Macrophage Tim-3 maintains intestinal homeostasis in DSS-induced colitis by suppressing neutrophil necroptosis

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, Tim3M - 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-kappa B pathway in Tim -3 knockdown macrophages mediates the TNF-alpha-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.