The Effect And Underlying Mechanism Of Exogenous IL-25 On Pulmonary Macrophage M1 Polarization And Airway Inflammation In Neutrophilic Asthma

Objective: To establish the mouse models of neutrophilic asthma and eosinophilic asthma,and to investigate the effects of Il-25 on macrophage polarization,Il-12,Il-23 expression and airway inflammation in neutrophilic asthma.Methods: 6-8 weeks female C57 BL,6J mice were divided into four groups(n=6-8): OVA/LPS group,OVA group,OVA/LPS/Il-25 group and control group.HE(Hematoxylin eosin)staining was performed on the paraffin embedded lung tissue sections of mice to show the infiltration of inflammatory cells around each airway,and the degree of inflammation was scored(0-4 points).The total number of BALF cells and the number of inflammatory cells were counted.RT-PCR was used to detect the m RNA expression levels of Il-12,Il-23,Il-25,macrophage polarization markers and Il-1β,Ifn-γ in lung tissues of mice in different groups.Flow cytometry was used to detect the M1 and M2 polarization of macrophages.Il-12 b and Cd80 positive cells were detected by immunofluorescence staining.Results: The pulmonary resistance of eosinophilic asthma group and neutrophilic asthma group increased.The results of HE staining and BALF inflammatory cell count showed that compared with the control group,and the airway inflammatory cell infiltration was obvious.Exogenous Il-25 significantly inhibited airway inflammatory infiltration and BALF neutrophil count in mice with neutrophil asthma.RT-PCR results showed that the m RNA expression of M1 markers(Cd80,inos,Tnf α),and effectors(Il-12,Il-23,Il-1β,Ifn-γ)in lung tissue of mice with neutrophil asthma increased significantly,while exogenous IL-25 significantly inhibited the expression of M1 markers and effectors The results of flow cytometry showed that macrophages in lung tissue and bronchoalveolar lavage fluid had obvious M1 polarization.Immunofluorescence showed that Il-12 b and Cd80 positive staining in bronchoalveolar lavage fluid increased significantly in the neutrophil asthma group.Compared with the neutrophil asthma group,the M1 polarization of macrophages and the expression of Il-12,Il-23 in the mouse model of neutrophil asthma were inhibited.Conclusion: There was obvious neutrophilic airway inflammation in the mouse model of neutrophilic asthma.The number of M1 macrophages and the expression of Il-12 and Il-23 were increased.Exogenous Il-25 can significantly reduce airway inflammation in mice with neutrophil asthma and inhibited M1 polarization and Il-12,Il-23 expression of macrophages.Objective: To observe whether Il-25 inhibited M1 polarization of macrophages and the expression of Il-12 and Il-23 in vitro.To investigate whether Il-25 inhibits LPS induced NF-κB nuclear translocation through STAT3 pathway,thereby inhibiting M1 polarization and Il-12 and Il-23 expression in macrophages.Methods: Mouse pulmonary macrophages were isolated and divided into four groups: control group,Il-25 group,LPS group and LPS,Il-25 group.RT-PCR was used to detect the m RNA expression of Il-12,Il-23.Western blot was used to detect the protein level of Il-12,Il-23 and the phosphorylation level of STAT3.Cytoplasmic and nuclear protein was extracted to detect the nuclear translocation of NF-κB.Immunofluorescence was used to detect the expression of Il-12,Il-23 and nuclear translocation of NF-κB in macrophages.Pretreat mouse lung macrophages with an effective inhibitor of p STAT3(Stattic)for 1 hour,and then intervene with LPS,Il-25 to observe the influence of nuclear translocation of NF-κB and Il-12,Il-23 expression by LPS and Il-25.Results: After LPS intervention,NF-κB nuclear translocation was enhanced,M1 marker expression and Il-12,Il-23 expression in macrophages were increased.After IL-25 intervention,STAT3 phosphorylation was enhanced,NF-κB nuclear translocation was inhibited,M1 marker expression was decreased,and Il-12,Il-23 expression was inhibited.Western blot and immunofluorescence results showed that the inhibitory effect of Il-25 on NF-κB nuclear translocation and Il-12,Il-23 was significantly decreased after stattic pretreatment.Conclusion: LPS induced nuclear translocation of c-Rel,a member of NF-κB family,promoted M1 polarization and Il-12 and Il-23 expression in macrophages.Exogenous Il-25 inhibits LPS induced nuclear translocation of c-Rel by activating STAT3 signaling pathway,thus blocking M1 polarization and the expression of Il-12 and Il-23 in macrophages.Objective: To collect airway brush,induced sputum and BALF cells of control group,eosinophilic asthma group and non-eosinophilic asthma group and detect the expressions of IL-12,IL-23 and IL-25.To verify the findings in animal experiment and cell experiment To verify the findings in animal experiments and cell experiments,and confirm that IL-25 can inhibit M1 polarization and the expression of IL-12 and IL-23 in macrophages.Methods: The airway brush specimens of control group(n=10),eosinophilic asthma group(n=20)and non eosinophilic asthma group(n=14)were collected.The m RNA levels of IL-25,IL-12 and IL-23 were detected by RT-PCR,and the correlation between IL-25 and IL-12,IL-23 was analyzed.The induced sputum of eosinophilic asthma group(n=17)and non eosinophilic asthma group(n=12)were collected.The m RNA levels of IL-25,IL-12 and IL-23,the m RNA levels of M1 macrophage markers(CD80,i NOS)and effector molecules(IL-1 β,IFN-γ)were detected by RT-PCR.The positive cells of CD80 and IL-12 B in BALF were detected by immunofluorescence.Results: The mRNA level of IL-25 in airway epithelial cells of eosinophilic asthma group was significantly higher than that of IL-12 and IL-23 in non-eosinophilic asthma group.In accordance with this,the m RNA level of IL-25 in induced sputum cells of eosinophilic asthma group was significantly higher than that of non eosinophilic asthma group,while the expression of IL-12 and IL-23 in non-eosinophilic asthma group was significantly higher than that of eosinophilic asthma group.At the same time,the expression of M1 macrophage marker(i NOS)and M1 macrophage effector molecules IL-1 β and IFN-γ in sputum cells of non eosinophilic asthma group increased.The results of BALF immunofluorescence showed that the number of CD80 positive cells and IL-12 B positive cells in induced sputum cells of patients with non eosinophilic asthma increased,and both expressed in the same cell.Conclusion: The expression of IL-12 and IL-23 in airway epithelial cells and induced sputum increased in patients with non-eosinophilic asthma.The expression of IL-25 in airway epithelial cells and induced sputum increased in eosinophilic asthma patients.The expression of IL-12,IL-23 and M1 macrophage markers CD80,i NOS in airway brush,induced sputum cells and BALF cells in patients with non-eosinophilic asthma was increased compared with those of patients with eosinophilic asthma.These results suggest that macrophage M1 polarization and up-regulated expression of IL-12 and IL-23 may be involved in the pathogenesis of non eosinophilic asthma.