TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

Transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) signaling can mediate inflammatory responses as well as tissue remodeling. Intestinal mucosal myofibroblast (IMF) activation drives gut fibrosis in Crohn's disease (CD); however, the molecular pathways involved are largely unknown. Thus we investigated the yet-unknown expression and function of TAK1 in human CD-associated fibrosis. Ileal surgical specimens, ileal biopsies, and IMF isolated from controls and CD patients were analyzed for TAK1 and its active phosphorylated form (pTAK1) by Western blotting, immunohistochemistry, and real-time quantitative PCR. TAK1 pharmacological inhibition and silencing were used to assess its role in collagen and inflammatory cytokine synthesis in IMF. TAK1 and pTAK1 levels increased in ileum specimens from CD patients compared with controls and correlated to tissue fibrosis. Similarly, TAK1 mRNA in ileal biopsies of CD patients correlated with fibrogenic marker expression but not inflammatory cytokines. CD-derived IMF showed higher TAK1 and pTAK1 expression associated with increased collagen1(α)1 mRNA levels compared with control IMF. TGF-ß1 promoted pTAK1 nuclear translocation and collagen synthesis. TAK1 inhibition or silencing significantly reduced TGF-ß1-stimulated collagen production and normalized the profibrogenic phenotype of CD-derived IMF. Taken together, these data suggest that TAK1 activation and nuclear translocation induce and maintain a fibrogenic phenotype in the IMF. Thus the TAK1 signaling pathway may represent a suitable target to design new, antifibrotic therapies.

Toll-like receptor 2 regulates intestinal inflammation by controlling integrity of the enteric nervous system.

BACKGROUND & AIMS: In the intestines, Toll-like receptor 2 (TLR2) mediates immune responses to pathogens and regulates epithelial barrier function; polymorphisms in TLR2 have been associated with inflammatory bowel disease phenotype. We assessed the effects of TLR2 signaling on the enteric nervous system (ENS) in mice. METHODS: TLR2 distribution and function in the ileal neuromuscular layer of mice were determined by immunofluorescence, cytofluorimetric analysis, immunoprecipitation, and immunoblot analyses. We assessed morphology and function of the ENS in Tlr2(-/-) mice and in mice with wild-type Tlr2 (wild-type mice) depleted of intestinal microbiota, using immunofluorescence, immunoblot, and gastrointestinal motility assays. Levels and signaling of glial cell line-derived neurotrophic factor (GDNF) were determined using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and immunoprecipitation analyses. Colitis was induced by administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid to Tlr2(-/-) mice after termination of GDNF administration. RESULTS: TLR2 was expressed in enteric neurons, glia, and smooth muscle cells of the intestinal wall. Tlr2(-/-) mice had alterations in ENS architecture and neurochemical profile, intestinal dysmotility, abnormal mucosal secretion, reduced levels of GDNF in smooth muscle cells, and impaired signaling via Ret-GFRα1. ENS structural and functional anomalies were completely corrected by administration of GDNF to Tlr2(-/-) mice. Wild-type mice depleted of intestinal microbiota had ENS defects and GDNF deficiency, similar to Tlr2(-/-) mice; these defects were partially restored by administration of a TLR2 agonist. Tlr2(-/-) mice developed more severe colitis than wild-type mice after administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid; colitis was not more severe if Tlr2(-/-) mice were given GDNF before dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid. CONCLUSIONS: In mice, TLR2 signaling regulates intestinal inflammation by controlling ENS structure and neurochemical coding, along with intestinal neuromuscular function. These findings provide information as to how defective TLR2 signaling in the ENS affects inflammatory bowel disease phenotype in humans.

Rectal administration of Lactobacillus casei DG modifies flora composition and Toll-like receptor expression in colonic mucosa of patients with mild ulcerative colitis.

BACKGROUND: An imbalance in gut microbiota seems to contribute to the development of chronic inflammatory disorders of the gastrointestinal tract, such as ulcerative colitis (UC). Although it has been suggested that probiotic supplementation is an effective approach to colitis, its effects on intestinal flora and on mucosal cytokine balance have never been explored. AIM: To evaluate the effect of Lactobacillus casei (L. casei) DG, a probiotic strain, on colonic-associated microbiota, mucosal cytokine balance, and toll-like receptor (TLR) expression. METHODS: Twenty-six patients with mild left-sided UC were randomly allocated to one of three groups for an 8-week treatment period: the first group of 7 patients received oral 5-aminosalicylic acid (5-ASA) alone, the second group of 8 patients received oral 5-ASA plus oral L. casei DG, and the third group of 11 patients received oral 5-ASA and rectal L. casei DG. Biopsies were collected from the sigmoid region to culture mucosal-associated microbes and to assess cytokine and TLR messenger RNA (mRNA) levels by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: 5-ASA alone or together with oral L. casei DG failed to affect colonic flora and TLR expression in a significant manner, but when coupled with rectally administered L. casei DG, it modified colonic microbiota by increasing Lactobacillus spp. and reducing Enterobacteriaceae. It also significantly reduced TLR-4 and interleukin (IL)-1ß mRNA levels and significantly increased mucosal IL-10. CONCLUSIONS: Manipulation of mucosal microbiota by L. casei DG and its effects on the mucosal immune system seem to be required to mediate the beneficial activities of probiotics in UC patients.

MiR-155 modulates the inflammatory phenotype of intestinal myofibroblasts by targeting SOCS1 in ulcerative colitis.

Abnormal levels of microRNA (miR)-155, which regulate inflammation and immune responses, have been demonstrated in the colonic mucosa of patients with inflammatory bowel diseases (IBD), although its role in disease pathophysiology is unknown. We investigated the role of miR-155 in the acquisition and maintenance of an activated phenotype by intestinal myofibroblasts (IMF), a key cell population contributing to mucosal damage in IBD. IMF were isolated from colonic biopsies of healthy controls, ulcerative colitis (UC) and Crohn's disease (CD) patients. MiR-155 in IMF was quantified by quantitative reverse transcription-PCR in basal condition and following exposure to TNF-α, interleukin (IL)-1ß, lipopolysaccharide (LPS) or TGF-ß1. The effects of miR-155 mimic or inhibitor transfection on cytokine release and suppressor of cytokine signaling 1 (SOCS1) expression were assessed by enzyme-linked immunosorbent assay and western blot, respectively. Regulation of the target gene SOCS1 expression by miR-155 was assessed using luciferase reporter construct. We found that miR-155 was significantly upregulated in UC as compared with control- and CD-derived IMF. Moreover, TNF-α and LPS, but not TGF-ß1 and IL-1ß, significantly increased miR-155 expression in IMF. Ectopic expression of miR-155 in control IMF augmented cytokines release, whereas it downregulated SOCS1 expression. MiR-155 knockdown in UC-IMF reduced cytokine production and enhanced SOCS1 expression. Luciferase reporter assay demonstrated that miR-155 directly targets SOCS1. Moreover, silencing of SOCS1 in control IMF significantly increased IL-6 and IL-8 release. In all, our data suggest that inflammatory mediators induce miR-155 expression in IMF of patients with UC. By downregulating the expression of SOCS1, miR-155 wires IMF inflammatory phenotype.