Takehide Fujimoto

Decreased abundance of Faecalibacterium prausnitzii in the gut microbiota of Crohn's disease.

Dysbiosis is thought to be relevant to the etiology and pathogenesis of Crohns disease (CD). In this study, we investigated the abundance of Faecalibacterium prausnitzii, as well as Bilophila wadsworthia, in the gut microbiota of Japanese CD patients.

Epithelial expression of interleukin-37b in inflammatory bowel disease

Interleukin (IL)‐37 is a member of the IL‐1 cytokine family. We investigated IL‐37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL‐37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL‐37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL‐37b mRNA and protein expression were determined by real time‐polymerase chain reaction (PCR) and Western blotting, respectively. IL‐37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL‐37b expression was increased markedly. In ulcerative colitis (UC) and Crohns disease (CD) patients, IL‐37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL‐37b mRNA and protein was enhanced by tumour necrosis factor (TNF)‐α. This IL‐37b induction by TNF‐α was mediated by nuclear factor (NF)‐κB and activator protein (AP)‐1 activation. Furthermore, IL‐37b inhibited TNF‐α‐induced interferon‐γ‐inducible protein (IP)‐10 expression significantly in human colonic SEMFs. Epithelial IL‐37b expression was increased in IBD patients, especially UC patients. IL‐37b may be involved in the pathophysiology of IBD as an anti‐inflammatory cytokine and an inhibitor of both innate and acquired immune responses.

Terminal Restriction Fragment Length Polymorphism Analysis of the Gut Microbiota Profiles of Pediatric Patients with Inflammatory Bowel Disease

Background/Aim: We analyzed the fecal microbiota profiles of pediatric patients with inflammatory bowel disease. Method: Terminal restriction fragment length polymorphism analysis was performed in 10 fecal samples from Crohn’s disease (CD), 14 samples from ulcerative colitis (UC) and 27 samples from healthy individuals. The bacterial diversity was evaluated by the Shannon diversity index. Result: In CD patients, a setting of similarity generated three major clusters. The majority of CD patients were classified into CD clusters I and II (9 out of 10), but the majority of healthy individuals (21 of 27) were classified into CD cluster III. In UC patients, a setting of similarity also generated three major UC clusters, but each cluster was not characteristic for UC patients or healthy individuals. The changes in simulated bacterial composition indicated that the class Clostridia, including the genus Faecalibacterium, was significantly decreased in CD patients as compared to UC patients and/or healthy individuals. The genus Bacteroides was also decreased as compared to healthy individuals. The bacterial diversity measured by the Shannon diversity index was significantly reduced in CD patients as compared to healthy individuals. Conclusion: The gut microbiota profile of pediatric CD patients was different from that of healthy children.

Tacrolimus (FK506) suppresses TNF-α-induced CCL2 (MCP-1) and CXCL10 (IP-10) expression via the inhibition of p38 MAP kinase activation in human colonic myofibroblasts.

In order to investigate the molecular mechanisms underlying the immunosuppressive effects of tacrolimus (FK506) on intestinal inflammation, we examined whether FK506 effects cytokine/chemokine secretion in human colonic myofibroblasts. Human colonic myofibroblasts were isolated from normal human colonic tissue. The mRNA and protein expression for human CCL2 and CXCL10 were analyzed by real-time PCR and ELISA, respectively. p38 MAP kinase activation was evaluated by western blotting. Tacrolimus (1 µM) suppressed tumor necrosis factor (TNF)-α-induced CCL2 and CXCL10 mRNA expression, but did not modulate TNF-α-induced interleukin (IL)-6 or CXCL8 mRNA expression. Dose-dependent, inhibitory effects of tacrolimus on CCL2 and CXCL10 expression were observed at the mRNA and protein levels. Significant inhibitory effects of tacrolimus were observed at concentrations as low as 0.5 µM for CCL2 and 0.1 µM for CXCL10, respectively. TNF-α-induced CCL2 and CXCL10 expression depended on p38 MAP kinase activation, and tacrolimus strongly inhibited the TNF-α-induced phosphorylation of p38 MAP kinase. Tacrolimus did not affect interferon (IFN)-γ-induced signaling transducer and activator of transcription (STAT)-1 phosphorylation, nor did it modulate CXCL10 mRNA and protein expression. In conclusion, tacrolimus suppressed CCL2 and CXCL10 expression in human colonic myofibroblasts. These inhibitory effects of tacrolimus may play key roles in the therapeutic effects of colonic inflammation in inflammatory bowel disease (IBD) patients.

Regulation of eotaxin-3/CC chemokine ligand 26 expression by T helper type 2 cytokines in human colonic myofibroblasts.

Eotaxins induce the trafficking of eosinophils to the sites of inflammation via CC chemokine receptor 3 (CCR3). In this study, we investigated eotaxin‐3/CC chemokine ligand 26 (CCL26) expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for eotaxin‐3 expression in human colonic myofibroblasts. Eotaxin‐3 mRNA and protein expression was evaluated by real time‐polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA), respectively. Eotaxin‐3 mRNA expression was elevated significantly in the active lesions of ulcerative colitis (UC) patients. Significant elevations were also observed in the active lesions of Crohns disease (CD) patients, but this was significantly lower than that detected in the active UC lesions. There were no significant increases in the inactive lesions of UC or CD patients. Colonic myofibroblasts were identified as a major source of eotaxin‐3 in the colonic mucosa, and interleukin (IL)‐4 and IL‐13 enhanced eotaxin‐3 mRNA and protein expression significantly in these cells. There was a significant positive correlation between mucosal eotaxin‐3 and IL‐4 mRNA expression in the active lesions of IBD patients. The IL‐4‐ and IL‐13‐induced eotaxin‐3 mRNA expression was regulated by the signal transducer and activator of transcription‐6 (STAT‐6) and suppressor of cytokine signalling (SOCS)1‐mediated pathways. Interferon (IFN)‐γ acts as a negative regulator on the IL‐4‐ and IL‐13‐induced eotaxin‐3 expression via STAT‐1 activation. Eotaxin‐3 expression was elevated specifically in the active lesions of IBD, in particular UC. Eotaxin‐3 derived from colonic myofibroblasts may play an important role in the pathophysiology of UC.

Terminal-Restriction Fragment Length Polymorphism (T-RFLP) Analysis for Changes in the Gut Microbiota Profiles of Indomethacin- and Rebamipide-Treated Mice

Background/Aims: We investigated the effects of indomethacin and rebamipide on the gut microbiota profiles using terminal restriction fragment polymorphism (T-RFLP) analysis. Materials and Methods: Female C57BL/6J mice were given indomethacin (10 mg/kg, s.c.) once a day and 2.5 mg rebamipide orally 3 times a day. After 7 days, they were sacrificed, and luminal contents were obtained from the ileum and cecum. The gut microbiota communities were analyzed by T-RFLP analysis with BslI digestion. Results: T-RFLP analyses showed that rebamipide and indomethacin had no significant effects on the gut microbiota profiles in the ileum and cecum. In contrast, the combination of rebamipide + indomethacin induced a significant change in the gut microbiota. The changes in the microbiota composition induced by the combination of rebamipide + indomethacin were characterized by the increase in the orders Bifidobacteriales and Lactobacillales, the genera Bacteroides and Prevotella and the family Clostridiaceae. The diversity of the gut microbiota community generated by the combination of rebamipide + indomethacin was significantly higher than those induced by either rebamipide or indomethacin alone. Conclusion: The combination of rebamipide + indomethacin induces remarkable changes in the gut microbiota composition and diversity. The clinical activity of rebamipide on nonsteroidal anti-inflammatory drug-induced intestinal injury may be exerted through a modulation of the gut microbiota.

Neutralization of complement component C5 ameliorates the development of dextran sulfate sodium (DSS)-colitis in mice

The complement system is a potent effector of innate immunity. To elucidate the pathophysiological role of the complement system in inflammatory bowel disease, we evaluated the effects of anti-C5 antibodies on the development of dextran sulfate sodium-induced colitis in mice. Dextran sulfate sodium-colitis was induced in BALB/c mice with intraperitoneal administrations of anti-C5 antibodies (1 µg/body) every 48 h. Tissue samples were evaluated by standard histological procedures. The mucosal mRNA expression of the inflammatory cytokines was analyzed by real-time PCR. Body weight loss in the mice was completely blocked by the administration of anti-C5 antibody. The disease activity index was significantly lower in the anti-C5 antibody-treated mice than the dextran sulfate sodium mice. The colonic weight/length ratio, histological colitis score and mucosal myeloperoxidase activity were significantly lower in the anti-C5 antibody-treated mice than the dextran sodium sulfate mice. The administration of the anti-C5 antibody significantly reduced the mucosal expression of mRNAs for tumor necrosis factor-α, interleukin-1β and interleukin-6. In conclusion, the complement system plays a role in the development of dextran sodium sulfate-induced experimental colitis.

Interleukin-36α Induces Inflammatory Mediators From Human Pancreatic Myofibroblasts Via a MyD88 Dependent Pathway

Objectives Interleukin-36 (IL-36) is a recently described proinflammatory cytokine, characterized by the induction of inflammatory mediators. In the present study, we investigated the biological activity and the signal transduction of IL-36&agr; in human pancreatic myofibroblasts. Methods The mRNA and protein expression of inflammatory mediators was evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of IL-36&agr; and its receptor in the pancreatic tissue was evaluated using immunohistochemical technique. Intracellular signaling pathways were evaluated using immunoblotting and specific small interference RNA–transfected cells. Results Interleukin-36&agr; and its receptor complex IL-36R/IL-1RAcP were detected in fibrotic tissue of chronic pancreatitis. Interleukin-36&agr; dose- and time-dependently induced the mRNA expression and protein secretion of CXCL1, CXCL8, MMP-1, and MMP-3 from human pancreatic myofibroblasts. Interleukin-36&agr; assembled MyD88 adaptor proteins (MyD88, TRAF6, IRAK1, and TAK1) into a complex. Furthermore, IL-36&agr; induced the phosphorylation of mitogen-activated protein kinases and the activation of nuclear factor &kgr;B and activator protein 1. Mitogen-activated protein kinase inhibitors and small interference RNAs specific for nuclear factor &kgr;B and activator protein 1 significantly suppressed the protein secretion of inflammatory mediators induced by IL-36&agr; stimulation. Conclusions It was suggested that IL-36&agr; plays an important role in the pathophysiology of inflammation and fibrosis in the pancreas via an autocrine function.

Eotaxin-3 (ccl26) Expression in Human Pancreatic Myofibroblasts

Objectives Eosinophil infiltration is a histological feature of autoimmune pancreatitis (AIP). However, little is known about the mechanisms underlying eosinophilic infiltration. In this study, we aimed to investigate the expression of the eosinophil chemotactic protein, eotaxin-3, in human pancreatic myofibroblasts. Methods Enzyme-linked immunosorbent assays and quantitative polymerase chain reactions were used to quantify eotaxin-3 protein and messenger RNA levels, respectively. Results Eotaxin-3 expression was induced by T helper type 2 cytokines, interleukin-4 (IL-4) and IL-13, in time- and dose-dependent manners. Both IL-4 and IL-13 induced the rapid phosphorylation of STAT6 (signal transducer and activator of transcription 6), and STAT6-specific small interfering RNA significantly blocked IL-4– and IL-13–induced eotaxin-3 expression, indicating involvement of STAT6 signaling pathways in eotaxin-3 induction. In contrast, SOCS (suppressor of cytokine signaling) protein–specific small interfering RNA experiments suggested that the SOCS family proteins are negative regulators of IL-4– and IL-13–induced eotaxin-3 expression in pancreatic myofibroblasts. Interferon-&ggr; significantly inhibited IL-4– and IL-13–induced eotaxin-3 expression, and this response was mediated by STAT1 activation. Conclusions Pancreatic myofibroblasts may be a cellular source of eotaxin-3 in the pancreas. The T helper type 2 cytokines, IL-4 and IL-13, are critical factors for the induction of eotaxin-3 in the pancreas.

A case of Crohn’s disease that developed anti-infliximab and anti-adalimumab antibodies

There are few reports about the rapid appearance of anti-adalimumab antibodies in patients with Crohn’s disease positive for anti-infliximab antibodies. We report the case of a 29-year-old female patient with a diagnosis of Crohn’s disease who revealed a loss of response to infliximab due to high levels of antibodies to infliximab, and did not respond to the subsequent therapy by adalimumab, with a rapid appearance of antibodies to adalimumab. As one of the possible mechanisms of non-response to adalimumab, immunologic reactivity of infliximab to adalimumab was suspected, since the patient’s IgG that was obtained just before the induction of adalimumab reacted with infliximab and adalimumab. We should pay attention to the easy appearance of anti-adalimumab antibodies in association with reactivity of anti-infliximab antibodies to adalimumab in patients with high levels of anti-infliximab antibodies.