Targeting CD30L Alleviates Airway Remodeling via JNK/p38 MAPK Pathway in OVA-Induced Asthmatic Mice

Abstract

Purpose

Airway remodeling is a key pathological feature of asthma. The CD30 ligand (CD30L), a member of the tumor necrosis factor (TNF) superfamily encoded by the Tnfsf8 gene, has been linked to immune-inflammatory pathologies. Nevertheless, the role of CD30L in airway remodeling has not been elucidated.

Methods

We investigated CD30L expression and localization in lung specimens from asthma patients and ovalbumin (OVA)-induced asthmatic mice. Subsequently, we established asthmatic mice with macrophage-specific Tnfsf8 knockout or intranasal administration of recombinant CD30L protein, and analyzed airway pathology using multiple techniques. In vitro, an indirect co-culture system of macrophages and bronchial epithelial cells was employed to investigate the impact of CD30L on airway epithelial remodeling. The mechanisms of CD30L in human bronchial epithelial (HBE) cells were explored using small interfering RNA targeting CD30 (the receptor for CD30L), c-Jun N-terminal kinase (JNK) inhibitor, and p38 inhibitor.

Results

CD30L expression was upregulated in asthmatic lung tissues (human/mice) and colocalized with macrophage markers. In vivo, macrophage-specific Tnfsf8 knockout attenuated extracellular matrix (ECM) deposition and epithelial-mesenchymal transition (EMT) during OVA-induced airway remodeling, whereas intranasal CD30L exacerbated these pathologies. Transcriptomic analysis of lung tissues revealed that CD30L regulated ECM deposition, cell adhesion, and epithelial cell migration. In vitro co-culture of macrophages with bronchial epithelial cells demonstrated that macrophage-specific CD30L silencing reversed remodeling-related proteins and EMT in bronchial epithelial cells. Furthermore, we found that CD30L significantly upregulated the JNK/p38 mitogen-activated protein kinase (MAPK) pathway in HBE cells. Silencing CD30 in HBE cells alleviated CD30L-induced remodeling and EMT, accompanied by downregulation of the JNK/p38 MAPK pathway. Treatment with JNK inhibitor (SP600125) or p38 inhibitor (SB203580) reversed the CD30L-induced pathological effects.

Conclusions

Collectively, these findings demonstrate that CD30L critically regulates asthma airway remodeling via the JNK/p38 MAPK pathway, strongly suggesting its therapeutic potential as a target for airway remodeling in asthma.

INTRODUCTION

Asthma, a chronic respiratory disease affecting approximately 300 million people globally, is characterized by reversible airway obstruction, persistent airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling.¹,² With the refinement of clinical guidelines, asthma management has become increasingly standardized. However, a subset of patients, particularly those with severe asthma, still exhibit suboptimal therapeutic responses despite high-dose medications and bronchodilator therapy.³ This highlights the urgent need to explore novel therapeutic strategies.

Airway remodeling, which is characterized by extracellular matrix (ECM) deposition, subepithelial fibrosis, and airway smooth muscle (ASM) hyperplasia, is nearly universal and strongly associated with increased exacerbation risks, accelerated lung function decline, and disease chronicity.⁴ Some patients with severe asthma, particularly those less responsive to high-dose inhaled corticosteroids (ICS) combined with long-acting β2-agonists (LABA), demonstrate marked airway remodeling.⁵,⁶ This pathological restructuring is associated with therapeutic resistance due to irreversible airflow obstruction.⁶,⁷ Indeed, excessive extracellular matrix deposition in small airways has been identified as a hallmark of fatal asthma.⁵ Hence, early detection combined with targeted interventions against airway remodeling may reduce acute asthma attacks and prevent irreversible structural damage. A significant goal in developing novel asthma therapies is the identification of airway remodeling biomarkers, which is essential for facilitating precise asthma phenotyping and enabling personalized treatment strategies.⁸

Macrophages, the most abundant immune cells in lung tissue, play a crucial role in airway remodeling.⁹,¹⁰ Specifically, macrophages interact with airway structural cells to promote asthma pathology and functional changes. For instance, activated macrophages are well-known producers of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, ¹¹ which can increase ASM proliferation and contractility.¹²,¹³,¹⁴ Interactions between macrophages and epithelial cells promote mucus hypersecretion and epithelial hyperplasia, thereby exacerbating airway obstruction.¹⁵,¹⁶ This highlights the crucial role of intercellular crosstalk and their secreted cytokines in asthmatic airway remodeling.

The CD30 ligand (CD30L), a member of the TNF superfamily encoded by the Tnfsf8 gene, is predominantly expressed on monocytes/macrophages and activated T cells.¹⁷,¹⁸ Previous studies have demonstrated the proinflammatory role of CD30L. In mouse models of allergic rhinitis, CD30L knockout significantly alleviated allergic inflammation.¹⁹ Similarly, in colitis models, CD30L was shown to exacerbate inflammatory responses.¹⁷ At present, some studies have shown that CD30L is closely related to the progression of asthma, and blocking the interaction between CD30L and its receptor suppresses eosinophilic inflammation.¹⁹,²⁰ While persistent airway inflammation is recognized as a key driver of asthmatic airway remodeling, the role of CD30L in this pathogenic process remains elusive. In the present study, we aim to explore the functional role of CD30L in asthmatic airway remodeling and elucidate its underlying molecular mechanisms, thereby providing meaningful insights for the development of precision therapeutic approaches in asthma management.

MATERIALS AND METHODS

Human samples

Serum samples were obtained from the respiratory clinic, with asthma diagnosis and severity stratification confirmed according to the Global Initiative for Asthma (GINA) guidelines (updated in 2024). Healthy control serum samples were collected from individuals undergoing routine health check-ups at the same clinic, with exclusion of any respiratory or allergic diseases. Lung tissues from asthmatic patients were collected through bronchoscopic biopsy. Control tissues of the healthy control group were obtained from patients with benign pulmonary nodules, with exclusion of respiratory system diseases. All participants provided written informed consent after full disclosure of study protocols, which were approved by the Institutional Review Board of The First Affiliated Hospital of Shandong First Medical University (approval No. 2022-S011).

Lung histopathology

Human and mouse lung tissues were fixed in 4% paraformaldehyde (PFA) at room temperature for 72 hours, paraffin-embedded, and sectioned into 5 μm slices. Following deparaffinization, Periodic acid-Schiff (PAS) and Masson trichrome staining were performed. Collagen deposition (collagen volume fraction) and mucus production (PAS score) were semiquantitatively evaluated according to previously described methods.²¹

Animal model construction

The asthma model was established via a 2-phase protocol: sensitization with intraperitoneal injections of 20 μg ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA) emulsified in 200 μL phosphate-buffered saline (PBS) containing 2 mg Al(OH)₃ on days 0, 7, and 14, followed by airway challenge with 3% OVA aerosol inhalation every other day from days 21 to 58. House dust mite (HDM)-induced asthma was modeled as previously described in our studies.²² The AHR test was performed to evaluate the successful establishment of the asthma model, following the experimental protocol consistent with that described in our previous publications.²³ Macrophage-specific Tnfsf8 knockout (Tnfsf8^fl/fl-Lyz2Cre) mice and their littermate controls (Tnfsf8^fl/fl) on a C57BL/6J background were generated by Cyagen Biosciences (Suzhou, China). Female C57BL/6J mice (6–8 weeks) were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). For OVA+CD30L experiments, recombinant mouse CD30L (rmCD30L; RD-Biotech, Besançon, France) was reconstituted in PBS and intranasally administered to mice after each nebulization.

Induction of THP-1-derived macrophages

THP-1 cells were cultured to sufficient density, centrifuged, and resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 100 ng/mL phorbol 12-myristate 13-acetate (GlpBio, Shanghai, China). Following a 24-hour incubation, the cells were differentiated into macrophages.

Culture of bone marrow-derived macrophages (BMDMs)

Bone marrow cells were isolated from Tnfsf8^fl/fl and Tnfsf8^fl/fl-Lyz2Cre mice as previously reported.²⁴ Red blood cells were lysed with red blood cell lysis buffer. The cells were then cultured in RPMI 1640 supplemented with 10% FBS and 20 ng/mL recombinant mouse macrophage colony-stimulating factor (Meilunbio, Dalian, China) for 7 days to generate BMDMs.

Cell culture and treatment

Human bronchial epithelial (HBE) cells and THP-1 cells were obtained from Fuheng Biotechnology (Shanghai, China). HBE cells were maintained in Keratinocyte Medium (KM; ScienCell, Carlsbad, CA, USA), enriched with 1% Keratinocyte Growth Supplement (KGS) from the same manufacturer. THP-1 cells were cultured in RPMI 1640 medium containing 10% FBS. Mouse bronchial epithelial cells (iCell Bioscience, Shanghai, China) were cultured in specialized medium (iCell-a008-002m) per the manufacturer’s protocol. THP-1-derived macrophages were stimulated with recombinant human IL-13 (rhIL-13, PRP1051; Abbkine, Wuhan, China) at different concentrations to assess CD30L expression. BMDMs from mice were treated with recombinant mouse IL-13 (rmIL-13, PRP1159; Abbkine). Recombinant human CD30L (rhCD30L, 0910250-2; PeproTech, Cranbury, NJ, USA) was used to stimulate HBE cells to evaluate airway remodeling and epithelial-mesenchymal transition (EMT)-related indicators.

For indirect co-culture experiments, macrophages were stimulated with IL-13 (20 ng/mL) for 24 hours. The conditioned medium (CM) was then collected by centrifugation (1,500 rpm, 10 minutes) and added to bronchial epithelial cells for 48 hours.

Cell transfection was performed using EndoFectin™-Max (GeneCopoeia, Rockville, MD, USA) according to the manufacturer’s protocol. All of the small interfering RNAs (siRNAs) were obtained from General Biol (Anhui, China) and their sequences were provided in Supplementary Table S1. To investigate potential mechanisms of CD30L, cells were pretreated with SP600125 (S1460; Selleck, Houston, TX, USA) or SB203580 (S1076; Selleck) for 1 hour to inhibit c-Jun N-terminal kinase (JNK) and p38 pathways, respectively, prior to rhCD30L stimulation.

Enzyme-linked immunosorbent assay (ELISA)

Bronchoalveolar lavage fluid (BALF) was collected by three sequential lavages with 1 mL of PBS administered through a tracheal cannula. The concentrations of CD30L in BALF were quantified using a commercial ELISA kit (EK0572; Boster Biological Technology, Pleasanton, CA, USA), while CD30L levels in serum and cell culture supernatant were measured with a FineTest ELISA kit (EH0120; FineTest, Wuhan, China) according to the manufacturer’s protocols.

RNA-sequencing and results analysis

The lung tissues from the OVA-Tnfsf8^fl/fl and OVA-Tnfsf8^fl/fl-Lyz2Cre groups were sent to Biomarker Technologies (Beijing, China) for RNA sequencing. The differentially expressed genes (DEGs) were downloaded from the Biomarker Technologies online platform (https://www.biocloud.net/). Subsequently, we utilized the Bioinformatics Cloud Platform (http://www.bioinformatics.com.cn) to perform enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA).

Western blotting

Western blotting was performed using standard protocol as previously described.²² The primary antibodies in this study were used: anti-CD30L (PA5-36365, 1:1,000; Invitrogen, Carlsbad, CA, USA), anti-CD30 (HA722093, 1:1,000; HUABIO, Woburn, MA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (R1210-1, 1:5,000; HUABIO), anti-E-cadherin (3195, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-fibronectin (ET1702-25, 1:1,000; HUABIO), anti-collagen I (ab260043, 1:1,000; Abcam, Cambridge, UK), anti-alpha smooth muscle actin (α-SMA, ET1607-53, 1:1,000; HUABIO), anti-vimentin (5741, 1:1,000; Cell Signaling Technology), anti-p38 (14064-1-AP, 1:1,000; Proteintech, Rosemont, IL, USA), anti-phospho-p38 (28796-1-AP, 1:1,000; Proteintech), anti-JNK(ET1601-28, 1:1,000; HUABIO), and anti-phospho-JNK (ET1609-42, 1:1,000; HUABIO). We used a targeted western blot approach in which we excised specific protein bands from the membrane before antibody incubation. The representative images of Western blots are provided in the main text, and all the original western blotting images used for the analysis are presented in Supplementary Data S1 and S2. The densitometric values and their normalization to loading controls for all protein bands included in the statistical analyses were provided in Supplementary Data S3.

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

Total RNA was extracted from cells using the RNA Fast 200 Extraction Kit (Fastagen Biotech, Shanghai, China) according to the manufacturer’s instructions. Subsequently, RNA was reverse-transcribed into cDNA using reverse transcription reagent (Vazyme, Nanjing, China). qRT-PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme) to amplify collagen I, fibronectin, vimentin, CD30L, CD30, and GAPDH. The primers used are described in Supplementary Table S2.

Immunohistochemistry (IHC) staining

Lung tissues were fixed in 4% PFA, paraffin-embedded, and sectioned into 5 μm slices. After antigen retrieval with citrate buffer, endogenous peroxidases were inactivated with 3% H₂O₂. Sections were blocked in 5% bovine serum albumin for 1 hour, then incubated overnight at 4°C with primary antibodies: anti-CD30L (PA5-36365, 1:200; Invitrogen), anti-E-cadherin (3195, 1:200; Cell Signaling Technology), and anti-collagen I (PB0980, 1:200; Boster Biological Technology). Following PBS washes, sections were incubated with biotinylated anti-rabbit immunoglobulin G for 1 hour at 37°C, and 3,3′-Diaminobenzidine staining was performed, followed by hematoxylin counterstaining, dehydration in graded ethanol, and xylene clearing.

Immunofluorescence (IF) staining

The experimental procedures for lung tissue IF staining and IHC staining were identical up to the primary antibody incubation step. Primary antibodies against collagen I (ab260043, 1:200; Abcam), α-SMA (ET1607-53, 1:200; HUABIO), vimentin (5741, 1:200; Cell Signaling Technology), F4/80 (HA721745, 1:200; HUABIO), CD68 (HA601115, 1:200; HUABIO), and CD30L (PA5-36365, 1:200; Invitrogen) were incubated with lung tissue sections overnight at 4°C. After PBS washing, sections were incubated with fluorescent secondary antibodies, counterstained with 4′,6′-diamidino-2-phenylindole, and imaged by fluorescence microscopy.

HBE cells were seeded on glass slides in 24-well plates, fixed with 4% PFA (30 minutes), and washed with PBS. Primary antibodies against collagen I (ab260043, 1:200; Abcam), vimentin (5741, 1:200; Cell Signaling Technology), and fibronectin (ET1702-25, 1:200; HUABIO) were incubated overnight at 4°C. Subsequent IF steps followed the lung tissue protocol.

Migration assay

Treated HBE cells (approximately 1 × 10⁶ cells/150 μL) in KGS-free KM medium were seeded into transwell inserts (8 μm pores), with the lower chambers containing 600 μL KM medium supplemented with 1% KGS. After 24 hours incubation, migrated cells were fixed in 4% PFA, stained with 5% crystal violet, non-migrated cells were removed, and migrated cells were counted by phase-contrast microscopy (100×) and quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA), with a significance threshold set at P < 0.05. Statistical analyses were conducted using Student’s t-test for normally distributed data or the Mann-Whitney U test for nonparametric comparisons between two groups. Multiple group comparisons with normal distributions were analyzed using one-way analysis of variance followed by Dunnett’s or Tukey’s post hoc tests. Correlation analysis was performed using Spearman’s rank correlation.

RESULTS

CD30L expression is elevated in asthmatic lung tissues and predominantly localized to macrophages

To explore the involvement of CD30L in asthma, we initially detected CD30L expression in the lungs of OVA-induced asthmatic mice. Western blot analysis demonstrated significantly elevated CD30L levels compared to controls (Fig. 1A). Consistently, IHC staining confirmed the upregulation of CD30L expression in the asthmatic lung tissues (Fig. 1B). To extend these findings, we quantified CD30L levels in BALF using ELISA, demonstrating a marked increase in asthmatic mice (127.4 ± 28.86 pg/mL, n = 8) compared to controls (96.87 ± 12.01 pg/mL, n = 8) (Fig. 1C). Consistent with this, CD30L expression was significantly increased in the lungs of HDM-induced asthmatic mice, as demonstrated by immunohistochemistry (Supplementary Fig. S1A and B). We further examined CD30L expression in extrapulmonary organs (heart, liver, kidney, spleen, lymph nodes, and thymus), but found no significant changes (Supplementary Fig. S2A-D). To further clarify the localization of CD30L in lung tissues, we performed co-immunostaining with the macrophage marker F4/80, revealing that CD30L was primarily localized to macrophage populations in OVA-induced asthmatic mice (Fig. 1D). A similar localization pattern was observed in the HDM-induced asthma model (Supplementary Fig. S3).

Fig. 1. Analysis of CD30L expression and localization in OVA-induced asthmatic mice and asthmatic patients. (A) Western blotting of CD30L expression in OVA-induced asthmatic mice (n = 8). (B) Detection of CD30L by IHC staining in OVA-induced asthmatic mice (n = 4). (C) The level of CD30L in the BALF of asthmatic mice was detected by ELISA (n = 8). (D) Co-immunostaining of CD30L (red) and mouse macrophage marker F4/80 (green) in OVA-induced asthmatic mice (n = 4). (E) IF staining of CD30L (red) and human macrophage marker CD68 (green) in the airways from asthmatic patients and controls (n = 4). (F) Serum CD30L levels were measured using ELISA in both asthmatic patients (n = 39) and healthy controls (n = 22). (G) Correlation analysis of serum CD30L levels and FEV1/FVC ratio (r = −0.4754, P = 0.0001). (H) Correlation analysis of serum CD30L levels and FEV1(%predicted) (r = −0.3686, P = 0.0035). Bars = 50 μm.

CD30L, CD30 ligand; OVA, ovalbumin; IHC, immunohistochemistry; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay; IF, immunofluorescence; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; FEV1(%predicted), forced expiratory volume at 1 second to predicted value ratio; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6′-diamidino-2-phenylindole.

^*P < 0.05, ^**P < 0.01 versus the control group.

Having established CD30L upregulation in murine models, we next validated its clinical relevance in human specimens. We evaluated the protein expression of CD30L in lung biopsies from 4 asthmatic patients (2 males and 2 females) and 4 controls (2 males and 2 females).All four asthmatic patients had moderate (n = 3) or severe disease (n = 1) and had been receiving long-term treatment with ICS/LABA. Detailed clinical characteristics of the biopsy donors were summarized in Table S3. Consistent with murine data, comparative analysis showed significantly elevated CD30L expression levels in asthmatic airways compared to control airways, and co-immunostaining with the human macrophage marker CD68 revealed colocalization of CD30L within the macrophage populations (Fig. 1E). Additionally, we quantified serum CD30L levels by ELISA in 39 asthma patients (20 males, 19 females) and 22 healthy controls (12 males and 10 females). All of the asthmatic patients had moderate (n = 24) or severe disease (n = 15) and were receiving long-term ICS/LABA therapy. Supplementary Table S4 presents the baseline characteristics of all participants. Our results demonstrated significantly elevated CD30L levels in serum from asthma patients (218.70 ± 71.81 pg/mL, n = 39) compared to controls (157.50 ± 38.41 pg/mL, n = 22) (Fig. 1F). Furthermore, serum CD30L levels exhibited significant negative correlations with key parameters of pulmonary ventilatory function, including the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) (r = −0.4754, P = 0.0001) and FEV1(%predicted) (r = −0.3686, P = 0.0035) (Fig. 1G and H). Moreover, asthma patients were stratified by peripheral blood eosinophil counts into high-eosinophil (> 300 cells/μL) and low-eosinophil groups. A significant negative correlation between CD30L and FEV1 was observed in the high-eosinophil group (r = −0.4872, P = 0.01), but not in the low-eosinophil group (r = −0.07, P = 0.8025) (Fig S4A and B). Similarly, stratification by serum total IgE (high-IgE: ≥ 100 IU/mL; low-IgE: < 100 IU/mL) revealed a significant inverse correlation in the high-IgE group (r = −0.5169, P = 0.0081) but not in the low-IgE group (r = 0.066, P = 0.823) (Supplementary Fig S4C and D). Further analysis by disease severity demonstrated a significant correlation between CD30L and FEV1 in moderate asthma (r = −0.5565, P = 0.0047), whereas no significance was observed in severe cases (r = −0.4143, P = 0.1262) (Supplementary Fig. S4E and F).

Macrophage-specific Tnfsf8 knockout attenuates AHR and airway remodeling in OVA-induced asthmatic mice

Given the increased expression of CD30L in both asthma patients and OVA-induced asthmatic mice, we conducted experiments to investigate its role in airway remodeling. The IF assay revealed that CD30L primarily co-localized with macrophages under asthmatic conditions (Fig. 1D and E). Therefore, we generated macrophage-specific Tnfsf8 knockout mice (Tnfsf8^fl/fl-Lyz2Cre) and their littermate controls (Tnfsf8^fl/fl) (Fig. 2A). Western blot analysis confirmed the absence of CD30L protein in BMDMs from Tnfsf8^fl/fl-Lyz2Cre (Fig. 2B). Subsequently, Tnfsf8^fl/fl-Lyz2Cre and Tnfsf8^fl/fl mice were subjected to OVA challenge to establish asthma models, as outlined in Fig. 2C. We found that macrophage-specific Tnfsf8 knockout significantly attenuated OVA-induced AHR in mice (Fig. 2D). Furthermore, Masson staining and PAS staining were performed on lung tissue sections. Notably, Masson staining showed marked peribronchial collagen deposition in OVA-induced asthmatic mice, which was significantly reduced by macrophage-specific Tnfsf8 knockout (Fig. 2E). Similarly, PAS staining demonstrated enhanced goblet cell proliferation and mucus production in OVA-induced asthmatic mice compared to controls, and these pathological alterations were substantially reversed by macrophage-specific Tnfsf8 knockout (Fig. 2E).

Fig. 2. Macrophage-specific deficiency of CD30L alleviates AHR and airway remodeling in OVA-induced asthmatic mice. (A) Schematic diagram of Tnfsf8^fl/fl-Lyz2Cre mice. (B) Western blotting detection of CD30L in BMDMs of Tnfsf8^fl/fl and Tnfsf8^fl/fl-Lyz2Cre mice (n = 6). (C) Flowchart for OVA-induced asthmatic mice. (D) Airway resistance was measured in each group of mice using increasing doses of methacholine. (E) Peribronchial collagen deposition was quantified by Masson staining, while goblet cell hyperplasia and mucus hypersecretion were analyzed using PAS staining (n = 8). Bars = 50 μm.

CD30L, CD30 ligand; AHR, airway hyperresponsiveness; OVA, ovalbumin; BMDM, bone marrow-derived macrophage; PAS, Periodic acid-Schiff; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

^*P < 0.05, ^**P < 0.01 versus the corresponding group.

Biological processes mediated by CD30L in OVA-induced asthmatic mice

Based on these findings, we further investigated transcriptome changes induced by macrophage-specific Tnfsf8 knockout. We performed RNA extraction from lung tissues of the OVA+Tnfsf8^fl/fl and OVA+Tnfsf8^fl/fl-Lyz2Cre groups, followed by RNA sequencing analysis. Applying thresholds of |fold-change (FC)| ≥ 1.2 and P < 0.05, we identified 1,766 DEGs (745 upregulated; 1,021 downregulated), visualized through volcano plot analysis (Fig. 3A). GO enrichment analysis of downregulated genes revealed the top 10 significantly enriched terms in biological processes, cellular components, and molecular functions, as depicted in Fig. 3B. Notably, the enriched terms are primarily associated with epithelial cell migration, cell adhesion, ECM structural constituent, and collagen-containing extracellular matrix. Remarkably, epithelial cell migration and cell adhesion are hallmark features of EMT. These results indicated that CD30L might play a significant role in the EMT process and ECM deposition. Thus, we performed Western blotting, IF, and IHC staining to determine the alterations in EMT indicators (vimentin, E-cadherin) and ECM indicators (collagen I). Western blot analysis revealed that macrophage-specific Tnfsf8 knockout markedly reversed the downregulation of E-cadherin protein expression and the upregulation of collagen I and vimentin protein levels in lung tissues of OVA-induced asthmatic mice (Fig. 3C and D). These findings were corroborated by IF and IHC staining, which revealed diminished vimentin and collagen I staining intensity concomitant with restored E-cadherin expression in airway epithelium of macrophage-specific Tnfsf8 knockout mice (Fig. 3E and F). Collectively, these data establish CD30L as a key mediator of airway remodeling through EMT and ECM deposition in allergic asthma.

Fig. 3. The results of transcriptomic analysis from the lung tissues of OVA-induced asthmatic mice between Tnfsf8^fl/fl-Lyz2Cre and Tnfsf8^fl/fl mice. (A) Volcano plot of RNA sequencing from lung tissues between OVA-Tnfsf8^fl/fl-Lyz2Cre and OVA-Tnfsf8^fl/fl mice (|FC| ≥ 1.2, P < 0.05). Red represents upregulated DEGs, while blue represents downregulated DEGs. (B) The top 10 significantly enriched GO terms (including biological processes, molecular functions, and cellular components). (C, D) Protein expression of ECM deposition and EMT indicator (collagen I, vimentin, and E-cadherin) and subsequent quantitative analysis (n = 5). (E) IF detection of collagen I and vimentin proteins and their quantitative analyses (n = 5). (F) Representative images of E-cadherin IHC staining in lungs of mice (n = 5). Bars = 50 μm.

OVA, ovalbumin; FC, fold-change; DEGs, differentially expressed genes; GO, Gene Ontology; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; IF, immunofluorescence; IHC, immunohistochemistry; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6′-diamidino-2-phenylindole.

^*P < 0.05, ^**P < 0.01 versus the corresponding group.

CD30L administration exacerbates AHR and airway remodeling in OVA-induced asthmatic mice

Extending these observations, we instilled CD30L intranasally into the OVA-induced asthmatic mice (Fig. 4A). Consistently, CD30L significantly exacerbated AHR in the OVA-induced asthma model (Fig. 4B). Furthermore, CD30L administration exacerbated goblet cell proliferation, mucus production in the airways, and peribronchial collagen deposition in these mice (Fig. 4C and D).

Fig. 4. CD30L administration exacerbated AHR and airway remodeling in OVA-induced asthmatic mice. (A) A schematic representation of intranasal CD30L administration in mice, with selected elements created using BioRender. (B) Airway resistance was measured in each group of mice using increasing doses of methacholine. (C, D) PAS and Masson staining were applied to evaluate histopathological changes after rmCD30L treatment in asthmatic mice (n = 8). (E, F) Protein expression of ECM deposition and EMT indicator (collagen I, α-SMA, vimentin, and E-cadherin) and quantitative analysis (n = 5). (G, H) IHC or IF detection of collagen I, E-cadherin, vimentin, and α-SMA followed by quantitative analyses (n = 5). Bars = 50 μm.

CD30L, CD30 ligand; AHR, airway hyperresponsiveness; OVA, ovalbumin; PAS, Periodic acid-Schiff; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; α-SMA, alpha smooth muscle actin; IHC, immunohistochemistry; IF, immunofluorescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6′-diamidino-2-phenylindole.

^*P < 0.05, ^**P < 0.01 versus the corresponding group.

Additionally, lung tissues from the control, OVA, and OVA+CD30L mice were homogenized, and proteins were extracted to evaluate markers of ECM deposition and EMT via western blot analysis. Western blot analysis demonstrated that compared with the OVA group, the expression levels of collagen I, α-SMA, and vimentin were significantly upregulated in lung tissues from the OVA+CD30L group, whereas E-cadherin expression was markedly downregulated (Fig. 4E and F). IF and IHC staining further demonstrated that the OVA+CD30L group exhibited upregulated vimentin and collagen I expression, concomitant with reduced E-cadherin immunoreactivity in airway epithelium compared to OVA group (Fig. 4G and H). These results collectively demonstrate that CD30L exacerbates airway remodeling in asthmatic mice.

IL-13 upregulates CD30L expression in macrophages, and silencing CD30L in macrophages attenuates the EMT process and remodeling of bronchial epithelial cells

Asthma is associated with T helper 2 (Th2) inflammation, and IL-13 is a key cytokine involved in mediating allergen-induced airway inflammation and remodeling.²⁵ RNA-seq analysis of GEO dataset GSE35979 (2 IL-13 transgenic vs. 4 wild-type mice) revealed elevated CD30L mRNA levels in IL-13 transgenic mouse lungs compared to wildtype controls (Fig. 5A). We used IL-13 to establish an in vitro asthmatic cellular model and examine CD30L expression. THP-1-derived macrophages treated with rhIL-13 (0, 10, 20, and 50 ng/mL) exhibited dose-dependent upregulation of CD30L: ELISA revealed elevated soluble CD30L in supernatants (Fig. 5B), while western blotting confirmed increased intracellular protein levels (Fig. 5C and D). Subsequently, three siRNAs targeting CD30L (CD30L-siRNA1, CD30L-siRNA2, and CD30L-siRNA3) were transfected into THP-1-derived macrophages to knock down CD30L expression. Based on the transfection efficiency assessed by Western blotting and qRT-PCR, CD30L-siRNA3 was chosen for further experiments (Supplementary Fig. S5A-C).

Fig. 5. The results of indirect co-culture of THP-1-derived macrophages and HBE cells using conditioned medium. (A) CD30L expression profile graph in GSE35979 and differential expression analysis between IL-13 transgenic mice and wildtype controls. (B) Levels of CD30L in cell culture supernatants after rhIL-13 stimulation were quantified by ELISA (n = 6). (C, D) Protein expression of CD30L in THP-1-derived macrophages with different concentrations of rhIL-13 treatment for 24 hours (n = 4). (E) The process of obtaining THP-1-derived macrophages conditioned medium for co-culture with HBE cells. (F, G) Protein expression of collagen I, fibronectin, and vimentin in HBE cells was examined following indirect co-culture with differentially treated THP-1-derived macrophages (n = 4). (H) IF detection of collagen I, fibronectin, and vimentin in HBE cells followed by quantitative analyses (n = 4). (I) The effect of CD30L knockdown in THP-1-derived macrophages on the migratory ability of HBE cells (n = 4). Bars = 50 μm.

HBE, human bronchial epithelial; CD30L, CD30 ligand; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; IF, immunofluorescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CM, conditioned medium; si-NC, small interfering RNA negative control.

^*P <0.05, ^**P <0.01 versus the corresponding group.

Airway epithelial cells serve as the initiating point of asthma airway remodeling and exacerbate this process through interactions with immune cells (e.g., macrophages) and other structural cells. Based on GO analysis demonstrating CD30L-mediated regulation of epithelial cell migration and cell adhesion (Fig. 3B), we co-cultured macrophages with epithelial cells (via CM) to further investigate the impact of CD30L on airway remodeling. We cultured THP-1-derived macrophages with CD30L knockdown and treated them with rhIL-13 for 24 hours. HBE cells were then cultured in the CM (Fig. 5E). Western blotting and IF staining revealed that the increase in collagen I, fibronectin, and vimentin expression in HBE cells caused by CM derived from IL-13-treated macrophages was significantly reversed by knockdown of CD30L (Fig. 5F-H). Furthermore, migration assays revealed a significantly higher number of migrated HBE cells in the IL-13-treated macrophage CM group compared to the control group, an effect that was reversed by CD30L knockdown (Fig. 5I). Similarly, we performed indirect co-culture experiments using BMDMs from Tnfsf8^fl/fl and Tnfsf8^fl/fl-Lyz2cre mice with mouse bronchial epithelial cells. Western blot analysis revealed that, compared to Tnfsf8^fl/fl, the expression of collagen I, fibronectin, and vimentin in mouse bronchial epithelial cells induced by CM derived from Tnfsf8^fl/fl-Lyz2cre BMDMs was significantly reduced (Supplementary Fig. S6). Overall, these results demonstrated that CD30L promotes EMT and remodeling of bronchial epithelial cells.

Exogenous CD30L enhances the EMT process and remodeling in HBE cells

Consistent with the in vivo findings, stimulation of HBE cells with rhCD30L yielded analogous results. Cells were incubated with rhCD30L at different concentrations (0, 50, 100, and 200 ng/mL) for 24 hours. The mRNA and protein levels of collagen I, fibronectin, and vimentin were significantly increased (Supplementary Fig. S7A-C). Similar to the concentration-dependent effects, the protein expression levels of collagen I, fibronectin, and vimentin were also elevated over time at the indicated time points (0, 12, 24, and 48 hours) (Supplementary Fig. S7D and E).

CD30 knockdown attenuates CD30L-induced EMT and remodeling in HBE cells

To investigate CD30L’s functional mechanisms, we designed 2 siRNAs targeting its receptor CD30 (CD30-siRNA1 and CD30-siRNA2) and validated knockdown efficiency in HBE cells via Western blotting and qRT-PCR (Supplementary Fig. S8A-F). As shown in Fig. 6A and B and Fig. S8G and H, Western blotting demonstrated CD30 knockdown significantly inhibited CD30L-induced EMT and remodeling in HBE cells. The results of IF staining corroborated the western blotting findings (Fig. 6C). Furthermore, transwell migration assays showed that CD30 knockdown effectively suppressed CD30L-enhanced migratory capacity in HBE cells (Fig. 6D).

Fig. 6. Knockdown of CD30 in HBE cells significantly alleviates the changes in remodeling and EMT markers, as well as alterations in migratory capacity induced by CD30L. (A, B) Protein expression of collagen I, fibronectin, and vimentin in HBE cells after CD30 knockdown. (C) IF detection of collagen I, fibronectin, and vimentin in HBE cells and their quantitative analysis. (D) The effects of CD30 knockdown on migration ability of HBE cells (n = 4). Bars = 50 μm.

CD30L, CD30 ligand; HBE, human bronchial epithelial; EMT, epithelial-mesenchymal transition; IF, immunofluorescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si-NC, small interfering RNA negative control; DAPI, 4′,6′-diamidino-2-phenylindole.

^*P < 0.05, ^**P < 0.01 versus the corresponding group.

CD30L enhances airway remodeling through JNK/p38 mitogen-activated protein kinase (MAPK) signaling pathway

We performed KEGG enrichment analysis and GSEA on RNA extracted from lung tissues of the OVA+Tnfsf8^fl/fl and OVA+Tnfsf8^fl/fl-Lyz2Cre mice. KEGG enrichment analysis demonstrated significant enrichment of the phosphatidylinositol 3-kinase/AKT and MAPK signaling pathways (Fig. 7A). Subsequently, GSEA showed that the MAPK signaling pathway activity was significantly downregulated in the OVA+Tnfsf8^fl/fl-Lyz2Cre group compared to the OVA+Tnfsf8^fl/fl group (normalized enrichment score = −1.771, false discovery rate < 0.05) (Fig. 7B). Consistently, macrophage-specific Tnfsf8 knockout significantly reduced OVA-induced phosphorylation of JNK (p-JNK) and p38 (p-p38) in mouse lung tissues (Fig. 7C). In contrast, the CD30L+OVA group showed significantly increased expression of p-JNK and p-p38 compared to the OVA group (Fig. 7D). In vitro, rhCD30L induced the phosphorylation of JNK and p38 in HBE cells, and this phosphorylation was reversed following the knockdown of CD30 (Fig. 7E).

Fig. 7. CD30L modulates the JNK/p38 MAPK signaling pathway through a CD30-dependent mechanism. (A) KEGG enrichment analysis of RNA-seq data showing the top 10 significantly enriched terms (P < 0.05). (B) GSEA enrichment plots comparing OVA-Tnfsf8^fl/fl-Lyz2Cre and OVA-Tnfsf8^fl/fl mice showing a negative enrichment of MAPK signaling pathway (NES = −1.771, FDR = 0.0). (C, D) Western blot analysis of p-JNK, JNK, p-p38, and p38 in the lungs of mice. (E) HBE cells were transfected with si-CD30 and then stimulated by rhCD30L for 24 hours. The expression levels of p-JNK and p-p38 were analyzed by western blotting (n = 4).

CD30L, CD30 ligand; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; NES, normalized enrichment score; FDR, false discovery rate; p-, phosphorylation of.

^*P < 0.05, ^**P < 0.01 versus the corresponding group.

To investigate the functional role of JNK/p38 MAPK signaling in CD30L-associated airway remodeling, HBE cells were pretreated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580 for 1 hour prior to rhCD30L stimulation. SP600125 treatment significantly reduced the CD30L-induced upregulation of collagen I, fibronectin, and vimentin (Fig. 8A), and reversed the enhanced migratory ability of HBE cells (Fig. 8B). Consistent with the effect of JNK inhibition, pharmacological blockade of p38 with SB203580 similarly attenuated the CD30L-induced upregulation of these EMT-related markers, airway remodeling indicators, and migratory capacity in HBE cells (Fig. 8C and D).

Fig. 8. The roles of CD30L are mediated via the JNK/p38 MAPK signaling pathway. (A) HBE cells were pretreated with the JNK inhibitor SP600126 (20 μm) for 1 hour before rhCD30L for 24 hours. The expression of collagen I, fibronectin, and vimentin was determined by western blotting. (B) The effects of SP600126 treatment on migration ability of HBE cells. (C) HBE cells were pretreated with the p38 inhibitor SB203580 (10 μm) for 1 hour before rhCD30L for 24 hours. The expression of collagen I, fibronectin, and vimentin was analyzed by western blotting. (D) The effects of SB203580 treatment on migration ability of HBE cells (n = 4).

CD30L, CD30 ligand; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; HBE, human bronchial epithelial; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

^*P < 0.05, ^**P < 0.01 versus the corresponding group.

DISCUSSION

Airway remodeling constitutes a critical pathological mechanism contributing to pulmonary function decline and acute exacerbations in asthmatic patients. This study provided the first demonstration that CD30L served as a critical mediator in exacerbating airway remodeling during asthma pathogenesis. CD30L orchestrated the EMT process and ECM deposition during asthma airway remodeling through the JNK/p38 MAPK signaling pathway activation. In summary, this study provided a comprehensive analysis of the role of CD30L in the pathogenesis of airway remodeling in asthma and explored its underlying mechanisms (Fig. 9).

Fig. 9. CD30L exacerbates airway remodeling in asthma by enhancing the EMT process and ECM deposition through the CD30-mediated JNK/p38 MAPK pathway. The schematic diagram was created using BioRender (https://app.biorender.com), with selected graphical elements adapted from its template library to ensure scientific accuracy and visual clarity.

CD30L, CD30 ligand; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; WT, wild-type; si-NC, small interfering RNA negative control; HBE, human bronchial epithelial.

Members of the TNF superfamily were initially recognized as mediators of inflammation, apoptosis, and cell survival.²⁶,²⁷ Recently, evidence has shown specific TNF superfamily members to be critical regulators of fibrosis and tissue remodeling across diverse organ systems and disease contexts.²⁶,²⁸ Among these, CD30L regulates cell proliferation, survival, and cytokine production—mechanisms linked to chronic inflammatory responses, a critical driver of airway remodeling in asthma.²⁹,³⁰ As we all know, CD4⁺ memory T cells play a crucial role in sustaining tissue inflammation and driving exacerbations of allergic diseases, including asthma. Notably, Sethi et al.³¹ have reported that dual blockade of CD30L and ICOSL effectively reduces lung-resident CD4⁺ memory T cell activity, thereby attenuating asthma exacerbations. Similarly, combined inhibition of CD30L and OX40L suppressed pulmonary eosinophilic inflammation.³² In addition, studies have shown that ICOSL and OX40L signaling contribute to fibrotic pathways.³³,³⁴ However, the role of CD30L in tissue fibrosis and airway remodeling remains unexplored. In this study, we demonstrated that intranasal administration of rmCD30L in OVA-induced asthmatic mice significantly exacerbated ECM deposition, evidenced by increased collagen I deposition and α-SMA expression compared to the OVA group. Concurrently, CD30L treatment enhanced the EMT process, characterized by up-regulated vimentin and down-regulated E-cadherin in lung tissues. Consistent with in vivo findings, in vitro stimulation of HBE cells with rhCD30L promoted collagen I and fibronectin synthesis, alongside enhanced EMT progression, as indicated by increased vimentin expression and cell migration capacity. These results collectively demonstrate that CD30L plays a crucial role in promoting airway remodeling in asthma. Consistent with our findings, Gupta et al.²⁹ have demonstrated that therapeutic blockade of CD30L alleviates collagen deposition in an allergen-induced atopic dermatitis mouse model. Additionally, an interesting observation was that serum CD30L levels in asthmatic patients were higher than BALF levels in murine models (Fig. 1C and F). First, interspecies variations between mice (BALF data) and humans (serum data) could influence baseline levels. Second, technical aspects, such as BALF dilution during lavage and differential ELISA sensitivity across species-specific kits, may underestimate alveolar CD30L. Biologically, serum integrates systemic CD30L pools, including soluble forms, shed from lung membrane-bound CD30L into circulation, whereas BALF primarily reflects localized pulmonary secretion. These combined technical and biological mechanisms likely explain the disparity.

Studies demonstrate that macrophages play a key role in fibrosis and serve as a critical source of ligands driving the fibrotic activation program of fibroblasts.³⁵ Transforming growth factor (TGF)-β1, a master regulator of fibrosis, is expressed by various cell types, including activated macrophages.³⁶ Genetic deletion of TGF-β1 in macrophages using LysM-Cre alleles significantly reduced pulmonary collagen content and overall fibrosis scores in bleomycin-induced pulmonary fibrosis models, highlighting the pivotal role of macrophage-derived TGF-β1 in fibrotic progression.³⁷ In this study, IF colocalization experiments showed that CD30L colocalized predominantly with macrophages in the lung tissues. Similar to TGF-β1, we identified macrophage-derived CD30L as a mediator of airway remodeling in asthma. We specifically knocked out Tnfsf8 in macrophages and found that it significantly alleviated peribronchial collagen deposition and mucus production in OVA-induced asthmatic mice.

Airway epithelial cells play a pivotal role in initiating airway remodeling in asthma.⁴,³⁸ Current therapeutic strategies for asthma that directly or indirectly target airway epithelial cells have shown not only to ameliorate clinical symptoms but also to improve pulmonary function and attenuate airway remodeling, thereby underscoring the central role of structural and functional epithelial dysregulation in asthma pathogenesis.⁴ The GO enrichment analysis results in this study demonstrated that CD30L regulates epithelial cell migration and cell adhesion. Notably, enhanced epithelial migratory capacity and loss of cell adhesion are the core hallmarks of EMT.²⁸,³⁹ To functionally validate this association, we employed an in vitro indirect co-culture system with macrophages and epithelial cells, which ultimately confirmed that CD30L knockdown effectively alleviated epithelial remodeling and suppressed the EMT process. CD30, the functional receptor of CD30L, was initially identified as a tumor-associated antigen.¹⁸,⁴⁰ In this study, we first demonstrated that CD30 was expressed on HBE cells. To investigate whether CD30L promotes remodeling and EMT through CD30, we used siRNA to silence CD30 in HBE cells and found that CD30 silencing significantly reversed the changes induced by CD30L. These findings demonstrate that CD30L blockade represents a viable therapeutic strategy for airway remodeling in asthma. However, further clinical experiments are needed to validate these findings.

Further KEGG enrichment analysis and GSEA of transcriptome data revealed significant enrichment of the MAPK signaling pathway. JNK and p38 represent 2 principal subfamilies within the MAPK family, which are involved in fibrosis and tissue remodeling.⁴¹,⁴²,⁴³,⁴⁴ In vivo experiments showed that macrophage-specific knockout of Tnfsf8 significantly suppressed p-JNK and p-p38 in asthmatic mice, whereas intranasal administration of recombinant CD30L protein had the opposite effect. In vitro, rhCD30L increased p-JNK and p-p38 in HBE cells, and this change was reversed after silencing CD30. The MAPK signaling pathway plays a pivotal role in promoting EMT through the precise regulation of downstream transcription factors. An important regulator in this process is the modulation of activator protein-1 (AP-1), a heterodimeric transcription factor composed of Fos, Jun, and ATF subunits, which drives fibrotic responses.³⁶ Specifically, JNK phosphorylates c-Jun, a key AP-1 component, while p38 MAPK enhances the transcriptional activity of ATF2, collectively amplifying AP-1-dependent expression of profibrotic mediators, such as α-SMA and fibronectin—hallmarks of lung fibrosis.⁴⁵ Furthermore, MAPK signaling regulates EMT-inducing transcription factors (EMT-TFs) such as Snail, Slug, and Twist, upregulating Snail and post-translationally stabilizing Slug/Twist.⁴⁶,⁴⁷,⁴⁸ These EMT-TFs orchestrate phenotypic reprogramming by suppressing epithelial markers (e.g., E-cadherin) and activating mesenchymal genes, thereby accelerating EMT initiation and progression. Given the regulatory role of the MAPK signaling pathway in modulating EMT-related transcription factors, we hypothesize that CD30L may contribute to airway remodeling by activating the MAPK pathway, thereby influencing the expression and stability of EMT-TFs. Future studies will be conducted to experimentally validate this proposed mechanism.

While this study highlights CD30L as a key mediator of airway remodeling, several limitations should be noted. First, our in vitro experiments focused on elucidating the effects and molecular mechanisms of CD30L on airway epithelial cells. Further investigations are warranted to explore the potential roles of this factor in other airway structural cells, such as smooth muscle cells and fibroblasts. Second, the OVA-induced model primarily reflects Th2-high asthma. Thus, future studies are required to validate the functional roles and underlying mechanisms of CD30L in non-Th2 asthma.

In conclusion, we have demonstrated that CD30L is significantly upregulated in asthmatic lung tissues and is primarily localized to pulmonary macrophages. Targeting CD30L can significantly alleviate airway remodeling potentially through the JNK/p38 MAPK signaling pathway. These findings further support the involvement of CD30L in airway remodeling, highlighting its significant potential as a therapeutic target in asthma.

SUPPLEMENTARY MATERIALS

Supplementary Data S1

All the original Western blotting images in the article (Figs. 1-7)

Supplementary Data S2

All the original western blotting images in the article (Fig. 8 and Supplementary Figs. S5, S6, S7, S8)

Supplementary Data S3

Densitometric values for all the protein bands included in the statistical analyses

Supplementary Table S1

Sequences of the siRNAs used in this study

Supplementary Table S2

Primers for qRT-PCR

Supplementary Table S3

The clinical characteristics of subjects that donated the bronchial biopsy specimens

Supplementary Table S4

Characteristics of the participants who provided serum samples

Supplementary Fig. S1

Detection of CD30L by IHC staining in HDM-induced asthmatic mice (n = 4). Bars = 50 μm.

Supplementary Fig. S2

Expression of CD30L in extrapulmonary tissues. (A, B) The IHC staining of CD30L in the liver, lymph node, and spleen. (C, D) The IHC staining of CD30L in the kidney, heart, and thymus (n = 4). Bars = 50 μm.

Supplementary Fig. S3

Co-immunostaining of CD30L (red) and mouse macrophage marker F4/80 (green) in HDM-induced asthmatic mice. Bars = 50 μm.

Supplementary Fig. S4

Associations between serum CD30L and FEV1(%predicted) stratified by asthma severity, peripheral blood eosinophil counts, and serum total IgE.

Supplementary Fig. S5

Evaluation of the knockdown efficiency of three siRNAs targeting CD30L. (A) mRNA expression levels of CD30L in THP-1-derived macrophages after small interference RNA transfection. (B, C) Protein expression levels of CD30L in THP-1-derived macrophages after small interference RNA transfection (n = 3).

Supplementary Fig. S6

The results of indirect co-culture of BMDMs and mouse bronchial epithelial cells using conditioned medium. (A) The process of obtaining BMDMs conditioned medium for co-culture with mouse bronchial epithelial cells. (B) The protein expression of collagen I, fibronectin, and vimentin in mouse bronchial epithelial cells was examined following indirect co-culture with BMDMs (n = 4).

Supplementary Fig. S7

Effects of the exogenous CD30L on EMT and remodeling in HBE cells. (A-C) mRNA and protein expression levels of collagen I, fibronectin, and vimentin after treatment with different concentrations of rhCD30L for 24 hours. (D, E) Protein expression levels of collagen I, fibronectin, and vimentin after treatment with 100 ng/mL CD30L at different time points (0, 12, 24, and 48 hours) (n = 3–4).

Supplementary Fig. S8

Evaluation of the knockdown efficiency of siRNA targeting CD30. (A-C) mRNA expression and protein expression level of CD30 in HBE cells after CD30-siRNA1. (D-F) mRNA expression and protein expression levels of CD30 in HBE cells after CD30-siRNA2. (G, H) Protein expression of collagen I, fibronectin, and vimentin in HBE cells after CD30 knockdown (n = 3).