MBD2 regulates differentiation and function of Th17 cells in neutrophils- dominant asthma via HIF-1α

Abstract

Background: T helper 17 (Th17) cells have proven to be crucial in the pathogenesis of neutrophils-dominant asthma. Hypoxia inducible factor-1α (HIF-1α) is involved in allergic responses in asthma. Our previous studies indicated that Methtyl-CpG binding domain protein 2 (MBD2) expression was increased in asthma patients. The aim of the present study is to understand how MBD2 interacts with HIF-1α to regulate Th17 cell differentiation and IL-17 expression in neutrophils-dominant asthma.

Methods: A neutrophils-dominant asthma mouse model was established using female C57BL/6 mice to investigate Th17 cell differentiation and MBD2 and HIF-1α expression regulation using flow cytometry, western blot or qRT-PCR. MBD2 and HIF-1α genes were silenced or overexpressed through lentiviral transduction to explore the roles of MBD2 in Th17 cell differentiation and IL-17 release in neutrophils-dominant asthma.

Results: A neutrophilic inflammatory asthma phenotype model was established successfully. This was characterized by airway hyperresponsiveness (AHR), increased BALF neutrophil granulocytes, activated Th17 cell differentiation, and high IL-17 levels. MBD2 and HIF-1α expression were significantly increased in the lung and spleen cells of mice with neutrophils-dominant asthma. Through overexpression or silencing of MBD2 and HIF-1α genes, we have concluded that MBD2 and HIF-1α regulate Th17 cell differentiation and IL-17 secretion. Moreover, MBD2 was also found to regulate HIF-1α expression.

Conclusions: Our findings have uncovered new roles for MBD2 and HIF-1α, and provide novel insights into the epigenetic regulation of neutrophils-dominant asthma.

Keywords: Hypoxia inducible factor-1α; Methtyl-CpG binding domain protein 2; Neutrophils-dominant asthma; T helper 17 cells.

Fig. 1

Establishment of a neutrophils-dominant asthma mouse model a Pulmonary resistance in control, conventional asthma, and neutrophils-dominant asthma groups, *p < 0.05 compared to control. #p < 0.05 compared with conventional asthma. b Total neutrophil and eosinophil cells in BALF from all groups. *p < 0.05 compared to other group. c Lung tissues stained with H&E and for neutrophil-specific antibody (anti-Gr1), eosinophil antibody (anti-ECP) in all groups. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05 or #p < 0.05)

Fig. 2

Neutrophils-dominant asthma mediated by Th17 cells a Th17 and Th2 cells in splenocytes, stained for intracellular APC-anti-IL-17 and PE-anti-IL-4 and assessed using flow cytometry. b and c qRT-PCR and western blot used to measure RORγt and GATA3 mRNA and protein expression in all groups. d Secreted IL-17 and IL-4 in splenic CD4⁺ T cells using ELISA. *p < 0.05 compared to other groups. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05)

Fig. 3

MBD2 expression in all groups a Lung tissues stained for anti-MBD2. b and c qRT-PCR and western blot used to measure MBD2 mRNA and protein expression in lungs. d and e qRT-PCR and western blot used to measure MBD2 mRNA and protein expression in splenocytes. *p < 0.05 compared to other groups. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05)

Fig. 4

MBD2 stimulates Th17 cell differentiation and IL-17 expression a western blot to verify MBD2 transfection. b Flow cytometry used to measure the ratio of positive Th17 cells in splenic CD4⁺ T cells of all groups with MBD2 gene silencing (M[−]) or overexpression (M[+]). c and d qRT-PCR and western blot used to measure RORγt mRNA and protein expression with MBD2 gene silencing or overexpression. e Secreted IL-17 in splenic CD4⁺ T cells measured using ELISA with MBD2 gene silencing (M[−]) or overexpression (M[+]).*p < 0.05 compared to other groups. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05)

Fig. 5

Expression of HIF-1α in all groups a Lung tissues were stained for anti- HIF-1α. b and c qRT-PCR and western blot analyses used to measure HIF-1α mRNA and protein expression in lung samples from all groups. d and e qRT-PCR and western blot analyses used to measure HIF-1α mRNA and protein expression in splenocytes from all groups. *p < 0.05 compared to other groups. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05)

Fig. 6

HIF-1α expression with MBD2 gene silencing or overexpression and MBD2 expression with HIF-1α gene silencing or overexpression; HIF-1α stimulates Th17 cell differentiation and IL-17 expression a western blot to measure HIF-1α protein expression in all groups with MBD2 gene silencing (M[−]) or overexpression (M[+]). b western blot to verify HIF-1α transfection. c western blot to measure MBD2 protein expression in all groups with HIF-1α gene silencing (H[−]) or overexpression (H[+]). d Flow cytometry used to measure the ratio of positive Th17 cells in splenic CD4⁺ T cells of all groups with HIF-1α gene silencing or overexpression. e and f qRT-PCR and western blot used to measure RORγt mRNA and protein expression in all groups with HIF-1α gene silencing or overexpression. g Secreted IL-17 in splenic CD4⁺ T cells as measured using ELISA with HIF-1α gene silencing or overexpression. *p < 0.05 compared to other groups. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05)

Fig. 7

Ratio of positive Th17 cells, RORγt expression, and IL-17 expression with MBD2 and HIF-1α gene silencing or overexpression a Flow cytometry used to measure the ratio of positive Th17 cells in splenic CD4⁺ T cells of all groups with MBD2/HIF-1α gene silencing or overexpression. b and c qRT-PCR and western blot used to measure RORγt mRNA and protein expression in all groups with MBD2 and HIF-1α gene silencing or overexpression. d Secreted IL-17 in splenic CD4⁺ T cells as measured using ELISA with MBD2 and HIF-1α gene silencing or overexpression. *p < 0.05 compared to control. #p < 0.05 compared with the M(+)H(+) group. $p < 0.05 compared with the M(−)H(−) group. One-way ANOVA with Bonferroni’s post hoc tests were applied to analyze the results for significant differences (*p < 0.05 or #p < 0.05 or $p < 0.05)