Abstract Background Inflammatory bowel disease (IBD) arises from dysregulated immune responses to intestinal microbiota in genetically susceptible hosts1. Macrophages play a central role in shaping mucosal inflammation, yet the contribution of C-type lectin receptors, particularly Mincle (Clec4e), to intestinal immune activation remains unclear2. This study aimed to elucidate how bacterial translocation and macrophage subsets influence colitis pathogenesis, and to define the functional role of Clec4e in macrophage-mediated inflammatory responses. Methods Fecal microbiota from patients with active Crohn’s disease were transplanted into germ-free mice to identify bacteria capable of translocating into mesenteric lymph nodes (CD-mLN-Bac). Peritoneal macrophages were stimulated with bacterial mixture of CD-mLN-Bac to assess inflammatory responses by qPCR. Dextran sulphate sodium (DSS) colitis was induced in mice, followed by single-cell RNA sequencing and spatial transcriptomics to characterise macrophage heterogeneity. Clec4e knockout (KO) mice were generated using CRISPR/Cas93, and disease severity was evaluated by body weight, colon length, histology, and cytokine expression in peritoneal macrophages stimulated with LPS or β-glucosylceramide. Results Three bacterial species were identified as CD-mLN-Bac that consistently translocated across the gut barrier in germ-free mice. Macrophages exhibited strong, time- and dose-dependent inflammatory gene induction upon stimulation by CD-mLN-Bac. Time lapse analyses revealed that macrophages phagocytosed CD-mLN-Bac. scRNA-seq and spatial analysis showed a marked expansion of inflammatory macrophages in DSS colitis, with Clec4e expression surpassing classical inflammatory markers such as Tnf and Il1b. Although Clec4e KO mice displayed similar body weight loss and colon length to wild-type mice, histology revealed more severe mucosal inflammation in KO animals. KO macrophages showed reduced Il10, Il6, and Tnf induction following β-glucosylceramide stimulation, while LPS stimulation provoked exaggerated Il6 responses, suggesting dysregulated TLR4–CLR crosstalk. Conclusion Mincle-expressing macrophages expand during colitis and respond robustly to bacterial and damage-associated signals. Loss of Clec4e alters cytokine regulation and worsens local intestinal inflammation despite unchanged systemic indices, indicating that Mincle has dual functions in promoting inflammation and supporting tissue protection, potentially through necrotic cell clearance. These findings highlight Clec4e as a key regulator of macrophage activation and intestinal immune homeostasis. References: 1.Neurath, M.F. (2024). Strategies for targeting cytokines in inflammatory bowel disease. Nat Rev Immunol 24, 559–576. 10.1038/s41577-024-01008-6. 2.Tanaka, M., Saka-Tanaka, M., Ochi, K., Fujieda, K., Sugiura, Y., Miyamoto, T., Kohda, H., Ito, A., Miyazawa, T., Matsumoto, A., et al. (2020). C-type lectin Mincle mediates cell death-triggered inflammation in acute kidney injury. J Exp Med 217. 10.1084/jem.20192230. 3.Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. 10.1126/science.1225829. Conflict of interest: Ito, Kotaro: Provision of writing assistance, medicines, equipment, or administrative support Tsunoda, Junya: Provision of writing assistance, medicines, equipment, or administrative support Tajima, Lili: Provision of writing assistance, medicines, equipment, or administrative support Mizushima, Ichiro: Provision of writing assistance, medicines, equipment, or administrative support Kaieda, Yuta: Provision of writing assistance, medicines, equipment, or administrative support Mikami, Yohei: Provision of writing assistance, medicines, equipment, or administrative support Kanai, Takanori: Provision of writing assistance, medicines, equipment, or administrative support
Ito et al. (Thu,) studied this question.