P0014 Mesenteric inflammation-associated fibroblasts are critical in the pathogenesis of Crohn’s disease

Abstract Background The Mesentery, initially considered as a structure support of abdominal organs, has emerged as an active participant in the inflammatory processes of Crohn's disease (CD). It serves as a central hub for immune cell activity, including macrophages, T cells, and B cells, driving the propagation of inflammation. Additionally, mesenteric fibroblasts play a crucial role in CD-associated fibrosis. These fibroblasts are activated by pro-fibrotic cytokines such as TGF-β and IL-13, leading to excessive extracellular matrix (ECM) deposition, which contributes to intestinal wall thickening and the formation of strictures. In this study, we aim to explore the specific role of mesenteric fibroblasts in the pathogenesis of CD. Methods Magnetic-activated cell sorting was utilized to isolate mesenteric inflammation-associated fibroblasts (IAFs) and normal fibroblasts (NFs) from surgical samples of patients with Crohn's disease (CD) and noncancerous colonic polyps (control group). Various cell-based assays, including cell viability, invasion, and collagen contraction assays, were conducted to investigate phenotypic differences between NFs and IAFs. To assess the impact of NFs and IAFs on colonic epithelium, Co-culture experiments were performed with the isolated fibroblasts and Caco-2 and HT-29 colorectal cancer cell lines using transwell and 3D Matrigel culture systems. Results IAFs exhibited a significantly faster growth rate than NFs (Figure 1B; fold change [FC] 1.60 ± 0.04, P < 0.001). Furthermore, IAFs showed markedly higher wound healing (Figure 1C; FC 1.44 ± 0.15, P = 0.004), invasion (Figure 1D; FC 1.45 ± 4.7, P = 0.035), and collagen contraction activities (Figure 1E; FC 1.26 ± 4.73, P = 0.007) compared to NFs. In a 3D Matrigel co-culture system with Caco-2 spheres, abnormal lumen formation with invasive structures of Caco-2 spheres was significantly higher in IAFs compared to NFs (Figure 2A; FC 1.3 ± 0.08, P < 0.001). Similarly, a fluorescein transport assay using a transwell co-culture system revealed that IAFs caused significantly greater epithelial disruption compared to NFs (Figure 2B; FC 1.25 ± 0.04, P = 0.04). Conditioned media from IAFs upregulated the expression of mesenchymal markers, such as ZEB1 (FC 2.67 ± 0.2, P < 0.01) and CDH2 (FC 2.11 ± 0.54, P < 0.05), in Caco-2 cells when compared with NFs (Figure 2C). Conclusion These findings suggest that mesenteric IAFs play an active role in the inflammatory and fibrotic processes in the pathogenesis of CD. Targeting mesenteric IAFs and their associated pathways may provide a promising therapeutic approach for managing CD.