Exploring the mechanism of interleukin-24 in regulating eosinophil differentiation in chronic obstructive pulmonary disease

Abstract

Chronic obstructive pulmonary disease (COPD) exacerbation is a common respiratory condition, particularly when accompanied by eosinophilia, which mediates inflammatory responses that significantly impair the integrity of the airway mucosal barrier. This study aims to elucidate the roles of interleukin-24 (IL-24) and eosinophils (EOS) in COPD-associated tissue remodeling, focusing on their effects on the expression of pro-inflammatory mediators and extracellular matrix (ECM) components in pulmonary fibroblasts, as well as the underlying molecular mechanisms. Based on the differential expression of IL-24 between healthy individuals and COPD patients and its correlation with EOS, we differentiated EOL-1 cells into EOS using butyrate and established a co-culture system with pulmonary fibroblasts. Concurrently, groups of fibroblasts were stimulated with varying concentrations of IL-24 alone. The regulatory effects on pro-inflammatory mediators and ECM expression were systematically analyzed using flow cytometry, real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and Western blotting. Under the present experimental conditions, IL-24 treatment was associated with enhanced fibrotic responses in pulmonary fibroblasts. Furthermore, compared with butyrate treatment alone, the combination of IL-24 and butyrate further increased the differentiation rate of EOL-1 cells into EOS cells. Differentiated EOS further stimulated pulmonary fibroblasts to secrete pro-inflammatory factors IL-6 and IL-8, as well as the tissue remodeling-related factor vascular endothelial growth factor (VEGF), thereby exacerbating the fibrotic process. This study confirms that IL-24 promotes the maturation and differentiation of EOL-1 cells and can induce a fibrotic phenotypic transformation in pulmonary fibroblasts. This interaction participates in regulating inflammatory responses and tissue remodeling during COPD progression, highlighting the critical role of eosinophil-fibroblast crosstalk in the pathological mechanisms of the disease.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12931-026-03687-2.

Introduction

Chronic Obstructive Pulmonary Disease (COPD) is characterized by persistent airflow obstruction and chronic inflammation affecting both the airways and lung parenchyma, frequently accompanied by emphysematous changes and fibrosis [1] .Eosinophilic COPD is an important inflammatory phenotype of chronic obstructive pulmonary disease, characterized by airway eosinophilic inflammation, manifested as elevated eosinophil counts in blood and/or sputum, and showing a better response to inhaled corticosteroid (ICS) therapy [2]. Although COPD has traditionally been considered predominantly neutrophilic inflammation, recent studies indicate that approximately 20%-40% of patients exhibit elevated sputum and/or blood eosinophil levels, suggesting that type 2 inflammation may play a significant role in a subset of patients [3]. Currently, due to the lack of standardized cut-off values for blood or sputum eosinophils across different studies, this phenotype lacks a universally accepted precise definition [4]. Most studies classify COPD patients with blood eosinophils ≥ 2% and/or sputum eosinophils >3% as having eosinophilic COPD [5]. Airway inflammation in this subgroup is primarily driven by eosinophils and has been recognized as a distinct clinical phenotype.

From a therapeutic strategy perspective, the identification of the eosinophilic COPD phenotype provides an important basis for precision intervention. Airway inflammation in this subgroup is predominantly driven by type 2 cytokines such as IL-5, IL-4, and IL-13, making biologics targeting these key pathways a rational treatment option. Anti-IL-5 pathway agents (benralizumab, mepolizumab) significantly reduce the risk of exacerbations in patients with elevated blood eosinophils and frequent exacerbations [6]; the anti-IL-4Rα antibody dupilumab similarly significantly reduces exacerbation rates and improves lung function in patients with BEC ≥ 300 cells/μ L [7]. These evidence indicate that patients with this phenotype can derive significant benefits from biological targeted therapy.

Fibroblasts serve as critical effector cells responsible for maintaining extracellular matrix (ECM) homeostasis within pulmonary tissue and play a central role in aberrant ECM remodeling observed in chronic lung diseases such as asthma, COPD,and pulmonary fibrosis [8]. Under conditions of sustained inflammation,various growth factors—most notably transforming growth factor-beta (TGF-β )—are released in substantial quantities. These factors act chemotactically to recruit and activate pulmonary fibroblasts,facilitating their differentiation into myofibroblast phenotypes that drive collagen deposition and airway wall fibrosis [9]. Eosinophils represent a significant source of TGF-β ,releasing this cytokine to stimulate fibroblast proliferation and activity,thereby promoting fibrosis and collagen accumulation within the airway wall and contributing to airway remodeling [10].

Interleukin-24 (IL-24), a member of the IL-20 cytokine subfamily—which also includes IL-19, IL-20, and IL-22—signals through two heterodimeric receptor complexes: IL-20Rα /IL-20Rβ and IL-22R/IL-20Rβ . These receptors are broadly expressed in tissues such as the skin, lungs, and reproductive organs [11]. Current research on IL-24 has predominantly focused on its roles in cutaneous wound healing and tumor suppression [12], while its potential functions in pulmonary injury repair and fibrosis remain largely unexplored. Evidence indicates that IL-24 facilitates extracellular matrix deposition and protease expression during skin wound healing, thereby activating fibroblasts and promoting tissue remodeling [13].

Notably, recent studies have revealed a close association between IL-24 and eosinophils. Wu et al., in a study published in Nature Communications, demonstrated for the first time that in asthma models, IL-24 is primarily secreted by infiltrating eosinophils and further enhances eosinophil recruitment to the lungs through the CXCL5/CXCR1/CXCR2 axis, forming a positive feedback loop; concurrently, IL-24 can directly disrupt airway epithelial integrity, promoting epithelial-mesenchymal transition and fibrotic processes [14]. Furthermore, Patel et al. found that IL-24 expression in human bronchial epithelial cells was upregulated up to 30.7-fold upon stimulation with cigarette smoke extract, suggesting that IL-24 may play an important role in COPD airway inflammation [15].

In summary, eosinophils play a central role in airway inflammation and remodeling in COPD, and significant progress has been made in biological therapies targeting this phenotype. However, IL-24, as a recently identified eosinophil-derived effector factor, has been confirmed to regulate eosinophil recruitment and promote fibrosis in asthma models, yet its mechanism of action in COPD, particularly in eosinophilic COPD, remains completely unknown. Based on this, the present study aims to investigate the effect of IL-24 on eosinophil precursor differentiation, elucidate its role in airway tissue remodeling and the inflammatory phenotype of pulmonary fibroblasts, reveal a novel mechanism by which eosinophils promote COPD progression and fibrosis, and provide a potential therapeutic target for precision treatment of this phenotype.

Methods

Human subjects

This study included 45 patients admitted to the Eighth Medical Center of the Chinese PLA General Hospital and 25 healthy individuals undergoing physical examinations as research subjects. Ethical approval for the study was granted by the Ethics Committee (approval number 309202109091005), and all participants provided informed consent prior to inclusion. The inclusion criteria for the chronic obstructive pulmonary disease (COPD) group were as follows: (1) Age equal to or greater than 40 years; (2) A clinical diagnosis of COPD established based on patient medical history, a smoking history of at least 10 pack-years, characteristic clinical signs and symptoms, and confirmation of irreversible airway obstruction via spirometric assessment.

EOL-1 cell culture

The human eosinophilic cell line EOL-1 (Biobw, bio-132912) was maintained in RPMI 1640 medium (Gibco, C11875500BT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 10270106) and penicillin-streptomycin (10,000 U/mL; Sigma). At approximately 90% confluence, cells were harvested by centrifugation, the supernatant was discarded, and the pellet was resuspended. Cells were seeded at  cells/mL into fresh flasks. Experimental treatments included: 50 μ M butyric acid (BA; Sigma, B103500) alone or combined with interleukin-24 (IL-24; MCE, HEK293) or interleukin-5 (IL-5; Abmole, M9979) at 1 or 10 ng/mL, as well as IL-24 alone at 1 or 10 ng/mL. Medium was refreshed every three days for up to nine days.

Lung fibroblast culture

Primary lung fibroblasts (Fuheng, FH-H033) were cultured in Minimum Essential Medium (MEM; Fuheng, PY-H033) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and penicillin-streptomycin (10,000 U/mL; Sigma). At approximately 80% confluence, cells were detached using trypsin, centrifuged, and resuspended before seeding at  cells/mL. Fibroblasts were then treated with varying concentrations of IL-24 for 24 h.

Cell viability assay using CCK-8

To evaluate potential cytotoxic effects, cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Abmole, M4839). EOL-1 cells were treated with: 50 μ M BA alone; 50 μ M BA with 1 or 10 ng/mL IL-24; 50 μ M BA with 1 or 10 ng/mL IL-5; or IL-24 alone at 1 or 10 ng/mL. After treatment, 10 μ L of CCK-8 reagent was added per well, and plates were incubated in the dark at C in a humidified 5% CO₂ atmosphere for 1 h. Optical density was measured at 450 nm to quantify viability.

Hematoxylin and eosin (H&E) staining

Bronchoalveolar lavage fluid (BALF) was collected from patients with eosinophilic COPD. Following centrifugation, the cell pellet was obtained and used to prepare smears for hematoxylin-eosin (HE) staining. The morphological characteristics of eosinophils (bilobed nuclei and eosinophilic cytoplasmic granules) were observed under a light microscope, and representative images were captured. EoL-1 cells cultured under standard conditions were subjected to various treatments, including 50 μ M BA alone; 50 μ M BA with 1 or 10 ng/mL IL-24; 50 μ M BA with 1 or 10 ng/mL IL-5; or IL-24 alone at 1 or 10 ng/mL for 3, 6, or 9 days. After treatment, cells were fixed and stained with hematoxylin and eosin. Eosinophil differentiation was assessed by light microscopy.

Flow cytometry analysis

Flow cytometry was used to assess eosinophil differentiation and IL-24 receptor expression. Untreated and treated EOL-1 cells were harvested at 3, 6, and 9 days post-treatment and stained with fluorescent antibodies: anti-IL-5R-PE (10 μ L per cells; BD Biosciences, 555902), anti-IL-20Rα -APC (10 μ L per cells; R&D Systems, FAB2770A), and anti-IL-22Rα 1-PE (10 μ L per cells; R&D Systems, FAB11762P). Staining was performed at C for 15 min. Data were acquired and analyzed using FlowJo software.

Co-culture of eosinophils and pulmonary fibroblasts

EOL-1 cells were cultured in RPMI-1640 medium with 10% FBS and treated with 50 μ M BA to induce eosinophilic differentiation. Differentiated eosinophils were then co-cultured with pulmonary fibroblasts at a 1:4 ratio in MEM containing 10% FBS. In 60 mm dishes, fibroblasts were seeded at cells, and EOL-1 cells at or cells. An indirect co-culture system was also established using Transwell chambers, with eosinophils in the upper chamber and fibroblasts in the lower chamber for 72 h.

Real-time quantitative PCR

Total RNA was isolated using Trizol reagent (Invitrogen,15596026CN) following the manufacturer’s instructions. Complementary DNA (cDNA) synthesis was performed using a premixed kit from Takara(Takara,RR047A) , according to the provided protocol. Quantitative PCR was performed using the SYBR Premix Ex Taq II Kit (Takara,RR820A) with 35 amplification cycles, and all reactions were conducted in triplicate. All kits were used following the manufacturers’instructions. Each assay was performed in triplicate, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) employed as the endogenous control.Primer sequences (synthesized by Sangon Biotech) were:

• GAPDH: Forward 5’-ACCACAGTCCATGCCATCAC-3’, Reverse 5’-TCCACCACCCTGTTGCTGTA-3’

• α -SMA: Forward 5’-CTATGAGGGCTATGCCTTGCC-3’, Reverse 5’-GCTCAGCAGTAGTAACGAAGGA-3’

• COL1A1: Forward 5’-AAAGATGGACTCAACGGTCTC-3’, Reverse 5’-CATCGTGAGCCTTCTCTTGAG-3’

• VEGF: Forward 5’-TTGCCTTGCTGCTCTACCTCCA-3’, Reverse 5’-GATGGCAGTAGCTGCGCTGATA-3’

Enzyme-linked immunosorbent assay (ELISA)

Peripheral blood samples were collected from all COPD patients into EDTA anticoagulant tubes. Plasma was separated by centrifugation at 4°C, and the supernatant was aliquoted and stored at -80°C for subsequent analysis. The levels of various cytokines in the plasma, including interleukin (IL)-5, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17, IL-1β , interferon (IFN)-α , IFN-γ , and tumor necrosis factor (TNF)-α , were measured using enzyme-linked immunosorbent assay (ELISA). All procedures were performed strictly in accordance with the manufacturer’s instructions. Plasma IL-24 concentrations were measured in blood samples from COPD patients and healthy controls using an ELISA kit (Cloud-Clone, SEC064Hu). To evaluate cytokine production, pulmonary fibroblasts ( cells) were co-cultured with EOL-1 cells at 1:2 ( cells) or 1:4 ( cells) ratios in 60 mm dishes. After 3 days, interleukin-6 (IL-6; BioLegend, 430507), interleukin-8 (IL-8; BioLegend, 431507), and interferon-γ (IFN-γ ; BioLegend, 430107) levels in supernatant were quantified by ELISA.

Western blot analysis

Fibroblasts were treated with various IL-24 concentrations or co-cultured with eosinophils at C in 5% CO₂. Cells were lysed in RIPA buffer with protease inhibitors. Protein concentration was determined using a BCA assay kit (Beyotime, CX00098). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 h at room temperature in TBS-T (TBS with 0.1% Tween-20 and 5% skim milk), then incubated overnight at C with primary antibodies: anti-GAPDH (1:1000; Santa Cruz Biotechnology, SC-47724), anti-collagen I (1:1000; Cell Signaling Technology, 72026), and anti-α -SMA (1:1000; Santa Cruz Biotechnology, SC-53015). After washing with TBS-T, membranes were incubated with HRP-conjugated secondary antibodies for 1.5 h at room temperature. Bands were visualized and quantified using ImageJ software.

Immunofluorescence staining

Immunocytochemical staining was conducted to evaluate protein expression and localization. Cell culture slides were pretreated by coating with poly-L-lysine for 4 h, followed by ultraviolet irradiation for 6 h prior to cell seeding. Pulmonary fibroblasts and EOL-1 cells were seeded onto these coated slides. Cells were fixed with 4% paraformaldehyde and permeabilized using 1% bovine serum albumin (BSA) containing 0.01% Triton X-100 for 10 min. Subsequently, cells were blocked with 3% BSA for 1 h. Fibroblasts were incubated with primary antibodies against α -SMA (1:200) and type I collagen (COL-I) (1:200), followed by incubation with secondary antibodies conjugated to Alexa Fluor 488 (anti-mouse, Abcam, AB150113) or Alexa Fluor 555 (anti-rabbit, Abcam, AB150078). Finally, nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, Beyotime, C1006) for 10 min, and fluorescence was visualized using a confocal laser scanning microscope.

Statistical analysis

GraphPad Prism 5.0 software was used for statistical analysis. Data normality was assessed using the Shapiro-Wilk test. Correlation analysis was performed using Spearman’s rank correlation. For comparisons between two groups, the independent samples t-test was used for normally distributed data, while the Mann-Whitney U test was used for non-normally distributed data. For comparisons among multiple groups, one-way or two-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test for multiple comparisons. All data were derived from at least three independent experiments. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

Expression levels of IL-24 in peripheral blood of patients with eosinophilic COPD

A total of 70 subjects were enrolled in this study, including patients with eosinophilic COPD (N = 45) and healthy controls (N = 25) (Table 1).To determine the expression of relevant cytokines in patients with eosinophilic COPD, we measured a panel of plasma cytokines. The results showed that among the 13 cytokines examined, both IL-5 and IL-24 were significantly positively correlated with peripheral blood eosinophil count and eosinophil percentage (Fig. 1A). Given the well-established association between IL-5 and eosinophils, this study further focused on the expression characteristics of IL-24 and its correlation with EOS.

Table 1.

Demographic and clinical characteristics of the study participants

	Control	IL-24	P value
Number of Subjects	N=25	N=45
Age (years)	70 ± 10	69 ± 9	0.720
Sex			0.642
Male	N=22 (88%)	N=37 (82.2%)
Female	N=3 (12%)	N=8 (17.8%)
FEV1/FVC	96 ± 4	59 ± 5	<0.01
Smoking History			0.726
Smoker	N=20 (80%)	N=36 (80%)
Non-smoker	N=5 (20%)	N=9 (20%)
Disease Duration
< 3 months	/	N=3 (6.6%)	/
3–6 months	/	N=5 (11.1%)	/
6 months–5 years	/	N=7 (15.6%)	/
5–10 years	/	N=13 (28.9%)	/
10–20 years	/	N=17 (37.8%)	/

Fig. 1.

A Expression of relevant cytokines in patients with eosinophilic COPD. B Comparative analysis of plasma IL-24 levels between the control group and the COPD group; p < 0.05 indicates a statistically significant difference. C, D Correlation analysis evaluating the relationships between absolute eosinophil count, eosinophil percentage, and IL-24 levels in the blood of healthy individuals. E, F Correlation analysis evaluating the relationships between absolute eosinophil count, eosinophil percentage, and IL-24 levels in the blood of patients with eosinophilic COPD. G, H Analysis of serum EOS and EOS% levels in COPD patients with low and high IL-24 concentrations

We first perform a Shapiro-Wilk normality test on the data. The results showed that the plasma IL-24 level (W = 0.8767, p = 0.002) and EOS count (W = 0.8612, p < 0.001) in the patient group (N = 45) were not normally distributed. The plasma IL-24 levels (W = 0.8767, p = 0.002) and EOS% (W = 0.6970, p < 0.001) in the patient group (N = 45) were not normally distributed. The plasma IL-24 level (W = 0.9353, p = 0.1153) and EOS count (W = 0.9536, p = 0.3020) in the control group (N = 25) were normally distributed. The plasma IL-24 levels (W = 0.9353, p = 0.1153) and EOS% (W = 0.9357, p = 0.1178) in the control group (N = 25) were normally distributed. Since the patient group data do not conform to the normal distribution, and the Pearson correlation requires the data to obey the normal distribution, we changed all correlation analyses to Spearman rank correlation (non-parametric method, no normal distribution required) to ensure methodological consistency and reliability of results.

ELISA results showed significantly increased plasma IL-24 levels in COPD patients compared to healthy controls (Fig. 1B, p = 0.002). Correlation analysis showed that plasma IL-24 levels were not correlated with eosinophil count or eosinophil percentage in healthy controls: IL-24 levels were not correlated with absolute eosinophil counts (Fig. 1C, r = -0.0084, p = 0.6889), nor with eosinophil percentage (Fig. 1D, r = -0.0508, p = 0.8096). In contrast, plasma IL-24 levels in COPD patients were significantly positively correlated with eosinophil count and eosinophil percentage: IL-24 levels were positively correlated with absolute eosinophil counts (Fig. 1E, r = 0.5055, p = 0.0004) and positively correlated with eosinophil percentage (Fig. 1F, r = 0.5977, p = 0.0001). The median plasma IL-24 concentration (5.95 pg/mL) of COPD patients was used as the cut-off value, and the patients were divided into low IL-24 group (n = 22) and high IL-24 group (n = 23). Serum EOS and EOS% levels were higher in patients with higher IL-24 concentrations in COPD than in patients with lower IL-24 concentrations (EOS p = 0.0023, EOS% p = 0.003) (Fig. 1G, H).

A multiple linear regression model was used to analyze the independent association between IL-24 and EOS%, with EOS% as the dependent variable, and IL-24 concentration, age, sex, and smoking history as independent variables. The results showed (Table 2): after adjusting for age, sex, and smoking history, IL-24 levels were significantly independently positively correlated with EOS count (β = 0.221, 95% CI: 0.065-0.376, p = 0.007), with a standardized regression coefficient β = 0.400, having the greatest impact among the four variables.

Table 2.

Multiple linear regression analysis of factors associated with blood eosinophil count in COPD patients

Independent Variables	β	95% CI	Standardized Cofficients β	P value
Constant	11.556	3.484 - 19.627	-	0.006
IL-24	0.221	0.065 - 0.376	0.400	0.007
Age (years)	-0.141	-0.249 to -0.033	-0.346	0.011
Male	0.950	-2.175 - 4.075	0.085	0.543
Smoking	0.167	-1.783 - 2.117	0.023	0.863

Role of IL-24 in enhancing differentiation of EOL-1 cells into eosinophils under experimental conditions

Previous research has established the use of IL-5 and BA as inducers of eosinophil differentiation. The present study explores the potential of IL-24 to facilitate the differentiation of EOL-1 cells. Cell viability was measured at multiple time points (24 hours, 48 hours, 3 days, 6 days, and 9 days) using the CCK-8 assay under varying concentrations of IL-24, BA, and IL-5. At 48 hours, the EOL-1 cells treated with 50 μ M BA and 1 ng/mL IL-5 exhibited the highest viability (139%), whereas other treatment groups demonstrated viability ranging from 89% to 113%. After six days, all treatment groups showed increased viability relative to controls, except for the group stimulated with 10 ng/mL IL-24, which exhibited the lowest viability at 88%. By day 9, all groups surpassed control viability levels, with the 10 ng/mL IL-24-treated group achieving the highest viability of 137% (Fig. 2A). These results indicate that BA in combination with various concentrations of IL-24 or IL-5 does not exert cytotoxic effects on EOL-1 cells, thereby permitting further differentiation assays.

Fig. 2.

Butyric acid differentiates EOL-1 cells into eosinophils. A Cell viability at 24 h, 48 h, 3 d, 6 d, and 9 d after stimulating EOL-1 cells with BA and different concentrations of IL-24 and IL-5. B Morphological appearance of native eosinophils in bronchoalveolar lavage fluid (BALF) from COPD patients. C H&E staining of EOL-1 cells stimulated with BA and different concentrations of IL-24 and IL-5 at 3 d, 6 d, and 9 d. Scale bar = 50 μ m. The square indicates differentiated eosinophils, while the horizontal line marks eosinophil precursors. D Eosinophil conversion rates from cell counts of ≥ 200 cells per group (** p < 0.01, *** p < 0.005, **** p < 0.001; two-way ANOVA)

To observe the typical morphology of eosinophils, bronchoalveolar lavage fluid (BALF) was collected from patients with eosinophilic COPD. Following centrifugation, the cell pellet was obtained and used to prepare smears for hematoxylin-eosin (H&E) staining. The morphological characteristics of eosinophils, including bilobed nuclei and eosinophilic cytoplasmic granules, were observed under a light microscope, and representative images were captured (Fig. 2B).

To evaluate whether IL-24 and BA induce eosinophilic differentiation of EOL-1 cells, we examined morphological changes and eosinophil-specific markers. EOL-1 cells were treated with: 50 μ M BA alone; 50 μ M BA combined with 1 or 10 ng/mL IL-24;50 μ M BA combined with 1 or 10 ng/mL IL-5; or IL-24 alone at 1 or 10 ng/mL for 3, 6, and 9 days.Morphological assessments were performed using inverted microscopy and hematoxylin & eosin (H&E) staining. Untreated EOL-1 cells measured approximately 15–20 μ m in diameter and exhibited myeloid morphology with round or oval nuclei. After 3 and 6 days of treatment, cell size decreased to 9–17 μ m, and distinct bilobed nuclei became apparent. Prolonged treatment increased the proportion of eosinophil-differentiated cells, accompanied by progressively pronounced morphological changes. After 9 days, prominent cytoplasmic granulation was observed in most cells (Fig. 2C).

Quantitative analysis of H&E-stained cells revealed a time-dependent increase in the conversion of EOL-1 cells to eosinophils. Notably, synergistic treatment with butyrate and varying concentrations of IL-24 or IL-5 significantly enhanced the differentiation rate compared to BA alone. Specifically, groups treated with 50  μ M BA alone, combined with 1 ng/mL or 10 ng/mL IL-24, or combined with 1 ng/mL or 10 ng/mL IL-5 exhibited statistically significant increases in eosinophil conversion at daysdays 3, 6, and 9 . Furthermore, the conversion rates in groups receiving combined BA and IL-24 or IL-5 were significantly higher than those treated with BA alone. In contrast, treatment with IL-24 alone did not produce significant differences in differentiation rates across time points (Fig. 2D, Table 3). Collectively, these findings suggest that, in this in vitro system, IL-24 synergized with BA to enhance the differentiation rate of EOL-1 cells toward an eosinophil-like phenotype.

Table 3.

Differentiation Rate of EOL-1 Cells toward Eosinophils (%)

Treatment	3 days	6 days	9 days
EOL-1	3.98	5.07	9.00
BA	5.94	15.6	29.1
50 μ M BA + 1 ng/mL IL-24	6.90	19.15	40.13
50 μ M BA + 1 ng/mL IL-5	7.56	13.97	42.27
50 μ M BA + 10 ng/mL IL-24	9.62	21.43	39.35
50 μ M BA + 10 ng/mL IL-5	10.05	21.5	40.23
1 ng/mL IL-24	5.39	10.7	18.07
10 ng/mL IL-24	8.51	14.33	19.57

The proportion of EOL-1 cells differentiating into eosinophils under different treatment conditions over time

Expression of IL-24 receptors in eosinophils

The present study demonstrates that IL-24 synergistically potentiates butyrate-induced differentiation of EOL-1 cells. On day 3 post-treatment with IL-24 and other agents, IL-5 receptor alpha (IL-5Rα ) expression remained minimal. By day 6, IL-5Rα expression reached 46.1% in cells treated with 50 μ M BA alone and 19.25% in cells treated with 50 μ M BA with 1 ng/mL IL-24. Additional treatment groups exhibited IL-5Rα expression levels of 15.2% (50 μ M butyrate with 1 ng/mL IL-5), 64.5% (50 μ M butyrate with 10 ng/mL IL-24), and 8.65% (50 μ M butyrate plus 10 ng/mL IL-5). Notably, IL-24 alone induced a lower conversion rate compared to its combination with butyrate, indicating that IL-24 enhances BA’s capacity to upregulate IL-5Rα expression and promote differentiation of EOL-1 cells into eosinophils (EOS) (Fig. 3A, Table 4).

Fig. 3.

Expression of IL-24 receptors by eosinophils. A IL-5Rα expression in EOL-1 cells after stimulation with butyric acid (BA) and various concentrations of IL-24 and IL-5. B, C Expression of the IL-24 receptor subunit IL-20Rα in EOL-1 cells treated with BA and different concentrations of IL-24 and IL-5 over 3, 6, and 9 d (*p < 0.05, ***p < 0.005, ****p < 0.001; two-way ANOVA)

Table 4.

IL-5Rα expression in EOL-1 cells (%)

Treatment	3 days	6 days	9 days
EOL-1	2.55	2.20	19.50
BA	3.75	46.10	66.50
50 μ M BA + 1 ng/mL IL-24	1.77	19.25	12.15
50 μ M BA + 1 ng/mL IL-5	8.00	15.20	15.05
50 μ M BA + 10 ng/mL IL-24	3.40	64.50	24.05
50 μ M BA + 10 ng/mL IL-5	2.70	8.65	26.90
1 ng/mL IL-24	3.50	5.59	14.65
10 ng/mL IL-24	2.40	18.25	25.75

Quantitative analysis of the proportion of IL-5Rα -positive cells across different treatment conditions

IL-22 receptor alpha (IL-22Rα ) and IL-20 receptor alpha (IL-20Rα ) serve as receptors for IL-24. This investigation evaluated the expression of IL-24 receptors on the surface of mature eosinophils. EOL-1 cells were stimulated with butyrate alongside varying concentrations of IL-24 and IL-5 for 3, 6, and 9 days, followed by assessment of surface receptor expression. IL-22Rα expression remained unchanged across conditions. Conversely, IL-20Rα expression increased progressively with stimulation duration at days 3, 6, and 9. Significant differences in IL-20Rα expression were observed in cells treated with 50 μ M butyrate combined with 1 ng/mL IL-24, 1 ng/mL IL-5, 10 ng/mL IL-24, or 10 ng/mL IL-5 at days 3 and 9. Moreover, IL-20Rα expression was significantly elevated in groups receiving 50 μ M BA with cytokines compared to BA alone (Fig. 3B, C, Table 5).

Table 5.

IL-20Rα Expression in EOL-1 Cells (%)

Treatment	3 days	6 days	9 days
EOL-1	2.09	13.50	12.79
BA	1.54	17.40	20.39
50 μ M BA + 1 ng/mL IL-24	1.85	16.20	19.53
50 μ M BA + 1 ng/mL IL-5	2.14	18.00	25.30
50 μ M BA + 10 ng/mL IL-24	1.93	17.10	21.03
50 μ M BA + 10 ng/mL IL-5	2.06	17.90	16.56
1 ng/mL IL-24	2.16	12.10	10.08
10 ng/mL IL-24	1.63	7.40	9.40

Quantitative analysis of the proportion of IL-20Rα -positive cells across different treatment conditions

Combined with H&E staining results, it was observed that BA stimulation promotes the transformation of EOL-1 cells into EOS. Furthermore, the addition of butyrate alongside different concentrations of IL-24 and IL-5 at varying stimulation durations enhances this transformation. Post-stimulation, EOS cells express IL-20Rα on their surface, indicating that IL-20Rα serves as a target for IL-24.

Under the conditions of this experiment, IL-24 is associated with fibrotic changes in lung fibroblasts

Previous studies have shown that IL-24 can induce the expression of pro-inflammatory cytokines and matrix metalloproteinases in fibroblasts, suggesting its involvement in tissue inflammation and remodeling processes. In the present study,the impact of varying concentrations of IL-24 on pulmonary fibroblast proliferation was investigated. Concentrations of 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, and 10 ng/mL IL-24 did not significantly affect fibroblast proliferation; however, a concentration of 100 ng/mL inhibited proliferation (Fig. 4A). To further elucidate the effects of IL-24 on fibrotic activity, RT-PCR analyses were conducted to measure the expression of fibrosis-associated markers, including α -SMA, COL-1, and VEGF, in pulmonary fibroblasts exposed to different IL-24 concentrations.

Fig. 4.

Different concentrations of IL-24 promote fibrosis levels in pulmonary fibroblasts. A Cell viability at 48 hours after stimulation of pulmonary fibroblasts with different concentrations of IL-24. B RNA extracted from fibroblasts stimulated with different concentrations of IL-24 was analyzed by PCR to assess expression of fibroblast-related genes. C, D Immunofluorescence analysis of fibroblast-related protein expression in pulmonary fibroblasts stimulated with different concentrations of IL-24. Scale bar = 20 μ M. E Western blot analysis of fibroblast-related protein expression in pulmonary fibroblasts stimulated with different concentrations of IL-24, following protein extraction from the fibroblasts. (* P<0.05, ** P<0.01, *** P<0.005, **** P<0.001 one-way ANOVA

The results demonstrated that treatment with 1 ng/mL IL-24 elicited the most pronounced increase in mRNA levels of extracellular matrix components α -SMA, COL-1, and VEGF. This concentration exhibited the strongest pro-fibrotic effect on pulmonary fibroblasts, whereas the lowest concentration tested (0.01 ng/mL) produced the minimal effect (Fig. 4B). All other treatment groups showed statistically significant differences when compared to the 1 ng/mL IL-24 group.

Subsequent co-culture experiments involving pulmonary fibroblasts and varying IL-24 concentrations revealed that both Western blot and immunofluorescence analyses detected the highest expression levels of COL-1 and TGF-β in fibroblasts co-cultured with 1 ng/mL IL-24. In contrast, fibroblasts cultured without IL-24 exhibited the lowest expression of these markers, indicating that, under the present experimental conditions, IL-24 treatment was associated with enhanced fibrotic responses in pulmonary fibroblasts (Fig. 4C–E). Furthermore, α -SMA and TGF-β expression levels were elevated most significantly in the group co-cultured with 1 ng/mL IL-24, with the fibroblast-only group showing the lowest expression, thereby suggesting a potential role for IL-24 in pulmonary fibroblast-mediated fibrotic processes in this experimental model.

Examines the influence of eosinophils on fibrosis in pulmonary fibroblasts

To investigate the effect of eosinophils on the fibrotic phenotype of pulmonary fibroblasts, EOL-1 cells, EOS, and pulmonary fibroblasts were co-cultured at a 4:1 ratio for three days, after which fibrosis-associated markers, including α -SMA, COL-1, and VEGF, were quantified in the fibroblasts. RT-PCR analysis indicated that co-culture with EOL-1 cells moderately elevated the RNA expression of extracellular matrix components (α -SMA and COL-1) and VEGF in fibroblasts. Notably, the EOS co-culture group exhibited significantly higher RNA levels of α -SMA, COL-1, and VEGF compared to other groups, suggesting a pronounced enhancement of pulmonary fibrosis.

Statistical analyses confirmed significant differences between the EOS-fibroblast co-culture and all other experimental groups (Fig. 5A). These findings were corroborated by immunofluorescence (Fig. 5B, C) and Western blot (Fig. 5D) assays, which demonstrated that the EOS-fibroblast co-culture group had the highest expression of COL-1, α -SMA, and TGF-β proteins, whereas fibroblasts cultured alone showed the lowest expression levels. Collectively, these results suggest that eosinophil co-culture potentiates fibrotic responses in pulmonary fibroblasts, potentially mediated through activation of the TGF-β signaling pathway.

Fig. 5.

Co-culture of eosinophils with pulmonary fibroblasts potentiates fibrotic activity. A RT-PCR analysis of fibrosis-associated gene expression in pulmonary fibroblasts after co-culture. B, C Immunofluorescence staining for collagen type I (COL-1) and α -smooth muscle actin (α -SMA) following eosinophil-fibroblast co-culture. Scale bar = 20 μ m. D Western blot analysis of fibrosis-related proteins in pulmonary fibroblasts after co-culture (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA)

Expression of IL-24 receptors in co-culture systems and their inflammatory profiles

Given that the IL-24 signaling pathway typically involves receptor expression on effector cells, we further examined the expression of IL-20IL-22Rα and IL-22Rα on the surface of eosinophils and fibroblasts in the co-culture system to investigate whether they also participate in the response.

Following co-culture of EOL-1 cells, eosinophils, and pulmonary fibroblasts at a 4:1 ratio, the expression levels of IL-24 receptor subunits on the cell surface were assessed in each group. The results showed that in the eosinophil-pulmonary fibroblast co-culture group, IL-22Rα expression on pulmonary fibroblasts was significantly elevated to 22.1% , whereas only 1.1% of pulmonary fibroblasts cultured alone expressed IL-22Rα (Fig. 6A, B).

Fig. 6.

Co-culture of eosinophils with pulmonary fibroblasts activates IL-24 receptors and inflammatory responses. A, B IL-22Rα expression in fibroblasts induced by eosinophil-fibroblast co-culture. C, D Expression of IL-22Rα and IL-20Rα on eosinophils following co-culture with fibroblasts. E Cytokine levels (IL-6, IL-8, IFN-γ ) in culture supernatant after eosinophil-fibroblast co-culture (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA)

We further examined IL-20Rα expression on eosinophils in the co-culture system. The results demonstrated that IL-20Rα expression on eosinophils was highest in the EOL-1 cell-pulmonary fibroblast co-culture group, reaching 5.45% , compared to only 0.37% in EOL-1 cells cultured alone. Statistical analysis revealed that the EOL-1 and pulmonary fibroblast co-culture group exhibited significant differences compared to all other groups (Fig. 6C, D).

Analysis of IL-6, IL-8, and IFN-γ levels in co-culture supernatants revealed highest IL-6 (3696 pg/mL) and IL-8 (3680 pg/mL) expression in the eosinophil–lung fibroblast co-culture group. The fibroblast-only group showed lowest IL-6 (1570 pg/mL) and IL-8 (2095 pg/mL) expression. Significant differences in IL-6 and IL-8 levels were observed between eosinophil–fibroblast and fibroblast-only groups. IFN-γ analysis showed highest expression in EOL-1–fibroblast co-culture (73.9 pg/mL) and lowest in eosinophil–fibroblast co-culture (35.6 pg/mL) (Fig. 6E).

Discussion

This study explored the influence of IL-24 and EOS on inflammatory responses and tissue remodeling in COPD. Clinical sample analysis revealed that serum IL-24 levels in patients with COPD were significantly positively correlated with both the absolute eosinophil count and the eosinophil percentage. Furthermore, in vitro experiments suggested that, under the present experimental conditions, IL-24 treatment was associated with the differentiation of EOL-1 cells toward an eosinophil-like phenotype but also stimulates lung fibroblasts to secrete various pro-inflammatory mediators. Concurrently, IL-24 promotes the excessive deposition of the extracellular matrix (ECM). These findings suggest that IL-24 may contribute to airway tissue remodeling and exacerbate the inflammatory response in COPD patients by promoting the proliferation and differentiation of eosinophils.

Regarding the effects of butyrate on eosinophils, there is some discrepancy in the literature, which may be closely related to the concentrations used. Studies have shown that lower concentrations of butyrate (e.g., 50 μ M) combined with IL-5 can induce EoL-1 cell differentiation toward eosinophils, promoting eosinophilic granule protein expression and nuclear lobulation [16]; whereas higher concentrations of butyrate tend to induce apoptosis, possibly through histone deacetylase inhibition [17]. Tai et al. also reported that butyrate alone does not significantly induce apoptosis but can promote further maturation and apoptosis of cAMP-induced eosinophils [18].

In the present study, 50 μ M butyrate combined with IL-5 was used to induce EoL-1 cell differentiation toward eosinophils. This concentration was selected based on the study by Wang et al., which confirmed that 50 μ M butyrate combined with IL-5 effectively induces morphological characteristics of eosinophils (nuclear lobulation, increased eosinophilic granules) and upregulation of related factors in EoL-1 cells. This concentration falls within the pro-differentiation range reported in the literature, distinct from the apoptosis-inducing effects of high concentrations of butyrate (typically in the mM range), reflecting the concentration-dependent nature of butyrate’s effects.

The present study confirms that combined exposure to IL-24 and BA similarly enhances EOL-1 differentiation into mature EOS and increases IL-5 receptor expression, thereby facilitating EOS maturation. Consistent with these findings, prior research has shown that combined treatment with BA and IL-5 promotes EOL-1 cell differentiation into eosinophils and upregulates IL-5 receptor expression on their surfaces [19]. IL-5 is recognized as a pivotal growth factor for EOS, essential for their activation and maturation [20]. Mature EOS express distinctive receptor complexes, including the IL-5 dimeric receptor, which is rarely found on other immune cells except for a limited number of differentiated EOS [21].

IL-24 likely activates downstream signaling pathways by binding to its receptor complexes (IL-20R1/IL-20R2 or IL-22R1/IL-20R2), thereby promoting eosinophil differentiation [22]. In this investigation, direct stimulation of fibroblasts with 1 ng/ml IL-24 significantly elevated fibrosis-associated markers relevant to COPD, such as α -SMA and COL-1, exacerbating both acute exacerbations and chronic inflammatory states. α -SMA is widely acknowledged as a hallmark of myofibroblast differentiation [23]. During fibrotic progression, synthesis of type I collagen predominates over type III collagen, and its uncontrolled deposition leads to tissue stiffening and scar formation [24]. EOS serve as critical immune effector cells with diverse functions, contributing to homeostasis and pathogenesis across multiple tissues, including the lungs. These cells infiltrate tissues and participate in cellular immune responses. Within the respiratory tract, eosinophils play a central pathological role in chronic inflammatory diseases such as asthma and COPD [25](21). Eosinophil-driven inflammation not only contributes to COPD onset and progression but also promotes airway remodeling, impaired pulmonary function, airway hyperresponsiveness, and increased frequency of acute exacerbations [26] .

Chronic eosinophilic inflammation can induce tissue remodeling and fibrosis [27]. Eosinophil-derived mediators and cytokines stimulate fibroblast proliferation and collagen deposition, culminating in irreversible tissue damage [28]. Activated eosinophils release cytotoxic proteins—including major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin—that directly mediate persistent tissue injury and inflammatory responses [29]. Additionally, eosinophils secrete a spectrum of cytokines such as IL-1β , IL-4, IL-5, IL-13, tumor necrosis factor-α (TNF-α ), and transforming growth factor-β (TGF-β ), which regulate immunoglobulin E (IgE) synthesis and inflammatory cascades [30]. Collectively, these factors contribute to airway hyperresponsiveness, mucus hypersecretion, and fibrotic remodeling.

Recent evidence suggests that elevated eosinophil counts in the airways are associated not only with inflammation but also with tissue remodeling through interactions with other cell types [31]. Fibroblasts are the principal effector cells responsible for collagen synthesis and tissue repair, and eosinophils can promote the fibrosis mediated by these cells. These eosinophils release profibrotic factors such as TGF-β , thereby promoting fibrosis and aberrant collagen deposition within the airway wall. They may also upregulate IL-24 receptor expression on pulmonary fibroblasts. Furthermore, pulmonary fibroblasts contribute to immune regulation by secreting cytokines [32].

This study holds significant theoretical and clinical implications for understanding the role of IL-24 in COPD, particularly within the eosinophilic phenotype. While IL-24 has been primarily implicated in autoimmune pathologies such as psoriasis [33] , the present findings extend its functional repertoire to include regulation of Th2-mediated airway inflammation, offering novel insights into the immunopathogenesis of eosinophilic COPD. The data suggest that IL-24 exacerbates airway inflammation and remodeling by promoting eosinophil precursor differentiation and modulating downstream inflammatory mediator release.

From a clinical perspective, IL-24 presents dual potential as both a biomarker and therapeutic target. Previous studies have demonstrated that cytokine profiles in sputum and serum can differentiate inflammatory phenotypes in COPD [34]. The current findings indicate that IL-24 levels correlate with clinical parameters such as peripheral blood eosinophil counts and acute exacerbation frequency, supporting its utility as a predictive biomarker. Moreover, therapeutic interventions targeting the IL-24 signaling axis—such as monoclonal antibodies or receptor antagonists—may attenuate its proinflammatory and profibrotic effects, thereby mitigating airway fibrosis progression [4].

However, this study has several limitations. Firstly, although we expanded the clinical sample size ( to 45 patients), the total sample size remains relatively limited, and this is a single-center study; therefore, the conclusions need to be validated in larger, multicenter cohorts. Secondly, this study primarily relied on the EoL-1 cell line for mechanistic exploration, which cannot fully recapitulate the complex inflammatory microenvironment in vivo—a limitation common to similar mechanistic studies [35]. Although we provided morphological validation for the cell line model through comparison with native eosinophils derived from BALF , EoL-1 cells cannot completely mimic the complex functions of primary human eosinophils; thus, key findings need to be verified in primary cells in future studies. Thirdly, the specific signaling pathways (e.g. JAK/STAT, p38 MAPK) through which IL-24 regulates IL-5Rα expression and eosinophil differentiation have not been elucidated and require further validation through gene knockdown and pathway inhibition experiments. Lastly, the lack of co-localization staining in lung tissues from COPD patients and in vivo experimental data represents a major limitation for extrapolating these conclusions to clinical settings.

In conclusion, this study provides preliminary evidence suggesting that, in this experimental model, IL-24 may play a proinflammatory role in the pathogenesis of eosinophilic COPD, highlighting potential interactions between eosinophils and pulmonary fibroblasts. Eosinophils stimulate fibroblasts to augment extracellular matrix production, ultimately contributing to tissue fibrosis. Furthermore, co-culture experiments revealed IL-24 receptor expression on eosinophil surfaces, including prominent IL-20Rα and detectable IL-22Rα expression, suggesting intrinsic connections among IL-24, eosinophils, and pulmonary fibroblasts. Future research should prioritize in vivo functional validation using animal models—such as IL-24 knockout mice or neutralizing antibody interventions—and establish clinical cohorts to systematically assess the relationship between IL-24 expression and disease severity.

Abbreviations

α -SMA

α -smooth muscle actin

BA

Butyrate acid

BSA

Bovine serum albumin

CCK-8

Cell Counting Kit-8

cDNA

Complementary DNA

COL-1

Collagen type I

COPD

Chronic obstructive pulmonary disease

CS

Cigarette smoke

DAPI

4’,6-diamidino-2-phenylindole

ECM

Extracellular matrix

ELISA

Enzyme-Linked Immunosorbent Assay

EOL-1

Human eosinophilic leukemia cell line

EOS

Eosinophils

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

H&E

Hematoxylin and Eosin

IFN-γ

Interferon-gamma

IgE

Immunoglobulin E

IL-1β

Interleukin-1 beta

IL-4

Interleukin-4

IL-5

Interleukin-5

IL-5Rα

Interleukin-5 receptor alpha

IL-6

Interleukin-6

IL-8

Interleukin-8

IL-19

Interleukin-19

IL-20

Interleukin-20

IL-20Rα

Interleukin-20 receptor alpha

IL-22

Interleukin-22

IL-22Rα

Interleukin-22 receptor alpha

IL-24

Interleukin-24

JAK/STAT

Janus kinase/Signal transducer and activator of transcription

MAPK

Mitogen-activated protein kinase

MEM

Minimum Essential Medium

OD

Optical density

PCR

Polymerase Chain Reaction

PVDF

Polyvinylidene fluoride

RIPA

Radioimmunoprecipitation assay

RNA

Ribonucleic acid

RT-PCR

Real-Time Polymerase Chain Reaction

TBS-T

Tris-buffered saline with Tween 20

TGF-β

Transforming growth factor-beta

VEGF

Vascular endothelial growth factor

Authors’ contributions

XC and RL conceived the study, designed and performed the experiments, analyzed the data, and wrote the manuscript.CL,BL,ZG and XK confirmed the data and discussed this study. LJ and LX supervised the research and reviewed the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This research was supported by Capital’s Funds for Health Improvement and Research (No.2022-2-5092)

Data availability

No datasets were generated or analysed during the current study.

Materials availability

Not applicable.

Code availability

Not applicable.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of the Eighth Medical Center, Chinese PLA General Hospital, which also authorized the research. It was conducted in accordance with the ethics committee’s approval (309202109091005). The patients/participants provided their written informed consent to participate in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.