Metformin-associated gut microbiota remodeling correlates with reinvigorated splenic immunity in aged mice: microbiome-immune crosstalk via the gut-spleen axis

BACKGROUND AND AIM: Immunosenescence involves age-related immune decline and chronic inflammation, with the spleen serving as a critical hub for immune dysregulation. While gut microbiota influences systemic immunity, its specific role and the potential existence of a gut-spleen axis in mediating splenic aging remains unclear. Therefore, we investigated whether metformin, a microbiota-modulating geroprotective drug, alleviates splenic immunosenescence in aged mice, specifically exploring the link between gut microbiota remodeling and splenic immune rejuvenation. METHODS: Aged C57BL/6 mice (15-month-old) received oral metformin (300 mg/kg/day) or vehicle for 5 months. Systemic toxicity and metabolism were monitored. Splenic immune subsets were analyzed using flow cytometry and immunohistochemistry. Gut microbiota composition (16S rRNA sequencing), cytokine levels (RT-qPCR), and functional pathways were assessed. RESULTS: Metformin caused no hepatorenal toxicity or weight changes. Treated mice exhibited increased cytotoxic T cells (Tc) and macrophages in the spleen, with reduced Th/Tc ratios and M1/M2 polarization. Pro-inflammatory cytokines (Ifng, Il17a, Il1b, Il6) decreased, while anti-inflammatory markers (Arg1, Tgfb1) rose. Gut microbiota showed enriched Akkermansia, Muribaculum, and Duncaniella, but reduced Lactobacillus. Akkermansia/Muribaculum negatively correlated with pro-inflammatory cytokines, whereas Lactobacillus and Lachnospiraceae linked to pro-inflammatory responses. Functional prediction analysis based on 16S rRNA sequencing data indicated upregulation of bile acid metabolism and oxidative phosphorylation pathways. CONCLUSION: Metformin reshapes the gut microbiota, which is associated with mitigation of age-associated splenic immune dysregulation, favoring anti-inflammatory macrophage polarization and cytotoxic T cell expansion. Critically, our findings establish the gut-spleen axis as a key mediator of splenic immunosenescence and a novel therapeutic target, which positions metformin as a promising microbiota-directed geroprotective agent. Future research should prioritize mechanistic dissection of gut-spleen communication and clinical validation of metformin's geroprotective efficacy in human populations.