O-030 YI Toll-like Receptor (TLR) 5 Promotes a Pro-fibrogenic Phenotype in Human Intestinal Fibroblasts (HIF) Via MyD88, Caspase-1 and Posttranscriptional Regulation

BACKGROUND: Intestinal fibrosis is caused by excessive extracellular matrix deposition by myofibroblasts. Activation of TLRs and Nod-like receptors (NLRs) by gut bacteria induces an abnormal innate immune response in inflammatory bowel disease (IBD). We investigated whether TLR or NLR ligands also induce a fibrogenic response in HIF and explored the mechanisms involved. METHODS: HIF from Crohn’s disease, ulcerative colitis and control normal mucosa were exposed to ligands for TLR2/6, 4, 5 and Nod1, and to TGF-&bgr;1, BzATP (extracellular ATP), S. typhimurium and its flagellin-negative FljB/FliC variant. Supernatants were assessed for fibronectin (FN), collagen I (Col1), TGF-&bgr;1 and IL-6 content. TLR signaling, VCAM1 and ICAM1 expression were investigated, and inhibition of TGF-&bgr;1, MyD88 and p38MAPK signaling or inhibition of caspase-1 activation was performed using siRNA and specific blocking agents, immunoblotting, flow cytometry and immunoprecipitation. Post-transcriptional regulation was investigated via RNA stability assays and polysome profiling. RESULTS: All TLR and NLR ligands failed to induce FN or Col1 production by HIF with the notable exception of flagellin which, alone or combined with Nod1, TLR2/6 or TLR4, significantly increased FN and Col 1 production. This TLR5-mediated response was independent of TGF-&bgr;1 signaling. Combined with TGF-&bgr;1, TLR5 augmented FN secretion and sensitized HIF to the subsequent action of TGF-&bgr;1. All TLR and NLR ligands induced IL-6 secretion and, while flagellin-positive S. typhimurium induced FN production by HIF, its flagellin-negative variant failed to do so, indicating that this was a TLR5 specific response. Interestingly, the inflammasome activator BzATP enhanced TLR5-induced FN production by HIF. The TLR5 ligand-induced FN secretion was dependent on MyD88, p38MAPK signaling and caspase-1 activation. Preliminary experiments show overrepresentation of FN and Col1 mRNA in the actively translated polysome fractions of HIF providing evidence for post-transcriptional regulation of the TLR5-induced FN and Col1 production. To investigate additional effects on fibrogenesis, we found that flagellin, alone or in combination with other TLR or NLR ligands, upregulated VCAM-1, but not ICAM-1, expression by HIF. This enhanced the ability of HIF to bind primary human monocytes, the predominant source of pro-fibrogenic TGF-&bgr;1. In fact, monocyte-derived TGF-&bgr;1 appeared to be critical for activation of HIF. CONCLUSIONS: Among multiple TLR and NLR ligands only activation of TLR5 by flagellin induces a fibrogenic response by HIF that is dependent on MyD88, p38MAPK and caspase-1, but independent of the TGF-&bgr;1 signaling pathway. TLR5 activity in HIF could be boosted by the inflammasome activator BzATP. The increased matrix production appears to be post-transcriptionally regulated. This direct fibrogenic response is distinct from the inflammatory response, but flagellin also induces fibrogenesis indirectly by promoting binding of TGF-&bgr;1-producing monocytes. These results suggest the existence of a complex network involving bacterial immunity and the inflammasome that leads to excessive matrix production in IBD-associated fibrosis.