Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

ß-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1ß and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Palmitic Acid and ß-Hydroxybutyrate Induce Inflammatory Responses in Bovine Endometrial Cells by Activating Oxidative Stress-Mediated NF-κB Signaling.

Ketosis is a nutritional metabolic disease in dairy cows, and researches indicated that ketonic cows always accompany reproductive problems. When ketosis occurs, the levels of non-esterified fatty acids (NEFAs) and ß-hydroxybutyrate (BHBA) in the blood increase significantly. Palmitic acid (PA) is a main component of saturated fatty acids composing NEFA. The aim of this study was to investigate whether high levels of PA and BHBA induce inflammatory responses and regulatory mechanisms in bovine endometrial cells (BEND). Using an enzyme-linked immunosorbent assay, quantitative real-time PCR, and western blotting, we evaluated oxidative stress, pro-inflammatory factors, and the nuclear factor (NF)-κB pathway in cultured BEND cells treated with different concentrations of PA, BHBA, pyrrolidinedithiocarbamate (PDTC, an NF-κB pathway inhibitor), and N-acetylcysteine (NAC, an antioxidant). The content of malondialdehyde was significantly higher, the content of glutathione was lower, and antioxidant activity-glutathione peroxidase, superoxide dismutase, catalase, and total antioxidant capacity-was lower in treated cells compared with control cells. PA- and BHBA-induced oxidative stress activated the NF-κB signaling pathway and upregulated the release of pro-inflammatory factors. Moreover, PA- and BHBA-induced activation of NF-κB-mediated inflammatory responses was inhibited by PDTC and NAC. High concentrations of PA and BHBA induce inflammatory responses in BEND cells by activating oxidative stress-mediated NF-κB signaling.

Pathway of programmed cell death and oxidative stress induced by ß-hydroxybutyrate in dairy cow abomasum smooth muscle cells and in mouse gastric smooth muscle.

The administration of exogenous ß-hydroxybutyrate (ß-HB), as well as fasting and caloric restriction, is a condition associated with ß-HB abundance and decreased appetite in animals. Increased ß-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of ß-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous ß-HB on the stomach, mice were injected intraperitoneally with ß-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of ß-HB. We found that ß-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. ß-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. ß-HB also promoted AIF, EndoG release and p53 expression. ß-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. ß-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with ß-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that ß-HB causes ROS generation through a Ca2+-dependent mechanism and that intracellular Ca2+ levels play a critical role in ß-HB -induced apoptotic cell death. The impact of ß-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of ß-HB on smooth muscle. We propose that ß-HB is a possible cause of some stomach diseases, including bovine LDA.