiology laboratory not only in patient management, but also in physician education. He became convinced that infectious disease clinicians must not only be excellent microbiologists, but also exercise technical and administrative control of the diagnostic microbiology laboratory. He demonstrated and taught this principle of medical practice and education for the next 35 years as director of diagnostic microbiology and chief of infectious diseases at the Texas Southwestern Medical School of the University of Texas from 1953-57, at the University of Pittsburgh from 1957 to 1969, and at the University of California at San Diego from 1967 until his death in December, 1984. He also was my mentor as a resident in 1972, and it was remarkable how he had developed his own antibiotic diffusion plates for sensitivity studies on bacterial isolates.
He discovered that the endotoxin of cold-growing bacteria that had contaminated refrigerated blood could cause vascular collapse and death of transfused people without bacterial multiplication in the body. This observation established the importance of endotoxin as the major virulence factor of Gram negative bacteria and set the stage for his later fundamental and applied research on the pathogenesis and treatment of life-threatening septic shock. This aspect of infection, in the host defense, is perhaps important in the development of the young Beutler.
During his years in Dallas, Pittsburgh, and San Diego, Braude made outstanding research contributions in three areas, anaerobic infections, pyelonephritis, and the endotoxin shock of Gram negative bacteremia. He devised a simple, effective method for culturing fastidious anaerobes, discovered Bacteroides penicillinase, and proved that so-called “sterile” brain abscesses are caused by anaerobes. His landmark papers on brain infections and sinusitis awakened us to the major role of anaerobes in infectious disease. His work on pyelonephritis ranged from devising realistic models of infection to elegant studies that helped explain the inadequacies of the immune responses of the kidney.
Beutler received his undergraduate degree from the University of California at San Diego in 1976, and his MD degree from the University of Chicago in 1981. After two years of residency at the University of Texas Southwestern Medical Center, he became a postdoctoral fellow and then an Assistant Professor at the Rockefeller University (1983-1986), where he isolated mouse tumor necrosis factor (TNF), and was the first to recognize TNF as a key executor of the inflammatory response. Returning to Dallas in 1986 as an HHMI investigator, he designed recombinant inhibitors of TNF that are widely used in the treatment of rheumatoid arthritis and other inflammatory diseases. He also used TNF as a biological endpoint in order to identify the receptor for bacterial lipopolysaccharide (LPS). This he achieved by positionally cloning the Lps mutation of mice, known to prevent all biological responses to LPS, including TNF production. He thus concluded that Toll-like receptor 4 (TLR4) acts as the signaling core of the LPS receptor and proposed that other TLRs might also recognize conserved molecular signatures of infection.
Moving in 2000 to the Scripps Research Institute, Beutler developed the largest mouse mutagenesis program in the world, and applied a forward genetic approach to decipher the signaling pathways activated by TLRs. He also identified many other molecules with non-redundant function in the immune response.
Beutler is currently a Regental Professor and Director of the Center for Genetics of Host Defense at the University of Texas Southwestern Medical Center. He also holds the Raymond and Ellen Willie Distinguished Chair in Cancer Research in honor of Laverne and Raymond Willie, Sr. He has authored or co-authored more than 300 papers, which have been cited more than 46,000 times. Before he received the Nobel Prize, his work was recognized by the Shaw Prize (2011), the Albany Medical Center Prize in Medicine and Biomedical Research (2009), election to the National Academy of Sciences and Institute of Medicine (2008), the Frederik B. Bang Award (2008), the Balzan Prize (2007), the Gran Prix Charles-Leopold-Mayer (2006), the William B. Coley Award (2005), the Robert-Koch-Prize (2004), and other honors.
“Monitored Saturation Mutagenesis of the Mouse Genome”
Bruce Beutler, University of Texas Southwestern Medical Center, TX USA
The availability of massively parallel sequencing platforms has accelerated the identification of induced mutations in the mouse genome. However, genetic mapping has remained essential to identify causative mutations, and for a time, became the rate-limiting step in mammalian forward genetics. We have recently described a procedure that permits real time identification of mutations responsible for phenotypes of any kind. By inducing mutations on a defined genetic background (C57BL/6J) and sequencing the whole exome of all G1 carriers of these mutations, we are able to identify all candidate aberrations that might cause phenotype in descendants of the G1. We then genotype all G3 mice to determine zygosity at each mutation site. Pre-genotyped G3 mice are distributed for extensive phenotypic screening. As quantitative phenotypic data are uploaded to the computer, an automated search for correlation between phenotype and genotype is initiated using dominant, recessive and semidominant models of transmission. The computer returns a list of statistical associations between specific mutations and phenotype, allowing immediate inference regarding causation. As allelic series develop at most loci, the strength of inference increases. CRISPR/Cas9 targeting is used to validate candidates identified by automated mapping. Presently, more than 65,000 point mutations disrupting the mouse coding region have been tested in this manner. To our best estimate, 18 percent of all mouse genes have been mutated to phenovariance and examined in 135 phenotypic screens. About 50,000 mutations can be surveyed annually. A list of robust associations between mouse genes and phenotypic anomalies will facilitate genetic investigations in humans, where a uniform genetic background is not available and a heavy mutation load confounds such analysis.
http://www.youtube.com/watch%3Fv%3D31rWtdpmjME Dec 9, 2014 … Professor Bruce Beutler, a 2011 recipient of the Nobel Prize in Medicine, was the Keynote speaker. Professor Beutler discovered the …
In the last few years of his life, Dr. Ralph Steinman made himself into an extraordinary human lab experiment, testing a series of unproven therapies – including some he helped to create – as he waged a very personal battle with pancreatic cancer.
The winner of the 2011 Nobel prize in medicine died only three days before the award was announced.
Immunologist Ralph Steinman was honored today by the Nobel Committee for his discovery of dendritic cells, a class of immune cells that help rally the body’s natural defenses to fight disease. However, the prize is bittersweet for all who knew the 68-year-old scientist during his long career as a researcher and mentor at Rockefeller University in New York City.
“Dendritic cells are the guys who are training the fighters,” says Pawel Kalinski, an immunologist at the University of Pittsburgh in Pennsylvania, about their role in activating T cells, the body’s immune sentries. Over decades, with Steinman often leading the way, the work transformed cancer research. Cancer vaccines either using or targeting dendritic cells are now the subject of numerous clinical trials, and the first-ever cancer vaccine to be approved in the United States—called Provenge, to treat prostate cancer—injects a patients’ own dendritic cells back into their body. It went on the market last year.
“He felt that human clinical investigation was the highest form of research, that it was critical to engage in it,” Dr. Sarah Schlesinger, Steinman’s clinical lab director and colleague at New York’s Rockefeller University, told Reuters. “He had great criticism of how slowly the process moved … he was impatient with data and mice,” she added.
Steinman spent his entire career on immunology research for which he won the Nobel Prize, an honor he shares with American Bruce Beutler and French biologist Jules Hoffmann for their contributions to explaining the immune system.
Steinman’s discovery of dendritic cells in 1973 led to the first therapeutic cancer vaccine, Dendreon’s Provenge, which treats men with advanced prostate cancer.
Jules A. Hoffmann (born 2 August 1941) is a Luxembourg-born French[1] biologist. During his youth, growing up in Luxembourg, he developed a strong interest in insects under the influence of his father, Jos Hoffmann. This eventually resulted in the younger Hoffmann’s dedication to the field of biology using insects as model organisms.[2] He currently holds a faculty position at the University of Strasbourg.[3] He is a research director and member of the board of administrators of the National Center of Scientific Research (CNRS) in Strasbourg, France. He was elected to the positions of Vice-President (2005-2006) and President (2007-2008) of the French Academy of Sciences.[3] Hoffmann and Bruce Beutler were jointly awarded a half share of the 2011 Nobel Prize in Physiology or Medicine for “their discoveries concerning the activation of innate immunity,”.[4] [More specifically,the work showing increased Drosomycin expression following activation of Toll pathway in microbial infection.]
Hoffmann and Lemaitre discovered the function of the fruit fly Toll gene in innate immunity. Its mammalian homologs, the Toll-like receptors, were discovered by Beutler. Toll-like receptors identify constituents of other organisms like fungi and bacteria, and trigger an immune response, explaining, for example, how septic shock can be triggered by bacterial remains.[5][6][7]
During his Ph.D. program under Pierre Joly, Hoffmann started his research in studying antimicrobial defenses in grasshoppers, inspired by the previous works done in the laboratory of Pierre Joly showing that no opportunistic infections were apparent in insects after the transplantation of certain organs from one to another.[2] Hoffmann confirmed discovery of phagocytosis done by Eli Metchnikoff, through injection of Bacillus thuringiensis and observation of increase of phagocytes.[2] In addition, he showed strong correlation between hematopoiesis and antimicrobial defenses by assessing the susceptibility of an insect to the microbial infection after X-ray treatment.[2] Hoffmann shifts from using grasshopper model to using dipteran species in the 80s. By using Phormia terranovae, Hoffmann and his colleagues were able to identify 82-residues long antimicrobial polypeptide named Diptericin which was glycine-rich, along with other polypeptides in Drosophila melanogaster such as defensin, cecropin, and attacin.[2] Further molecular genetic analysis revealed that the promoters for the genes encoding these antimicrobial peptides contained DNA sequences similar to the binding elements for NF-κB in mammalian DNA. Dorsal gene, critical in dorso-ventral patterning in the early embryo of Drosophila melanogaster was also identified to be in this NF-κB family. It was initially speculated by Hoffmann and colleagues that activity of Dorsal was directly linked to the expression of the Diptericin gene. However, it turned out that Diptericin was normally induced even in the loss-of-function Dorsal mutants. Further conducted research showed that Diptericin expression was dependent on the expression of imd gene. Identification of another antifungal peptide named Drosomycin and RNA blots demonstrated that two distinct pathways(Toll, Imd) exist, involving Drosomycin and Diptericin respectively. Similarities of structure and function between several members in the Drosophila embryo and members in mammals being noted, study “The Dorsoventral Regulatory Gene Cassette spảtzle/Toll/cactus Controls the Potent Antifungal Response in Drosophila Adults”[8] by Lemaitre and Hoffmann in 1996 illuminated the possible existing innate immunity in Drosophila in response to fungal challenge. Later works identified that Toll transmembrane receptors are present in a wide variety of phyla and are conserved through evolution along with conservation of NF-κB activating cascades.[2]
Hoffmann was a research assistant at CNRS from 1964 to 1968, and became a research associate in 1969. Since 1974 he has been a Research Director of CNRS. Between 1978 and 2005 he was Director of the CNRS research unit “Immune Response and Development in Insects”, and from 1994 to 2005 he was director of the Institute of Molecular and Cellular Biology of CNRS in Strasbourg.
The History of Toll-like receptors — Redefining Innate Immunity
The discovery of Toll-like receptors (TLRs) was an important event for immunology research and was recognized as such with the awarding of the 2011 Nobel Prize in Physiology or Medicine to Jules Hoffmann and Bruce Beutler, who, together with Ralph Steinman, the third winner of the 2011 Nobel Prize and the person who discovered the dendritic cell, were pioneers in the field of innate immunity. TLRs have a central role in immunity — in this Timeline article, we describe the landmark findings that gave rise to this important field of research.
oll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to microbial pathogens. TLR-mediated recognition of components derived from a wide range of pathogens and their role in the subsequent initiation of innate immune responses is widely accepted; however, the recent discovery of non-TLR PRRs, such as C-type lectin receptors, NOD-like receptors, and RIG-I-like receptors, suggests that many aspects of innate immunity are more sophisticated and complex. In this review, we will focus on the role played by TLRs in mounting protective immune responses against infection and their crosstalk with other PRRs with respect to pathogen recognition.
Invivogen – Review
Toll-Like Receptors (TLRs) play a critical role in the early innate immune response to invading pathogens by sensing microorganism and are involved in sensing endogenous danger signals.
TLRs are evolutionarily conserved receptors are homologues of the Drosophila Toll protein, discovered to be important for defense against microbial infection [1]. TLRs recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens,
or danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells.
Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB and interferon regulatory factors (IRFs). Signaling by TLRs result in a variety of cellular responses including the production of interferons (IFNs), pro-inflammatory cytokines and effector cytokines that direct the adaptive immune response.
The TLR Family
TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail that contains a conserved region called the Toll/IL-1 receptor (TIR) domain. The structure of the extracellular domain of TLR3 was revealed by crystallography studies as a large horseshoe-shape [2].
TLRs are predominantly expressed in tissues involved in immune function, such as spleen and peripheral blood leukocytes, as well as those exposed to the external environment such as lung and the gastrointestinal tract.
Their expression profiles vary among tissues and cell types. TLRs are located on the plasma membrane with the exception of TLR3, TLR7, TLR9 which are localized in the endosomal compartment [3].
Ten human and twelve murine TLRs have been characterized, TLR1 to TLR10 in humans, and TLR1 toTLR9, TLR11, TLR12 and TLR13 in mice, the homolog of TLR10 being a pseudogene.
TLR2 is essential for the recognition of a variety of PAMPs from Gram-positive bacteria, including bacterial lipoproteins, lipomannans and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detectsbacterial flagellin and TLR9 is required for response to unmethylated CpG DNA. Finally, TLR7 andTLR8 recognize small synthetic antiviral molecules [4], and single-stranded RNA was reported to be their natural ligand [5]. TLR11(12) has been reported to recognize uropathogenic E.coli [6] and a profilin-like protein from Toxoplasma gondii [7].
The repertoire of specificities of the TLRs is apparently extended by the ability of TLRs to heterodimerize with one another. For example, dimers of TLR2 and TLR6 are required for responses to diacylated lipoproteins while TLR2 and TLR1 interact to recognize triacylated lipoproteins [8]. Specificities of the TLRs are also influenced by various adapter and accessory molecules, such as MD-2 and CD14 that form a complex with TLR4 in response to LPS [9].
TLR Signaling
TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells.
The MyD88-dependent pathway is common to all TLRs, except TLR3 [10]. Upon activation byPAMPs or DAMPs, TLRs hetero- or homodimerize inducing the recruitment of adaptor proteins via the cytoplasmic TIR domain.
Adaptor proteins include the TIR-domain containing proteins, MyD88, TIRAP (TIR-associated protein), Mal (MyD88 adaptor-like protein), TRIF (TIR domain-containing adaptor protein-inducing IFN-β) and TRAM (TRIF-related adaptor molecule).
Recruitment of MyD88 for instance, in turn recruits IRAK1 and IRAK4. IRAK4 subsequently activates IRAK1 by phosphorylation. Both IRAK1 and IRAK4 leave the MyD88-TLR complex and associate temporarily with TRAF6 leading to its ubiquitination. Bcl10 and MALT1 form oligomers that bind to TRAF6 promoting TRAF6 self-ubiquitination [11].
Recently, IRAK2 was shown to play a central role in TRAF6 ubiquitination [12]. Following ubiquitination, TRAF6 forms a complex with TAB2/TAB3/TAK1 inducing TAK1 activation [13]. TAK1 then couples to the IKK complex, which includes the scaffold protein NEMO, leading to the phosphorylation of IκB and the subsequent nuclear localization of NF-κB. Activation of NF-κB triggers the the production of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-12.
Individual TLRs induce different signaling reponses by usage of the different adaptor molecules.
TLR4 and TLR2 signaling requires the adaptor TIRAP/Mal, which is involved in the MyD88-dependent pathway [14]. TLR3 triggers the production of IFN-β in response to double-stranded RNA, in a MyD88-independent manner, through the adaptor TRIF/TICAM-1 [15]. TRAM/TICAM-2 is another adaptor molecule involved in the MyD88-independent pathway [5] which function is restricted to the TLR4 pathway [16].
TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFNs. The signaling mechanisms leading to the induction of type I IFNs differ depending on the TLR activated. They involve the interferon regulatory
factors, IRFs, a family of transcription factors known to play a critical role in antiviral defense, cell growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3 and IRF7 [17], while TLR7 and TLR8 activate IRF5 and IRF7 [18]. Furthermore, type I IFN production stimulated by TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR [19].
TLR-Targeted Therapeutics
Significant progress has been made over the past years in the understanding of TLR function [20]. TLRs are essential receptors in host defense against pathogens by activating the innate immune system, a prerequisite to the induction of adaptive immune responses.
Although TLR-mediated signaling is paramount in eradicating microbial infections and promoting tissue repair, the regulation must be tight. TLRs are implicated in a number of inflammatory and immune disorders and play a role in cancer [21].
Many single nucleotide polymorphisms have been identified in various TLR genes and are associated with particular diseases. Several therapeutic agents targeting the TLRs are now under pre-clinical and clinical
evaluation [22].
However, the complexity lies in that TLRs act as double-edged swords either promoting or inhibiting disease progression. Furthermore, therapeutic agents targeting the TLRs must be able to antagonize the harmful effects resulting without affecting host defense functions.
Nonetheless, the potential of harnessing and directing the innate immune system with drugs targeting TLRs, to prevent or treat human inflammatory and autoimmune diseases as well as cancer, appears to be promising.
1. Medzhitov R. et al., 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388(6640):394-7.
2. Choe J. et al., 2005. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309; 581-585.
3. Nishiya T. & DeFranco AL., 2004. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling proper ties of the Toll-like receptors. J Biol Chem. 279(18):19008-17.
4. Jurk M. et al., 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol, 3(6):499.
5. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 303(5663):1526-9.
6. Zhang D. et al., 2004. A toll-like receptor that prevents infection by uropathogenic bacteria. Science. 303:1522-1526.
7. Lauw FN. et al., 2005. Of mice and man: TLR11 (finally) finds profilin. Trends Immunol. 26(10):509-11.
8. Ozinsky A. et al., 2000.The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS USA, 97(25):13766-71.
9. Miyake K., 2003. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. Int Immunopharmacol. 3(1):119-28.
10. Adachi O. et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1):143-50.
11. Sun L. et al., 2004. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell. 2004 14(3):289-301.
12. Keating SE. et al., 2007. IRAK-2 participates in multiple Toll-like receptor signaling pathways to NFκB via activation of TRAF6 ubiquitination. J Biol Chem. 282: 33435-33443.
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