| PART I The expression of TLR4 on myeloid dendritic cells of cigarette-associated emphysema miceObjective: To investigate the expression of TLR4 on myeloid dendritic cells of cigarette-associated emphysema mice.Method: Male C57BL/6J mice aged 6-8 weeks were randomly divided into air exposure group(AIR group)and chronic cigarette smoke exposure group(CS group).These mice were exposed to cigarette smoke or air for 24 weeks.After successful modeling,mice were sacrificed.The lower lobe of the right lung was retained in each group,and the pathological changes of the lung tissue were observed by HE staining,and the alveolar mean linear intercept(Lm)was calculated to evaluate the degree of emphysema in mice.The splenic and left lung were separated into single-cell,respectively.Flow cytometry was used to detect the expression of CD40,CD86 and TLR4 on myeloid dendritic cells;The expression of Th1 and Th17 cells in the two groups was detected by flow cytometry.Result:(1)Compared with the AIR group,the LM of the CS group was significantly increased,emphysema was formed.(2)Compared with the AIR group,the expression of TLR4,CD40 and CD86 on myeloid dendritic cells of spleen and lung tissues in CS group was increased,the difference was statistically significant.(3)The proportion of Th1 and Th17 cells in the spleen and lung tissue of the CS group was significantly higher than that of the AIR group,the difference was statistically significant.Conclusion: Chronic cigarette smoke exposure can activate the activation of myeloid dendritic cells and initiate the abnormal immune responses mediated by Th1 and Th17 cells,which may be related to the high expression of TLR4 on myeloid dendritic cells.PART II To investigate the effect of TLR4 on the activation of myeloid dendritic cells in mice with cigarette-associated emphysema by sequentially knocking out the TLR4Objective: To investigate whether TLR4 is involved in inducing the activation of myeloid dendritic cells in mice with cigarette-associated emphysema by constructing TLR4fl/flCD11C-cre mice.Method: TLR4fl/flCD11C-cre and TLR4fl/fl negative control mice were constructed and exposed to cigarette smoke for 24 weeks.The corresponding air exposure group was set up in each group.After successful modeling,mice were sacrificed.The lower lobe of the right lung was retained in each group,and the pathological changes of lung tissue were observed by HE staining,and the alveolar mean linear intercept(Lm)was calculated to evaluate the degree of emphysema in mice.The single-cell of spleen and lung were extracted from mice.Flow cytometry was used to detect the expression of CD40 and CD86 on myeloid dendritic cells in each group.The expression of Th1 and Thl7 cells was detected by flow cytometry.Immune magnetic beads were used to separate and purify the initial myeloid dendritic cells of the mouse spleen.The myeloid dendritic cells were immunized adoptively into TLR4fl/flCD11c-Cre mice by tail vein injection,and they were exposed to chronic cigarette smoke for 24 weeks.The expression of the related indexes was compared with that of TLR4fl/flCD11c-Cre mice.Result:(1)After 24 weeks of chronic cigarette smoke exposure,compared with the mice in the TLR4fl/fl group,the Lm of the mice in the TLR4fl/flCD11 cCre group decreased and the degree of emphysema was lighter.(2)Compared with the TLR4fl/fl group,the expression of CD40 and CD86 on myeloid dendritic cells in spleen and lung tissues was significantly down-regulated in the TLR4fl/flCD11C-cre group after 24 weeks of chronic cigarette smoke exposure.(3)The number of Th1 and Th17 cells in spleen and lung tissues of TLR4fl/flCD11 Ccre mice exposed to chronic cigarette smoke exposure and air exposure was significantly lower than that of TLR4fl/fl mice,and the difference was statistically significant.(4)After immunizing adoptive myeloid dendritic cells to TLR4fl/flCD11C-cre mice,Lm was significantly increased,the expression of TLR4,CD40 and CD86 on myeloid dendritic cells was up-regulated,and the proportion of Th1 and Th17 cells was increased.Conclusion: Conditionally knocking out TLR4 on myeloid dendritic cells can not only inhibit the activation of myeloid dendritic cells and the differentiation of Th1 and Th17 in chronic cigarette smoke-exposed mice,but also improve emphysema,which provides a basis for TLR4 to be a target for the treatment of cigarette-associated emphysema.PART III Cigarette-activated the TLR4-TRIF-dependent signaling pathway affects the activation of mouse bone-marrow derived dendritic cells(BMDC)Objective: To investigate whether the TLR4-TRIF-dependent signaling pathway affected the activation of BMDC.Methods: Using mouse bone marrow-derived dendritic cells DC2.4 as the research object,the CCK8 detection method was used to determine the survival rate of different cigarette smoke extract(CSE)concentrations,TLR4,TRIF,pIRF3 and IFN-β were detected by WB or real-time fluorescence quantitative assay(RT-PCR).Experimental groups included blank control group,CSE group,CSE+TLR4 antagonist group,and CSE+TRIF antagonist group.The expressions of TLR4,TRIF,TRAF3,IRF3,p-IRF3 and IFN-β in each group were detected by WB,and the percentage of CD40 and CD86 in each group was detected by flow cytometry,IL-1β and IL-6 in the cell culture supernatant were detected by ELISA.Results:(1)The results of CCK8 showed that there was no significant toxicity when the CSE concentration below 0.3% interfered with DC2.4.The protein or m RNA expression levels of TLR4,TRIF,p-IRF3 and IFN-β were increased after CSE intervention.(2)WB results showed that the expressions of TLR4,TRIF,TRAF3,p-IRF3 and IFN-β in the CSE group were higher than those in the blank control group.After the addition of TLR4 specific antagonist,it was significantly down-regulated compared with the CSE group.After the intervention of TRIF-specific antagonist,the expression of TLR4 showed no statistically significant difference compared with the CSE group,while the expression of TRAF3,p-IRF3 and IFN-β decreased,the difference was statistically significant.There was no significant difference in the IRF3 between each group,and there was no statistical significance.(3)Flow cytometry results showed that the percentages of CD40 and CD86 in the CSE group were higher than those in the blank group,but the percentages were decreased after the addition of TLR4 or TRIF antagonists.(4)The levels of IL-1β and IL-6 in the supernatant of CSE stimulated cell culture were increased compared with the blank group.After the intervention of TLR4 or TRIF antagonist,the inflammatory factors secreted by DC2.4 were down-regulated.Conclusion: TLR4 may be involved in the activation of myeloid dendritic cells through the TRIF-dependent signaling pathway,thereby affecting the inflammatory response of cigarette-associated.PART IV Effect inhibits the TLR4-TRIF-dependent signaling pathway and affects the activation of myeloid dendritic cells exposed to cigarette smokeObjective: To investigate whether the erythromycin could inhibit TLR4-TRIF dependent signaling pathway activated by cigarette,thereby affecting the activation of myeloid dendritic cells.Methods: Male C57BL/6J mice aged 6-8 weeks were used as the research objects.The experiment was divided into three groups: air exposure group(AIR group),chronic tobacco smoke exposure group(CS group),chronic tobacco smoke exposure + erythromycin intervention group(EM group).The lower lobe of the right lung was retained from each group,and the pathological changes of lung tissue were observed by HE staining,and the alveolar mean linear intercept(Lm)was calculated to evaluate the degree of emphysema in mice.The expression of TLR4 on myeloid dendritic cells was detected by flow cytometry;at the same time,a part of the tissue embedded section of the right lower lobe was used to detect the expression of CD11c+TLR4+ co-expression of the lung tissue by immunofluorescence.and Th1 and Thl7 cell in each group were detected by flow cytometry.The expression of CD80 on myeloid dendritic cells,Th1 and Th17 were detected by flow cytometry.In vitro,the DC2.4 cells were divided into three groups,including blank control group,CSE group and CSE+EM group.WB was used to detect the expression of TLR4,TRIF,TRAF3,IRF3,p-IRF3,IFN-β in each group.Flow cytometry was used to detect the ratio of CD40 and CD86 in each group.The expression of IL-1β and IL-6 in the cell culture supernatant of each group was determined by ELISA.Results:(1)Compared with the CS group,the Lm of the erythromycin group was reduced.(2)The expression of TLR4 on myeloid dendritic cells in the spleen and lung in the erythromycin group was lower than that of the CS group.Immunofluorescence results showed that the double positive expression rate of CD11c+TLR4+ in the CS group was higher than that in the AIR group,while the EM group decreased significantly.(3)Compared with the CS group,the ratio of CD80 on myeloid dendritic cells and the proportion of Th1,Th17 cells of the EM group was significantly decreased,and the difference was statistically significant.(4)In vitro,the expression of TLR4,TRIF,TRAF3,p-IRF3,IFN-β in the erythromycin group was down-regulated compared with the CSE group,and the difference was statistically significant;while there was no significant change in IRF3 among all groups.(5)The activation of DC2.4 was inhibited by erythromycin.(6)IL-1β and IL-6 of the EM group in vitro was lower than that of the CSE group,and the difference was statistically significant.Conclusion: Erythromycin may inhibit the activation of myeloid dendritic cells through the TLR4-TRIF-dependent signaling pathway,thereby reducing the chronic and acute inflammation of cigarette-associated emphysema. |
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