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.