The Role Of Neutrophil Pyroptosis Mediated By GSDMD In A Murine Model Of Acute Lung Injury

BackgroundGSDMD（Gasdermin D）mediated cell pyroptosis is an important antimicrobial immune defense mechanism discovered in the body in recent years,and cell pyroptosis can be involved in the occurrence,development and long-term outcome of a variety of diseases.Phorbol 12-myristate 13-acetate（PMA）was found to be the most effective in inducing neutrophil pyroptosis in vitro.PMA can induce neutrophil pyroptosis and increase the release of Neutrophil extracellular traps（NETs）.However,in the occurrence of acute lung injury,whether the neutrophils in the lung tissues will undergo pyroptosis.And if pyroptosis does occur,whether it plays an important role in the occurrence and development of acute lung injury and what is the potential mechanism,all this have not been reported.The purpose of this study was to explore the role of GSDMD mediated neutrophil pyroptosis in the inflammatory response of acute lung injury,and the potential mechanisms of neutrophil pyroptosis in acute lung injury.Methods1.Expression of GSDMD in neutrophils of ARDS patients and mice with acute lung injury1)Clinical samples:Peripheral blood samples from ARDS patients and healthy volunteers were collected to purify neutrophils and detected the expression level of GSDMD protein.2)In vitro experiments:C57BL/6 mice were randomly divided into two group:control group and LPS group（5mg/kg）,and the expression of GSDMD in neutrophils in lung tissues was detected by flow cytometry at 24 hours.2.Effect of GSDMD knockout in neutrophils on LPS-induced acute lung injury in miceIn vivo experiment,mice were randomly divided into four groups:GSDMD^WT/WTcontrol group,GSDMD^flox/flox control group,GSDMD^WT/WT LPS group and GSDMD^flox/flox LPS group.Acute lung injury model was established by intratracheal injection of LPS（5mg/kg）,while mice in the sham group were intratracheal injection of equal volume of PBS buffer solution.After 24 hours,blood was collected through heart and inflammatory factors（TNF-α,IL-6,IL-1βand IL-18）in serum were detected by ELISA.BALF was collected for analyzing total protein concentration and levels of inflammatory factors（TNF-α,IL-6,IL-1βand IL-18）.The ratio of neutrophils in BALF was analyzed and counted by flow cytometry.The left lung tissue was measured for wet/dry ratio,and the right lung tissues was stained H&E for detecting lung injury.3.Effect of increased GSDMD expression in neutrophils on the release of NETs1)Clinical samples:Peripheral blood samples from ARDS patients and healthy volunteers were collected.Neutrophils were cultured and treated with LDC7559,at indicated time,neutrophils were collected to detect the release of NETs.2)In vivo experiment:Mice were randomly divided into four group:GSDMD^WT/WT control group,GSDMD^flox/flox control group,GSDMD^WT/WT LPS group and GSDMD^flox/flox LPS group.The content of NETs in the lung tissues was measured byIF and the MPO-DNA complexes in serum and BALF were assayed by ELISA.4.Preliminary exploration on the mechanism of GSDMD expression in promoting acute lung injuryIn vivo experiment,mice were randomly divided into 8 groups:GSDMD^WT/WTcontrol group,GSDMD^WT/WT LPS group,GSDMD^WT/WT PMA group,GSDMD^WT/WTLPS+PMA group,GSDMD^flox/flox LPS control group,GSDMD^flox/flox PMA group and GSDMD^flox/flox LPS+PMA group.The injection of PMA to stimulate neutrophils to increase the release of NETs in vivo.We observed the effects of PMA on lung injury in GSDMD-knowout mice treated with LPS.Results1.The expression level about GSDMD in neutrophils in ARDS patients was significantly greater;the expression of GSDMD in neutrophils was significantly increased in ALI mice.2.The production of NETs in neutrophils from ARDS patients were significantly reduced after treatment with GSDMD inhibitor LDC7559;neutrophil-GSDMD-deficient mice significantly reduced the production of NETs in the lungs and the content of MPO-DNA complex in plasma and BALF after LPS treatment.3.GSDMD neutrophil knockout mice revealed that the indexes associated with lung tissue injury such as lung injury score,inflammatory factors in plasma and BALF,the W/D,the total protein concentrations and the number of neutrophils infiltrated in lung tissues were both obviously decreased.4.GSDMD neutrophil knockout mice were treated with LPS and then injected with PMA through tail vein.There was no significant increase in lung injury score,lung tissue NETs production,inflammatory cytokines concentration in plasma and BALF and total protein concentrations in BALF.ConclusionThe expression levels of GSDMD in neutrophils were significantly increased in ALI mice.In vitro experiments showed that neutrophils NET release could be inhibited by blocking GSDMD.The scores of lung injury,inflammatory response and release of NETs were significantly reduced in GSDMD neutrophil knockout mice.However,NETs activator PMA did not reverse the protective effect of GSDMD specific knockout on lung injury and NETs release,suggesting that the role of GSDMD on neutrophil NETs release occurs downstream of PKC.