Molecular and functional responses of milk-derived polymorphonuclear neutrophils from subclinical mastitis quarters to β-hydroxybutyric acid at the subclinical ketosis threshold in vitro

Subclinical ketosis (SCK), a prevalent metabolic disorder in dairy cattle characterized by elevated β-hydroxybutyric acid (BHBA) levels without clinical signs, is linked to increased susceptibility to postpartum infections such as mastitis. During mammary inflammation, circulating PMNs are recruited into the mammary gland, where they encounter a distinct, lipid-rich, and non-sterile microenvironment. However, the specific impact of BHBA on bovine milk-derived PMNs remains poorly understood. We hypothesized that high BHBA exposure would dysregulate the functional profile of milk PMNs, potentially leading to paradoxical effects. In this study, we aimed to determine the in vitro immunomodulatory effects of BHBA (1.2 mM, a commonly used blood-based threshold for SCK) on cells isolated from subclinical mastitis-positive quarters. The immunomodulatory effects of BHBA on bovine milk PMNs were investigated through a multifaceted method encompassing functional assays, gene and protein expression profiling, and molecular docking. BHBA exposure did not significantly alter milk-derived PMN migration, phagocytosis of S. aureus or E. coli , ROS or NO production, MPO degranulation, or NET release. In contrast, significant transcriptional alterations were observed, including upregulation of BAX and IL1B and downregulation of several genes associated with chemotaxis, pathogen recognition, phagocytosis, and oxidative regulation, including CXCL8 , CXCR1 , CXCR2 , TLR4 , CORO1A , RAB7A , RAC1 , and SOD1 . Decreased levels of ATP1B1, GST, TGF-β1, and MAPK14 were also observed. In silico analysis predicted potential docking interactions between BHBA and p38 MAPK14, NF-κB p65, and Nrf2. These predicted interactions may provide a basis for hypothesizing the involvement of these signaling proteins in downstream antioxidant and pro-inflammatory responses, although experimental validation is required. Collectively, these findings demonstrate that short-term in vitro BHBA exposure induces selected molecular alterations in SCM-derived milk PMNs without significant changes in the major cellular effector functions examined. The divergence between molecular responses and preserved functional outcomes suggests that BHBA may modulate the molecular state of mammary-conditioned PMNs without producing measurable functional impairment under the present experimental conditions. These findings should be interpreted specifically in the context of PMNs derived from subclinical mastitis-positive quarters, and further studies comparing healthy mammary and circulating neutrophils and incorporating additional metabolic factors are warranted.