The Mechanism Of Cell-free MtDNA Inducing NLRP3 Inflammasome Activation And Its Role In Acute Lung Injury

Background and ObjectiveSterile systemic inflammatory response syndrome(SIRS)and pulmonary inflammation is caused by pulmonary contusion or hemorrhagic shock are common complication of atrauma.It was shown that the incidence of SIRS was up to 30%-80%after polytrauma and SIRS was associated with sepsis,acute lung injury(ALI)and respiratory failure.It is well established that the interaction between the damage associated molecular patterns(DAMPs)and pattern recognition receptors(PRRs)is the strategic link involved in the pathogenesis of aseptic SIRS.DAMPs were a category of moleculars released by cell death or injury.Mitochondrial DNA(mtDNA)is one of the DAMPs discovered in the past decade and it contains high-frequency and low-methylated CpG repeat.It was demonstrated that mtDNA was capable of inducing the activation of MAPKs by interacting with TLR9 of neutrophils and macrophages.Inflammasomes are complexes composed of cytoplasmic PRRs and other moleculars including procaspase-1.The formation of inflammasome lead to activation of caspase-1 and mature of IL-1 family cytokines.The activation of inflammasome requires two stages which are "priming" and "activation".The priming of inflammasome is mainly mediated by TLRs and promote transcriptions of inflammasome component.Aprevious study indicated that the sterile inflammation and neutrophil infiltration in acute pancreatitis were mediated by NLRP3 inflammsome of pancreatic macrophages and TLR9 is a criticle molecular in the NLRP3 inflammsome priming.But they did not get the original DAMPs.This present study is designed to investigate the effect of cell-free mtDNA in the NLRP3 inflammasome activation in THP-1 macrophages and explore possible molecular mechanism.We also investigated the effect of mtDNA on pulmonary NLRP3 inflammasome activation and pulmonary inflammation and injury in mice.This study would provide theoretical basis for NLRP3 inhibitors in pulmonary inflammation and lung injury.Methods and MaterialsAfter exposure to mtDNA,IL-1 family cytokines in supernatant were analyzed by ELISA and caspase-1 activity in supernatant was analyzed by chemiluminescence.Transcription of NLRP,AIM2,ASC and caspase-1 was analyzed by real-time PCR.Caspase-1 p20 subunit in concentrated supernatant and NLRP3,procaspase-1,MAPKs and NF-?B in cells were analyzed by Western Blot.NLRP3-siRNA and AIM2-siRNA were used to verify which kind of inflammasome is involved in the cell-free mtDNA induced caspase-1 activation.TLR9-siRNA were used to verify whether or not TLR9 is involved in the inflammasome activation by cell-free mtDNA.p38-siRNA and SB203580(a p38 MAPK inhibitor)were used to confirmed its role in mtDNA induced inflammasome transcription.PDTC(a NF-?B inhibitor)was used to verify its role in inflammasome transcription.Intratracheal instillation of mtDNA in a volume of 60ul was performed to establish the animal model of topical inflammation within lung tissue induced by mtDNA.The wet/dry ratio of lung tissue was recorded.Lung tissue paraffin sections were stained with hematoxylin-eosin to observe the inflammatory morphological pathology induced by mtDNA exposure and TUNEL stain was used to detect apoptotic cells in lung.BALFs of mice were obtained and the number of cells in BALF was analyzed to reflect lung inflammation and injury.IL-1 family cytokines in BALF were analyzed by ELISA and caspase-1 activity in BALF was analyzed by chemiluminescence.Transcription of NLRP,ASC and caspase-1 was analyzed by real-time PCR.NLRP and caspase-1 p20 subunit in lung tissue were analyzed by western blot.Belnacasan(VX-765,a caspase-1 inhibitor)were used to verify the role of NLRP3 inflammasome in mtDNA induced lung inflammation and injury.Results1.Cell-free mtDNA induces NF-?B nuclear transfer and inflammasome activation in THP-1 macrophages via TLR9 and p38 MAPK and results in increasement of IL-1? and IL-18.Cell-free mtDNA induced signigicant increased release of IL-1? and IL-18 of THP-1 macrophages and the activaty and content of caspase-1 p20 subunit also significantly increased.Cell-free mtDNA induced significant increased transcription of NLRP,AIM2,ASC and caspase-1.It was shown that a significant increasement of NLRP3 and pro-caspase-1 in cell-free mtDNA stimulated THP-1 macrophages by Western Blot.Both caspase-1 siRNA and NLRP3-siRNA resulted in a drastical decreasement of cell-free mtDNA induced IL-1? release while AIM2-siRNA has no effect on it.The transfection of TLR9-siRNA significantly decreased the expression of NLRP3 induced by mtDNA.Both p38-siRNA and SB203580 would inhibitor the NF-?B nuclear transfer and transcription and expression of NLRP3 inflammasome.Inhibition of NF-?B also resulted a dramatical reduction of transcription and expression of NLRP3 inflammasome.And the NF-?B plays a more important role in the NLRP3 inflammasome transcription than p38 MAPK.2.Cell-free mtDNA induces inflammasome activation which results in lung inflammation and injury in mice.After intratracheal instillation of mtDNA,the wet/dry ratio of lung tissue in model animals was significantly increased the histological score of H&E stained lung tissue paraffin sections also significantly raise,demonstrating alveolar edema,vascular congestion and inflammatory injury.TUNEL stain indicated that mtDNA induced sinificant increased apoptosis of alveolar epithelial cell.Intratracheal instillation of mtDNA also induced significantly increased IL-1?,IL-18 and caspase-1 activity in BALF.It was shown that NLRP3 and caspase-1 p20 subunit also increased in mtDNA stimulated lung.Inhibitioon of caspase-1 by Belnacasan(VX-765)resulted in significant improvement of lung inflammation and injury by mtDNA.Conclusion1.We proved that cell-free mtDNA was capable of inducing NLRP3 inflammasome activation in THP-1 macrophages for the first time.Previous studies on mtDNA mainly focused on the TLR9 and we found thet cell-free mtDNA could also have an effect on other PRRs.2.We proved that cell-free mtDNA induced NLRP3 inflammasome activation was mediated through TLR9,p38 MAPK and NF-?B.The NF-?B nuclear transfer was partially mediated by p38 MAFK.We provided a new evidence of PRRs crosstalk by CpG containing DNA.3.We proved that cell-free mtDNA was capable of inducing NLRP3 inflammasome activation in mice lung and resulting in lung inflammation and injury.A recent study revealled that MCC950(a small molecular drug)was capable of selectively inhibit NLRP3 inflammasome and improve survival in mouse with NLRP3 activate mutation.This would provide evidence for future use of NLRP3 inhibitors in sterial lung inflammation and lung injury.