DOP41 Mucosal single-cell profiling of Crohn's-like disease of the pouch reveal unique pathogenesis and therapeutic targets

Abstract

Abstract Background After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), a chronic inflammatory condition that affects both the pouch body and extra-pouch organs including the pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients after IPAA with and without CDP using single cell analyses. Methods Endoscopic samples from the pouch body and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from both pouch body and pre-pouch ileum of patients with normal pouch/ileum and CDP using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis. Mucosal samples from patients with familial adenomatous polyposis (FAP) after colectomy and pouch formation were also analyzed by CyTOF. ScRNA-seq and CyTOF findings were independently validated using immunohistochemistry. Results We revealed distinct cell clusters in normal pouch body versus normal pre-pouch ileum in UC patients. Colitogenic immune cells expanded in normal UC pouch body compared to normal FAP pouch body. Compared to normal pouch/ileum, CDP pouch/ileum exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch/ileum (Figure 1). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch body and pre-pouch ileum of CDP. Ligand-receptor analysis further revealed a stromal – myeloid – lymphocyte circuit in CDP. Integrated analysis showed that upregulated immune mediators in CDP tissues were similar to those in CD and pouchitis, but not UC. In addition, the pouch body and pre-pouch ileum of CDP exhibited prominent activation of the unfolded protein response (UPR) across all major immune and non-immune cell compartments (Figure 2), which was not present in UC, CD or pouchitis based on reanalysis of published databases. Conclusion CDP demonstrates altered immune and non-immune cell populations/states in the pouch body and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones.