Phytosphingosine contributes gut microbiota metabolism to alleviate low-grade endotoxemia-induced mastitis

Recently, evidence has indicated that mastitis is closely associated with the ruminal dysbiosis caused by subacute ruminal acidosis (SARA) and subsequent low-grade endotoxemia (LGE). However, whether and how ruminal metabolic dysbiosis influences SARA-associated mastitis still unclear. Using untargeted metabolomics, we found that cows with SARA-associated mastitis exhibited altered ruminal metabolic profiles, particularly a reduced level of phytosphingosine (PS), compared to healthy cows. Oral administration of PS to mice alleviated LGE-induced mastitis, as evidenced by attenuated mammary injury and improved function of mammary tight junctions (TJs). Furthermore, we demonstrated that LGE induced significant gut dysbiosis, characterized by increased abundances of opportunistic pathogens such as Enterococcus, which was mitigated by PS treatment. Interestingly, transplantation of both fecal microbiota (FMT) and matched sterile supernatant (FST) from PS-treated mice alleviated LGE-induced mastitis, restored the blood-milk barrier, and modulated the gut microbiota in recipient mice following LGE exposure. Additionally, PS and PS-FMT treatments increased the abundances of fecal short-chain fatty acid (SCFA) producers, accompanied by elevated fecal SCFA levels, particularly butyric acid, in PS- and PS-FMT-treated mice. Butyric acid was negatively correlated with mammary inflammatory markers, and butyrate administration attenuated LGE-induced mastitis in mice. Mechanistically, butyrate promotes M2 macrophage polarization and inhibits NF-KB and NLRP3 inflammasome activation via G-protein-coupled receptor 41 (GPR41). Collectively, our results demonstrate that gut microbiota from PS-dosed mice alleviates LGE-induced mastitis in mice by promoting SCFA production and regulating macrophage polarization. Our findings provide deeper insights into gut dysbiosis-associated mastitis and highlight the potential of modulating gut microbiota and its metabolism as a strategy for managing mastitis and other related diseases.