The Mechanism Of ATP-induced Pyroptosis In NLRP3 Pathway-blocked Murine Macrophages

Aim:ATP acts as an canonical activator to induce NLRP3 inflammasome activation in macrophages,leading to caspase-1/gasdermin D-mediated pyroptosis.However,It remains unclear whether ATP can induce pyroptosis in macrophages where the NLRP3 pathway is blocked by pathogenic infection.In this study,we aimed to explore the cell death form in murine macrophages when the NLRP3 pathway was blocked by using RAW 264.7 cells,which is deficient in ASC,and bone marrow-derived macrophages(BMDM)in which NLRP3 inflammasome activation was blocked by specific inhibitor MCC950,as cell models in response to ATP stimulation.Methods:It is known that during pyroptosis there are obvious morphological changes including cell swollen,eventually leading to cell membrane rupture and release of cell contents.Therefore,we used propidium iodide(PI)assay to detect mecrosis in RAW 264.7 cells and BMDMs pre-treated with specific inhibitor MCC950,upon the stimulation of ATP.The protein levels of pro-IL-1β,pro-caspase-1,gasdermin D(GSDMD),gasdermin E(GSDME)and apoptosis-related caspases in the cells or the supernatants(released from the cells)were evaluated by western blotting.In order to further explore relevant mechanisms about cell death upon the stimulation of ATP,DEVD was used as a specific inhibitor of caspase-3.In addition to DEVD,siRNA was used to knock down the expression of caspase-3 Gasdermin E.Results:ATP treatment induced lytic cell death morphologically resembling pyroptosis phenotype but did not cause the activation of caspase-1 and GSDMD as in canonical pyroptosis,in BMDMs which NLRP3 inflammasome activation was blocked by specific inhibitor MCC950 and in RAW 264.7 cells deficient in ASC thus with the NLRP3 pathway being blocked.Instead,both apoptotic initiator(caspase-8 and-9)and executioner caspases(caspase-3 and-7)were evidently activated,resulting in the cleavage of GSDME,and thereby generating GSDME N-terminal fragment(GSDME-NT)which can execute pyroptosis.The GSDME-NT production and the lytic cell death induced by ATP were diminished by caspase-3 inhibitor.Knockdown of GSDME by small interfering RNA attenuated ATP-induced lytic cell death and HMGB1 release into culture supernatants.Knockdown of caspase-3 by small interfering RNA attenuated ATP-induced lytic cell death.Conclusions:When the tissues are damaged or infected,ATP is released as a common DAMP,and can induce pyroptosis through the caspase-3/GSDME axis when the canonical NLRP3 pathway is blocked,suggestive of an alternative mechanism to combat against pathogen evasion.