Shigeki Bamba

Increased expression of interleukin 17 in inflammatory bowel disease

Background and aim: Interleukin (IL) 17 is a cytokine which exerts strong proinflammatory activities. In this study we evaluated changes in IL-17 expression in the inflamed mucosa and in the serum of patients with inflammatory bowel disease (IBD). Methods: Tissue samples were obtained endoscopically or surgically from patients with ulcerative colitis (UC) (n=20), Crohn’s disease (CD) (n=20), infectious colitis (n=5), ischaemic colitis (n=8), and normal colorectal tissues (n=15). IL-17 expression was evaluated by a standard immunohistochemical procedure. Serum IL-17 levels were determined by ELISA. IL-17 mRNA expression was analysed by reverse transcriptase-polymerase chain reaction. Results: IL-17 expression was not detected in samples from normal colonic mucosa, infectious colitis, or ischaemic colitis. In the inflamed mucosa of active UC and CD patients, IL-17 expression was clearly detectable in CD3+ T cells or CD68+ monocytes/macrophages. The average number of IL-17+ cells was significantly increased in active UC and CD patients compared with inactive patients. IL-17 mRNA expression was not detected in normal mucosa but was detectable in the mucosa from active UC and CD patients. IL-17 was not detected in the sera from normal individuals, infectious colitis, or ischaemic colitis patients but IL-17 levels were significantly elevated in IBD patients. Conclusions: IL-17 expression in the mucosa and serum was increased in IBD patients. It is likely that IL-17 expression in IBD may be associated with altered immune and inflammatory responses in the intestinal mucosa.

Interleukin-33 expression is specifically enhanced in inflamed mucosa of ulcerative colitis

BackgroundInterleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 has been shown to elicit a Th2-like cytokine response in immune cells. In this study, we investigated IL-33 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for IL-33 expression in human colonic subepithelial myofibroblasts (SEMFs).MethodsIL-33 mRNA expression was determined by real-time polymerase chain reaction (PCR). IL-33 expression in the IBD mucosa was evaluated by immunohistochemical methods.ResultsIL-33 mRNA expression was significantly elevated in active lesions from patients with ulcerative colitis (UC), but was not detected in inactive lesions from UC patients or in lesions from patients with either active or inactive Crohn’s disease. Colonic SEMFs were identified as a major source of IL-33 in the mucosa. IL-1β and tumor necrosis factor-α (TNF-α) significantly enhanced IL-33 mRNA and protein expression in isolated colonic SEMFs. IL-1β and TNF-α did not affect IL-33 expression in intestinal epithelial cell lines (HT-29 and Caco-2 cells). This IL-1β- and TNF-α-induced IL-33 mRNA expression was mediated by p42/44 mitogen activated protein kinase (MAPK) pathway-dependent activation of nuclear factor (NF)-κB and activator protein (AP)-1.ConclusionsIL-33, derived from colonic SEMFs, may play an important role in the pathophysiology of UC.

Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn’s disease using terminal restriction fragment length polymorphism analysis

BackgroundTerminal restriction fragment length polymorphism (T-RFLP) analysis is a powerful tool to assess the diversity of a microbial community. In this study, we performed T-RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC) and those with Crohn’s disease (CD).MethodsThirty-one patients with UC, 31 patients with CD, and 30 healthy individuals were enrolled. The polymerase chain reaction (PCR) products obtained from the 16S rRNA genes of fecal samples were digested with BslI, and T-RF lengths were determined.ResultsThe fecal microbial communities were classified into 5 clusters. Twenty-eight of the 30 healthy individuals and 17 of the 18 patients with inactive UC were classified into clusters I, II, and III, but these clusters included a small number of patients with active UC and inactive/active CD. In contrast, 8 of the 13 patients with active UC and the majority of CD patients (12 of the 16 patients with inactive CD, and 11 of the 15 patients with active CD) were included in clusters IV and V. Based on the BslI-digested T-RFLP database, the bacteria showed a significant decrease in the Clostridium family in patients with active UC and inactive/active CD. In contrast, Bacteroides were significantly increased in CD patients. No significant differences were observed between patients with active UC and those with active CD.ConclusionThe fecal microbial communities of IBD patients were different from those of healthy individuals. The gut microbiota of patients with inactive UC tended to be closer to that of healthy individuals, suggesting different roles for the fecal microbiota in the pathophysiology of UC and CD.

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.

Curcumin Prevents the Development of Dextran Sulfate Sodium (DSS)-Induced Experimental Colitis

Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric). We evaluated the effects of curcumin on the development of dextran sulfate sodium (DSS)-induced experimental colitis. BALB/c mice were fed a chow containing either 3.5% (wt/wt) DSS or 3.5% DSS + 2.0% (wt/wt) curcumin. The body weight loss was more apparent in DSS-treated mice than in DSS + curcumin-treated mice. The disease activity index, histological colitis score, and MPO activity were all significantly higher in DSS-treated mice than in DSS plus curcumin-treated mice. Microscopically, mucosal edema, cellular infiltration, and epithelial disruption were much more severe in DSS-treated mice than in DSS + curcumin-treated mice. In DSS + curcumin-treated mice, NF-κB activation was blocked in the mucosa. In conclusion, the development of DSS-induced colitis was significantly attenuated by curcumin. Being a nontoxic natural dietary product, curcumin could be useful in treatment of IBD patients.

Colonic subepithelial myofibroblasts in mucosal inflammation and repair : contribution of bone marrow-derived stem cells to the gut regenerative response

mesenchymal cells that exhibit the ultrastructural features of both fibroblasts and smooth muscle cells, and they are characterized by positive immunoreactivity for a-smooth muscle actin (a-SMA) and vimentin, but negative immunoreactivity for desmin (Figs. 1A and B).4–6,7 SEMFs are distinguished from smooth muscle cells, which express a-SMA but are negative for vimentin. The location of SEMFs below the basement membrane suggests that these cells may play a role in the regulation of a number of epithelial cell functions, such as epithelial proliferation and differentiation and/ or extracellular matrix (ECM) metabolism, affecting the growth of the basement membrane. Intestinal SEMFs are classified as members of a family of functionally related cells, including hepatic Ito cells, glomerular mesangial cells, and orbital and synovial fibroblasts.8 Previous literature describes the existence of SEMFs as a syncytium that extends throughout the lamina propria of the gut, merging with the pericytes surrounding the blood vessels.5,9 In the region of the crypts, SEMFs are oval and scaphoid in appearance and overlap like shingles on a roof.5,10 They are attached to each other via both gap junctions and adherens junctions. In the upper regions of the crypts and the small intestinal villi, the SEMFs display a stellate morphology.9,11

Interleukin-1β and Tumor Necrosis Factor-α Induce Chemokine and Matrix Metalloproteinase Gene Expression in Human Colonic Subepithelial Myofibroblasts

Background: Colonic subepithelial myofibroblasts may play a role in the inflammatory responses and in extracellular matrix (ECM) metabolism. In this study, we investigated the effects of interleukin (IL)-1 β and tumor necrosis factor (TNF)- α on chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and ECM turnover (proliferation of subepithelial myofibroblasts, and secretion of ECM and matrix metalloproteinases (MMPs) in colonic subepithelial myofibroblasts. Methods: Human colonic sub-epithelial myofibroblasts were isolated using the method described by Mahida et al. (4). Chemokine and MMP expressions were determined by ELISA and Northern blotting. Nuclear factor (NF)- s B and NF-IL6 DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). Results: IL-1 β and TNF- α did not affect the proliferation of subepithelial myofibroblasts, but stimulated the secretion of types I and IV collagens weakly. Unstimulated subepithelial myofibroblasts secreted a large amount of MMP-2, but a small amount of IL-8, MCP-1 and MMP-1. IL-1 β and TNF- α both induced a dose- and time-dependent increase in IL-8, MCP-1 and MMP-1 secretion, and weakly stimulated MMP-2 secretion. IL-1 β and TNF- α both rapidly evoked NF- s B DNA-binding activity. The inhibition of NF- s B activation markedly blocked both IL-1 β - and TNF- α -induced IL-8 and MCP-1 mRNA expression, but did not affect MMP-1 mRNA expression. Conclusions: These observations indicate that chemokine secretion and ECM metabolism are collectively regulated by the proinflammatory cytokines, IL-1 β and TNF- α , in colonic subepithelial myofibroblasts. Thus, colonic subepithelial myofibroblasts may play an important role in the pathophysiology of inflammation in the colon.

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.

Expression of IL-24, an activator of the JAK1/STAT3/SOCS3 cascade, is enhanced in inflammatory bowel disease.

IL-24 is a member of the IL-10 family of cytokines. In this study, we investigated IL-24 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for IL-24 expression in human colonic subepithelial myofibroblasts (SEMFs). IL-24 expression in the IBD mucosa was evaluated by immunohistochemical methods. IL-24 mRNA and protein expression was determined by real-time PCR and ELISA, respectively. AP-1 and C/EBP DNA-binding activity and IL-24 promoter activity were assessed by EMSA analysis and a reporter gene assay, respectively. IL-24 mRNA expression was significantly elevated in active lesions from patients who have ulcerative colitis and Crohn’s disease. Colonic SEMFs were identified as a major source of IL-24 in the mucosa. IL-1β, but not IL-17A, TNF-α, or IFN-γ, significantly enhanced IL-24 mRNA and protein expression in isolated colonic SEMFs. The IL-1β-induced IL-24 mRNA expression was mediated by the activation of the transcription factors, AP-1 and C/EBP-β. Induction of IL-24 mRNA stabilization was also involved in the effects of IL-1β. IL-24 induced JAK1/STAT-3 phosphorylation and SOCS3 expression in HT-29 colonic epithelial cells. IL-24 did not modulate the proliferation of HT-29 cells, but significantly increased the mRNA expression of membrane-bound mucins (MUC1, MUC3, and MUC4). IL-24 derived from colonic SEMFs acts on colonic epithelial cells to elicit JAK1/STAT-3 activation and the expression of SOCS3 and mucins, supporting their suppressive effects on mucosal inflammation in IBD.

IL-17 Selectively Down-Regulates TNF-α-Induced RANTES Gene Expression in Human Colonic Subepithelial Myofibroblasts

IL-17 enhances the TNF-α-induced IL-6 and IL-8 secretion in human colonic subepithelial myofibroblasts. In this study, we investigated how IL-17 modulates RANTES secretion in these cells. TNF-α potently induced RANTES secretion, but IL-17 dose-dependently inhibited the TNF-α-induced RANTES secretion. This was also observed at the mRNA level. Even after pretreatment with TNF-α for 12 h, the inhibitory effect of IL-17 was detectable. IL-17 did not affect the TNF-α-induced stability of the RANTES gene. IL-17 significantly decreased the TNF-α-induced increase in RANTES promoter activity, and IL-17 actually blocked the TNF-α-induced RANTES gene transcription. EMSAs demonstrated that IL-17 did not modulate the TNF-α-induced NF-κB DNA-binding activity, but markedly decreased TNF-α-induced IFN regulatory factor-1 (IRF-1) DNA-binding activity. Because cooperation between NF-κB and IRF-1 is important in the TNF-α-induced RANTES gene expression, the major mechanism mediating the inhibitory effect of IL-17 may be achieved by the inhibition of IRF-1 DNA-binding activity.