Expression And Mechanism Of TREX1 In The Immunosuppressive Phase Of Sepsis

Part I: Organ changes and TREX1 expression in mice during sepsis immunosuppression Objective: The purpose of this study was to investigate the changes of organ,the possible role and related mechanisms of TREX1 in mice during sepsis immunosuppression.Methods: Male C57BL/6J mice aged 8-10 weeks were used in this experiment.Moderate to severe cecal ligation and puncture(CLP)model and LPS-induced mouse sepsis model were used as the main research objects.In the CLP group,cut the abdomen of the anesthetized mouse and fully expose the cecum.The 4-0 silk thread ligates the cecum at 1/3 from the ileocecal valve to ensure complete ligation,then use a 20 G puncture needle to ligate the far cecum twice.A small amount of stool is squeezed to ensure the perforation is unobstructed.In the LPS group,10 mg/Kg of LPS was injected intraperitoneally.All mice were divided into four groups:(1)Sham group: mice exposed the cecum only and no ligation and puncture;(2)LPS group: intraperitoneal injection of 10mg/Kg LPS for 24 hours;(3)CLP group:establishment of an animal sepsis model of CLP for 8 days;(4)CLP + LPS group: 7days after cecal ligation and puncture,surviving mice received "secondary" attack by intraperitoneal injection of 10mg/Kg LPS and observed for 24 hours;Sham group,LPS group,CLP group and CLP + LPS group were taken lung,liver and kidney of mice,HE staining was used to observe the damage of various organs under different sepsis conditions;the spleen of four groups of mice was prepared into a spleen cell suspension;Western Blot was used to detect TREX1,STING and TBK1 proteins in spleen cell changes.Results: Compared with the sham group,mild alveolar wall thickening was observed in the lungs of sepsis mice for 24 hours induced by 10mg/kg LPS;CLP 8d mice showed changes such as alveolar wall thickening and congestion;the lung condition of CLP mice after the "secondary" attack was significantly worse,with remarkablealveolar wall thickening and hemorrhage,alveolar collapse and destruction.With the aggravation of sepsis in mouse liver,the hepatic lobule structure was gradually destroyed,inflammatory cell infiltration and necrosis appeared in mouse liver cells after the "secondary" attack.Compared with LPS group,renal epithelial cells in CLP mice were disordered and interstitial edema was aggravated;after the "secondary" attack,the renal tubules were denaturated and the glomerulus showed severe sclerosis-like changes.The changes in viscera were consistent with the changes in the immunosuppressive phase of sepsis.In the Western Blot results,TREX1 in the CLP group was significantly increased compared to the Sham group(P <0.05);TREX1expression was more significant in the spleen cells of LPS+CLP mice(P<0.01).Compared with the mice at 24 hours after LPS injection,the expression of STING in the spleen of mice after CLP 8d and CLP+LPS group was significantly reduced(P<0.05).Compared with the mice at 24 hours after LPS injection,the expression of TBK1 in the spleen of mice at 8 days after CLP and CLP+LPS group was significantly reduced(P <0.01).Conclusion: During the occurrence and development of sepsis,the lung,liver,kidney,of mice were obviously damaged;TREX1 was gradually increased in the spleen cells of sepsis mice from early sepsis to immunosuppressive phase,while STING and TBK1 expression is down-regulated during the immunosuppressive phase compared with the early stage of sepsis.This may be the molecular mechanism by which TREX1 causes sepsis immune paralysis.Part II: Effects of DNA damage on TREX1 in macrophages during sepsis immunosuppressionObjective: To investigate the relationship among DNA damage and the secretion of related cytokines and TREX1,their changes during immune paralysis of sepsis in macrophages.Methods: In this experiment,the mouse mononuclear/macrophage Raw264.7 was used as the main research object,and the cells were randomly divided into:(1)Ctrl group;(2)LPS 4h group;(3)LPS 15 h group;(4)LPS15 + 4h group;Among them,Ctrl group was treated with DMEM medium for 21h;LPS 4h group was first cultured with DMEM for 17 h,then Raw264.7 cells were stimulated with 1 μg/m L LPS for 4 h;LPS15h group was pretreated with 10ng/m L LPS for 15 h,and then Raw 264.7 cells cultured with DMEM for 6 h;in the LPS15 + 4h group,Raw 264.7 cells were stimulated with 10 ng/m L LPS for 15 h to paralyze themselves,wash with DMEM for2 h,and then stimulated with LPS 1μg/m L LPS for 4 hours for "secondary" attack.ELISA was used to detected the expression of TNF-α and negatively regulated cytokine IL-10 in Raw264.7 cells at different stages of sepsis.Confocal imaging of immunofluorescence was used to observe the relationship between the DNA damage marker γH2A.X and TREX1 expression.Western Blot was used to detect the expression of TREX1,STING,and TBK1 in Raw264.7 cells of each group.Results: The ELISA results showed that compared with the Ctrl group,there was a significant increase in TNF-α in the LPS4 h and LPS15 h groups(P <0.01);compared with the LPS15 h group,the expression of TNF-α in the LPS15 h + 4h group had decreased significantly(P <0.01).Compared with the Ctrl group,the expression of IL-10 in Raw264.7 cells LPS4 h group and LPS15 h group was significantly reduced(P<0.01);IL-10 expression was significantly increased in LPS15 + 4h group compared to LPS15 h group(P <0.01).The fluorescence intensity of TREX1 in the LPS 15 h and LPS“secondary”strike group was significantly increased compared with Ctrl group and LPS 4h group.Meanwhile,the fluorescence intensity of DNA damage marker detector γH2A.X also increased with the increase of LPS induction time.Western Blot results showed that compared with Ctrl group,The expression of TREX1 increased with the aggravation of LPS stimulation time(P<0.01).LPS15 h Raw264.7 cells significantly decreased STING expression compared with LPS4 h group(P<0.01);compared to LPS 4h group,STING expression in LPS15 + 4h group was reduced(P<0.05).Compared to LPS4 h group,TBK1 expression in Raw264.7 cells of LPS15 h and 15 + 4h groupwas significantly reduced(P <0.05).Conclusion: In Raw264.7 macrophages,TNF-α expression increases in the early stage of sepsis and the expression of immunosuppression decreases;IL-10 expression in sepsis is decreased in the early stage of the disease,while the expression is increased in the immunosuppressive phase;DNA damage and TREX1 expression were significantly increased during the immunosuppressive phase of sepsis.In Raw264.7 macrophages,TREX1 expression increased with the severity of sepsis;compared with the early stage of sepsis,the expressions of STING and TBK1 were decreased in the immunosuppressive stage of sepsis.TREX1 plays an important role in sepsis immunosuppression.