Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Cytokines in IBD

Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Revised 2/14/2016

 

The following presentation explores the application of antisense oligonucleotide agents that modulate the activity of Il17 and Il23 signaling activity in the cell.

IL 17 & 23

United States Patent	9,238,042
Schnell ,   et al.	January 19, 2016

Antisense modulation of interleukins 17 and 23 signaling
Provided are antisense oligonucleotides and other agents that target and modulate IL-17 and/or IL-23 signaling activity in a cell, compositions that comprise the same, and methods of use thereof. Also provided are animal models for identifying agents that modulate 17 and/or IL-23 signaling activity.

Abes et al., “Arginine-rich cell penetrating peptides: Design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides,” J Pept Sci 14: 455-460, 2008. cited by applicant .
Abes et al., “Delivery of steric block morpholino oligomers by (R-X-R).sub.4 peptides: structure-activity studies,” Nucleic Acids Research 36(20): 6343-6354, Sep. 16, 2008. cited by applicant .
Abes et al., “Vectorization of morpholino oligomers by the (R-Ahx-R).sub.4 peptide allows efficient splicing correction in the absence of endosomolytic agents,” Journal of Controlled Release 116: 304-313, 2006. cited by applicant .
Lebleu et al., “Cell penetrating peptide conjugates of steric block oligonucleotides,” Advanced Drug Delivery Reviews 60: 517-529, 2008. cited by applicant .
Marshall et al., “Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing,” Journal of Immunological Methods 325: 114-126, 2007. cited by applicant .
Moulton et al., “Cellular Uptake of Antisense Morpholino Oligomers Conjugated to Arginine-Rich Peptides,” Bioconjugate Chem 15: 290-299, 2004. cited by applicant .
Summerton et al., “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Development 7: 187-195, 1997. cited by applicant .
Wright et al., “The Human IL-17F/IL-17A Heterodimeric Cytokine Signals through the IL-17RA/IL-17RC Receptor Complex,” The Journal of Immunology 181: 2799-2805, 2008. cited by applicant .

 

Immunity. 2015 Oct 20;43(4):739-50. doi: 10.1016/j.immuni.2015.08.019. Epub 2015 Sep 29.

Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation.

Maxwell JR¹, Zhang Y¹, Brown WA¹, Smith CL¹, Byrne FR², Fiorino M², Stevens E³, Bigler J⁴, Davis JA⁵, Rottman JB⁶, Budelsky AL¹, Symons A¹, Towne JE⁷.

 

Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn’s disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn’s disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn’s disease when IL-17A or IL-17RA is inhibited.

 

Immunity. 2015 Oct 20;43(4):727-38. doi: 10.1016/j.immuni.2015.09.003. Epub 2015 Sep 29.

Interleukin-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability.

Lee JS¹, Tato CM¹, Joyce-Shaikh B¹, Gulan F², Cayatte C¹, Chen Y¹, Blumenschein WM¹, Judo M¹, Ayanoglu G¹, McClanahan TK¹, Li X², Cua DJ³.

Whether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R(+) γδ T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing γδ T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa.

 

Gastroenterology. 2008 Apr;134(4):1038-48. doi: 10.1053/j.gastro.2008.01.041. Epub 2008 Jan 17.

Regulation of gut inflammation and th17 cell response by interleukin-21.

Fina D¹, Sarra M, Fantini MC, Rizzo A, Caruso R, Caprioli F, Stolfi C, Cardolini I, Dottori M, Boirivant M, Pallone F, Macdonald TT,Monteleone G.

Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation.

Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay.

High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes.

These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

 

Neurochem Res. 2010 Jun;35(6):940-6. doi: 10.1007/s11064-009-0091-9. Epub 2009 Nov 14.

Synergy of IL-23 and Th17 cytokines: new light on inflammatory bowel disease.

Shen W¹, Durum SK.

Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, involve an interplay between host genetics and environmental factors including intestinal microbiota. Animal models of IBD have indicated that chronic inflammation can result from over-production of inflammatory responses or deficiencies in key negative regulatory pathways. Recent research advances in both T-helper 1 (Th1) and T-helper 17 (Th17) effect responses have offered new insights on the induction and regulation of mucosal immunity which is linked to the development of IBD. Th17 cytokines, such as IL-17 and IL-22, in combination with IL-23, play crucial roles in intestinal protection and homeostasis. IL-23 is expressed in gut mucosa and tends to orchestrate T-cell-independent pathways of intestinal inflammation as well as T cell dependent pathways mediated by cytokines produced by Th1 and Th17 cells. Th17 cells, generally found to be proinflammatory, have specific functions in host defense against infection by recruiting neutrophils and macrophages to infected tissues. Here we will review emerging data on those cytokines and their related regulatory networks that appear to govern the complex development of chronic intestinal inflammation; we will focus on how IL-23 and Th17 cytokines act coordinately to influence the balance between tolerance and immunity in the intestine.

 

Eur J Immunol. 2007 Oct;37(10):2680-2.

IL-23 and IL-17 have a multi-faceted largely negative role in fungal infection.

Cooper AM¹.

The role of IL-23 and IL-17 in the response to fungal infection has been the focus of recent reports. In this issue of the European Journal of Immunology there is an article that reports an important role for IL-23 and IL-17 in limiting fungal control, promoting neutrophillic inflammation and regulating the killing activity of neutrophils. In the fungal model it appears that IL-23 and IL-17 are counter-productive for protection.

 

IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases

MWL Teng, EP Bowman, JJ McElwee,…, AM Cooper & DJ Cua
Nature Med July 2016; 21(7):719–729
http://www.nature.com/nm/journal/v21/n7/full/nm.3895.html

The cytokine interleukin-12 (IL-12) was thought to have a central role in T cell–mediated responses in inflammation for more than a decade after it was first identified. Discovery of the cytokine IL-23, which shares a common p40 subunit with IL-12, prompted efforts to clarify the relative contribution of these two cytokines in immune regulation. Ustekinumab, a therapeutic agent targeting both cytokines, was recently approved to treat psoriasis and psoriatic arthritis, and related agents are in clinical testing for a variety of inflammatory disorders. Here we discuss the therapeutic rationale for targeting these cytokines, the unintended consequences for host defense and tumor surveillance and potential ways in which these therapies can be applied to treat additional immune disorders.

IL-12 and IL-23 are produced by inflammatory myeloid cells and influence the development of T_H1 cell and IL-17–producing T helper (T_H17) cell responses, respectively. The rationale for developing IL-12 antagonists was prompted by observations that mice deficient in IL-12p40 are resistant to experimentally induced autoimmune conditions, including paralysis induction after immunization with brain-derived antigens, arthritis inflammation after immunization with a joint antigen, ocular disease after immunization with a retinal antigen and multiple gut disease models. This suggested that IL-12 could be an effective therapeutic target^{1, 2, 3, 4, 5}. Studies of neutralizing antibodies to IL-12p40 in multiple mouse strains seemed to confirm the importance of therapeutically targeting IL-12 to decrease immune pathology^{6, 7}. However, mice deficient in the other IL-12 subunit, IL-12p35, showed no protection or showed exacerbated disease in some models^{1, 2}. Following the recognition, in 2000, that IL-12 and IL-23 share the IL-12p40 subunit but only IL-23 uses the p19 subunit⁸, it was determined that mice deficient in IL-23 but not IL-12 are resistant to experimental immune-mediated disease^{1, 2, 3, 4, 5}. By 2000, the first anti–IL-12p40 therapy targeting IL-12—subsequently recognized to target IL-23 as well—was under evaluation in patients with Crohn’s disease⁹. Currently, at least 10 therapeutic agents targeting IL-12, IL-23 or IL-17A are being tested in the clinic for more than 17 immune-mediated diseases (Table 1). Here we discuss the preclinical and clinical data validating these therapeutic strategies and the potential consequences of targeting these immune pathways.

Figure 1: Schematic representation of IL-12 and IL-23, and their receptors and downstream signaling pathways

IL-12 is made up of the IL-12/23p40 and IL-12p35 subunits, and IL-23 comprises IL-23p19 and IL-12/23p40. IL-12 signals through the IL-12Rβ1 and IL-12Rβ2 subunits, and IL-23 signals through IL-12Rβ1 and IL-23R. IL-12 stimulation of JAK2…

Figure 4: Schematic representation of the mechanisms by which IL-23 indirectly or directly promotes tumorigenesis, growth and metastasis.

 

IL-23 is produced by myeloid cells in response to exogenous or endogenous signals such as damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs) or tumor-secreted factors such as prostaglandin E2 (PGE₂). IL-23 can act directly on tumor cells to promote their transformation, proliferation and/or metastasis. In mice, IL-23R is expressed on several innate and adaptive immune cell types, which are found in various proportions in tumors. Stimulation of IL-23R on these immune cells leads to production of cytokines such as IL-17 and/or IL-22, which can have direct proliferative effects on stromal or tumor cells. IL-17 and/or IL-22 also elicit a range of factors from various hematopoietic and nonhematopoietic cells, which can have direct effects on tumor proliferation and metastasis or induce the production of additional inflammatory cytokines, chemokines and mediators such as IL-6, IL-8, matrix metallopeptidases (MMPs) and vascular endothelial growth factor (VEGF), all of which can contribute to the generation of a tumor microenvironment in which CD8 and NK cell effector functions are suppressed. DC, dendritic cell; Mφ, macrophage.

IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases

Michele W L Teng, Edward P Bowman,…., & Daniel J Cua

Nature Medicine 21, 719–729 (2015) doi:10.1038/nm.3895

Familial genetic studies, large-scale genome-wide association studies (GWAS) and next-generation sequencing approaches have highlighted therapeutic indications where IL-23 may contribute to inflammatory disease risk. For example, a psoriasis GWAS reported a protective association for the single-nucleotide polymorphism (SNP) rs11209026 (c.1142G>A; p.Arg381Gln) residing in the IL-23R protein-coding sequence with a modest odds ratio (OR) of 0.67 (P = 7 × 10⁻⁷)²⁵. A GWAS in ileal Crohn’s disease also showed an association with rs11209026 (ref. 26), with the minor glutamine variant protective for Crohn’s disease risk with an OR of 0.26–0.45. The protective association of this variant (and other SNPs in linkage disequilibrium with it) in Crohn’s disease was also shown in ulcerative colitis^{27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41}. The largest meta-analysis of all inflammatory bowel disease GWAS to date (~40,000 cases and ~40,000 controls) indicates that carriage of the glutamine variant gives a modest reduction for disease risk (OR = 0.43, P = 8 × 10⁻¹⁶¹) (ref. 36). The rs11209026 allele is also associated with protection from ankylosing spondylitis^{42, 43}, psoriatic arthritis^{44, 45, 46, 47} and graft-versus-host disease^{48, 49, 50, 51}. Notably, this IL-23R variant has not been reliably associated with other common inflammatory diseases such as rheumatoid arthritis, type 1 diabetes or multiple sclerosis in GWAS powered to detect protective effects similar to those seen in Crohn’s disease and psoriasis^{52, 53, 54}. Although these GWAS findings are compelling, it is important to keep in mind the limitations of such studies; these common loci tend to additively explain only a small proportion of the narrow-sense heritability of disease risk⁵⁵.

Treatment of inflammatory disease with any immunosuppressive agent carries the theoretical risk of impaired host defense responses to pathogens and/or decreased tumor surveillance. Emerging data from human loss-of-function variants and mouse preclinical studies have informed the relative risks of targeting IL-12 and/or IL-23.

The theoretical risk of compromised immunity are of particular concern owing to immune defects discovered in patients with autosomal recessive deficiencies in IL-12/23p40 and IL-12Rβ1 (refs.105,106,107) (Fig. 3). Both deficiencies are genetic etiologies of Mendelian susceptibility to mycobacterial disease (MSMD) (genes involved in MSMD are listed at http://www.biobase-international.com), a rare condition in otherwise healthy patients who have a selective infection predisposition to weakly virulent mycobacteria such as Bacillus Calmette-Guerin (BCG) vaccines, nontuberculous environmental mycobacteria and virulent Mycobacterium tuberculosis (OMIM209950)^{108, 109, 110, 111, 112, 113}. Half of patients with MSMD also have nontyphoidal and, to a lesser extent, typhoidal Salmonella infection.

Owing to the roles of IL-12 and/or IL-23 in host defense and tumor surveillance, particular attention has been focused on infectious disease–related adverse events after anti–IL-12/23p40 treatment in humans. Meta-analysis of briakinumab’s phase 2, phase 3 and open-label extension (OLE) psoriasis databases in 2010 identified 14 cases of candidiasis (including mucocutaneous esophageal and oral candidiasis); no reports of mycobacteria or Salmonella were noted. With regard to the roles of IL-12 and/or IL-23 in tumorigenesis, malignancies were observed at a rate of 1.7 events per 100 patient years (PY), and were cancers commonly seen in the general population.

Concluding remarks

Clinical testing of IL-23 and IL-17A inhibitors have confirmed the initial hypotheses that IL-23–T_H17 pathways are indispensable in promoting immune-mediated diseases, and agents targeting these pathways work particularly well in specific disease settings. However, it is not clear why IL-17A and IL-17RA antagonists work well for psoriasis but exacerbate Crohn’s disease^{95, 96}. It appears that different classes of inhibitor targeting IL-23 and IL-17 pathways may have unique nonoverlapping attributes in different clinical settings. Investigators are still learning where the overlap occurs and what the differences are between targeting IL-23 and targeting other related pathway cytokines. For example, mouse innate lymphoid cells constitutively produce gut protective IL-17A and IL-22 in an IL-23–independent manner. The constitutive IL-17A and IL-22 expression levels generated in response to commensal gut organisms seem to be crucial for maintenance of epithelial barrier function¹⁸⁵ and tight junction formation (D.J.C., unpublished observation). However, high levels of IL-17A and IL-22 induced by IL-23 can be pathogenic during tissue injury responses in the presence of additional inflammatory cytokines such as IL-1, IL-6, GM-CSF and TNF. Therefore, targeting IL-23 via anti–IL-23p19 will partially suppress IL-17A and reduce inflammation, whereas anti–IL-17A therapy will neutralize all protective IL-17A.

The immune system’s function is to maintain balance in the face of insult from external pathogens and accumulation of genetic errors leading to cancer. Disruption of this balance toward immune-exuberance can lead to autoimmunity and immunopathology after infection, whereas inadequate immunity can allow pathogen evasion and breakdown in tumor surveillance. The common thread that connects autoimmunity, infection and cancer is inflammation, and the drivers of inflammation are intercellular messengers that enable cross-talk between immune cells and surrounding stromal tissues. We have underscored the importance of innate cell-produced IL-12 and IL-23 as intermediaries that act on T cells and NK cells to promote inflammation and highlighted that IL-12 and IL-23 have overlapping cellular immune functions. Whereas IL-12 is important in driving STAT1- and STAT4-mediated immune surveillance against specific intracellular pathogens and immunity against neoplasm, IL-23 promotes STAT3-dependent antifungal immunity and drives ‘sterile’ wound-healing responses in psoriatic lesions, which have a gene signature similar to that of many autoinflammatory conditions^{186, 187}. Strikingly, this signature of uncontrolled wound-healing response is also observed in many cancers¹⁸⁸. Although there is insufficient clinical data to determine the long-term safety of IL-23 inhibitors, preclinical models suggest that IL-23 paradoxically promotes tumorigenesis by enhancing skin and mucosal tissue inflammation associated with immune evasion mechanisms.

As the roles of IL-12 and IL-23 were elucidated in preclinical models, there was concern that inhibiting these factors could lead to profound immune suppression. Is it better to target factors capable of regulating a broad range of immune function and may leave patients unprotected against pathogens and cancers or to aim for a restricted pathway that may have limited efficacy for treatment of immune disorders? Although the efficacy and safety profiles of IL-12/23p40, IL-23p19 and IL-17A and IL-17RA therapies become clearer with each clinical trial, the decisions to progress these targets were made many years in advance, on the basis of limited data. Animal studies are important for elucidating the cellular and molecular mechanisms, but clinical testing is required to determine whether a specific disease mechanism also operates in humans. Immunological research is at an inflection point, where the basic concepts of molecular and cellular immunology are being translated into effective therapies for diseases that were considered intractable only a few years ago. Despite the challenges, efforts to translate basic disease mechanisms to the clinic are finally paying off. Although much work remains to be done, the fundamental question of which immune target will benefit which patient population is now being clarified. We optimistically await the answers that will change the lives of patients with serious immune-mediate conditions.

 

Cytokines in Crohn’s colitis.

Sher ME¹, D’Angelo AJ, Stein TA, Bailey B, Burns G, Wise L.
Am J Surg. 1995 Jan; 169(1):133-6.

Increasing evidence points to a pathologic role for cytokines in Crohn’s colitis. Levels of cytokines are increased in diseased segments of colon in Crohn’s colitis, but no one has studied the concentration of cytokines in clinically and histologically nondiseased segments.

Mucosal biopsies were obtained from 7 patients with active segmental Crohn’s colitis and from 7 controls without inflammatory bowel disease. The concentration of Interleukin (IL)-1 beta, IL-2, IL-6, and IL-8 in patients and controls were determined using enzyme linked immunosorbent assay and compared. Histologic sections were also performed to confirm diseased and nondiseased segments of colon.

The concentrations of IL-1 beta, IL-6, and IL-8 were significantly higher in the involved segments of colon (10.3 +/- 4.1, 3.7 +/- 1.0, 34.4 +/- 6.9 picograms [pg] per mg) when compared to controls (1.8 +/- 0.5, 1.1 +/- 0.5, 5.3 +/- 1.0 pg/mg). The concentrations of IL-1 beta, IL-2, and IL-8 (8.5 +/- 2.9, 5.3 +/- 1.2, 26.3 +/- 8.8 pg/mg) in normal appearing segments of colon of patients with Crohn’s colitis were also significantly higher than in controls, whose IL-2 level was 2.0 +/- 0.5 pg/mg. IL-1 beta and IL-8 were significantly more concentrated in both the involved and uninvolved colonic segments of patients with Crohn’s colitis compared to controls. IL-2 and IL-6 were also more concentrated in Crohn’s patients than in controls, but not significantly. The differences in interleukin concentrations between involved and uninvolved segments of colon in patients with segmental Crohn’s colitis were not significant.

Although Crohn’s colitis is often a segmental disease, concentrations of IL-1 beta and IL-8 are increased throughout the entire colon. These observations reinforce the hypothesis that Crohn’s colitis involves the whole colon even when this is not apparent clinically or histologically.

 

Clin Exp Immunol. 2000 May;120(2):241-6.

Increased production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by inflamed mucosa in inflammatory bowel disease.

Louis E¹, Ribbens C, Godon A, Franchimont D, De Groote D, Hardy N, Boniver J, Belaiche J, Malaise M.

Inflammatory bowel diseases (IBD) are characterized by a sustained inflammatory cascade that gives rise to the release of mediators capable of degrading and modifying bowel wall structure. Our aims were (i) to measure the production of matrix metalloproteinase-3 (MMP-3), and its tissue inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by inflamed and uninflamed colonic mucosa in IBD, and (ii) to correlate their production with that of proinflammatory cytokines and the anti-inflammatory cytokine, IL-10. Thirty-eight patients with IBD, including 25 with Crohn’s disease and 13 with ulcerative colitis, were included. Ten controls were also studied. Biopsies were taken from inflamed and uninflamed regions and inflammation was graded both macroscopically and histologically. Organ cultures were performed for 18 h. Tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-1beta, IL-10, MMP-3 and TIMP-1 concentrations were measured using specific immunoassays. The production of both MMP-3 and the TIMP-1 were either undetectable or below the sensitivity of our immunoassay in the vast majority of uninflamed samples either from controls or from those with Crohn’s disease or ulcerative colitis. In inflamed mucosa, the production of these mediators increased significantly both in Crohn’s disease (P < 0.01 and 0.001, respectively) and ulcerative colitis (P < 0.001 and 0.001, respectively). Mediator production in both cases was significantly correlated with the production of proinflammatory cytokines and IL-10, as well as with the degree of macroscopic and microscopic inflammation. Inflamed mucosa of both Crohn’s disease and ulcerative colitis show increased production of both MMP-3 and its tissue inhibitor, which correlates very well with production of IL-1beta, IL-6, TNF-alpha and IL-10.

 

Gut. 1997 Apr;40(4):475-80.

In vitro effects of oxpentifylline on inflammatory cytokine release in patients with inflammatory bowel disease.

Reimund JM¹, Dumont S, Muller CD, Kenney JS, Kedinger M, Baumann R, Poindron P, Duclos B.

Inflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta, have been implicated as primary mediators of intestinal inflammation in inflammatory bowel disease.

To investigate the in vitro effects of oxpentifylline (pentoxifylline; PTX; a phosphodiesterase inhibitor) on inflammatory cytokine production (1) by peripheral mononuclear cells (PBMCs) and (2) by inflamed intestinal mucosa cultures from patients with Crohn’s disease and patients with ulcerative colitis.

PBMCs and mucosal biopsy specimens were cultured for 24 hours in the absence or presence of PTX (up to 100 micrograms/ml), and the secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 determined by enzyme linked immunosorbent assays (ELISAs).

PTX inhibited the release of TNF-alpha by PBMCs from patients with inflammatory bowel disease and the secretion of TNF-alpha and IL-1 beta by organ cultures of inflamed mucosa from the same patients. Secretion of TNF-alpha by PBMCs was inhibited by about 50% at a PTX concentration of 25 micrograms/ml (IC50). PTX was equally potent in cultures from controls, patients with Crohn’s disease, and those with ulcerative colitis. The concentrations of IL-6 and IL-8 were not significantly modified in PBMCs, but IL-6 increased slightly in organ culture supernatants.

PTX or more potent related compounds may represent a new family of cytokine inhibitors, potentially interesting for treatment of inflammatory bowel disease.

 

Inflamm Bowel Dis. 2015 May;21(5):973-84. doi: 10.1097/MIB.0000000000000353.

Neutralizing IL-23 is superior to blocking IL-17 in suppressing intestinal inflammation in a spontaneous murine colitis model.

Wang R¹, Hasnain SZ, Tong H, Das I, Che-Hao Chen A, Oancea I, Proctor M, Florin TH, Eri RD, McGuckin MA.

IL-23/T(H)17 inflammatory responses are regarded as central to the pathogenesis of inflammatory bowel disease, but clinically IL-17A antibodies have shown low efficacy and increased infections in Crohn’s disease. Hence, we decided to closely examine the role of the IL-23/T(H)17 axis in 3 models of colitis.

IL-17A(-/-) and IL-17Ra(-/-) T cells were transferred into Rag1 and RaW mice to assess the role of IL-17A-IL-17Ra signaling in T cells during colitis. In Winnie mice with spontaneous colitis due to an epithelial defect, we studied the progression of colitis in the absence of IL-17A and the efficacy of neutralizing antibodies against the IL-17A or IL-23p19 cytokines.

In transfer colitis models, IL-17A-deficient T cells failed to ameliorate disease, and IL-17Ra-deficient T cells were more colitogenic than wild-type T cells. In Winnie mice with an epithelial defect and spontaneous T(H)17-dominated inflammation, genetic deficiency of IL-17A did not suppress initiation of colitis but limited colitis progression. Furthermore, inhibition of IL-17A by monoclonal antibodies did not reduce colitis severity. In contrast, neutralizing IL-23 using an anti-p19 antibody significantly alleviated both emerging and established colitis, downregulating T(H)17 proinflammatory cytokine expression and diminishing neutrophil infiltration.

Our results support clinical studies showing that IL-17 neutralization is not therapeutic but that targeting IL-23 suppresses intestinal inflammation. Effects of IL-23 distinct from its effects on maturation of IL-17A-producing lymphocytes may underlie the protection from inflammatory bowel disease conveyed by hypomorphic IL-23 receptor polymorphisms and contribute to the efficacy of IL-23 neutralizing antibodies in inflammatory bowel disease.

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IL-17A signaling in colonic epithelial cells inhibits pro-inflammatory cytokine production by enhancing the activity of ERK and PI3K.

Guo X¹, Jiang X², Xiao Y³, Zhou T², Guo Y⁴, Wang R², Zhao Z², Xiao H², Hou C², Ma L³, Lin Y², Lang X², Feng J², Chen G², Shen B², Han G², Li Y².
PLoS One. 2014 Feb 25;9(2):e89714. doi: 10.1371/journal.pone.0089714. eCollection 2014.

Our previous data suggested that IL-17A contributes to the inhibition of Th1 cell function in the gut. However, the underlying mechanisms remain unclear. Here we demonstrate that IL-17A signaling in colonic epithelial cells (CECs) increases TNF-α-induced PI3K-AKT and ERK phosphorylation and inhibits TNF-α induced expression of IL-12P35 and of a Th1 cell chemokine, CXCL11 at mRNA level. In a co-culture system using HT-29 cells and PBMCs, IL-17A inhibited TNF-α-induced IL-12P35 expression by HT-29 cells and led to decreased expression of IFN-γ and T-bet by PBMCs. Finally, adoptive transfer of CECs from mice with Crohn’s Disease (CD) led to an enhanced Th1 cell response and exacerbated colitis in CD mouse recipients. The pathogenic effect of CECs derived from CD mice was reversed by co-administration of recombinant IL-17A. Our data demonstrate a new IL-17A-mediated regulatory mechanism in CD. A better understanding of this pathway might shed new light on the pathogenesis of CD.

 

J Immunol. 2008 Aug 15;181(4):2799-805.

The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex.

Wright JF¹, Bennett F, Li B, Brooks J, Luxenberg DP, Whitters MJ, Tomkinson KN, Fitz LJ, Wolfman NM, Collins M, Dunussi-Joannopoulos K, Chatterjee-Kishore M, Carreno BM.

IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.

 

Am J Surg. 1995 Jan;169(1):133-6.

Cytokines in Crohn’s colitis.

Sher ME¹, D’Angelo AJ, Stein TA, Bailey B, Burns G, Wise L.

Increasing evidence points to a pathologic role for cytokines in Crohn’s colitis. Levels of cytokines are increased in diseased segments of colon in Crohn’s colitis, but no one has studied the concentration of cytokines in clinically and histologically nondiseased segments.

Mucosal biopsies were obtained from 7 patients with active segmental Crohn’s colitis and from 7 controls without inflammatory bowel disease. The concentration of Interleukin (IL)-1 beta, IL-2, IL-6, and IL-8 in patients and controls were determined using enzyme linked immunosorbent assay and compared. Histologic sections were also performed to confirm diseased and nondiseased segments of colon.

The concentrations of IL-1 beta, IL-6, and IL-8 were significantly higher in the involved segments of colon (10.3 +/- 4.1, 3.7 +/- 1.0, 34.4 +/- 6.9 picograms [pg] per mg) when compared to controls (1.8 +/- 0.5, 1.1 +/- 0.5, 5.3 +/- 1.0 pg/mg). The concentrations of IL-1 beta, IL-2, and IL-8 (8.5 +/- 2.9, 5.3 +/- 1.2, 26.3 +/- 8.8 pg/mg) in normal appearing segments of colon of patients with Crohn’s colitis were also significantly higher than in controls, whose IL-2 level was 2.0 +/- 0.5 pg/mg. IL-1 beta and IL-8 were significantly more concentrated in both the involved and uninvolved colonic segments of patients with Crohn’s colitis compared to controls. IL-2 and IL-6 were also more concentrated in Crohn’s patients than in controls, but not significantly. The differences in interleukin concentrations between involved and uninvolved segments of colon in patients with segmental Crohn’s colitis were not significant.

Although Crohn’s colitis is often a segmental disease, concentrations of IL-1 beta and IL-8 are increased throughout the entire colon. These observations reinforce the hypothesis that Crohn’s colitis involves the whole colon even when this is not apparent clinically or histologically.

 

Protein Pept Lett. 2015;22(7):570-8.

An Overview of Interleukin-17A and Interleukin-17 Receptor A Structure, Interaction and Signaling.

Krstic J, Obradovic H, Kukolj T, Mojsilovic S, Okic-Dordevic I, Bugarski D, Santibanez JF¹.

Interleukin-17A (IL-17A) and its receptor (IL-17RA) are prototype members of IL-17 ligand/receptor family firstly identified in CD4+ T cells, which comprises six ligands (IL-17A to IL- 17F) and five receptors (IL-17RA to IL-17RE). IL-17A is predominantly secreted by T helper 17 (Th17) cells, and plays important roles in the development of autoimmune and inflammatory diseases. IL-17RA is widely expressed, and forms a complex with IL-17RC. Binding of IL-17A to this receptor complex triggers the activation of several intracellular signaling pathways. In this review, we aimed to summarize literature data about molecular features of IL-17A and IL-17RA from gene to mature protein. We are also providing insight into regulatory mechanisms, protein structural conformation, including ligand-receptor interaction, and an overview of signaling pathways. Our aim was to compile the data on molecular characteristics of IL-17A and IL-17RA which may help in the understanding of their functions in health and disease.

 

Gut. 2014 Dec;63(12):1902-12. doi: 10.1136/gutjnl-2013-305632. Epub 2014 Feb 17.

Involvement of interleukin-17A-induced expression of heat shock protein 47 in intestinal fibrosis in Crohn’s disease.

Honzawa Y¹, Nakase H¹, Shiokawa M¹, Yoshino T¹, Imaeda H², Matsuura M¹, Kodama Y¹, Ikeuchi H³, Andoh A², Sakai Y⁴, Nagata K⁵, Chiba T¹.

Intestinal fibrosis is a clinically important issue in Crohn’s disease (CD). Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone involved in fibrotic diseases. The molecular mechanisms of HSP47 induction in intestinal fibrosis related to CD, however, remain unclear. Here we investigated the role of interleukin (IL)-17A-induced HSP47 expression in intestinal fibrosis in CD.

Expressions of HSP47 and IL-17A in the intestinal tissues of patients with IBD were determined. HSP47 and collagen I expressions were assessed in intestinal subepithelial myofibroblasts (ISEMFs) isolated from patients with IBD and CCD-18Co cells treated with IL-17A. We examined the role of HSP47 in IL-17A-induced collagen I expression by administration of short hairpin RNA (shRNA) to HSP47 and investigated signalling pathways of IL-17A-induced HSP47 expression using specific inhibitors in CCD-18Co cells.

Gene expressions of HSP47 and IL-17A were significantly elevated in the intestinal tissues of patients with active CD. Immunohistochemistry revealed HSP47 was expressed in α-smooth muscle actin (α-SMA)-positive cells and the number of HSP47-positive cells was significantly increased in the intestinal tissues of patients with active CD. IL-17A enhanced HSP47 and collagen I expressions in ISEMFs and CCD-18Co cells. Knockdown of HSP47 in these cells resulted in the inhibition of IL-17A-induced collagen I expression, and analysis of IL-17A signalling pathways revealed the involvement of c-Jun N-terminal kinase in IL-17A-induced HSP47 expression.

IL-17A-induced HSP47 expression is involved in collagen I expression in ISEMFs, which might contribute to intestinal fibrosis in CD.

 

 

Biochem Biophys Res Commun. 2011 Jan 14;404(2):599-604. doi: 10.1016/j.bbrc.2010.12.006. Epub 2010 Dec 6.

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis.

Kitamura H¹, Yamamoto S, Nakase H, Matsuura M, Honzawa Y, Matsumura K, Takeda Y, Uza N, Nagata K, Chiba T.

Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis.

HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo.

Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice.

Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.

 

Kidney Int. 2003 Sep;64(3):887-96.

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats.

Nishino T¹, Miyazaki M, Abe K, Furusu A, Mishima Y, Harada T, Ozono Y, Koji T, Kohno S.

Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG).

HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry.

In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels.

Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.

 

Trends Mol Med. 2007 Feb;13(2):45-53. Epub 2006 Dec 13.

The collagen-specific molecular chaperone HSP47: is there a role in fibrosis?

Taguchi T¹, Razzaque MS.

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. Recent studies have shown a close association between increased expression of HSP47 and excessive accumulation of collagens in scar tissues of various human and experimental fibrotic diseases. It is presumed that the increased levels of HSP47 in fibrotic diseases assist in excessive assembly and intracellular processing of procollagen molecules and, thereby, contribute to the formation of fibrotic lesions. Studies have also shown that suppression of HSP47 expression can reduce accumulation of collagens to delay the progression of fibrotic diseases in experimental animal models. Because HSP47 is a specific chaperone for collagen synthesis, it provides a selective target to manipulate collagen production, a phenomenon that might have enormous clinical impact in controlling a wide range of fibrotic diseases. Here, we outline the fibrogenic role of HSP47 and discuss the potential usefulness of HSP47 as an anti-fibrotic therapeutic target.

 

Arthritis Rheum. 2013 May;65(5):1347-56. doi: 10.1002/art.37860.

Interleukin-17A+ cell counts are increased in systemic sclerosis skin and their number is inversely correlated with the extent of skin involvement.

Truchetet ME¹, Brembilla NC, Montanari E, Lonati P, Raschi E, Zeni S, Fontao L, Meroni PL, Chizzolini C.

Levels of interleukin-17A (IL-17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL-17A-producing cells are present in affected SSc skin, and whether IL-17A exerts a role in the transdifferentiation of myofibroblasts.

Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo, including IL-17A+, IL-4+, CD3+, tryptase-positive, α-smooth muscle actin (α-SMA)-positive, myeloperoxidase-positive, and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples, and quantitative polymerase chain reaction, Western blotting, and solid-phase assays were used to quantify α-SMA, type I collagen, and matrix metalloproteinase 1 (MMP-1) production by the cultured fibroblasts.

IL-17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase-positive mast cells in the production of IL-17A was observed. Fibroblasts positive for α-SMA were found adjacent to IL-17A+ cells, but not IL-4+ cells. However, IL-17A did not induce α-SMA expression in cultured fibroblasts. In the presence of IL-17A, the α-SMA expression induced in response to transforming growth factor β was decreased, while MMP-1 production was directly enhanced. Furthermore, the frequency of IL-17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score).

IL-17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL-17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL-17A has a direct negative-regulatory role in the development of dermal fibrosis in humans.

 

Gut. 2014 Dec;63(12):1902-12. doi: 10.1136/gutjnl-2013-305632. Epub 2014 Feb 17.

Involvement of interleukin-17A-induced expression of heat shock protein 47 in intestinal fibrosis in Crohn’s disease.

Honzawa Y¹, Nakase H¹, Shiokawa M¹, Yoshino T¹, Imaeda H², Matsuura M¹, Kodama Y¹, Ikeuchi H³, Andoh A², Sakai Y⁴, Nagata K⁵, Chiba T¹.

Intestinal fibrosis is a clinically important issue in Crohn’s disease (CD). Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone involved in fibrotic diseases. The molecular mechanisms of HSP47 induction in intestinal fibrosis related to CD, however, remain unclear. Here we investigated the role of interleukin (IL)-17A-induced HSP47 expression in intestinal fibrosis in CD.

Expressions of HSP47 and IL-17A in the intestinal tissues of patients with IBD were determined. HSP47 and collagen I expressions were assessed in intestinal subepithelial myofibroblasts (ISEMFs) isolated from patients with IBD and CCD-18Co cells treated with IL-17A. We examined the role of HSP47 in IL-17A-induced collagen I expression by administration of short hairpin RNA (shRNA) to HSP47 and investigated signalling pathways of IL-17A-induced HSP47 expression using specific inhibitors in CCD-18Co cells.

Gene expressions of HSP47 and IL-17A were significantly elevated in the intestinal tissues of patients with active CD. Immunohistochemistry revealed HSP47 was expressed in α-smooth muscle actin (α-SMA)-positive cells and the number of HSP47-positive cells was significantly increased in the intestinal tissues of patients with active CD. IL-17A enhanced HSP47 and collagen I expressions in ISEMFs and CCD-18Co cells. Knockdown of HSP47 in these cells resulted in the inhibition of IL-17A-induced collagen I expression, and analysis of IL-17A signalling pathways revealed the involvement of c-Jun N-terminal kinase in IL-17A-induced HSP47 expression.

IL-17A-induced HSP47 expression is involved in collagen I expression in ISEMFs, which might contribute to intestinal fibrosis in CD.

 

Kidney Int. 2003 Sep;64(3):887-96.

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats.

Nishino T¹, Miyazaki M, Abe K, Furusu A, Mishima Y, Harada T, Ozono Y, Koji T, Kohno S.

Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG).

HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry.

In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels.

Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.

 

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2007 Aug;32(4):650-5.

[Effect of heat shock protein 47 on the expression of collagen I induced by TGF-beta(1) in hepatic stellate cell-T6 cells].

[Article in Chinese]

Li Y¹, Wu W, Jiang YF, Wang KK.

To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells.

We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay.

Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis.

Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis

 

Fibrogenesis Tissue Repair. 2013 Jul 8;6(1):13. doi: 10.1186/1755-1536-6-13.

The role of interleukin 17 in Crohn’s disease-associated intestinal fibrosis.

Biancheri P¹, Pender SL, Ammoscato F, Giuffrida P, Sampietro G, Ardizzone S, Ghanbari A, Curciarello R, Pasini A, Monteleone G,Corazza GR, Macdonald TT, Di Sabatino A.

Interleukin (IL)-17A and IL-17E (also known as IL-25) have been implicated in fibrosis in various tissues. However, the role of these cytokines in the development of intestinal strictures in Crohn’s disease (CD) has not been explored. We investigated the levels of IL-17A and IL-17E and their receptors in CD strictured and non-strictured gut, and the effects of IL-17A and IL-17E on CD myofibroblasts.

IL-17A was significantly overexpressed in strictured compared with non-strictured CD tissues, whereas no significant difference was found in the expression of IL-17E or IL-17A and IL-17E receptors (IL-17RC and IL-17RB, respectively) in strictured and non-strictured CD areas. Strictured CD explants released significantly higher amounts of IL-17A than non-strictured explants, whereas no difference was found as for IL-17E, IL-6, or tumor necrosis factor-α production. IL-17A, but not IL-17E, significantly inhibited myofibroblast migration, and also significantly upregulated matrix metalloproteinase (MMP)-3, MMP-12, tissue inhibitor of metalloproteinase-1 and collagen production by myofibroblasts from strictured CD tissues.

Our results suggest that IL-17A, but not IL-17E, is pro-fibrotic in CD. Further studies are needed to clarify whether the therapeutic blockade of IL-17A through the anti-IL-17A monoclonal antibody secukinumab is able to counteract the fibrogenic process in CD.

 

Int J Colorectal Dis. 2013 Jul;28(7):915-24. doi: 10.1007/s00384-012-1632-2. Epub 2012 Dec 28.

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn’s disease patients.

Romagnoli C¹, Marcucci T, Picariello L, Tonelli F, Vincenzini MT, Iantomasi T.

Intestinal subepithelial myofibroblasts (ISEMFs)(1) are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn’s disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα).

ISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit.

In cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients’ MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated.

For the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.

 

BMC Pulm Med. 2012 Jun 13;12:24. doi: 10.1186/1471-2466-12-24.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells.

Hisatomi K¹, Mukae H, Sakamoto N, Ishimatsu Y, Kakugawa T, Hara S, Fujita H, Nakamichi S, Oku H, Urata Y, Kubota H, Nagata K,Kohno S.

Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.

TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.

We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.