The methyl-CpG binding domain 2 regulates peptidylarginine deiminase 4 expression and promotes neutrophil extracellular trap formation via the Janus kinase 2 signaling pathway in experimental severe asthma.

Abstract

Objective: The prognosis for severe asthma is poor, and the current treatment options are limited. The methyl-CpG binding domain protein 2 (MBD2) participates in neutrophil-mediated severe asthma through epigenetic regulation. Neutrophil extracellular traps (NETs) play a critical role in the pathogenesis of severe asthma. This study aims to detect if MBD2 can reduce NETs formation and the potential mechanism in severe asthma.

Methods: A severe asthma model was established in C57BL/6 wild-type mice exposure to house dust mite (HDM), ovalbumin (OVA), and lipopolysaccharide (LPS). Enzyme-linked immunosorbent assay was used to measure the concentrations of IL-4, IL-17A, and IFN-γ in lung tissues. Flow cytometry was employed to determine the percentages of Th2, Th17, and Treg cells in lung tissues. Quantitative real-time polymerase chain reaction was utilized to assess the mRNA expression levels of MBD2, JAK2, and PAD4. Western blotting and immunofluorescence were conducted to detect the protein of MBD2, JAK2, PAD4, and CitH3. HL-60 cells were differentiated into neutrophil-like cells by culturing in a medium containing dimethyl sulfoxide and then stimulated with LPS. KCC-07, Ruxolitinib, and Cl-amidine were used to inhibit the expressions of MBD2, JAK2, and PAD4, respectively.

Results: Severe asthma mice were characterized by pulmonary neutrophilic inflammation and increased formation of neutrophil extracellular traps (NETs). The expression of MBD2, JAK2, and PAD4 was elevated in severe asthma mice. Inhibiting the expression of MBD2, JAK2, and PAD4 reduced NETs formation and decreased airway inflammation scores, total cell counts and neutrophil counts in BALF, and percentage of Th2 and Th17 cell in lung tissues, whereas increasing Treg cell counts. In both severe asthma mice and HL-60-differentiated neutrophil-like cells in vitro, inhibiting MBD2 reduced the mRNA and protein expression of JAK2 and PAD4, and inhibiting JAK2 reduced the expression of PAD4 mRNA and protein.

Conclusion: MBD2 regulates PAD4 expression through the JAK2 signaling pathway to promote NETs formation in mice with severe asthma. Further bench-based and bedside-based studies targeting the MBD2, PAD4, and JAK2 signaling pathways will help open new avenues for drug development of severe asthma.