Rutin triggers IRE1-mediated GSDMD-dependent pyroptosis in macrophages to suppress systemic Salmonella infection

• RT restored epithelial barrier integrity, reduced inflammation, and suppressed bacterial translocation into visceral tissues. • Rutin reduced the number of infected M1 and M2 type macrophages, but had no effect on uninfected macrophages. • Macrophage depletion reduced organ bacterial burden. • RT targets the GSDMD pathway, thereby driving pyroptosis and eliminating infected macrophages. • Rutin can promote endoplasmic reticulum stress through direct activation of IRE1 in infected macrophages. Rutin (RT), a flavonoid used clinically as a vasoprotective agent, exhibits anti-inflammatory and antioxidant activities. However, its immunomodulatory mechanisms, particularly during bacterial infection, remain unclear. Because macrophage pyroptosis is central to host defense against intracellular pathogens such as Salmonella Typhimurium ( S.Tm ), understanding how RT regulates this process is essential. To determine the effect of RT on macrophage death pathways during S.Tm infection and to elucidate the underlying mechanism and in vivo consequences. Fluorescence-labeled S.Tm was used to infect RT-treated macrophages. Pyroptosis, GSDMD activation, ER stress pathways, and macrophage polarization were assessed by flow cytometry and molecular assays. A mouse S.Tm infection model was used to evaluate bacterial burden, intestinal barrier integrity, immune cell profiles, and the impact of macrophage depletion. RT specifically enhanced pyroptosis in S.Tm-infected macrophages through GSDMD activation, with no effect on uninfected cells. RT reduced M1 and M2 macrophage populations and significantly decreased organ bacterial load. In vivo, RT improved intestinal barrier function and normalized immune cell numbers. Macrophage depletion alone also lowered bacterial burden. Mechanistically, RT triggered oxidative and ER stress, particularly activating the IRE1 pathway, thereby promoting pyroptosis in infected macrophages. This study reveals a context-dependent, ER stress–driven pro-pyroptotic effect of RT in S.Tm -infected macrophages, contributing to bacterial clearance and preservation of intestinal integrity without disturbing gut microbiota.