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-1beta to form mature IL-1beta. 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-1beta release. P2Y12 receptor-deficient microglia displayed markedly attenuated IL-1beta mRNA expression and release. P2Y12 receptor blockade also suppressed IL-6 production. Both IL-1beta and IL-6 responses were augmented by extracellular ADP or ADP-betaS and were abrogated by PSB0739. Mechanistically, ADP-betaS potentiated NF-kappaB 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-kappaB and the NLRP3 inflammasome to enhance microglial inflammation.