Single-cell RNA sequencing uncovers intestinal immune alterations and cellular diversity from chronic fluoride exposure in mice

<b>Rationale:</b> Chronic exposure to high-fluoride drinking water impairs intestinal structure and function, potentially damaging extraluminal tissues via the gut-organ axis. However, how lifelong exposure to naturally occurring moderate-to-high-fluoride water affects intestinal cells and their underlying mechanisms remain unclear. <b>Methods:</b> Single-cell RNA sequencing identified cellular heterogeneity and candidate risk genes in the mouse ileum after 56 weeks of 50-ppm fluoride exposure. Cellchart was employed to analyze fluoride-altered cell communication patterns, and gut bacterial richness was ablated using broad-spectrum antibiotics to validate high fluoride-disrupted intercellular signaling pathways. <b>Results:</b> Fluoride exposure disrupted enterocyte trans-differentiation, affected metabolic health by restricting nutrient absorption, and activated antibacterial activity in enterocytes at the villus base. Downregulation of genes associated with rapid goblet-cell emptying and transmembrane mucin 3 in goblet cell impairs mucus and glycocalyx formation. Antimicrobial peptides, lectins, and lysozymes were reduced in fluoride-exposed Paneth and goblet cells. Fluorescence <i>in situ</i> hybridization demonstrated bacterial invasion of the epithelium following mucus barrier damage. Immunologically, fluoride-exposed T cells exhibit high scores for apoptosis, cell cycle suppression, inflammation, and high gut-homing gene expression. Fluoride exposure promoted somatic hypermutation and affinity selection in B-lineage cells while expanding plasma cells with high developmental potential. Ligand-receptor analysis revealed that activated enterocytes presented antigens to T cells via the MHC-II L-R signaling pathway, triggering downstream responses such as upregulating proinflammatory factors and cytotoxic molecules, and remodeling B-lineage cells. Broad-spectrum antibiotics depleted gut microbiota, reducing fluoride-induced gut microbial overgrowth and suppressing MHCII signaling in enterocytes and T/B cell activation-thereby decreasing proinflammatory cytokines and immunoglobulins. <b>Conclusions:</b> High-fluoride exposure disrupts the intestinal mucus barrier and gut microbiota homeostasis, leading to bacterial invasion of the epithelium that activates MHC-II signaling in absorptive enterocytes. Upregulated MHC-II signaling triggers intestinal immune cell activation and inflammation. These results reveal new intercellular interactions and communication hubs in intestinal cells under fluoride exposure.