The Mechanisms Of Bacterial Lysates Nebulized Inhalation In The Prevention Of Bronchial Asthma Of Mice

Objective To investigate the effects of nebulized inhalation of bacterial lysates on preventing the airway inflammation and airway reactivity in asthmatic mice,and to explore the mechanism of nebulized inhalation of bacterial lysates regulates the regulatory T cells(Treg)and Th1/Th2 immune imbalance by acting on dendritic cells(DCs)in asthmatic mice.Methods We carried out the nebulized inhalation doses effects experiments of bacterial lysates to explore the optimal aerosol dose.After that,we used nebulized inhalation bacterial lysates to intervene mouse asthma model.In the adoptive-DCs-transfer intervention asthma group,after the last OVA sensitization,1×106 CD11c+DCs sorted and purified from the mouse lung which received Bacterial Lysates nebulization,and then DCs were injected into mice of DCs/OVA group.Airway responsiveness was detected after each model was made.Lung inflammation in mice were observed by HE staining and PAS staining.Inflammatory cells were counted in BALF,and the levels of IL-10,IL-17,IL-4 and OVA-IgE in BALF were detected by ELISA.The expression of Foxp3,Ki67 IL-17 and notch4 in lung were detected by immunohistochemistry.Flow cytometry detected the expression of CD40,CD80,PD-L1 in cDCs,the proportion of Ki67+Foxp3+CD25+CD4+T cells,Notch4+Foxp3+CD25+CD4+T cells,and the proportion of Th1,Th17,Th2,cDC1,cDC2,IRF4+MHCⅡ+CD11c+CD45+DCs,IRF8+MHC-Ⅱ+CD11 c+CD45+DCs,TLR4+MHCⅡ+CD11c+CD45+DCs in lung tissue.In vitro cell experiment:we isolated the DCs from bacterial lysates aerosol inhalation intervention mice,then we co-cultured the isolated DCs with the normal CD4+CD25+Treg cells,PD-L1 antibody was added or not.We detected the Foxp3 and Notch4 expressed in CD4+CD25+Treg cells and the concentrations of IL-10 in the co-culture supernatant.After that,we also isolated the mice lung DCs,and co-cultured DCs with CD4+T cells after the bacterial lysates intervened with DCs,in which TLR4 blocker was added to the co-culture system,we then detected the IFN-y Express of co-cultured CD4+T cells.Results The expression of CD40,CD80 and PD-L1 expression of in DCs of lungs were significantly increased in nebulized inhalation of bacterial lysates intervened mice.Nebulized inhalation of bacterial lysates inhibited the expression of Notch4 in Treg cells,resulted in inducing Treg cells proliferation and inhibit Th2 and Th17 immune responses in asthmatic mice.In addition,Nebulized inhalation of bacterial lysates up-regulated the expression of TLR4 and IRF8 in mouse cDC,inhibited the increase of IRF4 in asthmatic mice,increased the proportion of cDCl and decreased the proportion of cDC2,and extenuated the Th1/Th2 imbalance.Conclusion Nebulized inhalation of bacterial lysates stimulated the activation of DCs and induced the proliferation and activation of Treg cells to play an immune tolerance function,which leaded to the inhibition of Th2 and Th17 immune responses.Also,nebulized inhalation of bacterial lysates ameliorated the cDC1/cDC2 imbalance,resulted in relieving the Thl/Th2 imbalance through TLR4 signaling,thereby reducing airway inflammation and hyperresponsiveness in asthmatic mice.Part Ⅰ The effects of nebulized inhalation of bacterial lysates on regulating the dendritic cells and Treg in preventing asthmaObjective To investigate whether nebulized inhalation of bacterial lysates can act on DCs to regulate Treg cells and induce airway immune tolerance,which can reduce airway inflammation and airway reactivity in asthmatic mice.Methods We carried out the nebulized inhalation doses effects experiments of bacterial lysates to explore the optimal aerosol dose.After that,we used nebulized inhalation bacterial lysates to intervene mouse asthma models.Airway responsiveness was detected after each model was made.Lung inflammation in mice were observed by HE staining and PAS staining.Inflammatory cells were counted in BALF,and the levels of IL-10,IL-17,IL-4 and OVA-IgE in BALF were detected by ELISA.The expression of Foxp3,Ki67 and IL-17 in lung were detected by immunohistochemistry.Flow cytometry detected the expression of CD40 and CD80 in DCs,the proportion of Ki67+Foxp3+CD25+CD4+T cells,and the proportion of Th17 and Th2 cells of lungs.Results Nebulized inhalation of bacterial lysates reduced the airway reactivity and airway inflammation and decreased the levels of eosinophils,lymphocytes,OVA-IgE,IL-17 and IL-4 of mice in the Neb/OVA group.IL-10 in BALF of Neb/OVA group were significantly higher than the OVA group.Compared with NC group,IL-10 of Neb group increased.Nebulized inhalation of bacterial lysates increased the expression of Ki67 and Foxp3 in Neb group when compared with NC group.Nebulized inhalation of bacterial lysates reduced the expression of IL-17 in lung tissue of mice in OVA group.Nebulized inhalation of bacterial lysates increased the expression of CD40 and CD80 on lung DCs of mice indicated that inhalation of bacterial lysates could stimulate the activation and maturation of dendritic cells.Nebulized inhalation of bacterial lysates increased the proportion of Foxp3+CD25+CD4+T cells and Ki-67+Treg in normal mice and asthmatic mice,and decreased the proportion of IL-4+CD4+T cells and IL17+CD4+T cells in asthmatic mice.Conclusion Nebulized inhalation of bacterial lysates stimulated the activation of DCs and induced the proliferation and activation of Treg cells to play an immune tolerance function,which leaded to the inhibition of Th2 and Th17 immune responses,thereby reducing airway inflammation and airway hyperresponsiveness in asthmatic mice.Part Ⅱ The mechanism of nebulized inhalation of bacterial lysates on regulating the dendritic cells and Treg in preventing asthmaObjective To explore the mechanism of nebulized inhalation of bacterial lysates which can act on DCs to regulate Treg cells and induce airway immune tolerance.Methods We used nebulized inhalation bacterial lysates to intervene mouse asthma model.In the adoptive-DCs-transfer intervention asthma group,after the last OVA sensitization,1×106 CD11c+DCs sorted and purified from the mouse lung which received Bacterial Lysates nebulization,and then DCs were injected into mice of DCs/OVA group.Airway responsiveness was detected after each model was made,and lung tissue and bronchoalveolar lavage fluid were collected.Lung inflammation in mice were observed by HE staining and PAS staining.Inflammatory cells were counted in BALF,and the levels of IL-10,IL17,IFN-y,IL-4 and OVA-IgE in BALF were detected by ELISA.The expression of Notch4 protein in lung was detected by immunohistochemistry.The expression of PD-L1 of DCs and the proportion of Notch4+Foxp3+CD25+CD4+T cells were detected by flow cytometry.In vitro cell experiment:we isolated the DCs from bacterial lysates aerosol inhalation intervention mice,then we co-cultured the isolated DCs with the normal CD4+CD25+Treg cells,PD-L1 antibody was added or not.We detected the Foxp3 and Notch4 expressed in CD4+CD25+Treg cells and the concentration of IL-10 in the co-culture supernatant.Results DCs-transfer decreased the airway reactivity and airway inflammation in asthmatic mice.DCs-transfer decreased the total number of inflammatory cells,the proportion of eosinophils and lymphocytes,concentration OVA-IgE,IL-17 and IL-4 in the BALF of in asthmatic mice.increased the expressions of IL-10 and IFN-γ in normal mice and asthmatic mice.The expression of Notch4 in asthma mice were significantly higher than that in normal mice.The proportion of Notch4+Foxp3+CD25+CD4+T cells decreased and proportion of PD-L1+MHC-Ⅱ+CD11c+CD45+DCs increased in adoptive-DCstransfer intervention and nebulized inhalation bacterial lysates intervention asthma group.The results of the culture system showed and the expression of Foxp3 in CD4+CD25+Treg cells co-cultured with the DCs which isolated from bacterial lysates aerosol inhalation intervention mice were significantly increased.The expression of Foxp3 in CD4+CD25+Treg cells were significantly decreased.The expression of Notch4 in CD4+CD25+Treg cells which were co-cultured with DCs which isolated from bacterial lysates aerosol inhalation intervention mice were significantly decreased,while after co-culture with PD-L1 antibody,Notch4 in CD4+CD25+Treg cells which were increased.Conclusion nebulized inhalation of bacterial lysates intervenes upregulated DCs expressed PD-L1,which leaded to the inhibition expression of Notch4 in Treg cells,and induced the Treg proliferation and activation.That resulted in inhibiting the Th2 and Th17 immune responses in asthmatic mice.Part Ⅲ The effects of nebulized inhalation of bacterial lysates on regulating cDC1/cDC2 and Th1/Th2 immune imbalance in preventing asthmaObjective According to the role of bacterial lysates in the prevention of allergic reactions,while inhibiting the characteristics of Th2 immunity.The experiment mainly explored whether nebulized inhalation of bacterial lysates can intervene in the airway in the early stage with alleviated the imbalance of Thl/Th2 which by regulating cDC1/cDC2 subgroups to prevent and treat bronchial asthma.Methods We used nebulized inhalation of bacterial lysates to intervene in a model of asthma.Airway responsiveness was detected after each model was made.The levels of IFN-γ and IL-4 in BALF were detected by ELISA.Lung inflammation in mice were assessed by HE staining and PAS staining.Alveolar lavage fluid for differential cell counts.Flow cytometry was used to detect the proportions of CD103+CD11b-MHC-Ⅱ+CD11c+CD45+dendritic cells,CD103-CD11b+MHCⅡ+CD11c+CD45+dendritic cells,and Thl and Th2 cells.Inflammatory cells were counted in BALF,and the levels of IFN-γ and IL-4 in BALF were detected by ELISA.Flow cytometry was used to detect the proportions of CD103+CD11bMHC-Ⅱ+CD11C+CD45+dendritic cells,CD103-CD11b+MHCⅡ+CD11c+CD45+dendritic cells,and Th1 and Th2 cells of lungs.Results Nebulized inhalation of bacterial lysates significantly reduced inflammatory infiltration in lung tissue and reduced airway hyperresponsiveness in asthmatic mice.At the same time,it reduced the total number of inflammatory cells,the proportion of eosinophils,lymphocytes,and the level of IL-4 in BALF of asthmatic mice.Compared with normal mice,the proportion of IFN-γ+CD4+T cells in the lungs of asthmatic mice were decreased,and the proportion of IL4+CD4+T cells were increased.Nebulized inhalation of bacterial lysates relieved the imbalance in the ratio of IFN-γ+CD4+T cells to IL-4+CD4+T cells in the lungs of asthmatic mice.Asthmatic mice appeared CD103+CD11b-MHCⅡ+CD11c+CD45+dendritic cells,CD 103-CD11b+MHC-Ⅱ+CD11c+CD45+dendritic cells ratio imbalance,corresponding to Thl/Th2 imbalance,nebulized inhalation of bacterial lysates intervention relieved the asthma cDCl/cDC2 and Th1/Th2 imbalance.Conclusion nebulized inhalation of bacterial lysates can prevent bronchial asthma by alleviating the Thl/Th2 immune imbalance in lung tissue which was regulated the ratio of the cDCl and cDC2 in asthmatic mice.Part Ⅳ The mechanism of nebulized inhalation of bacterial lysates on regulating cDCl/cDC2 and Th1/Th2 immune imbalance in preventing asthmaObjective To investigate the mechanism of aerosol inhalation of bacterial lysates to regulate cDCl/cDC2,thereby reversing Th1/Th2 immune imbalance and preventing asthma.Methods We used nebulized inhalation of bacterial lysates to intervene in a model of asthma.Airway responsiveness was detected after each model was made.Flow cytometry was used to detect the proportion of IRF4+MHCⅡ+CD11c+CD45+,IRF8+MHC-Ⅱ+CD11c+CD45+,TLR4+MHC-Ⅱ+CD11c+CD45+dendritic cells in lung tissue.In vitro cell experiment:We also isolated the mice lung DCs,and co-cultured DCs with CD4+T cells after the bacterial lysates intervened with DCs,in which TLR4 blocker is added to the co-culture system,and we detected the IFN-y Express of co-cultured CD4+T cells with flow cytometry.The IFN-y concentration in the co-culture supernatant was detected by ELISA.Results The proportion of IRF4+MHC-Ⅱ+CD11c+CD45+dendritic cells increased,while,the proportion of IRF8+MHC-Ⅱ+CD11c+CD45+dendritic cells decreased in asthmatic mice.Nebulized inhalation of bacterial lysates increased the proportion of IRF8+MHC-Ⅱ+CD11c+CD45+dendritic cells in normal mice and asthmatic mice,also increased the expression of TLR4 in normal mice.Nebulized inhalation of bacterial lysates reduced the numbers of IRF4+MHCⅡ+CD11c+CD45+dendritic cells in asthmatic mice.CD4+T cells flow cytometry results in the co-culture system:CD4+T cells cultured alone had a lower IFN-γexpression,and bacterial lysates intervened DCs increased the IFN-γ expression in CD4+T cells.TLR4 blocker decreased the IFN-γ expression in co-cultured CD4+T cells.Conclusion Bacterial lysates increase of IRF8 expression,and decrease the IRF4 expression in asthmatic mice through TLR4 signaling.resulted in relieving the imbalance cDCl/cDC2 proportions,thereby extenuating the Th1/Th2 imbalance,which resulted in reducing airway inflammation and high reactivity in asthmatic mice.