The Proinflammatory Potential Of Extracellular Free MtDNA,involved Mechanism,and Its Ability To Early Predict Sterile SIRS

Background and ObjectiveSterile systemic inflammatory response syndrome(SIRS)is a common life-threatening complication which always arose following severity trauma and hemorrhagic shock,with a morbidity of up to 30%.Although great advances in the development of anti-inflammatory agents targeting modifying inflammatory processes,large-scale clinical studies frequently demonstrate that such modulators could not significantly improve the outcome of patients suffering SIRS.Early prediction of populations at high risk for SIRS and thoroughly understanding the upstream mechanisms involved in SIRS is important in order to allow clinicians to obtain optimal clinical intervention time and to improve patients outcome.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.Extracellular free mitochondrial DNA(mtDNA),a newly identified damage-associated molecular pattern,could activate the neutrophils p38 MAPK signal pathway via TLR9 and potentially contribute to the development of an inflammatory response.However,little is known concerning the proinflammatory effect of mtDNA on other immune cells or tissues.And the relationship between the extracellular free mtDNA levels and the risk of aseptic SIRS also remains to be determined.The aim of this study was to investigate the correlation between extracellular mtDNA concentration and aseptic SIRS in acute traumatic injury patients,and explore the proinflammatory effect and mechanism of mtDNA on THP-1 macrophages and on lung tissues,finally to provide the theoretical basis and molecular targets for early prediction and effective therapy in aseptic SIRS patients.Methods and Materials1.Blood samples were collected within 2 hours after admission and were used to extract the cell free plasma.Plasma extracellular free DNA was isolated with the QIAamp DNA Blood Kit(Qiagen),and the concentration of mtDNA was determined by real-time quantitative PCR.According to whether the post-traumatic SIRS was occurred,patients were stratified into SIRS(+)group and SIRS(-)group.And the correlation between plasma mtDNA levels and clinical characteristics and the prediction of aseptic SIRS in trauma patients was analyzed by statistical methods.2.In vitro,THP-1 macrophages were exposed to mtDNA,and the cytokine concentration in the cell conditioned medium was determined by ELISA.Meanwhile,the phosphorylation level of p38 protein was detected by Western blot.The specific inhibitor for p38 MAPK signal transduction pathway(SB203580)was used to exam whether the inhibition of the phosphorylation of p38 protein would reduce these proinflammatory cytokine levels in the THP-1 cell culture medium which was induced by mtDNA exposure.In order to clarify whether TLR9 or TLR4 is the key upstream molecular mediated the inflammation response induced by mtDNA in THP-1 macrophages,chloroquine(CQ)and TLR4-siRNA were respectively applied to down-regulate the interaction between mtDNA and TLR9,and between mtDNA and TLR4.And the effect of down-regulated interaction between mtDNA and TLR9(or TLR4)on the phosphorylation level of p38 protein and proinflammatory cytokine levels in cell conditioned medium following mtDNA exposure was detected by Western blot and ELISA,respectively.3.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.Several proinflammatory cytokine levels in lung tissue homogenate were examined by ELISA.Additionally,immunofluorescence experiment was carried out to demonstrate the infiltration of CD68+macrophages in lung tissue following mtDNA exposure,and subsequently clarify whether macrophages were involved in lung inflammation induced by mtDNA.Meanwhile,the phosphorylation level of p3 8 protein in lung tissue following mtDNA exposure was detected by Western blot,and to investigate whether the proinflammatory effect of mtDNA on lung tissue is accompanied with the activation of p38 MAPK signal pathway.Finally,in order to clarify whether TLR9 is the key upstream molecular mediated the inflammation response induced by mtDNA in in-vivo experiments,chloroquine(CQ)was applied to down-regulate the interaction between mtDNA and TLR9,and the subsequent effect on the phosphorylation level of p38 protein and proinflammatory cytokine levels in lung tissue following mtDNA exposure was detected by Western blot and ELISA,respectively.Results1.The median plasma mtDNA was significantly higher in trauma patients than in healthy controls,and was an independent predictors for post-traumatic SIRS.The present study was enrolled 86 acute trauma patients,the median plasma mtDNA was higher in trauma patients than in healthy controls(865.196(251.042-2565.40)pg/ml vs 64.2147(43.9049-80.6371)pg/ml,P<0.001)and was independently correlated with the ISS score(r=0.287,P<0.001).The plasma mtDNA concentration was also significantly higher in patients who developed post-traumatic SIRS than in patients who did not(1774.03(564.870-10901.3)pg/ml vs 500.496(145.415-1285.60)pg/ml,P<0.001).Further multiple logistic regression analysis revealed that the plasma mtDNA was an independent predictors for post-traumatic SIRS(OR,1.183(95%CI,1.015-1.379),P=0.032).ROC analysis demonstrated that a high plasma mtDNA level predicted post-traumatic SIRS with a sensitivity of 67%and a specificity of 76%,and the area under the ROC curves(AUC)was 0.725(95%CI 0.613-0.837),which indicated that plasma mtDNA was with moderate discriminative power to predict the risk of post-traumatic SIRS.2.TLR9-p38 MAPK signal transduction pathway mediated the proinflammatory potential of mtDNA on THP-1 macrophages,however,TLR4 is not the right key upstream molecule involved in the above phenomenon.ELISA assay demonstrated that mtDNA exposure significantly increased the inflammation cytokines concentration in the THP-1 macrophage culture medium.Meanwhile,western blot assay showed that the phosphorylation level of p38 protein in THP-1 macrophages was dramatically up-regulated following mtDNA exposure,the specific inhibitor for p38 MAPK signal transduction pathway(SB203580)could inhibit the phosphorylation of p38 protein and subsequently reduce these proinflammatory cytokine levels in cell conditioned medium which was induced by mtDNA exposure.We further respectively applied chloroquine(CQ)and TLR4-siRNA to down-regulate the interaction between mtDNA and TLR9,and between mtDNA and TLR4.And the results showed that the pretreatment of CQ would significantly decrease the phosphorylation level of p38 protein and proinflammatory cytokine levels in cell conditioned medium following mtDNA exposure.However,although the TLR4-siRNA successfully knocked down TLR4 protein expression in THP-1 cells,the down-regulated interaction between mtDNA and TLR4 did not substantially affect the proinflammatory potential of mtDNA on THP-1 macrophages.3.mtDNA exposure successfully induced topical inflammation response in mice lung tissue in in-vivo experiments,and it was mediated by the activation of TLR9-p38 MAPK signal transduction pathway.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 cells(including CD68+ macrophages)infiltration.Additionally,ELISA assay demonstrated that mtDNA exposure significantly increased the inflammation cytokines expression in lung tissues.Meanwhile,Western blot assay showed that the proinflammatory effect of mtDNA on lung tissue is accompanied with the up-regulation of phosphorylation level of p38 protein in lung tissues induced by mtDNA exposure.We further applied chloroquine(CQ)to down-regulate the interaction between mtDNA and TLR9,in order to determine whether the decreased interaction between mtDNA and TLR9 would affect the proinflammatory potential of mtDNA in lung tissue.And the results showed that the pretreatment of CQ would significantly decrease the phosphorylation level of p38 protein and the severity of inflammation response in lung tissues following mtDNA exposure.It is suggested that the activation of TLR9-p38 MAPK signal transduction pathway is responsible for the inflammation of the lung tissue following intratracheal instillation of mtDNA.Conclusion1.The plasma mtDNA is an independent predictor for post-traumatic sterile SIRS.Statistical analysis based on the assessment of clinical specimens revealed that plasma mtDNA concentration was correlated with post-traumatic SIRS,and was an independent predictor which was with moderate discriminative power to predict the risk of post-traumatic SIRS.It provides a new molecular biomarker for the early prediction and diagnosis in post-traumatic SIRS,with prospects of clinical translation and industrialization.2.TLR9-p38 MAPK signal transduction pathway mediated the proinflammatory potential of mtDNA on THP-1 macrophages,however,TLR4 is not the right key upstream molecule involved in the above phenomenon.It provides a new theoretical basis for the study of proinflammatory mechanism of mtDNA in THP-1 macrophage.3.mtDNA exposure successfully induced topical inflammation response in mice lung tissue in in-vivo experiments,and we firstly determined that the above phenomenon was mediated by the activation of TLR9-p38 MAPK signal transduction pathway.It provides a new molecular target and theoretical basis for the effective therapy of aseptic inflammation in lung tissues following the pathophysiological condition of SIRS.