Lipids drive epithrlial mesenchymal transition through the activation of S1PR3 signaling exacerbating intestinal fibrosis in Chron's disease

The transdifferentation of epithelial into mesenchymal phenotype (EMT) has been pathologically linked to intestinal fibrosis, a major complication of Crohn’s disease (CD). However, its mechanisms remain largely unknown. In cancer, high fat diet (HFD) is a key player of EMT, but there no evidences if it may be a driver of intestinal fibrotic processes. Sphingosine-1-phosphate (S1P), a lipid mediator involved in inflammatory processes, have been reported to promote fibrosis in different organs by activating S1P receptor 3 (S1PR3), but its involvement in the intestinal fibrotic processes remains unknown. Here, we explored the role of the lipids in promoting intestinal fibrosis and S1PR3 activation. Spatial lipidomics was evaluated in surgical specimens from fibrotic and non-fibrotic area of CD patients. S1PR3 expression was analyzed in tissue and in primary human epithelial cells (ECs) isolated from 14 fibrotic CD patients by immunohistochemistry and WB. Experimental model of intestinal fibrosis was induced by TNBS administration in S1PR3 knock out mice and C57BL/6N mice that were fed for 12 weeks with 45% of high-Fat diet (HFD) or treated for 5 weeks with CAY-10444 inhibitor. Clinical parameters, collagen deposition, pro-fibrotic factors and EMT markers were evaluated by RT-PCR, and S1PR3-mediated signaling by WB. Induced pluripotent stem cell-derived intestinal organoids (iHOs) were treated for 3 weeks with a cytokines cocktail or lipids (2%) and analyzed by RNA-seq. Fibrotic tissue was characterized by an accumulation of very long-chain fatty acids. Intestinal fibrosis was highly amplified and accelerated by HFD consumption that led to EMT and a marked expression of sphingonsine-1 receptor 3 (S1PR3) in WT mice. S1PR3, unknown to be expressed by intestinal epithelium, was upregulated on intestinal epithelial cells upon fibrotic injury reprograming the expression of key transcriptional factors, including SNAIL, switching into a mesenchymal phenotype and promoting intestinal fibrosis progression. S1PR3 was significantly upregulated in 7 out of 14 ECs of fibrotic CD patients compared to non-fibrotic ones. Particularly, patients not responding to conventional and biological therapies showed elevated levels of epithelial S1PR3 almost 18 times higher than responsive patients. Knocking out S1PR3 in mice or blocking S1PR3 with a specific inhibitor in mice normalized TNBS-induced fibrosis in vivo and prevented effects of HFD. Lipids promoted in iHOs an increase in S1PR3 and in transcription factors ZEB2, TWIST, SNAI2, SMARCA2 related to the EMT process similarly to inflammatory cytokines. Therefore, inflammatory mediators and lipids drive EMT through the activation of S1PR3 signaling supporting fibroblastmediated pro-fibrotic activity in CD patients. S1P3 receptor may be a new therapeutic target for the treatment of intestinal fibrosis