Vitamin D metabolites reshape bovine mammary epithelial responses relevant to mastitis through host-directed mechanisms

IntroductionBovine mastitis remains one of the most prevalent diseases in dairy cattle and is largely managed through antibiotic therapy, which contributes to antimicrobial resistance. Host-directed strategies that enhance mammary epithelial defenses may complement conventional treatments. Although vitamin D3 metabolites can modulate immune responses, their comparative effects on bovine mammary epithelial cells have not been fully characterized. This study compared the antimicrobial and host-directed effects of vitamin D3 (D), 25-hydroxyvitamin D3 (25D), and 1α,25-dihydroxyvitamin D3 (1,25D) through an integrated in vitro approach.MethodsAntimicrobial and antibiofilm activity were assessed against mastitis-associated Staphylococcus spp. isolates. Bovine mammary epithelial MAC-T cells were then treated to evaluate cellular tolerance, transcriptional regulation of vitamin D-related enzymes and receptors, global proteomic alterations, and Staphylococcus aureus internalization.ResultsD and 25D were well tolerated by MAC-T cells. They exhibited limited antimicrobial or antibiofilm activity, but pretreatment of the cells with 25D significantly reduced S. aureus internalization. Enzymes involved in vitamin D metabolism were differentially regulated by 25D and 1,25D: while 24-hydroxylase was upregulated, vitamin D receptor expression remained largely unchanged. Proteomic profiling identified 2,203 proteins common to all conditions, as well as compound-specific signatures related to epithelial homeostasis, innate immunity, vitamin D catabolism, vesicular trafficking, endocytic processes, and mitochondrial organization.ConclusionThe findings indicate that vitamin D3 metabolites primarily act through host-directed mechanisms rather than by direct antimicrobial activity. They could thus potentially be used as complementary strategies to manage mastitis in alignment with One Health principles.