P093 Creeping fat-derived long chain free fatty acids drive intestinal muscularis propria muscle cell proliferation via carnitine palmitoyltransferase 1 (CPT-1) – a relevant mechanism for stricturing Crohn’s disease

Abstract

Background Mesenteric fat wrapping around the intestinal wall, so called ‘creeping fat’ (CF), is spatially linked with stricture formation in Crohn’s disease (CD). Intestinal muscularis propria (MP) smooth muscle cell (HIMC) hyperplasia is a major contributor to luminal narrowing in stricturing CD. We investigated CF derived factors and their effect on HIMC hyperplasia in vitro and in vivo using human tissues, primary human cells and a colitis model. Methods Secretion of free fatty acids (FFA) by mesenteric fat (MF) or CF organ cultures was determined via lipidomic mass spectrometry. Effects of different length and types of FFA as well as CF and MF conditioned medium on proliferation of primary HIMF was assessed. Next generation sequencing (NGS) and lipidomics on HIMF was performed and relevant pathways inhibited with small molecules or siRNA knockdown. In the dextrane sodium sulfate (DSS) induced colitis model CPT-1 blockade was achieved via the small molecule etomoxir. Results Histopathology analysis of intestinal resection tissues revealed CD CF being located in the subserosa and its presence was linked with dramatic thickening of the MP. CF conditioned medium markedly upregulated HIMC proliferation compared to mesenteric fat from CD, UC and normal controls. CF released higher amounts of total, saturated and poly-unsaturated FFA with elevated levels of five long-chain (LC-)FFA, including palmitate. LC, but not medium or short chain FFA selectively increased proliferation in HIMC and fibroblasts, but not other intestinal cell types. NGS revealed gene regulation suggesting LC-FFA transport as a putative mechanism. Lipidomic analysis indicated the majority of palmitate being converted into phospholipids, predominantly phosphatidylcholine. Inhibition of LC-FFA uptake into cells via CD36, metabolism through inhibition of acyl-CoA synthetase, choline-phosphate cytidylyltransferase & choline kinase or blockade of LC-FFA uptake into mitochondria through CPT-1A reduced palmitate and CF conditioned medium induced HIMC proliferation. MP thickness increased in acute DSS colitis. Prophylactic inhibition of CPT-1 with etomoxir in acute DSS colitis did not reduce histopathologic inflammation or inflammatory cytokine gene expression, but reduced MP thickness and gene expression of the smooth muscle cell genes desmin and Sm22. Conclusion Subserosal CF releases LC-FFA inducing a selective proliferative response by HIMC. This effect was dependent on CD36, acyl-CoA synthetase and CPT-1. LC-FFA in HIMC are converted into phospholipids. Inhibtion of CPT-1 in DSS colitis reduced the increased MP thickness. These results point to CF as a novel contributor to stricture formation in CD.