Diverging in vitro inflammatory responses toward Streptococcus uberis in mouse macrophages either preconditioned or continuously treated with β-hydroxybutyrate

Abstract

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 Streptococcus uberis. A secondary objective was to determine whether the inflammatory response to the S. uberis 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 mM) for 24 h; the medium was then replaced with a standard cell culture medium, and the cells were challenged or not with S. uberis 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 mM) for 24 h, the medium was replaced with fresh medium containing the same concentration of BHB, and cells were either challenged or not with S. uberis for 6 h. In both experiments, relative transcript abundance of cell membrane receptors (Tlr2 and Gpr109a), cytokines (Il1b, Il10, Tnf, and Tgfb1), and chemokines (Cxcl2 and Ccl5) were determined using quantitative real-time PCR and normalized against the geometric mean of Hprt and B2m. Data were analyzed using a linear mixed model, and orthogonal contrasts were conducted to examine the effect of S. uberis challenge and BHB treatment. Streptococcus uberis activated the macrophages, noted by greater transcript abundance of analyzed genes. Intriguingly, in both experiments, the S. uberis challenge increased expression of Gpr109a, which encodes a receptor that is ligated by BHB. Paradoxically, preconditioning macrophages with BHB increased transcript abundance of the immunosuppressive cytokine Tgfb1 and increased that of the neutrophil chemoattractant Cxcl2. Preconditioning decreased Tlr2 and tended to decrease Il10 transcript abundance. In opposition to the preconditioning experiment, continuous treatment of BHB during the S. uberis challenge linearly increased abundance of Tlr2 and Il10 transcripts. Continuous BHB treatment also increased expression of Il1b. In conclusion, BHB treatment altered macrophage inflammatory responses during an S. uberis challenge; however, the direction of this response was dependent on whether BHB was added to the medium during the S. uberis challenge. Future studies should be conducted using bovine macrophages and in vivo approaches to examine BHB effects during an S. uberis challenge.