HUMAN ADIPOSE STEM CELLS FROM CREEPING FAT EXHIBIT REDUCED APOPTOSIS AND SECRETE LESS PRO-INFLAMMATORY CYTOKINES BASED ON BULK RNA SEQUENCING

BACKGROUND: Mesenteric fat is increasingly believed to exert an important influence on intestinal inflammation. A hallmark of severe Crohn's disease (CD) is the accumulation of mesenteric fat around the inflamed intestine, typically in the terminal ileum. This so-called "creeping fat" is an important indicator of regional disease activity and is often used by surgeons as a measure of disease severity. Creeping fat correlates with intestinal transmural inflammation, muscular hypertrophy, and fibrosis/stricture formation. The pathologic function of creeping fat, however, remains unclear. We investigated this matter through the lens of adipose-derived stem cells (ASCs). ASCs are mesenchymal cells with the capacity for self-renewal and multipotential differentiation. They are critical to the adipose function and, importantly, can be cultured and studied ex vivo in a controlled and robust fashion. Here we aimed to characterize the function of ASCs in CD. METHODS: ASCs were isolated from the creeping fat of patients with CD and the mesenteric fat of control patients (n =4). After purifying and testing via fluorescence-activated single-cell sorting (FACS), bulk RNA sequencing and gene set enrichment analysis (GSEA) was performed to investigate the distinctive gene pattern of ASCs. The levels of various pro-inflammatory cytokines (TNF-a, IL-6, and IL8) were measured by ELISA and the apoptosis was measured by immunostaining of cleaved- caspase-3. RESULTS: Principal component analysis (PCA) and gene heat mapping revealed a distinctive gene pattern in ASCs from creeping fat compared to those from controls. ASCs from creeping fat were characterized by less colony formation as well as reduced differentiation and migration capacity. GSEA results suggested an up-regulated pathway for inflammatory response and apoptosis in ASCs from creeping fat. ELISA assays confirmed that ASCs from the creeping fat secreted less pro-inflammatory cytokines, including TNF-α, IL-6, and IL-8. ASCs from the creeping fat showed a lower level of cleaved caspase-3, suggesting that ASCs from CD patients experience reduced levels of apoptosis. CONCLUSIONS: Our study provides an explanation of the accumulation of creeping fat in CD patients - namely, that ASCs from creeping fat undergo less apoptosis and have reduced differentiation and migration capacity, resulting in an accumulation of this distinctive mesenteric fat. Moreover, we found that ASCs from CD patients secrete less pro-inflammatory cytokines, lending support to the emerging consensus that creeping fat likely serves a protective function in CD, by serving as a barrier surrounding the inflamed intestine. As such, our study may assist in the development of novel, adipose-based CD therapies.