Extracellular ADP augments microglial inflammasome and NF‐κB activation via the P2Y12 receptor

The NLRP3 inflammasome is a molecular complex that translates signals from pathogens and tissue damage into inflammatory responses, and plays crucial roles in numerous neurological diseases. Activation of the NLRP3 inflammasome leads to caspase‐1 dependent cleavage of pro‐IL‐1β to form mature IL‐1β. By acting on the P2X7 purinergic receptor, extracellular ATP is one of the major stimuli that activates the NLRP3 inflammasome. Although microglia express multiple purinergic receptors, their roles in inflammasome‐mediated inflammation are largely unknown. We studied the role of the P2Y12 receptor, a metabotropic P2Y receptor enriched in microglia, on inflammation in vitro. Inhibition of the microglial P2Y12 receptor by PSB0739 or siRNA knockdown suppressed IL‐1β release. P2Y12 receptor‐deficient microglia displayed markedly attenuated IL‐1β mRNA expression and release. P2Y12 receptor blockade also suppressed IL‐6 production. Both IL‐1β and IL‐6 responses were augmented by extracellular ADP or ADP‐βS and were abrogated by PSB0739. Mechanistically, ADP‐βS potentiated NF‐κB activation. In addition, ADP altered mitochondrial membrane potential in combination with ATP and increased the number of caspase‐1 positive cells through the P2Y12 receptor. These results elucidate a novel inflammatory mechanism by which extracellular ADP acts on the P2Y12 receptor to activate NF‐κB and the NLRP3 inflammasome to enhance microglial inflammation. We found that the extracellular ADP plays a pivotal role in activation of microglial NLRP3 inflammasome and NF‐κB via the P2Y12 receptor. Strong potassium efflux produced by combined stimulation of ATP and ADP via the P2X7, P2Y12 receptors, triggers to augment the NLRP3 inflammasome and caspase‐1 activations to release IL‐1β.