β-Hydroxybutyrate impairs neutrophil migration distance through activation of a protein kinase C and myosin light chain 2 signaling pathway in ketotic cows

Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)—a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction—was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.