MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS
Authors: Cao Siyan , Davidson Nicholas , Ciorba Matthew , Deepak Parakkal , Colonna Marco 
Published: January 2024

Abstract

BACKGROUND & AIMS
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), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including 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 with and without CDP using single cell analyses.

METHODS
Endoscopic samples from the pouch 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 pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8).

RESULTS
We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). 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 and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases.

CONCLUSIONS
CDP demonstrates altered immune and non-immune cell populations/states in the pouch 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.

Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p&lt;0.01.

Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.