The contribution of T cell-derived cytokines and proteases to chronic inflammation in the human intestine

This Thesis explores aspects of disease pathogenesis in Crohn’s disease (CD) and ulcerative colitis (UC), investigates mechanisms of responsiveness to biologic treatment in inflammatory bowel disease (IBD), and describes the clinical and immunologic phenotype associated with a homozygous deletion in a disintegrin and a metalloproteinase (ADAM)17 gene. The role of interleukin (IL)-17A, IL-17E and IL-13 in human intestinal inflammation is not clear. IL-13 plays an important role in experimental colitis and in intestinal experimental fibrosis. However, contrasting observations exist on the levels and the role of IL-13 in inflamed IBD mucosa, and limited information is available on the role of IL-13 in CD intestinal fibrosis. We observed that IL-13 is not up-regulated in UC intestinal mucosa, and that it is unlikely to play a functional role in the mucosal pro-inflammatory response in the majority of patients with UC. Conversely, IL-17A is up-regulated in fibrostenosing CD intestine, and may contribute to intestinal fibrosis in CD. Our results indicate that IL-17E and IL-13 are not up-regulated in CD intestinal strictures, and are unlikely to play a role in intestinal fibrosis in CD. A considerable proportion of IBD patients do not respond to anti-tumour necrosis factor (TNF)- agents. Anti-TNF- agents exert their action in inflamed tissues, rich in matrix metalloproteinase (MMP)-3 and MMP-12, which in turn can degrade immunoglobulin (Ig)G1. We observed that MMP-3, MMP-12, and protein extracts from inflamed IBD mucosa, but not MMP-9, degrade the anti-TNF- agents infliximab, adalimumab and etanercept, however etanercept shows a higher susceptibility than infliximab and adalimumab. We also observed that a subgroup of IBD patients who did not respond to anti-TNF- agents have particularly high serum levels of MMP-3-/MMP-12-cleaved endogenous IgG and anti-hinge autoantibodies compared to IBD patients who subsequently responded to biologic therapy. 8 Finally, we observed that homozygous deletion in ADAM17 in humans is associated with a complex, neonatal-onset, multi-organ syndrome affecting mainly the skin, the intestine, and the cardiovascular system. In this condition, ADAM17 expression is down-regulated in the skin and in the duodenum, and soluble TNF- release by peripheral blood mononuclear cells (PBMCs) is substantially impaired. These results underline the heterogeneity characterising chronic intestinal inflammation, and may form the basis for subsequent studies with the aim to identify accurate serum biomarkers of disease progression and responsiveness to biologic therapy, and ultimately to develop effective strategies of patient stratification in IBD

Epithelial down-regulation of the miR-200 family in fibrostenosing Crohn's disease is associated with features of epithelial to mesenchymal transition

Intestinal mesenchymal cells deposit extracellular matrix in fibrotic Crohn's disease (CD). The contribution of epithelial to mesenchymal transition (EMT) to the mesenchymal cell pool in CD fibrosis remains obscure. The miR‐200 family regulates fibrosis‐related EMT in organs other than the gut. E‐cadherin, cytokeratin‐18 and vimentin expression was assessed using immunohistochemistry on paired strictured (SCD) and non‐strictured (NSCD) ileal CD resections and correlated with fibrosis grade. MiR‐200 expression was measured in paired SCD and NSCD tissue compartments using laser capture microdissection and RT‐qPCR. Serum miR‐200 expression was also measured in healthy controls and CD patients with stricturing and non‐stricturing phenotypes. Extra‐epithelial cytokeratin‐18 staining and vimentin‐positive epithelial staining were significantly greater in SCD samples (P = 0.04 and P = 0.03, respectively). Cytokeratin‐18 staining correlated positively with subserosal fibrosis (P < 0.001). Four miR‐200 family members were down‐regulated in fresh SCD samples (miR‐141, P = 0.002; miR‐200a, P = 0.002; miR‐200c, P = 0.001; miR‐429; P = 0.004); miR‐200 down‐regulation in SCD tissue was localised to the epithelium (P = 0.001‐0.015). The miR‐200 target ZEB1 was up‐regulated in SCD samples (P = 0.035). No difference in serum expression between patient groups was observed. Together, these observations suggest the presence of EMT in CD strictures and implicate the miR‐200 family as regulators. Functional studies to prove this relationship are now warranted

Fever During Anti-integrin Therapy: New Immunodeficiency

Background: The causes of inflammatory bowel disease (IBD) have not yet been clearly elucidated, but it is known that genetic susceptibility, altered gut microbiota and environmental factors are all involved, and that a combination of these factors causes an inappropriate immune response, resulting in impaired intestinal barrier function. With regard to the treatment of IBD, the use of conventional immunosuppressive drugs has been complemented by more specific therapeutic agents, including biological drugs. Systemic immune suppression is a risk factor for cytomegalovirus (CMV) infection, which is associated with considerable morbidity and mortality in immunocompromised hosts. Case Report: A 33-year-old male patient was admitted to our medical unit complaining of a 10-day history of fever, fatigue and headache. He had been suffering from ulcerative colitis and primary sclerosing cholangitis for five years and was currently being treated with azathioprine and vedolizumab. In the past he had already taken infliximab, adalimumab and golimumab without any clinical response. After the exclusion of systemic infectious diseases, his serology was consistent with a primary CMV infection. This was successfully treated with intravenous ganciclovir therapy. Conclusion: Vedolizumab is an anti-integrin biological agent approved for IBD treatment. Its gut-selective mechanism of action would appear to increase its safety profile, however data on this are still limited. Moreover, it should always be remembered that IBD patients have an increased risk of CMV infection, both primary and reactivation, because of their concurrent immunosuppression

Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis

Background: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition. Methods: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa. Results: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays. Conclusions: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease

Proteolytic cleavage and loss of function of biologic agents that neutralize tumor necrosis factor in the mucosa of patients with inflammatory bowel disease

BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti–tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn’s disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNFneutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn’s disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn’s disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab’)2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents

Abnormal thymic stromal lymphopoietin expression in the duodenal mucosa of patients with coeliac disease

OBJECTIVE: The short isoform of thymic stromal lymphopoietin (TSLP), a cytokine constitutively expressed by epithelial cells, is crucial in preserving immune tolerance in the gut. TSLP deficiency has been implicated in sustaining intestinal damage in Crohn's disease. We explored mucosal TSLP expression and function in refractory and uncomplicated coeliac disease (CD), a T-cell-mediated enteropathy induced by gluten in genetically susceptible individuals. DESIGN: TSLP isoforms-long and short-and receptors-TSLPR and interleukin (IL)-7Rα-were assessed by immunofluorescence, immunoblotting and qRT-PCR in the duodenum of untreated, treated, potential and refractory patients with CD. The ability of the serine protease furin or CD biopsy supernatants to cleave TSLP was evaluated by immunoblotting. The production of interferon (IFN)-γ and IL-8 by untreated CD biopsies cultured ex vivo with TSLP isoforms was also assessed. RESULTS: Mucosal TSLP, but not TSLPR and IL-7Rα, was reduced in untreated CD and refractory CD in comparison to treated CD, potential CD and controls. Transcripts of both TSLP isoforms were decreased in active CD mucosa. Furin, which was overexpressed in active CD biopsies, was able to cleave TSLP in vitro. Accordingly, refractory and untreated CD supernatants showed higher TSLP-degrading capacity in comparison to treated CD and control supernatants. In our ex vivo model, both TSLP isoforms significantly downregulated IFN-γ and IL-8 production by untreated CD biopsies. CONCLUSIONS: Reduced mucosal TSLP expression may contribute to intestinal damage in refractory and untreated CD. Further studies are needed to verify whether restoring TSLP might be therapeutically useful especially in refractory patients with CD

Interleukin 6 increases production of cytokines by colonic innate lymphoid cells in mice and patients with chronic intestinal inflammation

Background & Aims: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods: ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. Conclusions: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells

Proteases and small intestinal barrier function in health and disease

PURPOSE OF REVIEW: To summarize the recent knowledge regarding intestinal proteases and the gut barrier. RECENT FINDINGS: It is now well established that intestinal proteases, such as matrix metalloproteinase (MMP)-1, MMP-3, MMP-10 and MMP-12, are key players in the development of ulcers in inflammatory bowel disease, have direct effects on epithelial barrier function and are involved in epithelial restitution. However, more recent work has suggested that the membrane-anchored epithelial cell serine protease matriptase is critical in maintaining the gut barrier, and roles have also been described for elastase, MMP-13, gelatinases, mast cell proteases and proteases derived from parasites and gut bacteria. Interestingly, epithelial proteases often co-localize with epithelial adherens junctions, and nonepithelial-derived proteases have junctional proteins as targets. SUMMARY: The role of proteases in controlling normal barrier function in the gut is now becoming very clear, to go alongside their role in intestinal inflammation