| Objective To observe the expression of HMGB1,dsDNA and their complexes in mice exposed to tobacco smoke and the effect of RAGE on activation of mDC and differentiation of CD4+T cells in vivo.To investigate whether HMGB1-dsDNA complex promotes the activation of bone marrow derived dendritic cells(BMDC)through the RAGE-PI3K/AKT signaling pathway in vitro.Methods C57BL/6 mice(male,6-8 weeks)and RAGE conditioned knockout mice were randomly exposed to air and chronic cigarette smoke for 24 weeks.HE staining was used to observe the pathological changes of lung tissues in each group,and the mean linear intercept(Lm)was measured to evaluate the degree of emphysema.The expression of HMGB1 in BALF and sRAGE in serum were detected by ELISA.The expression of dsDNA in BALF was detected by Picogreen fluorescence quantitative method.The peripheral blood neutrophils of mice exposed to air and cigarette smoke were stimulated with cigarette smoke extract(CSE),and the induced sputum of COPD patients was collected for immunofluorescence detection to determine the presence of HMGB 1-dsDNA complexes.The expression of RAGE,activation marker(CD40,CD80,CD86),p-PI3K,p-AKT in myeloid dendritic cells(mDCs),lung Thl and Th17 cells were detected by flow cytometry and immunofluorescence.Bone marrow derived dendritic cells DC2.4 were used as the study object.Experimental groups:control group,HMGB1 group,dsDNA group,HMGB1-dsDNA complexes group,complexes+RAGE antagonist group,complexes+PI3K inhibitor group,CSE positive control group.The expressions of RAGE,p-PI3K,AKT,p-AKT,NF-κB,p-NF-κB,MHC-II,CD40,CD80 and CD86 were detected by WB and flow cytometry.Results 1.Compared with the air control group,the alveoli of mice exposed to chronic cigarette smoke were enlarged,ruptured and fused,forming obvious emphysema-like changes,and Lm was significantly increased,with statistical significance.Compared with RAGEfl/flCD11c-Cre-AIR,Lm in RAGEfl/flCD11cCre-CS was increased,but significantly lower than WT-CS.There was no difference between RAGEfl/flCD11c-Cre-AIR and WT-AIR.2.Compared with the air control group,the levels of HMGB1 and dsDNA in BALF of mice exposed to cigarette smoke were increased,while the levels of sRAGE in serum was decreased.3.Compared with the air control group,neutrophils in mice exposed to cigarette smoke produced more HMGB1-dsDNA complexes after CSE stimulation,and a large number of HMGB1-dsDNA complexes were also present in the sputum of COPD patients.4.Compared with the air control group,lung mDCs aggregation and activation,RAGE expression on mDCs were increased,and Thl and Th17 cells were also significantly increased in cigarette smoke exposure group.Compared with RAGEfl/flCD11c-Cre-AIR,the expression of RAGE on mDCs from RAGEfl/flCD11c-Cre-CS group did not increase,but the expression of p-PI3K,p-Akt,CD40,CD86 and CD80 all increased.However,it was still significantly lower than WT-CS(except p-Akt).Compared with RAGEfl/flCD11c-Cre-AIR,the expression of RAGE on mDCs from RAGEfl/flCD11c-Cre-CS group did not increase,but the expression of p-PI3K,p-Akt,CD40,CD86 and CD80 all increased.However,it was still significantly lower than WT-CS(except p-Akt).5.In vitro,HMGB1 and dsDNA alone or complexes can stimulate the expression of RAGE,p-PI3K,p-Akt,and p-NF-κB,and induce the increase of mature marker MHC-Ⅱ and activation markers CD40,CD80 and CD86 in DC2.4.The effect of complexes was stronger.Pretreatment of RAGE inhibitor FPS-ZM1 decreased the activation of RAGE/PI3K/AKT pathway and the maturation and activation of DC2.4 after complexes stimulated.PI3K inhibitor LY294002 did not affect the expression of RAGE,but decreased the activities of p-Akt and p-NF-KB,and decreased the maturation and activation of DC2.4 after complexes stimulated.The difference was statistically significant.Conclusion Chronic cigarette smoke exposure leads to the release of endogenous DAMPs molecules HMGB1,dsDNA and HMGB1-dsDNA complexes,accompanied by the aggregation and activation of mDCs,and the increase of Thl and Th17 cells.Conditional knockout of RAGE on mDCs has protective effect on emphysema in mice exposed to chronic tobacco smoke.The expression of RAGE in dendritic cells is essential for the maturation,activation and differentiation of CD4+T cells into Th17.In vitro,HMGB1,dsDNA or HMGB1-dsDNA complexes can stimulate the maturation and activation of dendritic cells,and the activation of RAGE/PI3K/AKT pathway.The effect of complexes was stronger.The complex induces PI3K/AKT pathway activation by binding to RAGE,which is essential for dendritic cell activation.PART ⅠObjective To observe the expression of HMGB1 and dsDNA and the formation of HMGB1-dsDNA complexes in air and cigarette smoke exposed mice,and to observe the activation of myeloid dendritic cells and the expression of RAGE.Methods C57BL/6 mice(male,6-8 weeks)were randomly exposed to air and chronic cigarette smoke for 24 weeks.HE staining was used to observe the pathological changes of lung tissues,and the mean linear intercept(Lm)was measured to evaluate the degree of emphysema.The expression of HMGB1 in BALF and sRAGE in serum were detected by ELISA.The expression of dsDNA in BALF was detected by Picogreen fluorescence quantitative method.The peripheral blood neutrophils of mice exposed to air and cigarette smoke were stimulated with cigarette smoke extract(CSE),and the induced sputum of COPD patients was collected for immunofluorescence detection to determine the presence of HMGB1-dsDNA complexes.The expression of RAGE,activation marker(CD40,CD80,CD86)in myeloid dendritic cells(mDCs),lung Thl and Th17 cells were detected by flow cytometry and immunofluorescence.Results 1.Compared with the air control group,the alveoli of mice exposed to chronic cigarette smoke were enlarged,ruptured and fused,forming obvious emphysema-like changes,and Lm was significantly increased,with statistical significance(P<0.05).2.Compared with the air control group,the levels of HMGB1 and dsDNA in BALF of mice exposed to cigarette smoke were increased,while the levels of sRAGE in serum was decreased,with statistical significance(P<0.05).3.Compared with the air control group,neutrophils in mice exposed to cigarette smoke produced more HMGB1-dsDNA complexes after CSE stimulation,and a large number of HMGB1-dsDNA complexes were also present in the sputum of COPD patients.4.Compared with the air control group,lung mDCs aggregation and activation,RAGE expression on mDCs were increased,and Thl and Th17 cells were also significantly increased in cigarette smoke exposure group(P<0.05).Conclusion Chronic cigarette smoke exposure leads to the necrosis of resident lung cells and the release of endogenous DAMPs molecules HMGB1,dsDNA and HMGB1-dsDNA complexes,accompanied by the aggregation and activation of mDCs,and the increase of Thl and Th17 cells.Chronic cigarette smoke may promote dendritic cell activation and initiate acquired immunity through HMGB1,dsDNA or HMGB1-dsDNA complexes,which may involve dendritic cell membrane receptor RAGE.PART ⅡObjective To investigate whether HMGB1-dsDNA complexes promotes the activation of bone marrow derived dendritic cells(BMDC)through the RAGE-PI3K/AKT signaling pathway.Methods Bone marrow derived dendritic cells DC2.4 were used as the study object.Experimental groups:control group,HMGB1 group,dsDNA group,HMGB1-dsDNA complexes group,complexes+RAGE antagonist group,complexes+PI3K inhibitor group,CSE positive control group.The expressions of RAGE,p-PI3K,AKT,p-AKT,NF-κB,p-NF-κB,MHC-11,CD40,CD80 and CD86 were detected by WB and flow cytometry.Results 1.HMGB1 and dsDNA alone or complexes can stimulate the expression of RAGE,p-PI3K,p-Akt,and p-NF-KB,and induce the increase of mature marker MHC-11 and activation markers CD40,CD80 and CD86 in DC2.4.The effect of complexes was stronger.The difference was statistically significant(P<0.05).2.Pretreatment of RAGE inhibitor FPS-ZM1 decreased the activation of RAGE/PI3K/AKT pathway and the maturation and activation of DC2.4 after complexes stimulated,with statistical significance(P<0.05).PI3K inhibitor LY294002 did not affect the expression of RAGE,but decreased the activities of p-Akt and p-NF-KB,and decreased the maturation and activation of DC2.4 after complexes stimulated,with statistical significance(P<0.05).Conclusion HMGB1,dsDNA or HMGB1-dsDNA complexes can stimulate the maturation and activation of dendritic cells,and the activation of RAGE/PI3K/AKT pathway.The effect of complexes was stronger.The complex induces PI3K/AKT pathway activation by binding to RAGE,which is essential for dendritic cell activation.PART ⅢObjective To investigate whether conditional knockout of RAGE on mDCs has an effect on mDC activation after cigarette smoke exposure.Methods Mice were divided into wild-type air group(WT-AIR),wild-type cigarette smoke exposure group(WT-CS),RAGE conditioned knockout air group(RAGEfl/flCD11c-Cre-AIR),RAGE conditioned knockout cigarette smoke exposure group(RAGEfl/flCD11c-Cre-CS).HE staining was used to observe the pathological changes,and the Lm was measured to evaluate the degree of emphysema.The expression of RAGE,p-PI3K,p-Akt,CD40,CD80 and CD86 in lung mDCs,Thl and Th17 cells were detected by flow cytometry.Results 1.Compared with RAGEfl/flCD11c-Cre-AIR,Lm in RAGEfl/flCD11cCre-CS was increased,but significantly lower than WT-CS(P<0.05).There was no difference between RAGEfl/flCD11c-Cre-AIR and WT-AIR.2.Compared with RAGEfl/flCD11c-Cre-AIR,the expression of RAGE on mDCs from RAGEfl/flCD1lc-Cre-CS group did not increase,but the expression of p-PI3K,p-Akt,CD40,CD86 and CD80 all increased.However,it was still significantly lower than WT-CS(except p-Akt)(P<0.05).3.Compared with RAGEfl/fl 1CD11c-Cre-AIR,the percentage of CD4+IFN-γ+T cells in RAGEfl/flCD11cCre-CS was increased,and there was no difference between RAGEfl/flCD11cCre-CS and WT-CS,while CD4+IL17+T cells were not increased.Conclusion Conditional knockout of RAGE on mDCs has protective effect on emphysema in mice exposed to chronic tobacco smoke.The expression of RAGE in dendritic cells is essential for the maturation,activation and differentiation of CD4+T cells into Th17. |
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