Including Inflammatory Bowel Diseases Patients Research Articles

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified.In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Abnormal Genetic and Epigenetic Changes in Signal Transducer and Activator of Transcription 4 in the Pathogenesis of Inflammatory Bowel Diseases

Changes in the expression of signal transducer and activator of transcription 4 (STAT4) contribute to the development of a variety of autoimmune diseases including inflammatory bowel diseases (IBDs). Moreover, epigenetic modifications, including DNA methylation, are considered a basis for differentiation of T helper cells and regulation of cytokines. In this study, we investigated the methylation status of STAT4 gene in IBD patients and the associations between its genetic and epigenetic alterations in IBD patients. Blood and colonic mucosa samples were obtained from Korean patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated, and total RNA and genomic DNA were isolated from the PBMCs and colon mucosa tissues. The mRNA level and DNA methylation status of the promoter were determined by real-time RT-PCR and pyrosequencing, respectively. The chosen SNPs (rs11889341, rs7574865, rs8179673, rs6752770, rs925847, rs10168266, rs10181656, and rs11685878) were genotyped using the TaqMan nuclease assay. Elevated expression of STAT4 was observed in the colonic mucosa and PBMCs of IBD patients. IBD patients showed a lower degree of methylation of the STAT4 promoter than did the healthy controls. Moreover, a significant correlation between risk alleles and methylation status at -172 of the STAT4 promoter was observed, and mRNA levels of STAT4 in IBD patients were correlated inversely with the T-risk allele (rs7574865). Our data demonstrated that the DNA methylation status of STAT4 is associated with genetic polymorphisms, providing insights into the interactions between genetic and epigenetic aberrances in STAT4 that contribute to the development of IBD.

Efficacy of a potent and safe vitamin D receptor agonist for the treatment of inflammatory bowel disease

Deficiency in 1α,25-dihydroxyvitamin D 3 (1,25D 3) has been suggested as an important environmental factor for immuno-mediated disorders including inflammatory bowel diseases (IBD), comprising Crohn's disease and ulcerative colitis, both characterized by chronic intestinal inflammation. Administration of vitamin D receptor (VDR) agonists can ameliorate spontaneous and induced animal models of colitis, but hypercalcemia is a dose-limiting adverse event. Previous work in our laboratory has identified 1α,25(OH) 2-16-ene-20-cyclopropyl-vitamin D 3 (BXL-62) as a potent anti-inflammatory VDR agonist with a low calcemic activity. In the present study, we confirm the marked anti-inflammatory properties of BXL-62 and show its capacity to induce VDR primary response genes, like CYP24A1 and CAMP, at lower concentrations than 1,25D 3, in PBMCs from IBD patients. Its higher anti-inflammatory potency compared to 1,25D 3 was demonstrated by the significantly more potent inhibition in PBMCs and in lymphocyte-enriched lamina propria mononuclear cells of the pro-inflammatory cytokines TNF-α, IL-12/23p40, IL-6 and IFN-γ, both at mRNA and protein level. The therapeutic efficacy of intra-rectal administration of BXL-62 in experimental IBD is shown by its beneficial effects, significantly higher than 1,25D 3, to induce recovery of clinical symptoms of colitis at normocalcemic doses in mice undergoing dextran sodium sulfate-induced colitis. These results confirm the therapeutic efficacy of VDR agonists in experimental colitis, and suggest BXL-62 as a promising compound for IBD treatment.

Helminth infections and intestinal inflammation

Evidence from epidemiological studies indicates an inverse correlation between the incidence of certain immune-mediated diseases, including inflammatory bowel diseases (IBD), and exposure to helminths. Helminth parasites are the classic inducers of Th2 responses. The Th2-polarized T cell response driven by helminth infection has been linked to the attenuation of some damaging Th1 driven inflammatory responses, preventing some Th1-mediated autoimmune diseases in the host, including experimentally induced colitis. Helminth parasites (the porcine whipworm, Trichuris suis) have been tested for treating IBD patients, resulting in clinical amelioration of the disease. As a result, there is a great deal of interest in the research community in exploring the therapeutic use of helminth parasites for the control of immune-mediated diseases, including IBD. However, recent studies have provided evidence indicating the exacerbating effects of helminths on bacterial as well as non-infectious colitis in animal models. Therefore, a better understanding of mechanisms by which helminths modulate host immune responses in the gut may reveal novel, more effective and safer approaches to helminth-based therapy of IBD.