Mechanism Of Mycobacterium Vaccae Aerosol Inhalation Regulating Hippo Signaling Pathway To Prevent And Treat Asthma

Objectives The purpose of this study was to explore the therapeutic targets of asthmatic mice using transcriptomic sequencing(RNA-Seq)on mouse lung tissue and to explore the mechanism of aerosol inhalation of Mycobacterium vaccae(M.vaccae)regulating Hippo signaling pathway to prevent and treat asthma.Methods Twelve Balb/c mice were randomly divided into normal control group and asthma group.A mouse model of asthma was established.Airway reactivity,bronchoalveolar lavage fluid(BALF)cell count and classification,and histopathology were measured.Lung tissues were collected from mice and pooled for mRNA sequencing and bioinformatics analysis.The relative mRNA expression of Hippo signaling pathway-related proteins Yap1,Lef1 and Ctgf was also measured.Double immunofluorescence staining was performed on lung tissues to evaluate macrophage marker F4/80 expression and Yap1/Lef1/Ctgf expression.Twenty-four Balb/c mice were randomly divided into 4 groups:normal control group,asthma group,and intervention group during the allergen sensitization period of M.vaccae(aerosol inhalation of M.vaccae during the first allergen sensitization period for 5 consecutive days)or during the allergen challenge period of M.vaccae(aerosol inhalation of M.vaccae for 5 consecutive days during the first allergen challenge).The concentrations of cytokines IL-4,IFN-y,IL-10 and IL-17 in BALF were detected.Eighteen Balb/c mice were randomly divided into 3 groups:normal control group,asthma group,and M.vaccae group(M.vaccae was inhaled 60 minutes before each allergen challenge for 7 days,once a day.).The expression of TLR2 in lung tissue was detected by RT-PCR and immunohistochemistry,and the expression of Yap1 was detected by immunohistochemistry,and the correlation between the positive rate of TLR2 and Yap1 protein was analyzed.Results Airway reactivity,the total number of inflammatory cells and the proportion of eosinophils in the BALF,and airway inflammation in the lung tissue were increased in the asthma group.Results of the RNA-Seq of lung tissues demonstrated that the Hippo signaling pathway was down-regulated in OVAinduced allergic asthma.Using the cytoHubba tool kits in Cytoscape,the following top 10 hub genes of Hippo signaling pathway were identified:Yap1,Lef1,Ctgf,Ccnd1,Axin2,Smad7,Wnt4,Wnt3a,Pard6b,and Wwc1.Using the seq-ImmuCC(http://218.4.234.74:3200/immune/),a negative correlation was found between macrophages and Hippo signaling pathway activity(R2=0.93).The mRNA expression levels of pulmonary Yapl,Lefl,and Ctgf were downregulated in the mice model of OVA-induced allergic asthma.Moreover,doublestained immunofluorescence for F4/80 and Yapl,Lefl,Ctgf in mouse lung sections respectively revealed that macrophage proliferation was correlated with downregulation of the Hippo signaling pathway in the mice model of OVAinduced allergic asthma.Compared with the asthma group,aerosol inhalation of M.vaccae for 5 consecutive days during allergen sensitization or challenge in mice suppressed airway hyperresponsiveness and airway inflammation,decreased IL-4 expression,and increased IFN-γ,IL-10 expression.Compared with the asthma group,aerosol inhalation of M.vaccae 60 minutes before each allergen challenge can reduce airway hyperresponsiveness,airway inflammation and airway remodeling,and increase the expression of TLR2 gene and protein in the lung tissue of mice.Moreover,TLR2 and Yap1 protein expression were strongly negatively correlated(correlation coefficient r=-0.812),Yap 1 protein expression was decreased,and the distribution in the nucleus was less.Conclusions These results demonstrated that the Hippo signaling pathway was down-regulated in asthma mice,and the proliferation of macrophages was associated with downregulation of the Hippo signaling pathway.These findings reveal novel insights into the pathogenesis and treatment of asthma.M.vaccae aerosol inhalation can suppress airway hyperresponsiveness and inflammation during allergen sensitization or challenge,and modulates Th1/Th2 and Treg/Th17 immune imbalances.Aerosol inhalation of Mycobacterium vaccae reduce asthma may be related to activation of TLR2 and Hippo signaling pathways.This may be an important mechanism of Mycobacterium vaccae in the treatment of asthma.Part 1 Transcriptomic Analysis Reveals a Link Between Hippo Signaling Pathway and Macrophages in Lungs of Mice with OVAInduced Allergic AsthmaObjectives The Hippo signaling pathway participates in the restriction of cell proliferation and organ growth.Activated macrophages have been implicated in the pathogenesis of allergic asthma.Recent studies have shown that Hippo signaling pathway may also be involved in the regulation of asthma.However,the link between Hippo signaling pathway and macrophages in the context of allergic asthma has not been investigated.The purpose of this study was to explore the link between Hippo signaling pathway and macrophages using a mice model of OVA-induced allergic asthma.Methods Mice models of asthma were established.Lung tissues were collected from mice and pooled for mRNA sequencing and bioinformatics analysis.The relative mRNA expression of Hippo signaling pathway-related proteins Yap1,Lef1 and Ctgf was also measured.Double immunofluorescence staining was performed on lung tissues to evaluate macrophage marker F4/80 expression and Yap1/Lefl/Ctgf expression.Results Results of the RNA-Seq of lung tissues demonstrated that the Hippo signaling pathway was down-regulated in OVA-induced allergic asthma.Using the cytoHubba tool kits in Cytoscape,the following top 10 hub genes of Hippo signalling pathway were identified:Yap1,Lef1,Ctgf,Ccnd1,Axin2,Smad7,Wnt4,Wnt3a,Pard6b,and Wwc1.Using the seq-ImmuCC(http://218.4.234.74:3200/immune/),a negative correlation was found between macrophages and Hippo signaling pathway activity(R2=0.93).The mRNA expression levels of pulmonary Yap1,Lef1,and Ctgf were down-regulated in the mice model of OVA-induced allergic asthma.Moreover,double-stained immunofluorescence for F4/80 and Yap1,Lef1,Ctgf in mouse lung sections respectively revealed that macrophage proliferation was correlated with downregulation of the Hippo signaling pathway in the mice model of OVAinduced allergic asthma.Conclusions These results demonstrated that the Hippo signaling pathway was down-regulated in asthma mice,and the proliferation of macrophages was associated with downregulation of the Hippo signaling pathway.These findings reveal novel insights into the pathogenesis and treatment of asthma.Part 2 Effects of Mycobacterium Vaccae Aerosol Inhalation on Airway Inflammation and cytokine IL-4,IL-10,IL-17 and IFN-γ in Asthma Mouse ModelObjectives Mycobacterium vaccae vaccine(M.vaccae),a composition of Mycobacterium proteins,has been known to have bidirectional immunomodulatory functions.Recent studies have shown that M.vaccae has a therapeutic potential for treating asthma.However,little is known regarding the effect of M.vaccae aerosol inhalation during allergen sensitization or challenge on asthma.The purpose of this study was to explore the effect of M.vaccae aerosol inhalation during allergen sensitization or challenge on airway inflammation in an asthma mouse model.Methods Twenty-four Balb/c mice were randomly divided into 4 groups:normal control group,asthma group,and intervention group during the allergen sensitization period of M.vaccae(aerosol inhalation of M.vaccae during the first allergen sensitization period for 5 consecutive days)or during the allergen challenge period of M.vaccae(aerosol inhalation of M.vaccae for 5 consecutive days during the first allergen challenge).Asthma mouse models were established.Mice received aerosol inhalation with M.vaccae once daily during allergen sensitization or challenge for 5 days successively.Airway responsiveness,bronchoalveolar lavage fluid(BALF)cell count,histology,and cytokine concentrations(IL-4,IFN-γ,IL-10,and IL-17)were measured.Results Compared with the asthma group,aerosol inhalation of M.vaccae for 5 consecutive days during allergen sensitization or challenge in mice suppressed airway hyperresponsiveness,the total number of inflammatory cells and the proportion of eosinophils in BALF,and inhibit the infiltration of inflammatory cells around the airway and the secretion of airway mucus,reduced levels of IL-4,upregulated expression of IFN-γ and IL-10 in BALF during allergen sensitization or challenge.Conclusions M.vaccae aerosol inhalation can suppress airway hyperresponsiveness and inflammation during allergen sensitization or challenge,and modulate Th1/Th2 and Treg/Th17 immune imbalances.Part 3 Aerosol Inhalation of Mycobacterium Vaccae Reduce Asthma Airway Inflammation and Remodeling by Regulating Hippo Signaling PathwayObjectives To observe the effect of aerosol inhalation of Mycobacterium vaccae(M.vaccae)on asthma-like symptoms(airway hyperresponsiveness,airway inflammation and airway remodeling)in asthmatic mice,and to investigate the role of Hippo signaling pathway in the prevention and treatment of asthma by aerosol inhalation of M.vaccae.Methods Eighteen Balb/c mice were randomly divided into 3 groups:normal control group,asthma group,and M.vaccae group(M.vaccae was inhaled 60 minutes before each allergen challenge for 7 days,once a day.).Asthma mouse models were established.Airway responsiveness,bronchoalveolar lavage fluid(BALF)cell count,and histology were measured.The expression of TLR2 in lung tissue was detected by RT-PCR and immunohistochemistry,and the expression of Yap1 was detected by immunohistochemistry,and the correlation between the positive rate of TLR2 and Yap1 protein was analyzed.Results Compared with the asthma group,aerosol inhalation of M.vaccae can reduce airway hyperresponsiveness,airway inflammation and airway remodeling,and increase the expression of TLR2 gene and protein in the lung tissue of mice.Moreover,TLR2 and Yapl protein expression were strongly negatively correlated(correlation coefficient r=-0.812),Yap1 protein expression was decreased,and the distribution in the nucleus was less.Conclusions Aerosol inhalation of M.vaccae reduces asthma may be related to activation of TLR2 and Hippo signaling pathways.This may be an important mechanism of M.vaccae in the treatment of asthma.