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

Introduction Bovine 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 D 3 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 D 3 (D), 25-hydroxyvitamin D 3 (25D), and 1α,25-dihydroxyvitamin D 3 (1,25D) through an integrated in vitro approach. Methods Antimicrobial 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. Results D 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. Conclusion The findings indicate that vitamin D 3 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.