17 SINGLE-CELL SEQUENCING OF CREEPING FAT IN CROHN’S DISEASE REVEALS DISTINCT IMMUNE CLUSTERS AND CELLULAR RESTRUCTURING IN THE PRESENCE OF LIVE BACTERIA AND FUNGI

Abstract

Crohn’s Disease and ulcerative colitis present distinct characteristics, one being the expansion of mesenteric adipose tissue (MAT) surrounding the inflamed intestinal tract (“creeping fat”-CF), which is typical for CD and absent in UC. To-date, creeping fat has been accepted as a mystery with very little published data, and no determination of whether this is a pro-inflammatory or protective response, or both. The current study explores whether microorganism translocation from the inflamed gut to the adjacent mesenteric adipose drives the MAT restructuring observed as CF. We cultivated bacteria and fungi anaerobically and aerobically from CF and adjacent aseptically resected large bowel from seven CD patients as well as uninvolved bowel and MAT from the same patient. Additional controls included paired involved and uninvolved bowel and MAT from UC patients (n=7). In addition to cultivation, the 16s rRNA and ITS regions of microbiota and fungi from mucosal scrapings and MAT were sequenced to assess the total microbiome community differences between paired samples. A distinct cultivable microbiome was consistently identified in MAT of all patients, with greater numbers of distinct species found in CF of CD patients compared to paired uninvolved tissue, and UC controls. Single cells from the stromal vascular fraction of CF and uninvolved paired MAT was isolated and encapsulated for RNA sequencing to identify cell populations unique to creeping fat and individual gene expression that may suggest the functional role of CF. Results revealed more numerous cell types in creeping fat compared to uninvolved MAT, with an upregulation within specific immune clusters of antimicrobial genes. Together these data suggest that live bacteria exist within CF and illicit a host immune response within this tissue that is not present in uninvolved tissue.