MBD2 Regulates Th17/IL-17 And Its Role In Airway Inflammatory Phenotype Of Asthma In Mice

Objective:To observe the changes of Methyl-Cp G-binding protein 2(MBD2)and DNA methylation in asthmatic mice with different weeks old and different airway inflammatory phenotypes,to define the regulatory effects of MBD2,DNA methylation and ovalbumin(OVA)on the differentiation and function of Th17 cells(Th17/IL-17),and to preliminarily elaborate Epigenetic mechanism regulatory protein MBD2 regulates Th17/IL-17 and its role in the airway inflammatory phenotype of asthma in mice.Methods:1.In vivo experiment:C57BL/6 mice aged 4 weeks,8 weeks and 6～8 months were randomly divided into control group,eosinophil inflammatory phenotype group and neutrophil inflammatory phenotype group for modeling and intervention.The behavioral changes and pathological changes of lung tissue of mice in each group were observed.The airway hyperreactivity(AHR)of mice was detected by noninvasive small animal lung function instrument.The expression of MBD2,IL-4 and IL-17 in bronchoalveolar lavage fluid of mice in each group were detected by Enzyme-linked immunosorbent assay(ELISA).Western blot(WB)was used to detect the expression of MBD2protein in lung tissue of mice in each group.Immunohistochemistry(IHC)staining was used to detect the distribution of MBD2,eosinophils and neutrophils in lung tissue.Immunofluorescence(IF)staining was used to detect the secretion of IL-4 and IL-17 in lung tissue,and the overall level of DNA methylation in lung tissue of mice in each group was also detected.To observe the expression of MBD2 and DNA methylation in the airway of mice with different weeks old and different asthma airway inflammatory phenotypes.2.In vitro experiment:Using spleen derived CD4~+T lymphocytes from C57BL/6mice and lentivirus transfection silences MBD2 treated CD4~+T cells to simulate the differentiation microenvironment of Th17 cells in vitro.5-Aza-2’-deoxycytidine(5-Aza-Cd R),a DNA methylation inhibitor,was added in groups to stimulate the culture.Flow cytometry(FCM)was used to detect the differentiation ratio of Th17 cells in each group,ELISA was used to detect IL-17secretion level in the culture supernatant of each group,and the overall level of DNA methylation in each group was also detected.To observe the regulatory effects of MBD2 and DNA methylation on Th17/IL-17.Mice spleen derived mononuclear cells and MBD2 silenced mononuclear cells were divided into groups and added to OVA stimulated culture.The differentiation ratio of Th17cells,the secretion level of IL-17 and the overall level of DNA methylation were also detected.To observe the regulatory effect of OVA on Th17/IL-17 and the effect on DNA methylation level.Results:1.Successfully constructed and simulated mice asthma models with different weeks old and different airway inflammatory phenotypes.Compared with eosinophil inflammatory phenotype group,MBD2,IL-17 and DNA methylation level were more highly expressed in the airways of mice in the neutrophil inflammatory phenotype group(P<0.05),and showed weeks old related differences.2.Compared with the induced differentiation group,5-Aza-Cd R intervention reduced Th17 cell differentiation along with IL-17secretion level(P<0.05),and MBD2 silencing could enhance this effect(P<0.05).Th17/IL-17 was highly expressed in the OVA group when compared with control group(P<0.05).Conclusions:1.MBD2 and DNA methylation were increased locally in the airway of asthmatic mice,and were more pronounced in asthmatic mice with neutrophil inflammatory phenotype,and were correlated with weeks old.2.MBD2 silencing and DNA methylation inhibition could affect Th17/IL-17expression,showing a trend of reduced Th17 cells and decreased IL-17 secretion.3.OVA could stimulate Th17/IL-17 signal response,which can be attenuated when MBD2 function is deficient.Combined with the previous research results,it is suggested that MBD2 can regulate Th17/IL-17,which may be one of the regulatory mechanisms of MBD2 involved in the epigenetic pathogenesis of asthma and the phenotypic changes of airway inflammation.