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4. Pro-fibrogenic action of toll-like receptor and NOD-like receptor ligands on human intestinal myofibroblasts

F. Rieder1, A. Schirbel2, Z. Ouyang1, G. West1, H. Rho1, C. De la Motte1, C. Fiocchi1

1Cleveland Clinic Foundation, Cleveland, OH, United States; 2Charité University, Berlin, Germany

Aim: In inflammatory bowel disease (IBD) gut bacteria induce an abnormal innate immune response mediated by toll-like receptors (TLRs) and Nod-like receptors (NLRs). Since fibrosis is caused by excessive extracellular matrix deposition by mesenchymal cells, we investigated whether TLR or NLR ligands can induce a fibrogenic response in HIF as a novel fibrogenic pathway in IBD.

Material and Methods: Human intestinal myofibroblasts (HIF) from IBD and control mucosa were exposed to ligands for TLR2/6, 4, 5, and Nod1, TGF-β1 or their combinations, and supernatants assessed for fibronectin (FN), collagen I (Col 1), TGF-β1 and IL-6 content by ELISA or RIA. TGF-β1 signaling was inhibited by SB431542 and p38MAPK signaling blocked by SB203580. To assess whether TLR/NLR activation sensitizes HIF to TGF-β1 fibrogenic activity, HIF were pre-stimulated with TLR or NLR ligands followed by TGF-β1. TLR/NLR-mediated modulation of αSMA, ICAM-1 and VCAM-1 was investigated by flow cytometry and immunocytochemistry, and adhesion assays were performed using primary human blood monocytes.

Results: Stimulation of HIF with TLR5 ligand alone or TLR5 in combination with Nod1, TLR2/6 or TLR4 induced a marked and significant increase in FN and Col 1 production (p < 0.03–0.001). In contrast, the same TLR or Nod1 ligands, either individually or in combination, failed to trigger TGF-β1 signaling, boost secretion of TGF-β1, or upregulate levels of αSMA expression in HIF. TLR5 induced FN secretion was dependent on p38MAPK signaling. When combined with TGF-β1, TLR5 augmented FN secretion by HIF (p < 0.01) and pre-stimulation by Nod1 ligand or combined TLR5 + Nod1 ligands sensitized HIF to the subsequent action of TGF-β1 (p < 0.05). All TLR and NLR ligands induced the secretion of IL-6 by HIF, however with the maximal levels after incubation with TLR2/6. Individual TLR2/6 and TLR5 ligands, or combinations of Nod1, TLR2/6 or TLR5 ligands upregulated VCAM-1 (p < 0.05), but not ICAM-1, expression by HIF. The ability of HIF to bind monocytes was significantly increased by pre-activation with TLR2/6, 4 and 5 ligands, either individually or in combination, or individual TLR combined with Nod1 ligand (p < 0.03–0.001). Monocyte derived TGF-β1 was critical in inducing FN and Col 1 production by HIF. The above results were largely independent of the IBD or control origin of HIF.

Conclusions: TLR and NLR ligands, alone and combined, induce a fibrogenic response by HIF as demonstrated by a significant TGF-β1-independent, but p38MAPK dependent, increase in FN and Col 1 production. This process is distinct from the inflammatory response. Moreover, TLR5 and Nod1 ligands sensitize HIF to the fibrogenic activity of TGF-β1. This response also involves upregulation of VCAM-1 expression and adhesiveness for monocytes, a critical source of TGF-β1 in intestinal fibrosis. These results suggest, for the first time, the existence of a direct link between innate immunity to bacteria and fibrosis in human IBD.