P0130Roseomonas mucosa drives epithelial EMT and profibrotic fibroblast phenotypes, revealing a microbiota-dependent mechanism of intestinal fibrosis

Abstract Background Intestinal fibrosis remains a major unmet need in Crohn’s disease (CD). Current therapies target inflammation but do not prevent or reverse fibrotic progression, suggesting that additional drivers may be involved. Using multi-omic analyses, we identified Roseomonas mucosa as enriched in fibrotic CD mucosa. This study investigates whether R. mucosa contributes to epithelial barrier failure and stromal activation—two key events in fibrosis development. Methods Organoid-derived epithelial monolayers and primary human colonic fibroblasts were stimulated with R. mucosa lysates (10 µg/mL). EMT (Epthelial-to-Mesenchimal Transition) and profibrotic markers (TJP1, OCLN, CLDN1/2/3/4, CDH2, VIM, ACTA2, FN1, FAP, PDPN) were assessed by qRT-PCR and immunofluorescence; epithelial function was measured by TEER. A two-chamber co-culture model was used to study epithelial–stromal crosstalk under microbial stimulation. Results R. mucosa induced a combined loss of epithelial barrier components and activation of EMT-related genes. Tight-junction markers (TJP1, OCLN, CLDN1/3/4) were reduced, whereas VIM, ACTA2, FN1, CDH2, CLDN2, and FAP increased. Functionally, TEER decreased and ZO-1 localization was disrupted, indicating early epithelial destabilization. Fibroblasts responded with a strong profibrotic signature. Alongside ACTA2, FN1, COL4A1/2, and FAP, we observed marked induction of PDPN, a stromal marker associated with reticular fibroblast expansion in fibrotic CD lesions. Immunofluorescence confirmed activation toward myofibroblast/reticular phenotypes. In the epithelial–fibroblast co-culture, preliminary qRT-PCR and IF data indicate synergistic enhancement of both responses:epithelial cells exhibit a stronger EMT/tight-junction alteration signature and fibroblasts show amplified profibrotic gene induction. Together, these findings indicate that R. mucosa initiates a coordinated epithelial–stromal activation process, in which barrier disruption and fibroblast activation reinforce one another, ultimately magnifying mucosal remodeling. Conclusion Roseomonas mucosa impairs epithelial barrier function and activates PDPN+ profibrotic fibroblasts, with amplified effects through epithelial–stromal crosstalk, supporting a microbiota-driven mechanism of mucosal remodeling in Crohn’s disease. Conflict of interest: Dr. Nicolo’, Sabrina: No conflict of interest Massimino, Luca: No conflict of interest Bozzetti, Valentina: No conflict of interest Errico, Carmela: No conflict of interest Spanò, Salvatore: No conflict of interest Solitano, Virginia: Speaker’s fees from Pfizer, Takeda, Giuliani, Tillotts Pharma consulting fees from J & J travel grant from Abbvie Tommaso Lorenzo, Parigi: No conflict of interest Cagliani, Stefania: No conflict of interest Ungaro, Federica: No conflict of interest Danese, Silvio: Personal Fees: AbbVie, Alimentiv, Allergan, Amgen, Applied Molecular Transport, AstraZeneca, Athos Therapeutics, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Dr Falk Pharma, Eli Lilly, Enthera, Ferring Pharmaceuticals Inc., Gilead, Hospira, Inotrem, Janssen, Johnson & Johnson, Morphic, MSD, Mundipharma, Mylan, Pfizer, Roche, Sandoz, Sublimity Therapeutics, Takeda, Teladoc Health, TiGenix, UCB Inc., Vial, Vifor Lecture fees from Abbvie, Amgen, Ferring Pharmaceuticals Inc., Gilead, Janssen, Mylan, Pfizer, Takeda