Diverging in Vitro Inflammatory Responses Toward in Mouse Macrophages Either Preconditioned or Continuously Treated with β-hydroxybutyrate

Hyperketonemia is a common condition in early-lactation dairy cows that has been associated with an increase in the risk of infectious disease. Recent mouse studies have elucidated an anti-inflammatory effect of the ketone body β-hydroxybutyrate (BHB). Therefore, the objective of this study was to determine whether BHB altered inflammatory responses in macrophages challenged with the common mastitis pathogen . A secondary objective was to determine whether the inflammatory response to the challenge was dependent on whether BHB was present in the medium during the challenge (i.e., preconditioned vs. continuous treatment). Two cell culture experiments were conducted. In the first experiment, mouse macrophages (RAW 264.7 line) were preconditioned with BHB (0, 0.6, 1.2, and 1.8 m) for 24 h; the medium was then replaced with a standard cell culture medium, and the cells were challenged or not with for an additional 6 h. In the second experiment, a similar protocol was used; however, cells were preconditioned with BHB (0, 0.6, 1.2, and 1.8 m) for 24 h, the medium was replaced with fresh medium containing the same concentration of BHB, and cells were either challenged or not with for 6 h. In both experiments, relative transcript abundance of cell membrane receptors ( and ), cytokines (, and ), and chemokines ( and ) were determined using quantitative real-time PCR and normalized against the geometric mean of and . Data were analyzed using a linear mixed model, and orthogonal contrasts were conducted to examine the effect of challenge and BHB treatment. activated the macrophages, noted by greater transcript abundance of analyzed genes. Intriguingly, in both experiments, the challenge increased expression of , which encodes a receptor that is ligated by BHB. Paradoxically, preconditioning macrophages with BHB increased transcript abundance of the immunosuppressive cytokine and increased that of the neutrophil chemoattractant . Preconditioning decreased and tended to decrease transcript abundance. In opposition to the preconditioning experiment, continuous treatment of BHB during the challenge linearly increased abundance of and transcripts. Continuous BHB treatment also increased expression of . In conclusion, BHB treatment altered macrophage inflammatory responses during an challenge; however, the direction of this response was dependent on whether BHB was added to the medium during the challenge. Future studies should be conducted using bovine macrophages and in vivo approaches to examine BHB effects during an challenge.