P-196 YI Human Intestinal Mesenchymal Cells Exhibit a Pro-Fibrogenic Phenotype in Response to Adipose Tissue Derived-Mediators—Linking Fat to Intestinal Fibrosis

Abstract

Mesenteric adipose tissue, mainly consisting of preadipocytes and adipocytes, is increasingly considered an active player in the pathogenesis of inflammatory bowel disease (IBD). Adipocyte hyperplasia and creeping fat in Crohn's disease (CD) are associated with stricture formation, which is caused by excessive extracellular matrix (ECM) deposition by myofibroblasts and muscle cells. We therefore investigated whether mesenteric adipocyte/preadipocyte-derived mediators induce a pro-fibrogenic response by human intestinal myofibroblasts (HIF) and muscle cells (HIMC) and the mechanisms involved. HIF and HIMC derived from IBD and control tissues were exposed to 6 different adipokines (leptin, chemerin, resistin, visfatin, vaspin, nesfatin), free fatty acids (FFA) of different length (palmitate, stearate, oleate, decanoate, butyrate) as well as TNF, IL-1β, TGF-β1, bFGF, or their combinations, and cell proliferation was assessed by 3H-thymidine incorporation and cell counting. Modulation of αSMA was investigated by immunoblot; fibronectin (FN), collagen I (Col 1) and IL-6 content in the supernatants was measured by ELISA, RIA or immunoblot; and migration by Boyden chamber assay. p38MAPK, PKC, PI3K, NFκB, TLR2, TLR4 or MyD88 signaling was blocked by specific inhibitors or siRNA, respectively. Primary human intestinal endothelial cells, preadipocytes and adipocytes as well as the intestinal epithelial cell line CaCo2 served as controls. bFGF and long chain, but not short chain, FFA increased proliferation of HIF and HIMC 2.2 and 3.4-fold, respectively (P < 0.001). When mediators were combined a striking synergistic effect of bFGF and long-chain FFA was observed, with a dramatic 12-fold increase in HIF and HIMC proliferation. Synergy was dependent on p38MAPK, PKC and PI3K, but not MyD88, TLR2, TLR4, NFκB or CD36. Supernatants of human intestinal adipocytes, but not preadipocytes increased proliferation of HIMC. The effect of palmitate, the most abundant long-chain FFA, on mesenchymal cell proliferation was specific, since it failed to induce proliferation in intestinal epithelial, endothelial cells as well as preadipocytes and adipocytes. TGF-β1, bFGF and TNF induced a marked increase in FN secretion, while TGF-β1, bFGF and IL-1β enhanced the production of Col-1 (P < 0.05–0.001) by HIF. The HIF inflammatory response, as measured by IL-6, was increased by IL-1β, TNF and TGF-β1 (P < 0.05), and TGF-β1 upregulated αSMA expression. HIF- and HIMC-conditioned medium induced HIF and HIMC migration with further increase after exposure to TGF-β1 and bFGF (P < 0.01). Adipokines and FFA failed to increase matrix production, IL-6 secretion, αSMA expression or cell migration. The above results were largely independent of the IBD or control origin of HIF or HIMC. Mesenteric adipocyte/preadipocyte-derived mediators induce a differential and selective fibrogenic response by HIF and HIMC. bFGF and long-chain, but not short chain, FFA synergistically increased HIF and HIMC proliferation in a p38MAPK-, PKC- and PI3K-dependent, but NFκB-, MyD88-, TLR2-, TLR4- and CD36-independent manner. Adipocyte-, but not preadipocyte-conditioned medium enhanced HIMC proliferation. TGF-β1, bFGF, TNF and IL-1β, but not adipokines or FFA increased ECM secretion, IL-6 production, αSMA expression and cell migration. These results indicate that fat-derived mediators actively contribute to intestinal fibrosis.