β-Hydroxybutyrate impairs the functional response of bovine neutrophils to mammary pathogenic Staphylococcus aureus and a TLR2/1 agonist by limiting glucose metabolism

Ketosis is a common metabolic disease affecting dairy cows during early lactation. β-Hydroxybutyrate, the predominant ketone body in the bloodstream of ketotic cows, has been linked to neutrophil dysfunction and a higher incidence of mastitis. Neutrophils, the first line of cellular defense against bacteria, rely heavily on carbohydrate metabolism. In this study, we investigated the effect of high BHB concentrations on glycolysis and on the functional response of bovine neutrophils to Staphylococcus aureus-a leading cause of mastitis worldwide-and to Pam3CSK4, a synthetic agonist of toll-like receptor 2/1 (TLR2/1), which is critical for S. aureus recognition by immune cells. At both 2.5- and 5.0-mM concentrations, BHB reduced basal glycolysis and restricted the glycolytic capacity of neutrophils following Pam3CSK4 stimulation. By pharmacological inhibition, we confirmed that bovine neutrophils depend on both glycolysis and glycogenolysis to mount effective responses to S. aureus and Pam3CSK4. Interestingly, both 2.5- and 5.0-mM BHB similarly impaired neutrophil responses against S. aureus and Pam3CSK4, including respiratory burst, neutrophil extracellular traps formation, matrix metallopeptidase 9 release, phagocytosis, and chemotaxis. Our findings suggest that BHB-mediated glycolytic restriction may constitute a central mechanism contributing to neutrophil dysfunction during ketosis, thereby increasing the susceptibility of dairy cows to mastitis in early lactation.