A NOVEL ORALLY ACTIVE METABOLITE REVERSES CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS

Abstract

Abstract BACKGROUND Intestinal fibrosis is a debilitating complication of Crohn’s disease (CD) patients. Our groups and others discovered that intestinal metabolites are associated with intestinal fibrosis. We selected the most relevant metabolites and determined their anti-fibrogenic efficacies in three mouse models of intestinal fibrosis. METHODS We compared the intestinal metabolite profiles between non-IBD, stricturing CD, and non-stricturing CD patients in inflammatory bowel disease (IBD) multi-omics database to identify CD stricture-related metabolites. We compared the fecal metabolite profiles between trinitrobenzene sulfonic acid (TNBS)-treated mice with and without anti-fibrogenic drug CSA13 treatment to identify mouse intestinal fibrosis-related metabolites. RESULTS Compared to non-stricturing CD patients and non-IBD patients, stricturing CD patients had reduced fecal levels of hydrocinnamate (H), inosine (I), taurine (T), tauroursodeoxycholate (TUDCA), and sphingosine (S). Oral CSA13 increased fecal levels of inosine (I) and sphingosine (S) in the TNBS-treated mice. Overall disease activity (ODA) is a comprehensive assessment of histology and fibrosis scores and intestinal mRNA expression of fibrosis and inflammation markers. Sphingosine (10mg/kg/day for 7-14 days via oral gavages) showed the best anti-fibrogenic effects against spontaneous ileal fibrosis in 42-week-old SAMP1/YitFc mice (11% ODA), cecal fibrosis in Salmonella-infected mice (10% ODA), and colonic fibrosis in TNBS-treated mice (9% ODA). The other four metabolites were ineffective in reversing intestinal fibrosis. Sphingosine (10 microM), but not other metabolites, directly inhibited collagen expression in human colonic CCD-18Co fibroblasts and primary human intestinal fibroblasts from CD patients (CD-HIF). RNA-seq and proteomics identified that low MAPK3 and high ZEB1 mRNA expression in intestines are associated with intestinal fibrosis in CD patients. Sphingosine activated ERK1/2 phosphorylation and inhibited TGF-b1-induced ZEB1 expression in CCD-18Co fibroblasts. The sphingosine-mediated inhibition of collagen synthesis was reversed by ERK1/2 inhibitor PD98059 and ZEB1 overexpression. The anti-fibrogenic effect of sphingosine in the TNBS model was reversed by Zeb1 overexpression. Stricturing CD patients have higher circulating granulocyte-colony stimulating factor (G-CSF) levels than non-stricturing CD patients. Csf3 promoted collagen expression in CD-HIF, while Csf3 inhibition diminished cecal fibrosis in Salmonella-infected mice. Sphingosine diminished G-CSF secretion in primary human peripheral blood mononuclear cells (PBMCs). Sphingosine also inhibited cecal Csf3 mRNA expression and reduced circulating G-CSF levels in Salmonella-infected mice. CONCLUSIONS Sphingosine exerts the most potent anti-fibrogenic effects by modulating ERK1/2, ZEB1, and G-CSF expression.