Acetoacetate is a trigger of NLRP3 inflammasome activation in bovine peripheral blood mononuclear cells

Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, β-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1β secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1β secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1β in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1β secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1β secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1β secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1β may be associated with repeat breeding in cattle.