Hirotsugu Imaeda

NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice.

To explore induced islet neogenesis in the liver as a strategy for the treatment of diabetes, we used helper-dependent adenovirus (HDAD) to deliver the pancreatic duodenal homeobox-1 gene (Ipf1; also known as Pdx-1) to streptozotocin (STZ)-treated diabetic mice. HDAD is relatively nontoxic as it is devoid of genes encoding viral protein. Mice treated with HDAD-Ipf1 developed fulminant hepatitis, however, because of the exocrine-differentiating activity of Ipf1. The diabetes of STZ mice was partially reversed by HDAD-mediated transfer of NeuroD (Neurod), a factor downstream of Ipf1, and completely reversed by a combination of Neurod and betacellulin (Btc), without producing hepatitis. Treated mice were healthy and normoglycemic for the duration of the experiment (>120 d). We detected in the liver insulin and other islet-specific transcripts, including proinsulin-processing enzymes, β-cell–specific glucokinase and sulfonylurea receptor. Immunocytochemistry detected the presence of insulin, glucagon, pancreatic polypeptide and somatostatin-producing cells organized into islet clusters; immuno-electron microscopy showed typical insulin-containing granules. Our data suggest that Neurod-Btc gene therapy is a promising regimen to induce islet neogenesis for the treatment of insulin-dependent diabetes.

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.

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.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)‐17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL‐17 and C3 mRNA expressions in the IBD mucosa were evaluated by real‐time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme‐linked immunosorbent assay. IL‐17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohns disease (CD) patients. There was a significant positive correlation between IL‐17 and C3 mRNA expression in the IBD mucosa. IL‐17 stimulated a dose‐ and time‐dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115‐kDa α‐chain linked to a 70‐kDa β‐chain by disulphide bonds, which was identical to serum C3. The IL‐17‐induced C3 mRNA expression was blocked by p42/44 mitogen‐activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL‐17‐induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL‐17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL‐17.

Expression of interleukin 1-like cytokine interleukin 33 and its receptor complex (ST2L and IL1RAcP) in human pancreatic myofibroblasts

Objective Interleukin 33 (IL33) is a cytokine belonging to the IL1 family and it binds to a complex of the ST2L/IL1 receptor accessory protein (IL1RAcP). To define the role of IL33 in fibrogenesis of the pancreas, the expression of IL33, ST2L and IL1RAcP was examined in chronic pancreatitis tissues. The effects of IL33 on the functions of human pancreatic myofibroblasts were also investigated. Methods Tissue samples were obtained surgically. The expression of IL33, ST2L and IL1RAcP was evaluated by standard immunohistochemical procedures. Messenger RNA expression for IL33, ST2L and IL1RAcP was analysed by northern blotting and real-time PCR analyses, and protein expression was assessed by western blotting and ELISA. Cell proliferation and migration were assessed by a 3H-thymidine incorporation assay and the modified Boyden chamber assay, respectively. Results IL33, ST2L and IL1RAcP were expressed by α-SMA-positive myofibroblasts in the fibrosis of chronic pancreatitis. In human pancreatic myofibroblasts, IL33 was weakly immunoexpressed without any stimuli, and this was markedly enhanced by IL1β, tumour necrosis factor α (TNFα) and lipopolysaccharide (LPS) via the mitogen-activated protein kinase (MAPK)-dependent AP-1 activation pathway. ST2L mRNA was weakly detected in unstimulated cells, and IL4 and interferon γ (IFNγ) strongly enhanced ST2L expression via STAT6 and STAT1 signalling, respectively. IL33 rapidly induced the phosphorylation of MAPKs and IκBα, and enhanced the expression of inflammatory mediators (IL6, IL8, IP-10, Gro-α, Gro-β and MCP-1) in IL4- or IFNγ-pretreated cells. IL33 stimulated the proliferation and migration of pancreatic myofibroblasts. Conclusions IL33 and its receptor complex (ST2L and IL1RAcP) constitute a novel signalling system which may play an important role in the pathogenesis of chronic pancreatitis.

Interleukin-33 suppresses Notch ligand expression and prevents goblet cell depletion in dextran sulfate sodium-induced colitis

Interleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 plays an important role in Th2 immune responses, and induces goblet cell hyperplasia in the intestinal mucosa. In this study, to elucidate the molecular mechanisms underlying IL-33-induced goblet cell hyperplasia, we investigated how IL-33 modulates the Notch signaling pathway in dextran sulfate sodium (DSS)-induced experimental colitis. DSS colitis was induced in BALB/c mice with intraperitoneal administrations of IL-33 (1 µg/body) every 48 h. Tissue samples were evaluated by standard immunohistochemical procedures. The mucosal mRNA expression of the Notch ligands was analyzed by a real-time polymerase chain reaction. The mucosal mRNA expression of Notch ligands [Jagged1 (Jag1) and Delta-like (Dll) 1 and 4] was significantly increased in DSS-colitis mice. IL-33-induced goblet cell hyperplasia in the control mice. In the DSS-colitis mice, the goblet cells were depleted in the colon, but IL-33 completely prevented goblet cell depletion in the DSS-colitis mice. IL-33 induced a significant decrease in Jag1 and Dll4 mRNA expression in the mucosa of the control mice. Mucosal mRNA expression for Jag1, Dll1 and 4 was significantly elevated in the DSS-colitis mice, but this elevation was significantly blocked by the administration of IL-33. IL-33 dose-dependently decreased Jag1 mRNA expression in mouse colonic subepithelial myofibroblasts. In contrast to its preventive effects on goblet cell depletion, IL-33 aggravated DSS colitis. IL-33 prevented goblet cell depletion via its inhibitory actions against Notch ligand expression in DSS colitis, but exacerbated the disease activity. IL-33 plays two counter actions in mucosal inflammation; the first is a protective action via goblet cell induction, and the second is a pro-inflammatory action as a Th2 cytokine.

Comparison of the gut microbial community between obese and lean peoples using 16S gene sequencing in a Japanese population

Altered gut microbial ecology contributes to the development of metabolic diseases including obesity. In this study, we performed 16S rRNA sequence analysis of the gut microbiota profiles of obese and lean Japanese populations. The V3–V4 hypervariable regions of 16S rRNA of fecal samples from 10 obese and 10 lean volunteers were sequenced using the Illumina MiSeqTMII system. The average body mass index of the obese and lean group were 38.1 and 16.6 kg/m2, respectively (p<0.01). The Shannon diversity index was significantly higher in the lean group than in the obese group (p<0.01). The phyla Firmicutes and Fusobacteria were significantly more abundant in obese people than in lean people. The abundance of the phylum Bacteroidetes and the Bacteroidetes/Firmicutes ratio were not different between the obese and lean groups. The genera Alistipes, Anaerococcus, Corpococcus, Fusobacterium and Parvimonas increased significantly in obese people, and the genera Bacteroides, Desulfovibrio, Faecalibacterium, Lachnoanaerobaculum and Olsenella increased significantly in lean people. Bacteria species possessing anti-inflammatory properties, such as Faecalibacterium prausnitzii, increased significantly in lean people, but bacteria species possessing pro-inflammatory properties increased in obese people. Obesity-associated gut microbiota in the Japanese population was different from that in Western people.

Increased Expression of Interleukin-36, a Member of the Interleukin-1 Cytokine Family, in Inflammatory Bowel Disease.

Background:Interleukin (IL)-36 (IL-36&agr;, IL-36&bgr;, and IL-36&ggr;) is a recently reported member of the IL-1 cytokine family. In this study, we investigated IL-36 expression in the inflamed mucosa of patients with inflammatory bowel disease and characterized the proinflammatory actions of IL-36 cytokines in human colonic epithelial cells. Methods:IL-36 mRNA expression was evaluated using real-time PCR. IL-36 protein expression was analyzed using immunoblotting and immunohistochemical technique. Intracellular signaling pathways were evaluated by immunoblotting and by specific siRNA-transfected cells. Results:The mRNA expression of IL-36&agr; and IL-36&ggr;, but not of IL-36&bgr;, was enhanced in the inflamed mucosa of patients with inflammatory bowel disease, in particular, in ulcerative colitis. Immunohistochemical analysis showed that T cells, monocytes, and plasma cells are the source of IL-36&agr; and IL-36&ggr; in colonic mucosa. DNA microarray analysis indicated that IL-36&agr; induces the mRNA expression of CXC chemokines and acute phase proteins in intestinal epithelial cell line, HT-29 cells. IL-36&agr; and IL-36&ggr; dose-dependently and time-dependently induced the mRNA and protein expression of CXC chemokines (CXCL1, CXCL2, CXCL3 etc.) in HT-29 and Widr cells. Stimulation with IL-36&agr; and IL-36&ggr; assembled MyD88 adaptor proteins (MyD88, TRAF6, IRAK1, and TAK1) into a complex and induced the activation of NF-&kgr;B and AP-1 and also the phosphorylation of MAPKs. MAPK inhibitors and siRNAs specific for NF-&kgr;B and c-Jun AP-1 significantly reduced IL-36–induced CXC chemokine expression. Conclusions:IL-36&agr; and IL-36&ggr; may play a proinflammatory role in the pathophysiology of inflammatory bowel disease through induction of CXC chemokines and acute phase proteins.