| Summary: | T-cell immunoglobulin domain and mucin domain-3 (Tim-3) is a versatile immunomodulator that protects against intestinal inflammation. Necroptosis is a type of cell death that regulates intestinal homeostasis and inflammation. The mechanism(s) underlying the protective role of macrophage Tim-3 in intestinal inflammation is unclear; thus, we investigated whether specific Tim-3 knockdown in macrophages drives intestinal inflammation via necroptosis. Tim-3 protein and mRNA expression were assessed via double immunofluorescence staining and single-cell RNA sequencing (sc-RNA seq), respectively, in the colonic tissues of patients with inflammatory bowel disease (IBD) and healthy controls. Macrophage-specific Tim3-knockout (Tim-3M−KO) mice were generated to explore the function and mechanism of Tim-3 in dextran sodium sulfate (DSS)-induced colitis. Necroptosis was blocked by pharmacological inhibitors of receptor-interacting protein kinase (RIP)1, RIP3, and reactive oxygen species (ROS). Additionally, in vitro experiments were performed to assess the mechanisms of neutrophil necroptosis induced by Tim-3 knockdown macrophages. Although Tim-3 is relatively inactive in macrophages during colon homeostasis, it is highly active during colitis. Compared to those in controls, Tim-3M−KO mice showed increased susceptibility to colitis, higher colitis scores, and increased pro-inflammatory mediator expression. Following the administration of RIP1/RIP3 or ROS inhibitors, a significant reduction in intestinal inflammation symptoms was observed in DSS-treated Tim-3M−KO mice. Further analysis indicated the TLR4/NF-κB pathway in Tim-3 knockdown macrophages mediates the TNF-α-induced necroptosis pathway in neutrophils. Macrophage Tim-3 regulates neutrophil necroptosis via intracellular ROS signaling. Tim-3 knockdown macrophages can recruit neutrophils and induce neutrophil necroptosis, thereby damaging the intestinal mucosal barrier and triggering a vicious cycle in the development of colitis. Our results demonstrate a protective role of macrophage Tim-3 in maintaining gut homeostasis by inhibiting neutrophil necroptosis and provide novel insights into the pathogenesis of IBD.
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