P0120 Study of the Implication of the Epithelium in the Development and Progression of Fibrosis in Ulcerative Colitis
Authors: Stepniak M , Adam P , Salée C , Massot C , Loly J P , Vieujean S , Reenaers C , Coimbra Garcia Marques C , Decker E , Delvenne P , Blétard N , Speca S , Beq S , Louis E , Meuwis M A 
Published: January 2025

Abstract

Background
Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) with diffuse colonic mucosal inflammation. Fibrosis has been observed in the submucosa and muscularis mucosae even without active inflammation2, yet remains underexplored in UC. No anti-fibrotic treatments currently exist for IBD4. At the epithelial level, IBD-related endoplasmic reticulum stress (ERS) and inflammation upregulate ER chaperones like AGR2, which, when secreted extracellularly (eAGR2), can induce fibroblast-to-myofibroblast transition (FMT), suggesting a role in fibrosis3. Our aim is to use a pro-fibrotic organoid model from UC patients and controls to study the epithelium’s role in fibrosis, focusing on AGR2’s paracrine action

Methods
Apical-out organoids were derived from intestinal stem cells of UC patients and controls1. ERS was induced with Tunicamycin (Tm), followed by recovery in fresh medium, to observe epithelial responses post-stress, assessing paracrine effects. Transcripts, proteins, and supernatants were analyzed for ERS markers, epithelial-mesenchymal transition (EMT), pro-fibrotic markers (TGF-β, eAGR2), and cytokines. Immunofluorescence (IF) was performed for AGR2 and sXBP1. Conditioned supernatants were used to expose intestinal fibroblasts (CCD18-Co) to assess FMT by IF (α-SMA increase), confirming the pro-fibrotic phenotype of UC organoids. Forty-four UC patients (198 slides) were scored for inflammation (Geboes score), fibrosis2, and ERS markers (AGR2/BiP staining)

Results
Organoids from UC patients and controls showed increased ERS markers (RT-qPCR: BiP/CHOP/sXBP1 fold change 6-16, 4-9.5, and 3-8), with minimal variation in pro-inflammatory cytokines and TGF-β. AGR2 expression increased post-ERS compared to control (Tm/CTL: 2-10). Intracellular AGR2, BiP, and ERP72 rose in parallel with ERS induction. IF showed increased AGR2 and sXBP1, with AGR2 redistributing from the apical pole and diffusing across the organoid under ERS. Active eAGR2 levels in supernatants increased post-ERS (Tm/CTL eAGR2 range: 1.5 to 3). CCD18-Co assays with supernatants showed elevated α-SMA, confirming FMT. ERS was confirmed in UC tissues, with stronger chaperone signals associated with inflammation and fibrosis.

Conclusion
We developed a model for studying ERS-induced epithelial responses in UC. Apical-out UC organoids exposed to ERS demonstrate a pro-fibrotic paracrine effect independent of pro-inflammatory/pro-fibrotics markers, suggesting that ERS in IBD may promote intestinal fibrosis. The specific role of eAGR2 in this phenotype is under investigation, focusing on transcriptomic and secretomic shifts accompanying ERS responses in UC organoids

References
1)Co, J. Y., et al. (2021). Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases. In Nature Protocols. https://doi.org/10.1038/s41596-021-00607-0
2)Gordon, I. O., et al. (2018). Fibrosis in ulcerative colitis is directly linked to severity and chronicity of mucosal inflammation. Alimentary Pharmacology and Therapeutics. https://doi.org/10.1111/apt.14526
3)Vieujean, S., et al. (2021). Potential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homologue in Crohn’s Disease Fibrosis. Journal of Crohn’s and Colitis. https://doi.org/10.1093/ecco-jcc/jjab061
4)F. C. EDWARDS and S. C. TRUELOVE, “the Course and Prognosis of Ulcerative Colitis. Iii. Complications.,” Gut, vol. 5, pp. 1–22, 1964