Mucosal Single-Cell Profiling of Crohn's-Like Disease of the Pouch Reveals Unique Pathogenesis and Therapeutic Targets

Abstract

The pathophysiology of Crohn's-like disease of the ileal pouch-anal anastomosis (CDP) in patients with a history of ulcerative colitis (UC) is unknown. We examined mucosal cells from patients with and without CDP using single cell analyses. Endoscopic samples were collected from pouch body and pre-pouch ileum (pouch/ileum) of 50 patients with an IPAA. Single-cell RNA sequencing (scRNA-seq) was performed on pouch/ileal tissues of patients with normal pouch/ileum and CDP. Mass cytometry was performed on mucosal immune cells from patients with UC with normal pouch/ileum, CDP, pouchitis, as well as those with familial adenomatous polyposis (FAP) after pouch formation. Findings were independently validated using immunohistochemistry. The cell populations/states in pouch body differed from those in pre-pouch ileum, likely secondary to increased microbial burden. Compared to FAP pouch, UC pouch was enriched in colitogenic immune cells even without inflammation. CDP was characterized by increases in Th17 cells, inflammatory fibroblasts, inflammatory monocytes, TREM1+ monocytes, clonal expansion of effector T cells, and overexpression of Th17-inducing cytokine genes such as IL23, IL1B and IL6 by mononuclear phagocytes (MNPs). Ligand-receptor analysis further revealed a stromal-MNP-lymphocyte circuit in CDP. Integrated analysis showed that upregulated immune mediators in CDP were similar to those in CD and pouchitis, but not UC. Additionally, CDP pouch/ileum exhibited heightened endoplasmic reticulum (ER) stress across all major cell compartments. CDP likely represents a distinct entity of inflammatory bowel disease with heightened ER stress in both immune and non-immune cells, which may become a novel diagnostic biomarker and therapeutic target for CDP.