Abstract Background The mesentery contributes to Crohn’s disease (CD) progression, promoting immune cell infiltration and fibrosis. Mesenteric fibroblasts, activated by pro-fibrotic cytokines such as TGF-β, stimulate fibrosis through excessive extracellular matrix (ECM) deposition, leading to intestinal thickening and stricture formation. In this study, we aim to explore the specific role of the mesenteric inflammation-associated fibroblasts in the development of CD. Methods Mesenteric inflammation-associated fibroblasts (IAFs) and normal fibroblasts (NFs) were isolated from the mesenteries of patients with Crohn’s disease (CD) and individuals with non-cancerous colonic polyps (control samples) using magnetic-activated cell sorting. Phenotypic differences between the fibroblast populations were analyzed using cell viability, invasion, and collagen contraction assays. Co-culture systems, including transwell and 3D Matrigel co-cultures, were employed to evaluate the effects of NFs and IAFs on the behavior of colonic epithelial cancer cell lines (Caco-2 and HT-29). RNA-seq followed by gene set enrichment and gene ontology analyses identified IAF-specific genes associated with CD progression. Candidate genes were further validated by immunofluorescence staining in mesenteric tissues from CD patients. Results IAFs exhibited higher cell growth, wound healing, invasion, and collagen contraction activities compared with NFs. In a 3D Matrigel co-culture system, IAFs induced abnormal lumen formation and invasive structures in Caco-2 spheres. Consistently, IAFs promoted epithelial barrier disruption in a fluorescein transport assay using a transwell co-culture system. Conditioned media from IAFs upregulated mesenchymal markers in Caco-2 cells. Furthermore, transcriptomic analyses revealed enrichment of ECM organization chemokine- and cytokine-mediated signaling pathways in IAFs. Among the upregulated genes, we identified gene X as an IAF-specific chemokine and confirmed that its expression was significantly higher in mesenteric tissues from CD patients compared with controls. Conclusion These findings indicate that mesenteric IAFs actively contribute to epithelial barrier dysfunction and disease progression in CD through the secretion of chemokines. Targeting IAF-derived chemokine may represent a novel therapeutic strategy. Conflict of interest: Ms. Park, Jihye: No conflict of interest Joo, Yang Hee: No conflict of interest Song, Eun Mi: No conflict of interest Park, Yehyun: No conflict of interest Byeon, Ju Ran: No conflict of interest Choe, A Reum: No conflict of interest Noh, Gyoung Tae: No conflict of interest Kim, Ho Seung: No conflict of interest Choi, Euno: No conflict of interest Byun, Jieun: No conflict of interest Ahn, Young-Ho: No conflict of interest Jung, Sung-Ae: No conflict of interest
Park et al. (Thu,) studied this question.
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