ATP-induced Inflammasome Activation And Pyroptosis Is Regulated By AMP-activated Protein Kinase In Macrophages

Aim: Macrophages are one of the most important innate immune cells,and also the link between innate immunity and adaptive immunity.This is achieved by recognition of the pathogen-associated molecular patterns(PAMP)by their surface or internal pattern recognition receptor(PRR),which initiates the inflammatory response and increases the expression of inflammasome components(such as NLRP3,pro-caspase-1 and pro-IL-1?).When the macrophagesare further stimulated with a damage-associated molecular pattern(DAMP)signal,inflammasome components can be assembled into active inflammasomes,resulting in the release of mature caspase-1p10 and IL-1?,or induction of programmed cell death(pyroptosis).Lipopolysaccharide(LPS),which is a cell wall component of gram-negative bacteria,is one of such PAMPs.ATP is a product of invaded bacteria orthe host cells during bacterial infection as well as a sterile tissue injury,and it can be a DAMP signal molecule.Further stimulation of LPS-treated macrophages with ATP can induce the activation of inflammasomes and further induce pyroptosis of the macrophages.Previous studies have shown that ATP treatment leads to AMP-activated protein kinase(AMPK)activation.AMPK is a key kinase in maintaining the balance of ATP generation and consumption in eukaryotic cells by feeling the energy state of cells,which can be activated by elevated AMP/ATP ratio.However,it is unclear whether AMPK signaling is involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis.To explore this issue,we used the macrophages which were activated by lipopolysaccharide(LPS)as an inflammatory model,including in mouse macrophage cell line J774 A.1,thioglycolate(TG)-elicited mouse peritoneal macrophages and bone marrow-derivedmacrophages(BMDMs).In this project,we also explored the AMPK signaling pathway in regulating the survival rate of sepsis model with bacterial infection,to verify the mechanism of AMPK signaling pathway on the activation of inflammasome and pyroptosis.Methods: Previous studies have reported that LPS induces the expression of inflammasome components(including NLRP3,pro-caspase-1,pro-IL-1?)increased in macrophages.Under further stimulation with ATP,NLRP3 proteins recruit the apoptosis associated speck like protein containing a CARD(ASC)to assemble an active inflammasome,which activated caspase-1,cleaved pro-IL-1?,and subsequently pyroptosis take place,leading to the release of mature IL-1?and HMGB1.The formation of pores in the cell membrane due to pyroptosis impairs the integrity of the cell membrane.In this study,the pyroptotic cells were observed after propidium iodide(PI)staining.The protein levels of IL-1?,caspase-1 and HMGB1 in the cells or the supernatants(released from the cells)were evaluated by western blotting.Immunofluorescent microscopy was recruited to detect the subcellular distribution of the purinergic P2X7 receptor(P2X7R)and ASC,their fluorescence intensisty being calculated.Cytometric bead array(the beads embedded with the corresponding antibody)was used to investigate the expression of IL-1? in the supernatants of culture medium and blood serum of C57BL/6 mice.The proportions of neutrophils in the peritoneal cavity were analyzed by flow cytometry.Metformin and compound C were used as AMPK agonist and inhibitor,respectively.In addition to compound C,siRNA was used to knockdown the expression of AMPK?1 and thus suppress the AMPK activity.Moreover,inflammasome activation was detected in C57BL/6mouse sepsis models with abdominal bacterial infection,and the effect of metformin(by gastric gavage)on the inflammasome activation and mouse survival were analyzed in these animal models.Results: Pyroptosis assay found that metformin per se did not induce pyroptosis in LPS-activated peritoneal macrophages,but it significantly and dose-dependently increased the pyroptosis induced by ATP treatment.And compound C inhibited the pyroptosis and the effect of metformin on pyroptosis.Western blot showed that,at protein level,maturated IL-1?(17 kDa),active caspase-1(10 kDa)and HMGB1 could not be released from the cells upon single LPS or LPS plus metformin stimulation.But after the macrophages were further stimulated with ATP,inflammosomes were activated and the maturated IL-1? was released into the supernatants of thecell culture medium.Moreover,metformin dose-dependently increased the release of maturated IL-1?,active caspase-1and HMGB1 upon LPS and ATP stimulation.But ATP induced inflammasome activation and cell pyroptosis were significantly inhibited by AMPK inhibitor compound C or AMPK?1 knockdown.Immunofluorescence microscopy found that ASC gathered into specks in LPS-activated BMDMs after ATP stimulation,and metformin treatment promoted the formation of ASC specks.Mechanically,metformin significantly promoted the redistribution of P2X7 receptor(P2X7R)in peritoneal macrophages activated by LPS.Animal model experiments also showed that metformin increased the mortality of septic mice,suggesting that metformin increased the acute inflammation caused by bacterial infection through activating the AMPK signaling,resulting in enhanced inflammasome activation and cell pyroptosis.Conclusion:The AMPK activity in macrophages was inhibited under LPS,but significantly activated after ATP treatment,and then it induced inflammasome activation and pyroptosis.Our results suggested that AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis,highlighting AMPK-targeting therapies for acute and chronic inflammatory diseases.