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. 2025 Jan 27;57(1):2458207. doi: 10.1080/07853890.2025.2458207

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

Biao Peng a,*, Mu-Yun Yan a,*, Yun-Rong Chen b,*, Fei Sun c, Xu-Dong Xiang d, Da Liu a,✉
PMCID: PMC11774153  PMID: 39865866

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.

Keywords: Severe asthma, methyl-CpG-binding domain 2, Janus kinase 2, peptidylarginine deiminase 4, neutrophil extracellular traps

1. Introduction

Asthma is a chronic inflammatory airway disease characterized by increased airway responsiveness and infiltration of inflammatory cells [1]. Asthma can be classified into eosinophilic-, neutrophilic-, mixed granulocytic-, and paucigranulocytic asthma, according to the type of inflammatory cells [2]. Eosinophilic asthma is typically associated with Th2 cell-mediated allergen exposure, whereas Th17 cell-mediated neutrophilic asthma is often triggered by infections and air pollution. The latter exhibits poor responsiveness to glucocorticoids, carries a higher risk of progression to severe asthma, and involves a more complex pathophysiological mechanism.

Previous studies have indicated that globally 5%–10% asthma patients with asthma suffer from severe asthma [3,4]. Therefore, investigating the pathogenesis of severe asthma is of great importance. Epigenetic modifications, including DNA methylation, histone modifications, and miRNAs, play a critical role in the pathogenesis of asthma [5]. The methyl-CpG-binding domain 2 (MBD2) recognizes DNA methylation markers involved in various epigenetic processes [6,7]. It has been linked to several immune-related diseases, including experimental colitis, rheumatoid arthritis, and lupus nephritis [8–11]. A study from Australia [12] reported elevated DNA methylation in individuals with neutrophilic asthma. Furthermore, Zhong et al. [13] demonstrated that the absence of MBD2 gene in experimental autoimmune encephalomyelitis disrupted DNA methylation markers and inhibited Th17 cell differentiation. In recent years, the role of DNA methylation in the pathogenesis of severe asthma has gained significant attention, particularly for its role in mediating the Th17 differentiation. A clinic study [14] found a synchronous relationship between the number of Th17 cells in the peripheral blood of patients with severe asthma and the expression level of MBD2. Chen et al. [15] further confirmed that MBD2 promotes naive T-lymphocyte differentiation into Th17 cells through methylation modification of misshapen-like kinase 1 (MINK1), further interpreting the mechanism of MBD2 in neutrophil-mediated severe asthma.

Neutrophil extracellular traps (NETs), composed of double-stranded DNA (dsDNA), citrullinated histone H3 (CitH3), and neutrophil granular proteins, are products of activated neutrophils [16]. Initially discovered for their ability to engulf and eliminate pathogenic microorganisms [17], NETs have been implicated in driving immune dysregulation in multiple autoimmune diseases [18–22]. NETs have been shown to play a crucial role in the development of severe asthma. Our previous study [23] demonstrated that the peptidylarginine deiminase 4 (PAD4) inhibitor, Cl-amidine, reduced NET formation and alleviated airway resistance and inflammation in mice with severe asthma. This effect is attributed to PAD4 chromatin remodeling from a dense to a more relaxed structure, thereby facilitating the release of NETs [24]. Wolach et al. [25] discovered that Janus kinase 2 (JAK2) knock-in mice exhibited a significant increase in NETs formation in myeloproliferative neoplasms. Cheng et al. [26] confirmed that the MBD2 deletion inhibited the transduction of the JAK2 signaling pathway in mice with chronic myeloid leukemia mice. Additionally, another study reported reduced JAK2 expression in dendritic cells derived from MBD2−/− mice [27].

In a previous study, we queried the Bloodspot public database (www.bloodspot.eu.) and found that MBD2 was highly expressed in granulocytes (Supplementary 1A), suggesting that MBD2 may play an important role in granulocytes. We also used an online CpG methylation island prediction tool (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi.) and identified a CpG island in the promoter region of mouse JAK2 (Supplementary 1B). We hypothesized that MBD2, being an island reader, may regulate the expression of JAK2 by recognizing the CpG island in the JAK2 promoter region. According to these findings, we speculated that MBD2 may regulate PAD4 expression and promote NET formation via the JAK2 signaling pathway in individuals with severe asthma.

In this study, we assessed the expression levels of MBD2, JAK2, and PAD4 in severely asthmatic mice and in HL-60-derived neutrophils using real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunofluorescence. Ruxolitinib that inhibits JAK2 expression, is currently used as a therapeutic drug for myelofibrosis and multiple myeloma. KCC-07 and Cl-amidine inhibit MBD2 and PAD4, respectively, and are currently only used in preclinical studies. After using KCC-07, Ruxolitinib, and Cl-amidine to interfere with the expression of their target genes, we measured CitH3 levels by Western blotting and immunofluorescence as indicators of NET formation. In addition, we evaluated the airway inflammation and mucus secretion levels in the mice through histopathology, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, as well as the inflammatory cytokine levels (IL-4, IL-17A, INF-γ) and T cell subpopulation counts (Th2, Th17, Treg) in the bronchoalveolar lavage fluid (BALF).

2. Material and methods

2.1. Animal

Female C57BL/6 wild-type mice, aged 8 weeks and weighing 18–20 g, were procured from GemPharmatech Co., Ltd (Jiangsu, China). All mice were provided sufficient food and subjected to a 12-h light/12-h dark circadian cycle within a Specific Pathogen-Free facility.

2.2. Establishment of asthma models

Both conventional and severe asthma models were established following previously described protocols [23] (Figure 1). In brief, 36 mice were randomly assigned to six groups. In the severe asthma (OVA+LPS) group, mice received intraperitoneal sensitization on days 0, 1, and 2, with injections of 25 µg LPS, 100 µg HDM, 100 µg OVA, and 2 mg aluminum hydroxide suspension. Afterward, on days 14, 15, 18, and 19, mice were exposed to an atomized 6% OVA solution for 30 min, followed by intranasal exposure to 100 µg HDM. In the conventional asthma (OVA) group, mice were sensitized on days zero and seven with 25 µg OVA and 1 mg aluminum hydroxide suspension, followed by 30-min challenges with an atomized 6% OVA solution from days 14 to 20. The control group received only saline injections. All mice were euthanized on day 21.

Figure 1.

Figure 1.

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Asthma mouse model establishment method and drug intervention flowchart.

2.3. Drugs intervention

In the OVA+LPS mice, KCC-07 was administered via intraperitoneal injection at a dosage of 100 mg/kg in 200 µL of phosphate buffer saline (PBS), twice daily from day -one through day two. This treatment regimen was continued from days 13–20. Similarly, Ruxolitinib was administered via intraperitoneal injection at a dose of 90 mg/kg in 200 µL of PBS, twice daily over the same intervals as KCC-07. Concurrently, Cl-amidine was administered intraperitoneally at a dosage of 10 mg/kg in 200 µL PBS, once daily from day 1 to day 2, and then from days 13–20 (Supplementary 2).

2.4. Measurement of airway resistance

On day 21, precisely 24 h after the final challenge, airway resistance induced by methacholine (Mch) was evaluated using direct plethysmography (Buxco Electronics, RC System, USA) following established protocols [14]. Mice were anesthetized, and subjected to tracheotomy, and invasive mechanical ventilation. Subsequently, aerosolized Mch at various concentrations (0, 3, 10, 30, and 100 mg/mL) was introduced through the inhalation port for 10 s. Finally, we assessed and normalized the airway resistance in response to different Mch doses relative to the saline baseline.

2.5. Assessment of airway inflammation

Two pathologists independently assessed peribronchial cellular infiltration using a light microscope, applying a graded scoring system to measure the severity of pulmonary inflammation using the following criteria: 0, denoting the absence of infiltrates; 1, indicating the presence of a few inflammatory cells; 2, signifying the presence of a single layer of inflammatory cells; 3, indicating the presence of two to four layers of inflammatory cells; and 4, representing the presence of more than four layers of inflammatory cells. To quantify mucus production, the percentage of periodic acid-Schiff (PAS)-stained goblet cells relative to the total epithelial cells in randomly selected bronchi was determined. The assessment followed a methodology established in prior research [28].

2.6. BALF cell counts

A catheter adapter was inserted into the pulmonary organ of a mouse, and sequentially lavaged was performed on the lung with saline three times, with each volume measuring 3 mL. The lavage-retrieved cells were concentrated onto glass slides by centrifugation and allowed to dry in open air. These cells were then stained following the May-Grunwald-Giemsa protocol, facilitating the enumeration of the total cell count and specifically, eosinophils and neutrophils cell counts from the cytospin samples, using a light microscope to calculate the absolute numbers of these immune cell subsets.

2.7. Flow cytometry

After the bronchoalveolar lavage, the lung tissue was minced and digested with 0.5 mg/mL collagenase I (Sigma, USA) and 10 μg/mL DNase I (Roche, Switzerland) in RPMI medium at 37 °C in a shaking water bath for 1 h. The lung cells were filtered through a 70 μm cell strainer and treated with red blood cell lysis buffer (eBioscience, USA). They were then stained with FITC-anti-CD4 antibodies (Invitrogen, USA), fixed, and permeabilized with eBioscience fixation and permeabilization buffer. After washing, intracellular markers (PerCP-Cy5.5-anti-IL-17A and APC-anti-IL-4 antibodies; Invitrogen, USA) were added for 20 min. Flow cytometry was performed using a FACS Calibur, and the data were analyzed with FlowJo software.

2.8. Immunofluorescence staining

To assess the expression of MBD2, JAK2, and PAD4 in mouse lung tissues, we performed immunofluorescence staining on paraffin-embedded sections of mouse lung tissue. The slides were incubated overnight with primary antibodies. Afterward, they were treated with secondary antibodies, Alexa Fluor 488 and Alexa Fluor 594 (Invitrogen), for one hour. Following this, the slides were mounted with Vectashield (Vector Laboratories, USA) containing 4′,6′-diamidino-2-phenylindole (DAPI). Imaging and cell counting were carried out using the LSM 510 confocal microscope (Zeiss, Germany).

For the detection of NETs in lung tissues, the slides were incubated overnight with primary antibodies. A rabbit anti-mouse antibody targeting CitH3 (R&D Systems) was used at a 1:50 dilution in a blocking buffer. After PBS washing, the following secondary antibodies were applied: CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) antibody (Proteintech, USA) and CoraLite488–Fluorescein (TRITC)-conjugated Affinipure Donkey Anti-Goat IgG (H + L) antibody (Proteintech, USA), both at a 1:200 dilution in blocking buffer (1:1,000). The samples were incubated for one hour and then mounted using AY89-2 (Yuantai, China) containing DAPI. Imaging and cell counting were performed using the BA210T confocal microscope (Motic, China).

Single lung cells were resuspended in HEPES-buffered RPMI medium, and neutrophils were isolated using a mouse neutrophil isolation kit (Solarbia, China) for identifying NETs in neutrophils from lung tissue. After isolation, the neutrophils were fixed with 2% paraformaldehyde, permeabilized, and blocked. Staining was carried out using a rabbit anti-mouse antibody targeting CitH3 and CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) antibody (1:200 in blocking buffer). The neutrophils were then counterstained with DAPI and mounted with Vectashield (Vector Laboratories, USA). Imaging and cell counting were conducted using the LSM 510 confocal microscope (Zeiss, Germany).

2.9. HL-60 cell culture and differentiation

The HL-60 human acute promyelocytic leukemia cell line (Honorgene, China) was cultured in phenol-red free RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco, Australia). Cells were cultured at 37 °C in a 5% CO2 atmosphere. We added 1.25% dimethyl sulfoxide (DMSO) to induce the differentiation of HL-60 cells into neutrophil-like cells, and this differentiation process lasted for 5 days. Differentiation of HL-60 into neutrophil-like cells was conducted using flow cytometry, specifically by assessing CD11b expression. This analysis was performed using a BD LSRFortessa Flow Cytometer and BD FACSDiva Software. Following differentiation into neutrophil-like cells, HL-60 cells were incubated with KCC-07, Ruxolitinib, Cl-amidine, or PBS for 2 h. Subsequently, cells were stimulated with LPS (100 ng/mL) or PBS for 2 h. Further stimulation with PMA (100 nM) was performed for an additional 2 h.

2.10. Enzyme-linked immunosorbent assay (ELISA)

The BALF supernatant was collected to analyze the cytokine levels. Specifically, the concentrations of IL-1β, IL-4, and IL-17A in the BALF were determined using a mouse cell-specific quantitative factor ELISA kit from Proteintech (USA), following the manufacturer’s instructions. The ELISA was performed according to the manufacturer’s protocols to quantify the levels of these cytokines in the collected BALF supernatant.

2.11. Western blot

Lung tissues and neutrophil-like cells were homogenized in radioimmunoprecipitation assay buffer, supplemented with a protease inhibitor cocktail sourced from (Honorgen, China). Protein concentration in the resulting homogenates was quantified using a bicinchoninic acid protein assay kit Pierce. Next, the proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred subsequently onto a nitrocellulose membrane. For the detection of total protein levels, the membranes were probed with specific primary antibodies, followed by incubation with an HRP-conjugated secondary antibody. Notably, the primary antibodies used to the detect MBD2, JAK2, PAD4, and CitH3 were consistent with those previously mentioned.

2.12. Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagents (Invitrogen, USA), followed by reverse transcription using the PrimeScript RT reagent kit (Thermo Fisher Scientific). Real-time PCR was performed on an ABI StepOne Plus Detection System (Applied Biosystems) with Power SYBR Green PCR Master Mix. The primers used were designed in the lab and synthesized by Sangon Biotech (Shanghai, China). The primers were designed as follows – MBD2: forward, 5′-AGC GAT GTC TAC TAC TTC AGT CC-3′, and reverse 5′-CGG TCC TGA AGT CAA AAC TGC TA-3′; JAK2: forward 5′-ACT GGA CTA TAT GTG CTA CGA-3′, and reverse 5′-GTT CCT CTT AGT CCC GCT GA-3′; PAD4: forward, 5′-GAT GCC TTT GGG AAC CTG GA-3′, and reverse, 5′-GCT GCT GGA GTA ACC GCT AT-3′.

2.13. Statistical analysis

Unless otherwise stated, data are reported as mean ± standard deviation (SD). The in vitro experiments were independently repeated thrice. The Shapiro-Wilk test and Z-scores were used for normality testing, and the results showed that each group of data followed a normal distribution. One-way and two-way ANOVA were performed using GraphPad Prism 9. Statistically significance was defined as p < 0.05, with significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. Results

3.1. Inhibition of MBD2, JAK2, and PAD4 ameliorates pulmonary inflammation in asthma mice

A model of severe asthma mice model was successfully established in mice sensitized to OVA and LPS. Compared with normal control and conventional asthma mice, severely asthmatic mice exhibited higher airway resistance upon stimulation with 100 mg/mL Mch (Figure 2a). Cell analysis showed that mice with severe asthma had higher total cell counts and neutrophil counts (Figure 2b and c), but lower eosinophil counts (Figure 2d) in BALF. The ELISA results indicated that severely asthmatic mice had higher concentrations of IL-17A (Figure 2e), but lower concentrations of IL-4 (Figure 2f). Histological analysis of the lungs revealed that severely asthmatic mice had higher airway inflammation scores (Figure 2h and j) and increased airway mucus secretion levels (Figure 2i and k). Flow cytometry results confirmed that, the percentage of Th17 cells was higher (Figure 2n and o), whereas the percentages of Th2 and Treg cells (Figure 2l, m, p, and q) were lower in the lung tissues of mice with severe asthma.

Figure 2.

Figure 2.

Figure 2.

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Inhibition of methyl-CpG-binding domain 2, janus kinase 2, and peptidylarginine deiminase 4 ameliorates pulmonary inflammation in asthma mice. (a) Methacholine-induced airway dynamic resistance. (b–d) Total cells, neutrophil and eosinophil counts in BALF from all groups. (e–g) Concentration of IL-17A, IL-4 and INF-γ in BALF. (h–k) Representative H&E and PAS staining of lung tissue sections from mice. (l–q) Flow cytometry analysis of the proportions of Th2, Th17, and Treg lymphocytes in lung tissue. (OVA: ovalbumin; LPS: lipopolysaccharide; Mch: methacholine; BALF: bronchoalveolar lavage fluid; HE: hematoxylin and eosin; PAS: periodic acid-Schiff).

Severe asthma mice treated with KCC-07 (MBD2 inhibitor), Ruxolitinib (JAK2 inhibitor), and Cl-amidine (PAD4 inhibitor) showed milder asthma features, including reduced airway resistance (Figure 2a), lower total cell and neutrophil counts (Figure 2b and c), and decreased concentrations of IL-17A and INF-γ (Figure 2e and g) in BALF, reduced airway inflammation scores (Figure 2h and j) and airway mucus secretion levels (Figure 2i and k), lower percentage of Th2 and Th17 cells (Figure 2l–o), but a higher percentage of Treg cells (Figure 2p and q) in the lung tissue.

3.2. Inhibition of MBD2, JAK2 and PAD4 reduces NETs formation in asthma mice

In a previous study [22], we demonstrated that NET volumes were significantly higher in mice with severe asthma mice than in mice with conventional asthma. In this study, we found that CitH3 expression was significantly reduced (Figure 3a) in the lung tissues of mice with severe asthma treated with KCC-07, Ruxolitinib, and Cl-amidine. Immunofluorescence analysis also demonstrated decreased CitH3 and myeloperoxidase (MPO) staining intensities (Figure 3b) in these three groups.

Figure 3.

Figure 3.

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Inhibition of methyl-CpG-binding domain 2, janus kinase 2, and peptidylarginine deiminase 4 reduces the formation of neutrophil extracellular traps in asthma mice. (a) Representative immunofluorescence images of PAD4 and 4′,6′-diamidino-2-phenylindole (DAPI) staining of lung sections. (b) Confocal microscopy staining of CitH3+ MPO+ DAPI+ NETs released from the lung tissues of the indicated groups of mice. KCC-07, Ruxolitinib, and Cl-amidine reduced the fluorescence intensity of CitH3 and MPO in the lung tissues of severe asthmatic mice. (OVA: ovalbumin; LPS: lipopolysaccharide; DAPI: 4′,6′-diamidino-2-phenylindole; CitH3: citrullinated histone; MPO: myeloperoxidase).

3.3. The expression of MBD2 increases in asthma mice

To investigate the expression of MBD2 in severe asthma, we performed RT-qPCR and Western blotting to measure MBD2 mRNA and protein levels in lung tissue. Immunofluorescence was used to detect the fluorescence intensity of MBD2. Compared to normal control and conventional asthmatic mice, severe asthmatic mice had significantly increased levels in MBD2 (Figure 4a and e) staining intensity, as well as a marked increase in MBD2 mRNA and protein (Figure 4b–d).

Figure 4.

Figure 4.

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The expression of methyl-CpG-binding domain 2 (MBD2) increased in asthma mice. (a) Representative immunofluorescence images of MBD2 and 4′,6′-diamidino-2-phenylindole (DAPI) staining of lung sections. The fluorescence intensity of MBD2 increased in the lung tissues of severe asthmatic mice, and no significant change in fluorescence intensity was observed after treatment with KCC-07, Ruxolitinib, or Cl-amidine. (b) Western blotting detected MBD2 protein expression in the indicated groups of mice. (c) Real-time quantitative polymerase chain reaction (RT-qPCR) detected MBD2 mRNA in lung homogenates of mice. (d) Quantification of normalized MBD2 levels in lung protein extracts of mice. (e) The fluorescence intensity ratio of MBD2 to nucleus. (OVA: ovalbumin; LPS: lipopolysaccharide; DAPI: 4′,6′-diamidino-2-phenylindole; MBD2: methyl-CpG-binding domain 2).

3.4. MBD2 regulates the expression of JAK2 in asthma mice

Compared to normal control and conventional asthma mice, the staining intensity of JAK2 in the lung tissues of severely asthmatic mice was markedly stronger (Figure 5a and e). Additionally, severely asthmatic mice showed significantly higher JAK2 mRNA expression and increased JAK2 protein levels (Figure 5b–d) in the lung tissue.

Figure 5.

Figure 5.

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The methyl-CpG-binding domain 2 regulates the expression of janus kinase 2 in severe asthma mice. (a) Representative immunofluorescence images of JAK2 and 4′,6′-diamidino-2-phenylindole (DAPI) staining of lung sections. The fluorescence intensity of JAK2 increased in the lung tissues of severe asthmatic mice. KCC-07 reduced the fluorescence intensity of JAK2, while Ruxolitinib and Cl-amidine had no significant difference. (b) Western blotting detected JAK2 protein expression in the indicated groups of mice. (c) Real-time quantitative polymerase chain reaction (RT-qPCR) detected JAK2 mRNA in lung homogenates of mice. (d) Quantification of normalized JAK2 levels in lung protein extracts of mice. (e) The fluorescence intensity ratio of JAK2 to nucleus. (OVA: ovalbumin; LPS: lipopolysaccharide; DAPI: 4′,6′-diamidino-2-phenylindole; JAK2: janus kinase 2).

To understand the mechanistic relationship between MBD2 and JAK2 in severe asthma, we measured the expression levels of JAK2 in KCC-07 treated mice. In the lung tissue of severe asthmatic mice treated with KCC-07, JAK2 staining intensity (Figure 5a and e) was significantly reduced, along with a decrease in JAK2 mRNA and protein levels (Figure 5b–d).

3.5. MBD2 promotes NETs formation by regulating PAD4 expression via JAK2 signal pathway in severe asthma mice

Consistent with our previous findings, severe asthmatic mice exhibited significantly increased levels of PAD4 mRNA and protein (Figure 5b–d) compared with normal control and conventional asthmatic mice.

It is necessary to explore the relationship between MBD2, JAK2, and PAD4, given that PAD4 regulates NETs formation, along with the inhibition of MBD2 and JAK2 with KCC-07 and Ruxolitinib respectively, resulting in reduced NETs formation in the lung tissue of mice with severe asthma. KCC-07-treated and Cl-amidine-treated severe asthma mice showed various degrees of reduction in PAD4 mRNA and protein levels (Figure 6b–d), and a significant decrease in PAD4 staining intensity (Figure 6a and e).

Figure 6.

Figure 6.

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The methyl-CpG-binding domain 2 promotes the formation of neutrophil extracellular traps by regulating peptidylarginine deiminase 4 expression through JAK2 signal pathway in severe asthma mice. (a) Representative immunofluorescence images of PAD4 and 4′,6′-diamidino-2-phenylindole (DAPI) staining of lung sections. The fluorescence intensity of PAD4 increased in the lung tissues of severe asthmatic mice. KCC-07 and Ruxolitinib reduced the fluorescence intensity of PAD4, while Cl-amidine had no significant difference. (b) Western blotting detected PAD4 protein expression in the indicated groups of mice. (c) The expression levels of PAD4 mRNA examined by real-time quantitative polymerase chain reaction in lung homogenates of mice. (d) Quantification of normalized PAD4 levels in lung protein extracts of mice. (e) The fluorescence intensity ratio of PAD4 to nucleus. (OVA: ovalbumin; LPS: lipopolysaccharide; DAPI: 4′,6′-diamidino-2-phenylindole; PAD4: peptidylarginine deiminase 4).

3.6. MBD2 promotes NETs formation by regulating PAD4 expression via JAK2 signal pathway in neutrophils

To confirm that MBD2 regulates PAD4 expression via JAK2 to promote NETs formation, we conducted in vitro experiments by culturing HL-60 cells stimulated to differentiate into neutrophil-like cells. Flow cytometry analysis of CD11b expression on HL-60 cells showed that 80.42% of HL-60 cells differentiated into neutrophil-like cells (Figure 7a) upon stimulation with 1.25% DMSO.

Figure 7.

Figure 7.

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The formation of neutrophil extracellular traps is significant reduced when methyl-CpG-binding domain 2 (MBD2), janus kinase 2 (JAK2), and peptidylarginine deiminase 4 (PAD4) was inhibited, respectively. (a) Flow cytometry analysis of CD11b expression on HL-60 cells after 5 days of culture with dimethyl sulfoxide (DMSO). (b) Western blotting detected MBD2, JAK2, and PAD4 proteins expression in the indicated groups of HL-60 differentiated neutrophil-like cells. (c) Quantification of normalized CitH3 levels in the HL-60-differentiated neutrophil-like cells. (d) Real-time quantitative polymerase chain reaction (RT-qPCR) detected JAK2 mRNA in the indicated groups of HL-60 differentiated neutrophil-like cells. (e) Quantification of normalized JAK2 protein levels in the HL-60-differentiated neutrophil-like cells. (f) RT-qPCR detected PAD4 mRNA in the indicated groups of HL-60 differentiated neutrophil-like cells. (g) Quantification of normalized PAD4 levels in the HL-60-differentiated neutrophil-like cells. (h) Representative immunofluorescence images of CitH3 and 4′,6′-diamidino-2-phenylindole (DAPI) staining of HL-60-differentiated neutrophil-like cells. The fluorescence intensity of CitH3 increased in HL-60 differentiation-derived neutrophil-like cells. KCC-07, Ruxolitinib, and Cl-amidine significantly reduced the fluorescence intensity of CitH3. (i) The average CitH3 fluorescence intensity per cell. (j) Representative immunofluorescence images of JAK2 and DAPI staining of HL-60-differentiated neutrophil-like cells. The fluorescence intensity of JAK2 increased in HL-60 differentiation-derived neutrophil-like cells. KCC-07 significantly reduced the fluorescence intensity of JAK2, while Ruxolitinib and Cl-amidine had no significant difference. (k) The average JAK2 fluorescence intensity per cell. (l) Representative immunofluorescence images of PAD4 and DAPI staining of HL-60-differentiated neutrophil-like cells. The fluorescence intensity of PAD4 increased in HL-60 differentiation-derived neutrophil-like cells. KCC-07 and Ruxolitinib significantly reduced the fluorescence intensity of PAD4, while Cl-amidine had no significant difference. (m) The average PAD4 fluorescence intensity per cell. (LPS: lipopolysaccharide; DAPI: 4′,6′-diamidino-2-phenylindole; CitH3: citrullinated histone; JAK2: janus kinase 2; PAD4: peptidylarginine deiminase 4).

CitH3 levels significantly increased (Figure 7b, c, h and i) in LPS-treated neutrophil-like cells, whereas CitH3 levels were significantly reduced (Figure 7b, c, h, and i) in LPS-treated neutrophil-like cells pretreated with KCC-07, Ruxolitinib, and Cl-amidine. In LPS-treated neutrophil-like cells pretreated with KCC-07, the levels of JAK2 and PAD4 (Figure 7d–g) were significantly reduced, as were the average staining intensities of JAK2 and PAD4 per cell (Figure 7j–m). In LPS-treated neutrophil-like cells pretreated with Ruxolitinib, PAD4 mRNA and protein levels were significantly reduced (Figure 7b, f, and g), as was a decrease in the average staining intensity of PAD4 per cell (Figure 7l and m).

4. Discussion

Asthma is a heterogeneous, chronic inflammatory disease of the airway. It can be classified into subtypes based on the degree of airway inflammation [29]. Neutrophilic asthma constitutes 10–15% of severe asthma cases and is characterized by late onset, non-atopy, and poor response to corticosteroids [30]. Limited treatment options and high medical costs are the greatest challenges for these patients. NETs have been reported to associate with the exacerbation of asthma [31,32]. In this study, we successfully established a mouse model of severe asthma that was sensitized with HDM, OVA, and LPS. Severe asthma mice had developed characteristics of conventional asthma, including higher airway resistance and mucus secretion levels, total cell counts and neutrophils counts in BALF, elevated concentration of INF-γ and IL-17. Furthermore, we successfully induced the differentiation of HL-60 cells into neutrophil-like cells in vitro by adding 1.25% DMSO to the medium. LPS-stimulated neutrophil-like cells were found to have higher levels of CitH3 compared to PBS-treated neutrophil-like cells, which is an important marker of NET formation [33].

MBD2 binds to the methylated regions of target gene promoters as an important epigenetic protein, inducing post-transcriptional histone modifications, altering chromatin structure, and ultimately regulating target genes expression. It is also widely expressed in various diseases. MBD2 significantly promoted the proliferation, migration, invasion, and circulation of renal carcinoma cells, contributing to a poor prognosis and tumor progression [34]. Furthermore, MBD2 was found to inhibit Erdr1 expression, promote fibroblast differentiation into myofibroblasts and exacerbate pulmonary fibrosis [35]. In this study, mice with severe asthma exhibited up-regulated MBD2 expression. Airway inflammation was alleviated, and NET formation was significantly reduced in severe asthmatic mice treated with KCC-07. Similarly, higher expression levels of CitH3 were observed in LPS-exposed neutrophil-like cell, but these levels were significantly reduced upon exposure to KCC-07. These findings suggested that MBD2 play a critical role in promoting NET formation in severe asthma.

JAK2, a member of the tyrosine kinase family, mediates cytokine production via the STAT signaling pathway [36]. The JAK2-STAT3 pathway drives CD4+ naive T lymphocytes to differentiate into Th17 cells by modulating the transcription factor receptor-related orphan receptor γt (RORγt). Conversely, the JAK2-STAT5 pathway facilitates Treg cell differentiation by regulating forkhead box protein P3 (FOXP3) [37]. The JAK2-STAT pathway has been implicated in exacerbating chronic myeloid leukemia and pulmonary reactive airway disease [26,38]. A recent study found that pan-JAK inhibitors interfere with the neutrophil activation process in severe asthma [39]. JAK2 may influence NET formation, as NETs are produced by activated neutrophils. In the present study, JAK2 was highly expressed in mice with severe asthma. NET formation and severe asthma symptoms were reduced when JAK2 was inhibited with Ruxolitinib. In vitro, LPS-stimulated neutrophil-like cells showed higher JAK2 expression, and Ruxolitinib significantly reduced NET formation. Furthermore, PAD4 expression was markedly down-regulated in both severe asthmaic mice and LPS-stimulated neutrophil-like cells exposed to Ruxolitinib. These results suggested that JAK2 regulates PAD4 expression and influences NET formation in case of severe asthma.

PAD4 was initially identified in differentiating human HL-60 leukemia cells, where it catalyzes the citrullination of histone arginine residues to form CitH3 [40,41]. Arneth et al. [42] reported that Cl-amidine-exposed mice exhibited reduced NET formation and milder vasculitis symptoms. Thiam et al. [22] observed that PAD4 knockout in mice with severe asthma resulted in reduced NETs levels, thereby mitigating the symptoms of severe asthma. In this study, PAD4 expression was up-regulated in mice with severe asthma. Furthermore, alleviation of asthma features and reduced NET formation were found in Cl-amidine-treated mice with severe asthma. Similarly, in vitro PAD4 was highly expression in LPS-stimulated neutrophil-like cells, while Cl-amidine down-regulated CitH3 expression. These results suggested that PAD4 regulates the formation of neutrophil-mediated NETs in severe asthma.

This study elucidated a novel pathogenic pathway for severe asthma (the MBD2-JAK2-PAD4 axis) that plays a positive role in advancing the exploration of targeted drug therapies for severe asthma that is resistant to conventional treatments. However, this study has some limitations to this study. Further investigation into the specific effects of HDM on severe asthma and measurement of granulocytes in mouse lung tissues are beneficial. The neutrophils used in this study were derived from differentiated HL-60 cells. This study was a preclinical investigation, and its clinical applicability requires further confirmation. Additionally, we found an imbalance of Th17/Treg cells in the lung tissue of mice with severe asthma after the inhibition of MBD2, JAK2, and PAD4 expression. Previous studies [43] have confirmed that MBD2 promotes demethylation of FOXP3, thereby inhibiting Treg cell function. It has been reported that the JAK2-STAT3/5 pathway is involved in the directed differentiation of CD4+ T lymphocytes [37]. This suggests that MBD2, JAK2, and PAD4 may play crucial roles in the Th17/Treg cell transdifferentiation or the initial differentiation of CD4+ T lymphocytes in patients with severe asthma. Notably, since PAD4, as a downstream component, does not influence the expression of MBD2 and JAK2 in this study, PAD4-induced Th17/Treg cell imbalance may involve a specific mechanism (Supplementary 2).

5. 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.

Supplementary Material

Supplementary Fig1.jpg
IANN_A_2458207_SM1084.jpg (2.9MB, jpg)
Supplementary Fig2.jpg
IANN_A_2458207_SM1083.jpg (1.5MB, jpg)

Acknowledgement

We would like to thank Editage (www.editage.cn) for English language editing.

Funding Statement

This work was supported by the National Natural Science Foundation of China (82300042), Hunan Province Science and Technology Plan Project (2021SK53401), Changsha Natural Science Foundation of China (kq2202047).

Ethical approval

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Changsha Central Hospital Affiliated to University of South China. This study has adhered to ARRIVE guidelines.

Author contributions

Conceptualization, DL and CYR; Data curation, BP and CYR; Formal analysis, BP and CYR; Investigation, DL; Methodology, BP, CYR and DL; Project administration, DL; Resources, CYR; Visualization, BP and CYR, Writing, original draft, BP; Writing, review and editing, FS, XXD, YMY and DL. All authors have read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data in this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Fig1.jpg
IANN_A_2458207_SM1084.jpg (2.9MB, jpg)
Supplementary Fig2.jpg
IANN_A_2458207_SM1083.jpg (1.5MB, jpg)

Data Availability Statement

The data in this study are available from the corresponding author upon reasonable request.

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