Desmoglein-2 dysregulation impairs epithelial barrier integrity in eosinophilic asthma

Abstract

Background

Eosinophilic asthma, affecting more than 50% of adult asthma patients, demonstrates airway inflammation severity directly correlated with eosinophil infiltration levels. While transepithelial eosinophil migration constitutes a pivotal step in airway inflammation pathogenesis, the regulatory mechanisms remain undefined. We aim to investigate how desmoglein-2 (DSG2), which is a key desmosomal cadherin mediating epithelial intercellular adhesion, regulates eosinophil transmigration across the bronchial epithelial barrier.

Methods

DSG2 protein levels were measured by ELISA in asthma patients’ and healthy volunteers’ serum. In vitro, a lower level of DSG2 in bronchial cells weakened barrier integrity, boosting eosinophil migration. To further investigate the role of DSG2 in eosinophilic asthma, in vivo mouse studies were conducted. These studies employed an adeno-associated virus to modulate DSG2 expression (either downregulation or upregulation) or utilized exogenous supplementation with recombinant DSG2 protein, followed by assessment of their effects on ovalbumin (OVA)/house dust mite (HDM)-induced eosinophilic asthma.

Results

DSG2 was primarily expressed in human bronchial epithelial cells and was decreased in asthma patients compared to healthy individuals. The serum levels of DSG2 protein were significantly lower in asthmatic patients compared to healthy subjects. A negative correlation was observed between serum DSG2 protein levels and the count of peripheral blood eosinophils in asthma patients. Furthermore, Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) downregulated the expression of DSG2 protein. In bronchial epithelial cells, DSG2 protein played a crucial role in maintaining epithelial barrier function and cell adhesion. A reduction in its expression led to enhanced transepithelial migration of eosinophils in vitro. Consistent with these findings, a murine model of eosinophilic asthma exhibited decreased DSG2 expression and increased eosinophil infiltration in the airways. Notably, this eosinophil infiltration was exacerbated by the downregulation of DSG2. Critically, in both in vivo and in vitro experiments, overexpression of DSG2 in bronchial epithelial cells or exogenous addition of recombinant DSG2 protein partially reversed asthma-associated epithelial barrier impairment and alleviated eosinophilic airway inflammation.

Conclusion

We elucidated the crucial role of DSG2 in bronchial epithelial cells during eosinophilic asthma development, focusing on its maintenance of desmosome structure and facilitation of eosinophil migration across the airway epithelium.

Graphical Abstract

During the development of asthma, type 2 inflammatory cytokines (such as IL-4 and IL-13) downregulate the expression of DSG2 protein in bronchial epithelial cells, leading to impaired desmosome structure and widened intercellular gaps. These changes subsequently cause epithelial barrier dysfunction and increased permeability, resulting in enhanced eosinophil infiltration into the airway lumen and increased allergen exposure beneath the epithelium. This condition further exacerbates the release of type 2 inflammatory cytokines and continuously decreases DSG2 expression in epithelial cells, forming a vicious cycle that aggravates eosinophilic airway inflammation in asthma.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-026-03510-y.

Introduction

Asthma is a common chronic airway disease worldwide, and affects around 300 million individuals globally [1]. Chronic airway inflammation is a central mechanism of asthma. Based on the types and proportions of inflammatory cells in induced sputum from asthma patients, asthma can be classified into four subtypes: eosinophilic asthma, neutrophilic asthma, mixed granulocytic asthma, and paucigranulocytic asthma. Among these, eosinophilic asthma is the most common phenotype, accounting for 50%-80% of cases [2, 3].

Notably, a series of studies have consistently demonstrated a significant correlation between the number of eosinophils infiltrating the airway lumen and the severity of asthma [4–6]. In asthma, the trafficking of eosinophils into the airways encompasses a comprehensive migratory pathway extending from the bone marrow through the peripheral blood, into lung tissue, and ultimately within the airway lumen. However, the precise mechanisms governing eosinophil migration in asthma remain elusive. Specifically, it is unclear how these cells traverse from lung tissue into bronchial epithelial interepithelial spaces, and subsequently migrate into the airway lumen. This intricate process ultimately drives the clinical manifestations of the disease.

Previous investigators have postulated that the shedding of bronchial epithelial cells during the asthmatic process serves as a primary mechanism for the penetration of allergens into the subepithelial layer and the transepithelial migration of inflammatory cells, such as eosinophils and neutrophils, into the airway lumen [7, 8]. Emerging evidence indicates structural preservation of bronchial epithelium in most eosinophilic asthma patients, though morphological alterations may persist; in contrast, epithelial shedding is primarily observed in subsets with severe asthma. Notably, epithelial shedding is not a prevalent feature in these patients [9, 10]. Thus, in general, the process of eosinophil transmigration into the airway lumen involves an intricate interaction with bronchial epithelial cells, constituting a transepithelial migration.

Tight junctions, adherens junctions, and desmosomes constitute the key intercellular adhesion complexes maintaining bronchial epithelial integrity [11–14]. Among these junctions, desmosomes are positioned as the deepest anchoring structures, forming the critical barrier eosinophils must penetrate during epithelial transmigration. Their hyper-adhesive nature renders desmosomes the most robust stabilizers of epithelial architecture [15]. Experimental evidence demonstrates that targeted disruption of desmosomal components through monoclonal antibodies or genetic knockout compromises both desmosome formation and the integrity of tight junctions and adherens junctions, confirming their central role in junctional complex stabilization [16, 17]. These findings collectively implicate desmosomal disruption as a key facilitator of eosinophil transepithelial migration in asthma pathogenesis.

Desmogleins (DSGs) and desmocollins (DSCs), belonging to the cadherin family, constitute the core adhesive components of desmosomes. The human DSG family comprises four isoforms (DSG1-4), while three DSC isoforms (DSC1-3) exist, distinguished by their distinct numbers of repeated unit domains [18]. In bronchial epithelium, DSG2 emerges as the predominant desmosomal cadherin [19]. Experimental evidence establishes the critical role of DSG2 in desmosomal integrity: DSG2-deficient mice exhibit myocardial membrane disruption, aberrant intercalated disc morphology, and ultrastructural desmosome loss [20–22]. Truncation of the DSG2 led to a visible change on the ultrastructural level and to a significant reduction in desmosomes per cell [23]. The adhesion function and transepithelial electrical resistance (TER) of intestinal epithelial cells were decreased when DSG2 was knocked out in vitro [17]. Therefore, we aimed to explore the expression of DSG2 in asthmatic bronchial epithelium and its role in maintaining epithelial barrier function in eosinophilic asthma.

Materials and methods

Detailed information can be found in the supplementary materials (Table S1-3 and Fig. S1).

Patients’ lung tissue, serum collection and transcriptomic profiling

Lung tissues were obtained from individuals who underwent surgery for lung cancer. Additionally, human serum samples were collected from asthma patients visiting the respiratory outpatient clinic at the First Affiliated Hospital of Nanjing Medical University, as well as from healthy volunteers within the community. The clinical and demographic characteristics of the patients are shown in Table S2 and S3.

To comprehensively explore gene expression patterns, we analyzed bronchial epithelial brushings obtained from participants in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) cohort [24]. After RNA extraction, the raw transcriptomic data underwent robust multi-array average (RMA) normalization to guarantee data consistency and comparability. mRNA expression levels were assessed using the Limma package. Additional details are provided in the data supplement.

Animal experiments

Eosinophilic asthma models were established using 6-8-week-old female BALB/c mice. For the HDM model, mice were sensitized intranasally on day 0, followed by daily challenges from days 7 to 11, and sacrificed on day 14, as previously described [25]. In the OVA model, mice received intraperitoneal sensitization on days 0, 7, and 14, followed by intranasal challenges on days 21 to 23, and were euthanized on day 24, following established protocols [26]. The DSG2 recombinant protein used in this study was rmDSG2 (absin, China). Bronchoalveolar lavage fluid (BALF) was collected for cellular analysis, and lung inflammation was assessed via hematoxylin and eosin (H&E)-stained paraffin sections using standardized histopathology scoring systems.

DSG2 knockdown or overexpression cohorts received 1 × 10¹¹ viral genomes (vg) of AAV6 adeno-associated virus (WZ Biosciences In, China) expressing short hairpin RNA (Recombinant, for konckdown) or AAV6 vectors encoding murine Dsg2 cDNA under the CMV promoter (for overexpression) via intratracheal instillation 21 days prior to sensitization. Detailed experimental procedures are provided in the supplementary methodology.

Measurement of tracheal epithelial permeability in mice

Tracheal epithelial permeability was assessed by intratracheal instillation of 50 µL Evans blue dye-albumin solution (4% albumin) for 20 min. After PBS lavage, tracheal segments were homogenized in formamide and incubated at 60 °C for 18 h. Following centrifugation (5000×g, 30 min), fluorescence intensity of 100 µL supernatant aliquots was quantified spectrophotometrically at 620 nm using serially diluted standards.

Enzyme-linked immunosorbent assay (ELISA)

Human serum DSG2 concentrations were measured using ELISA kits (RayBiotech, USA) with a detection range of 16-4000 pg/mL and a detection limit of 16 pg/mL. Mouse BALF and serum DSG2 levels were quantified with kits (FineTest, China), showing a detection range of 31.2–2000 pg/mL and a detection limit of 18.75 pg/mL.

Immunofluorescence and immunohistochemistry

After dewaxing and washing, paraffin sections of human and mouse lung tissues were fixed with 4% paraformaldehyde. Additional details are provided in the data supplement.

Quantitative real-time PCR and Western blotting

The mRNA and proteins were analyzed as described in the data supplement.

Cell transfection for gene knockdown and overexpression

Cells were transfected with lentiviruses encoding for a control shRNA or human DSG2-shRNA (GenePharma, China), and also with lentiviruses carrying human DSG2-overexpressing or empty-vector control plasmids for overexpression studies (Genechem, China). The lentiviruses were diluted in enhanced infection solution (10 mg/mL) containing polybrene (5ug/mL) and added to the cells at 37 °C for 24 h and freshly prepared complete medium. Additional details are provided in the data supplement.

Measurement of transepithelial electrical resistance (TER) and epithelial permeability

The TER of the 16HBE cells was measured daily with a Millicell-ERS system (Millipore Co., USA). Paracellular flux across the epithelium was measured with fluorescein isothiocyanate (FITC)-labeled 4-kD dextran [27]. Additional details are provided in the data supplement.

Cell treatment

Cells were treated for 24 h with recombinant human cytokines (PeproTech, USA) at escalating concentrations: IL-4 (0, 3, 30, and 100 ng/mL), IL-13 (0, 3, 30, and 100 ng/mL). In some cases, bronchial epithelial cells were preincubated with either the Janus kinase 2/3 (JAK2/3) inhibitor AT9283 (30 nM) or the signal transducer and activator of transcription 6 (STAT6) phosphorylation inhibitor AS1517499 (30 nM) for 1 h. After preincubation, these cells, along with non - preincubated control cells, were stimulated with either IL-4 or IL-13 at a concentration of 30 ng/mL.

In experimental setups that necessitate stimulation by inflammatory factors, when recombinant human DSG2 protein (rhDSG2, sourced from CUSABIO, China) was included, the cells underwent a 24-hour co-treatment with both 30 ng/mL of IL-4/IL-13 and 30 ng/mL of rhDSG2. For DSG2 knockdown cells, confirm knockdown, group, and stimulate with 30 ng/mL IL-4/IL-13 for 24 h. For DSG2 overexpression cells, confirm overexpression, group DSG2-OE and Ctrl-OE cells, and stimulate similarly. After 24 h, TER and FITC-dextran permeability were measured per protocols.

Eosinophil extraction and migration

Human eosinophils were isolated from EDTA-anticoagulated venous blood and then were isolated by negative selection with magnetic beads (eosinophil isolation kit, Miltenyi Biotec, Germany) according to the manufacturer’s protocol. Sample purity was analyzed via flow cytometry following co-staining with CD16 and CCR3 antibodies (Biolegend, USA). Eosinophil populations were defined as CD16-negative/CCR3-positive (Fig. S2). Eosinophils were added into each upper compartment of transwells at a 5 × 10⁵ eosinophils in 200 µL of RPMI 1640 medium supplemented with 10% FBS. Each lower chamber was added with the same medium 500 µL containing 20% FBS and 50 ng/mL recombinant IL-5 (PeproTech, USA). The cells were incubated at 37℃ and 5% CO₂ for 2 h. At the end of incubation, the medium in the lower chamber was collected and assessed for the number of eosinophils present [27].

Statistical analysis

All data are presented as mean (± SEM), and P less than 0.05 was considered significant. The statistical analyses were performed in GraphPad Prism software v9.0 (Graphpad software, USA). For two-group comparisons, normally distributed parameters were analyzed with either a standard independent samples t-test (assuming equal variances) or Welch’s corrected t-test (for unequal variances), while nonnormally distributed parameters were assessed using the Mann-Whitney U test. Experiments with multiple comparisons were evaluated with one-way ANOVA followed by Tukey’s post hoc test or Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test (for normally distributed parameters) and the Kruskal-Wallis test (for non-normally distributed parameters) for multiple data sets.

Results

DSG2 expression is decreased in airway epithelium and serum of asthmatic patients and negatively correlates with the blood eosinophils

Transcriptomic analysis using the Human Protein Atlas database identified DSG2 as the predominant desmosomal cadherin in human lung tissue (Fig. 1A) [19]. Immunohistochemical staining revealed significantly reduced DSG2 protein expression in bronchial epithelial cells of asthma patients compared to healthy controls (Fig. 1B-C). ELISA confirmed decreased serum DSG2 levels in asthmatic patients (Fig. 1D). Asthmatics with high peripheral blood eosinophil counts (> 300/µL) showed lower serum DSG2 than those with low counts (Fig. 1E), and serum DSG2 levels negatively correlated with eosinophil counts (Fig. 1F).

Fig. 1.

The expression levels of DSG2 protein in bronchial epithelia and serum of asthmatic patients are decreased. A RNA expression analysis of desmosomal cadherin family members in human lung tissue using normalized transcripts per million (nTPM) values from the Human Protein Atlas database revealed DSG2 as the predominant isoform. Data were presented as mean ± SEM using Kruskal-Wallis test. **P < 0.01 and ****P < 0.0001. B Representative images showing reduced DSG2 protein immunostaining (brown color) in patients with asthma (n = 3) compared with that seen in healthy subjects (n = 4). The red arrow represents bronchial epithelial cells. Scale bar: 50 μm. C DSG2 protein relative expression was analyzed and presented graphically. Data were presented as mean ± SEM using Unpaired test. **P < 0.01. D-E Much more decreased serum DSG2 protein levels in asthmatic patients (n = 42) than in healthy controls (n = 24), especially in patients with high blood eosinophils (blood eosinophil>300 cells/µL). F Negative correlation between serum DSG2 levels and peripheral blood eosinophil counts in asthmatic patients. The data were analyzed using Mann-Whitney test (D, E) and Nonparametric Spearman correlation (F). ***P < 0.001 and ****P < 0.0001. G The expression of DSG2 mRNA in U-BIOPRED bronchial brushings array data was shown after log2 transformation and normalization. H DSG2 protein expression was decreased in the serum of nonsmoking severe asthma patients (SA.ns) compared to healthy volunteers (HV) and mild/moderate nonsmoking asthma patients (MMA) in the U-BIOPRED data using somaSCAN platform. I Serum DSG2 protein expression was decreased in patients with TAC1 asthma subtype compared to other asthma subtypes in the U-BIOPRED data. *P < 0.05 and ****P < 0.0001. EOS, Eosinophil; HV, healthy volunteers; SA.ns: severe asthma nonsmokers; MMA: mild/moderate nonsmoking asthma; TAC: Transcriptome-associated cluster

Furthermore, we identified significantly lower DSG2 mRNA levels in bronchial epithelial cells of nonsmoking severe asthma patients compared to healthy volunteers in U-BIOPRED cohort (Fig. 1G, P = 0.039). Serum proteomics (somaSCAN) confirmed decreased DSG2 protein in severe asthma patients (Fig. 1H). Among sputum transcriptomics-defined clusters (TACs), TAC1 patients (enriched for IL-13/Th2 signatures) showed the lowest serum DSG2 levels (Fig. 1I).

In asthmatic mice, bronchial epithelia exhibit disrupted desmosomes and DSG2 expression

We established eosinophilic asthma mouse models using OVA and HDM extracts, respectively (Fig. 2A, H and Fig. S3). Electron microscopic examination of mouse lung tissues revealed that, compared with healthy control mice, eosinophilic asthma mice exhibited damaged desmosomal structures in bronchial epithelia, with widened desmosomal gaps (Fig. 2B-C and I-J). Immunohistochemistry showed reduced DSG2 expression in asthmatic bronchial epithelium (Fig. 2D-E and K-L). ELISA confirmed reduced levels of DSG2 in serum and BALF of asthmatic mice (Fig. 2F-G and M-N). Regarding other desmosomal or junctional proteins, our immunohistochemical analysis confirmed negligible expression of desmoglein-3 (DSG3) and desmocollin-2 (DSC2) in both human and murine lung tissues (Fig. S4). Furthermore, expression levels of desmoglein-1 (DSG1), desmoglein-4 (DSG4), and γ-Catenin in murine bronchial epithelium showed no significant differences between control and asthmatic groups (Fig. S5).

Fig. 2.

In asthmatic mice, the desmosome structure of bronchial epithelia is disrupted, and the expression level of DSG2 protein is decreased. A Schematic of ovalbumin (OVA)-induced murine eosinophilic asthma model establishment. B Transmission electron micrographs comparing desmosomal ultrastructure: intact junctions in controls vs. fragmented complexes (red arrow indications) in OVA-induced asthma mouse models. Scale bar: 500 nm. C Intercellular distances at desmosome levels were determined by electron microscopy (OVA-Control, n = 5; OVA, n = 5). D Immunohistochemical analysis of DSG2 protein (brown color) expression in PBS-exposed control mice (OVA-Control, n = 7), OVA-treated asthma mice (OVA, n = 8). The red arrow represents bronchial epithelial cells. Scale bar: 50 μm. E Comparative DSG2 protein expression analysis with graphical representation. F Serum levels of DSG2 protein were significantly decreased in the OVA group (n = 8) compared with the control group (n = 7). G BALF DSG2 protein levels in OVA group (n = 8) were decreased compared with control group (n = 7). H Protocol schematic for HDM-induced eosinophilic asthma modeling. I Representative electron micrographs showing fragmented desmosomal junctions (red arrows highlight desmosomes) in bronchial epithelial cells from controls and HDM-induced asthma mouse models. Scale bar: 500 nm. J Desmosome-level intercellular spacing measurements (HDM-Control, n = 5; HDM, n = 4). K Immunohistochemical analysis of DSG2 protein (brown color) expression in PBS-exposed control mice (n = 8), HDM-induced asthma mice (n = 7). The red arrow represents bronchial epithelial cells. Scale bar: 50 μm. L DSG2 protein relative expression was analyzed and presented graphically. M Serum DSG2 protein levels in HDM group (n = 7) were decreased compared with control group (n = 8). N BALF levels of DSG2 protein were significantly decreased in the HDM group (n = 7) compared with the control group (n = 8). Data were presented as mean ± SEM using either the standard independent samples t-test (C, F, G, J, L, N) or Welch’s corrected t-test (E, M). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

IL-4 and IL-13 reduce DSG2 expression in bronchial epithelial cells

Stimulation of bronchial epithelial cells with the type 2 inflammatory cytokines IL-4 (Fig. 3A-B) and IL-13 (Fig. 3C-D) for 24 h resulted in a reduction of DSG2 protein expression. The JAK2/3 inhibitor AT9283 effectively attenuated the downregulation of DSG2 induced by both IL-4 (Fig. 3E-F) and IL-13 (Fig. 3G-H). Furthermore, the exogenous addition of the STAT6 phosphorylation inhibitor AS1517499 also partially reversed the suppression of DSG2 expression mediated by IL-4 (Fig. 3I-J) and IL-13 (Fig. 3K-L).

Fig. 3.

Regulatory mechanism of DSG2 expression in bronchial epithelial cells. A-B The effect of varying concentrations (0-100 ng/mL) of IL-4 on DSG2 protein expression in 16HBE cells was evaluated after 24 h of treatment using Western blotting and semiquantitative analysis (n = 3 per group). C-D Similarly, the impact of IL-13 (0-100 ng/mL) on DSG2 protein expression in 16HBE cells was assessed after 24 h using the same methods (n = 3 per group). E-F 16HBE cells were pretreated with the JAK2/3 inhibitor AT9283 (30 nM) for 1 h prior to IL-4 (30 ng/mL) stimulation. DSG2 expression was measured after 24 h using Western blotting and semiquantitative analysis. G-H 16HBE cells were pretreated with AT9283 (30 nM) for 1 h before IL-13 (30 ng/mL) stimulation, and DSG2 protein levels were evaluated after 24 h (n = 4 per group). I-J To explore the role of STAT6 signaling in IL-4-induced DSG2 expression, 16HBE cells were pretreated with the STAT6 inhibitor AS1517499 (30 nM) for 1 h prior to IL-4 (30 ng/mL) stimulation. After 24 h of stimulation, DSG2 expression levels were assessed by Western blotting and semiquantitative analysis (n = 4 per group). K-L 16HBE cells were pretreated with STAT6 inhibitor AS1517499 (30 nM) for 1 h before IL-13 (30 ng/mL) stimulation. DSG2 expression were assessed by Western blotting and semiquantitative analysis (n = 4 per group). Data were presented as mean ± SEM using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

DSG2 protein preserves the epithelial barrier integrity in bronchial epithelial cells

Electron microscopic examination revealed significant disruption of the desmosomal structures in the airway epithelia of mice in the DSG2 knockdown group (Fig. S6A-C, Fig. 4A). Lentiviral vectors were used to knock down or overexpress DSG2 in bronchial epithelial cells in vitro, confirmed by qPCR/WB (Fig. S6D-I). Light microscopy revealed widened intercellular spaces in bronchial epithelium (Fig. 4B), indicating impaired barrier integrity. DSG2 knockdown exacerbated this effect, significantly reducing transepithelial resistance (TER) and increasing permeability (Fig. 4C, D). Conversely, under IL-4 or IL-13 stimulation, DSG2 overexpression (DSG2-OE) enhanced barrier function compared to control (Ctrl-OE), elevating TER and reducing permeability (Fig. 4E-H). Similarly, exogenous recombinant human DSG2 protein restored barrier integrity in cytokine-stimulated cells, increasing TER and decreasing permeability versus controls (Fig. 4I-L). Furthermore, DSG2 modulated eosinophil transepithelial migration: knockdown (DSG2-shR) enhanced transmigration versus control (Ctrl-shR), while overexpression (DSG2-OE) suppressed it relative to Ctrl-OE (Fig. 4N-Q).

Fig. 4.

DSG2 regulates airway epithelial barrier integrity and eosinophil transmigration under Th2 inflammation. A Intratracheal instillation of AAV for observation of changes in desmosome structure under electron microscope (n = 3 per group, red arrows indicate desmosomes). B DSG2-deficient bronchial epithelia (DSG2-shR) displayed expanded intercellular junctions relative to control (Ctrl-shR) counterparts in 16HBE cells (n = 4 per group, red arrows indicate intercellular spaces). C DSG2-shR epithelia showed significantly reduced transepithelial resistance (TER) versus Ctrl-shR (n = 6 per group). D FITC-dextran permeability was markedly elevated in DSG2-shR epithelia compared to Ctrl-shR (n = 6 per group). E-F TER values and FITC-dextran permeability measurements in the IL-4-stimulated control group (Ctrl-OE) and DSG2-overexpressing group (DSG2-OE), with n = 4 per group. G-H Assessment of TER and FITC-dextran permeability in the IL-13-stimulated Ctrl-OE and DSG2-OE groups (n = 4 per group). I-J Compared with the group of normal bronchial epithelial cells stimulated by IL-4, the group with exogenous addition of recombinant human DSG2 protein (rhDSG2) exhibited an increase in TER and a decrease in FITC-dextran permeability (n = 5 per group). K-L In comparison with the group of normal bronchial epithelial cells stimulated by IL-13, the group supplemented with exogenous rhDSG2 showed an elevation in TER and a reduction in FITC-dextran permeability (n = 5 per group). M Schematic illustration of eosinophil transmigration across bronchial epithelium. N-O Eosinophil transmigration through DSG2-shR epithelia demonstrated significant enhancement relative to Ctrl-shR (n = 4 per group). P-Q Eosinophil transmigration across DSG2-overexpressing epithelial cells was significantly reduced compared to the control group (Ctrl-OE) (n = 4 per group). The data were analyzed using either the standard independent samples t-test or Welch’s corrected t-test. *P < 0.5, **P < 0.01, ***P < 0.001 and ****P < 0.0001

DSG2 deficiency exacerbates eosinophilic asthma pathology

Compared to non-knockdown controls, bronchial epithelial DSG2 knockdown significantly exacerbated asthma pathology in both OVA- and HDM-induced eosinophilic models (Fig. 5A, H: model schematics). In OVA-induced mice, DSG2 deficiency amplified peribronchial inflammatory cell infiltration (Fig. 5B-C), elevated total inflammatory cells (Fig. 5D) and eosinophil counts (Fig. 5E) in BALF, heightened airway hyperresponsiveness to acetylcholine (0–50 mg/mL, Fig. 5F), and increased bronchial epithelial permeability (Fig. 5G). Strikingly, these pathological intensifications were fully recapitulated in the HDM model: DSG2 knockdown mice exhibited augmented inflammatory cell accumulation (Fig. 5I-J), enhanced eosinophil recruitment (Fig. 5K-L), exaggerated airway reactivity (Fig. 5M), and compromised epithelial barrier integrity (Fig. 5N).

Fig. 5.

Epithelial DSG2 deficiency amplifies eosinophilic inflammation and barrier dysfunction in asthma models. A Schematic of OVA-induced eosinophilic asthma model with intratracheal AAV6 delivery. B-C H&E-stained lung sections showing peribronchial inflammatory infiltrates in adeno-associated virus treated control group (AAV-Ctrl-PBS), adeno-associated virus treated OVA group (AAV-Ctrl-OVA), DSG2 downexpression control group (AAV-DSG2-PBS) and DSG2 downexpression OVA group (AAV-DSG2-OVA) (n = 5 per group). Scale bars: 50 μm. D-E Total cell and eosinophil counts in BALF (n = 5 per group). F The resistance of the central airways (Rn) following 0-50 mg/mL acetylcholine challenge, n = 5–7 per group. The data were analyzed using Unpaired test or Welch’s test. *P < 0.05 (AAV-Ctrl-OVA vs. AAV-DSG2-OVA), # P < 0.05 (AAV-Ctrl-PBS vs. AAV-Ctrl-OVA), ✝ P < 0.05 (AAV-DSG2-PBS vs. AAV-DSG2-OVA). G Quantification of bronchial epithelial barrier integrity via Evans blue albumin extravasation, n = 3–5 per group. H Schematic diagram of establishing HDM-induced eosinophilic asthma model following intratracheal viral instillation in mice. I-J H&E staining evaluated inflammatory cell infiltration in pulmonary tracheal tissues in AAV-Ctrl-PBS, AAV-Ctrl-HDM, AAV-DSG2-PBS and AAV-DSG2-HDM group, n = 5–6 per group. K-L The total cell and eosinophil counts in BALF (n = 5–6 per group). M Airway responsiveness of mice assessed with 0–50 mg/mL acetylcholine (n = 5–6 per group). *P < 0.05 and **P < 0.01 (AAV-Ctrl-PBS vs. AAV-DSG2-PBS), # P < 0.05 (AAV-Ctrl-PBS vs. AAV-Ctrl-HDM), ✝ P < 0.05 and ✝✝ P < 0.01 (AAV-DSG2-PBS vs. AAV-DSG2-HDM). N Assessment of airway epithelial permeability by Evans Blue dye extravasation, n = 3–5 per group. Data were presented as mean ± SEM using one-way ANOVA followed by Tukey’s post hoc test or Brown-Forsythe test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (C, D, E, G, J, K, L, N)

DSG2 overexpression or exogenous replenishment attenuates eosinophilic airway pathology in asthma models

Airway instillation of adeno-associated virus AAV6 for DSG2 overexpression (Fig. S6J-L, Fig. 6A) significantly attenuated pulmonary tissue inflammatory infiltration (Fig. 6B-C), reduced the total cell counts and eosinophil counts in BALF (Fig. 6D-G), and notably decreased bronchial epithelial permeability in OVA- or HDM-induced asthmatic mice (Fig. 6H-I). Exogenous addition of recombinant mouse DSG2 protein during the allergen challenge phase (Fig. 6J) also alleviated pulmonary tissue inflammatory infiltration (Fig. 6K-L), decreased the total cell counts and eosinophil counts in BALF (Fig. 6M-P) in OVA- or HDM-induced asthmatic mice. Additionally, it led to a certain degree of reduction in bronchial epithelial permeability associated with asthma (Fig. 6Q-R).

Fig. 6.

Bronchial epithelial DSG2 overexpression or recombinant DSG2 protein supplementation attenuates eosinophilic airway inflammation and barrier dysfunction in asthma. (A) Schematic of OVA-or HDM-induced eosinophilic asthma model with intratracheal AAV6 delivery. B-C H&E-stained lung sections showing peribronchial inflammatory infiltrates in OVA group treated with control AAV (AAV-Ctrl-OVA), OVA group with over-expression adeno-associated virus treatment (AAV-OE-OVA), HDM group with control adeno-associated virus treatment (AAV-Ctrl-HDM), HDM group with over-expression adeno-associated virus treatment (AAV-OE-HDM) (n=7-8 per group). Scale bars: 50 μm. D-G The total cell and eosinophil counts in BALF (n=7-8 per group). H-I Quantification of bronchial epithelial barrier integrity via Evans blue albumin extravasation, n=5 per group. (J) Schematic representation of the eosinophilic asthma model induced by OVA or HDM, with or without the incorporation of recombinant mouse DSG2 protein. K-L H&E-stained lung sections showing peribronchial inflammatory infiltrates in OVA group, OVA with recombinant mouse DSG2 protein (OVA+rmDSG2), HDM group and HDM+rmDSG2 group (n=7-8 per group). Scale bars: 50 μm. M-P The total cell and eosinophil counts in BALF (n=6-7 per group). Q-R Quantification of bronchial epithelial barrier integrity via Evans blue albumin extravasation, n=5 per group. The data were analyzed using either the standard independent samples t-test or Welch’s corrected t-test. *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001

Discussion

While substantial progress has been made in understanding eosinophil trafficking from bone marrow to pulmonary compartments during eosinophilic asthma progression, the precise mechanism governing tissue-resident eosinophil recruitment to peribronchial regions and subsequent epithelial transmigration remains undefined. Three intercellular junctions-tight junctions, adherens junctions, and desmosomes-collectively maintain bronchial epithelial integrity and cytoarchitecture [28]. Asthma-related factors damage airway epithelial cells, leading to the disruption of intercellular connections and barrier dysfunction, which plays a significant role in the transepithelial migration of eosinophils.

Desmosomes, strategically positioned at the basolateral junctional interface, constitute the primary structural defense against eosinophil transmigration [29]. Their distinct hyperadhesive properties endow these complexes with exceptional mechanical resilience, making them critical guardians of intercellular cohesion and barrier stability. Our clinical data revealed reduced expression of desmosomal cadherin DSG2 in asthmatic bronchial epithelium and serum, with more pronounced reduction in patients exhibiting elevated peripheral blood eosinophil counts. In eosinophilic asthma mouse models induced by OVA and HDM, respectively, the levels of DSG2 protein in bronchial epithelial cells, BALF and serum of asthmatic mice were significantly lower compared to the control group. Importantly, we found that other desmosomal components showed distinct expression patterns: DSG3 and DSC2 were negligibly expressed in human/murine lung tissues, while DSG1, DSG4, and γ-Catenin remained unchanged in asthmatic murine bronchial epithelium. Therefore, we hypothesize that the expression of epithelial DSG2 protein in asthma is closely associated with the development of the eosinophilic asthma phenotype.

While prior evidence indicated DSG2 suppression by IL-1β and TNF-α exclusively in intestinal epithelium [30], the regulatory mechanisms governing DSG2 expression in bronchial epithelium remained undefined. We now demonstrate, for the first time, that type 2 cytokines directly downregulate DSG2 in bronchial epithelial cells. In vitro, IL-4 and IL-13 significantly reduced DSG2 expression—an effect partially attenuated by JAK2/3 or STAT6 inhibitors, indicating involvement of the JAK/STAT6 signaling pathway. Critically, this mechanism is reinforced by clinical evidence among asthma patients stratified by sputum TACsb, the TAC1 subgroup characterized by dominant IL-13/Th2/ILC2 signatures exhibited the most pronounced reductions in serum DSG2 levels. Collectively, these findings establish IL-4/IL-13-mediated JAK/STAT6 signaling as a novel regulatory axis suppressing DSG2 in asthma, thereby compromising epithelial barrier function.

Bronchial epithelial cells are located at the interface between the external environment and the internal body, forming a tightly arranged bronchial epithelial barrier [31]. Although DSG2 represents the predominant desmosomal cadherin in pulmonary epithelia, its functional contribution to barrier maintenance remains incompletely characterized. Through integrative in vitro and in vivo experimental validation, we definitively establish the essential role of DSG2 in preserving bronchial epithelial structural and functional integrity.

In further in vitro and in vivo experiments, we knocked down DSG2 protein in bronchial epithelial cells, resulting in a significant increase in eosinophil transepithelial migration, accompanied by desmosome damage. Based on these findings, we speculate that abnormal desmosome function in asthmatic epithelial cells is a primary factor driving eosinophils to cross the airway epithelium and contribute to the eosinophilic inflammatory phenotype.

Expanding on these insights, we conducted additional investigations. In vitro studies revealed that restoring DSG2, whether through the use of recombinant protein or overexpression techniques, could reverse the barrier dysfunction induced by IL-4/IL-13 in human airway epithelium. Moreover, this restoration also effectively suppressed eosinophil migration across the epithelium. Moving to in vivo experiments using murine asthma models, we found that overexpressing DSG2 intratracheally or administering recombinant DSG2 intranasally during antigen challenge led to a significant reduction in airway inflammation, eosinophil infiltration, and barrier leakage.

These comprehensive findings collectively unveil a self-reinforcing pathogenic cycle. Initially, type 2 cytokines downregulate DSG2, which subsequently causes barrier disruption. This compromised barrier then allows allergens to penetrate more easily and facilitates the influx of eosinophils. As a result, inflammation is amplified, which in turn leads to further loss of DSG2. Therefore, it becomes evident that DSG2 deficiency is not merely a consequence of epithelial barrier failure and inflammatory progression. Instead, it acts as a causal initiator, setting off this vicious cycle that underlies the pathogenesis of asthma.

Previous studies demonstrate that DSG2-mediated intercellular adhesion depends on extracellular domain crosslinking, with exogenous DSG2 administration shown to rescue desmosomal adhesion defects [32]. Recently, proteomic sequencing conducted by scholars on primary bronchial epithelial cells from healthy individuals cultured at the air-liquid interface revealed that DSG2 is abundantly expressed and secreted by basal cells, suggesting its crucial role in maintaining epithelial cell homeostasis [33]. In our study, bronchial epithelial cells were cultured in vitro and subjected to DSG2 knockdown. Subsequently, the epithelial cell gaps widened, cells became deformed, and cell adhesion decreased. Taken together, DSG2, which is a component of desmosomes, plays a pivotal role in stabilizing intercellular adhesion among bronchial epithelial cells and maintaining the structural integrity of the bronchial epithelium, potentially participating in the pathogenesis and progression of asthma.

This study has several limitations, a prominent one being that the experiments conducted in vitro utilized 16HBE cells in submerged culture, rather than primary human bronchial epithelial cells grown at the air-liquid interface. However, 16HBE is a cell line extensively used in research focused on bronchial epithelial cell junctions [27, 34, 35]. We applied gene editing techniques to construct DSG2 knockdown and overexpression models in 16HBE cells. In the DSG2 knockdown model, we clearly observed a significant decline in epithelial barrier integrity. Conversely, when DSG2 was overexpressed, the epithelial barrier function was notably improved. We also employed knockdown and overexpression techniques, along with exogenous recombinant proteins, in animal models. Through these diverse approaches, we were able to draw clear and definitive conclusions about the role of DSG2. Furthermore, research has established DSG2 as a predictive biomarker for lung cancer, with its expression in tissue being upregulated by the disease [36]. In our study, lung tissues were obtained from locations situated more than 5 centimeters away from the tumor border.

Conclusions

This study elucidates the pivotal role of DSG2 in bronchial epithelial cells during the formation of eosinophilic asthma, specifically its role in maintaining desmosome structure and facilitating the migration of eosinophils across the airway epithelium into the airway lumen. This study establishes desmosome dysfunction as a critical driver in the pathogenesis of eosinophilic airway inflammation in asthma, providing mechanistic insights that inform targeted diagnostic strategies and therapeutic development for this asthma endotype.

Abbreviations

AAV

Adeno-associated virus

DSG

Desmoglein

DSC

Desmocollin

HDM

House dust mite

IL

Interleukin

JAK

Janus kinase

OVA

Ovalbumin

TAC

Transcriptome-associated cluster

TER

Transepithelial electrical resistance

STAT

Signal transducer and activator of transcription

Authors’ contributions

Lei Yao, Xin Yao, Man Jia and Ian M Adcock designed this study. Lei Yao, Xijing Yuan, and Peng Sun performed the animal experiments. Lei Yao performed the ex vivo experiments. Lei Yao and Heng fu recruited the patient. Lei Yao and Xijing Yuan performed the analysis of data and interpretation. Nazanin Zounemat Kermani analyzed the data from the U-BIOPRED cohort. Lei Yao wrote the manuscript. Yi Liu assisted in collecting lung tissue specimens. Xin Yao and Ian M Adcock critically revised the manuscript. All the authors read and approved the final paper.

Funding

This work was supported by the National Natural Science Foundation of China (grants 82400027, 82200035, 82170040 and 82370035), the Jiangsu Funding Program for Excellent Postdoctoral Talent, the Natural Science Foundation of Jiangsu Province (grant number BK20210957) and the China Postdoctoral Science Foundation (grant number 2025M772061).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Human subject protocols received approval from the Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University (2019-SR-371&2024-SR-811). Written informed consent was systematically obtained from all participating patients prior to enrollment. Animal experiments were conducted in strict compliance with institutional guidelines under approval from the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-2410025), with all procedures designed to minimize animal distress.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.