Lactobacillus reuteri-derived extracellular vesicles alleviate Staphylococcus aureus-induced mastitis in mice

Mastitis is one of the most severe diseases in dairy cows worldwide, causing substantial economic losses to the dairy industry. However, effective prevention and control strategies, remain to be further elucidated. This study aims to investigate the protective effects of Lactobacillus reuteri (L. reuteri) and its extracellular vesicles (EVs) against S. aureus-induced mastitis and to explore the potential molecular mechanisms. We established a murine mastitis model induced by S. aureus, followed by treatment with extracellular vesicles secreted by L. reuteri. Histopathological alterations in mammary tissues were assessed using hematoxylin and eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the concentrations of pro-inflammatory cytokines. Western blotting was utilized to characterize the expression levels of key proteins involved in the NF-κB signaling cascade. We demonstrate that L. reuteri effectively ameliorates S. aureus-induced mastitis in mice, accompanied by inhibited inflammatory responses and restored integrity of the blood-milk barrier in mammary tissues. Further studies revealed that EVs derived from L. reuteri exert similar protective effects, significantly alleviating pathological injury in mammary tissues. Mechanistically, S. aureus infection was associated with upregulation of NF-κB/NLRP3 signaling components and increased TLR2 expression in mammary tissues, whereas L. reuteri-derived EVs markedly suppressed this inflammatory cascade in parallel with reduced TLR2 expression, attenuating inflammatory cytokine release and maintaining blood-milk barrier integrity Collectively, our findings indicate that L. reuteri-derived EVs effectively alleviate S. aureus-induced mastitis in association with suppression of the NF-κB/NLRP3 inflammatory cascade and restoration of mammary barrier integrity. This study highlights the protective role of probiotics and their derived EVs against mastitis pathogenesis, providing promising preventive and therapeutic strategies as well as candidate biomaterials for the management of this disease.