DOP140Gut Microbiota-dependent Regulation of Intestinal Epithelial Fucosylation by CX3CR1+ Macrophages in Crohn’s Disease

Background Crohn’s disease(CD) pathogenesis involves genetic susceptibility (e.g., loss-of-function mutations in the fucosyltransferase 2 (FUT2) gene) and gut microbiota dysbiosis. The FUT2 gene directly controls the level of intestinal epithelial fucosylation. The lectin UEA-1, a robust marker for FUT2 enzymatic activity, exhibits binding signals that co-localize with FUT2 expression. Within the gut, CX3CR1+ macrophages (Mφ) are central to gut homeostasis but can also drive disease progression in inflammatory bowel disease (IBD). However, the specific impact of CX3CR1+ Mφ on intestinal fucosylation remains unclear. Methods We enrolled 20 participants (10 CD and 10 non-IBD controls). Intestinal samples from CD patients included both inflamed/non-inflamed margins, whereas controls were obtained from functioning stomas in non-IBD. We performed immunofluorescence (IF) and qPCR analyses across the three sample groups. In mice, CX3CR1 knockout and wild-type (WT) mice underwent microbiota depletion. Mice were then treated with water, DSS, or DSS+E. coli gavage, and intestinal FUT2 expression (by IF) and cytokine levels (by ELISA) were assessed. Results In CD patients, the inflamed tissue exhibited markedly reduced α-1,2-fucosylation, which was accompanied by a significant increase in CX3CR1+ macrophage infiltration (Fig.A). FUT2 mRNA expression was markedly downregulated in CD inflamed tissues compared to their matched non-inflamed regions (Fig.B). Concurrently, CX3CR1 expression was notably upregulated in CD inflamed tissues compared to both non-inflamed areas and controls. The mRNA levels of key cytokines from CX3CR1+ Mφ, particularly IL-23 and IL-1β, were markedly higher in inflamed areas, indicating a pronounced pro-inflammatory phenotype at the disease sites. In mouse, depletion of CX3CR1+ Mφ resulted in a pronounced decrease in FUT2 expression (Fig.D). This decrease was further intensified by DSS-induced colitis. Notably, FUT2 expression was partially restored in DSS+ E. coli -treated WT mice. This indicates that CX3CR1+ Mφ and gut bacteria collaboratively regulate FUT2 expression. ELISA confirmed elevated IL-23 and IL-1β in colitic mice. However, IL-1β levels increased more in WT mice, while IL-23 was more strongly elevated in CX3CR1-deficient mice. This suggests that IL-1β, rather than IL-23, may be a key mediator through which CX3CR1+ Mφ regulate FUT2 expression (Fig.E). Conclusion A significant negative correlation between intestinal inflammation and FUT2-mediated fucosylation. CX3CR1+ Mφ and intestinal bacteria co-regulate FUT2 expression, potentially through the key cytokine IL-1β. Aberrant activation of pro-inflammatory CX3CR1+ Mφ impair protective fucosylation, thereby fueling the initiation and progression of inflammation in CD. Conflict of interest: Lin, Jue: No conflict of interest Cui, Yitong: No conflict of interest Zhang, Huoyan: No conflict of interest Zhang, Qi: No conflict of interest Xu, Zhaoyuan: No conflict of interest Zhang, Min: No conflict of interest Wang, Wei: No conflict of interest Hu, Jun: No conflict of interest Zhi, Min: No conflict of interest