CREEPING FAT-DERIVED FIBROBLASTS PARTICIPATE IN INTESTINAL FIBROSIS IN A NOVEL MOUSE MODEL OF INTESTINAL STRICTURES

Abstract

Abstract INTRODUCTION An intestinal stricture is a common morbidity in Crohn's disease, but its pathogenesis is poorly understood. CF forms adjacent to strictures and is associated with stricture recurrence, but whether it promotes stricture formation is unclear. Adipocytes display remarkable plasticity, and adipocyte-derived fibroblasts (ADFs) play important roles in tissue wound healing and fibrosis. Here, we show that CF ADFs may promote intestinal stricture formation. METHODS We developed a novel mouse model of CF and intestinal fibrosis by creating anti-mesenteric colotomies. We then created colotomies in Adipoq-Cre; mTmG mice and harvested the mice at post-operative day (POD) 14. This system exclusively labels adipocytes and their derivatives GFP+, whereas all other cells are TdTomato+ (Figure 1A-B). We immunostained sham and colotomy bowel for mature adipocyte marker PLIN1. We quantified GFP+ lineage-negative ADFs (CD45-CD31-Terr119-EPCAM-ECadherin-) isolated from either sham or colotomy bowel at POD 14. We then isolated ADFs (GFP+) and non-ADFs (nADFs) (TdTomato+) by flow cytometry and performed single-cell RNA-seq (scRNA-seq) (Figure 1F). RESULTS The creation of a colotomy is sufficient to induce localized mesenteric adipose tissue (MAT) expansion around the colotomy site, consistent with CF formation (Figure 1C). We observed the presence of GFP+ cells with a fibroblast morphology that lost the expression of adipocyte markers and acquired the expression of fibroblast markers. (Figure 1D). Furthermore, we observed that GFP+ ADFs expanded in colotomy bowel compared to sham (Figure 1E). scRNA-seq of ADFs and nADFs revealed 5 major fibroblast clusters, including a Clu+ pro-fibrotic population and a Pi16+ progenitor population, which were enriched in bowel GFP+ ADFs (Figure 1G-H). Finally, quantification of gene expression revealed that GFP+ ADFs lacked mature adipocyte markers, expressed fibroblast markers, and were enriched for various ECM proteins. CONCLUSIONS An anti-mesenteric model of intestinal strictures and CF in combination with immunostaining and scRNA-seq revealed that ADFs from CF infiltrate the bowel wall and expand at the site of fibrosis. ADFs exhibit fibroblast heterogeneity and are enriched for pro-fibrotic and stromal progenitor populations. Finally, ADFs produce collagen and fibronectin, suggesting that they may be pro-fibrotic. Altogether, these data highly suggest that CF contains pro-fibrotic stromal cells that infiltrate the stricture site to promote fibrosis.