IL-5 signaling in asthmatic derived fibroblasts exacerbates airway remodeling through ECM dysregulation and apoptosis resistance

Abstract

Background: Airway remodelling, a critical feature of severe asthma, involves fibroblast-driven extracellular matrix (ECM) dysregulation. While IL-5 is pivotal in eosinophilic inflammation, its direct role in fibroblast-mediated fibrosis remains undefined.

Methods: Primary lung fibroblasts from asthmatic and healthy donors were stimulated with 0.5 ng/ml of IL-5 for several time points. ECM components, Matrix metalloproteinases (MMPs), Tissue inhibitor of metalloproteinases (TIMPs), and cytokines were analysed via quantitative real time-PCR (qRT-PCR), Western blot, ELISA, and flow cytometry. RNA sequencing and absolute gene set enrichment analysis (absGSEA) identified signaling pathways. Apoptosis was assessed using Annexin V/PI staining.

Results: IL-5 shown to markedly increase the expression of ECM proteins, including collagen I and fibronectin, in asthmatic fibroblasts. It also upregulated MMP-2 and MMP-3 expression, alongside increased levels of TIMP-1 and TIMP-2. Moreover, IL-5 promoted the secretion of IL-6 and TGF-β. RNA-seq analysis identified 472 differentially expressed genes in asthmatic fibroblasts, highlighting activation of the MAPK pathway and suppression of apoptosis through NR4A1 upregulation. IL-5 further reduced fibroblast apoptosis and enhanced IL-5Rα expression, indicating potential autocrine signalling.

Conclusion: IL-5 directly activates lung fibroblasts to drive airway remodelling in severe asthma through ECM deposition, MMP/TIMP imbalance, and pro-fibrotic cytokine secretion, positioning it as a dual mediator of inflammation and fibrosis with novel therapeutic potential.

Keywords: Airway Remodeling; Asthma; Fibrosis; Interlukin-5 (IL-5).

Fig. 1

Evaluating the impact of fibroblasts stimulation with IL-5 on the levels of ECM genes and proteins. A-F mRNA levels of ECM genes in normal and asthmatic derived lung fibroblasts post stimulation with IL-5. 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2.^−ΔΔct. Data shown are the mean fold changes ± SD of five separate experiment. Significance was assessed using two-tailed paired- student’sTest, *p < 0.05. (A) COL1A1, (B) COL3A1, (C) COL5A1, (D) FN1, (E) Tenascin C, and (F) Lumican. G-I Western immunoblots and densitometric analysis of expression in normal and asthmatic derived lung fibroblasts. G Collagen I, (H) fibronectin, (I) Tenascin C. Data shown are mean ± SD after normalization to the untreated control and are representative of five different experiments; Tubulin was used as loading control, significance was assessed using two-tailed paired t-Test, *p < 0.05

Fig. 2

Evaluating the stimulatory effect of IL-5 on the levels of MMPs. A-D Showing the mRNA levels of the four MMPs in normal and asthmatic derived lung fibroblasts post stimulation with IL-5. Data shown is representative for five different experiments; 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2.^−ΔΔct, Significance was assessed using two tailed paired student’s t-Test, *p < 0.05. A MMP2, (B) MMP-3, (C) MMP-7, and (D) MMP9. E–H Western immunoblots and densitometric analysis of expression in normal and asthmatic derived lung fibroblasts post 48 h of IL-5 stimulation. Data shown are mean ± SD after normalization to the untreated control and are representative of five different experiments; Tubulin was used as loading control, significance was assessed using two tailed paired student’s t-Test, *p < 0.05. E MMP-2, (F) MMP-3, (G) MMP-7, and (H) MMP-9. I Showing the secreted levels of MMP-3 from normal and asthmatic derived fibroblasts supernatant measured by ELISA. J Showing the activity levels of MMP-3 in both normal and asthma derived fibroblasts respectively measured by ELISA in a mU/ml. Data shown is the mean ± SD of at least four separate experiments; two tailed pared student’s t-Test statistical analysis was done *p < 0.05

Fig. 3

Lung fibroblasts stimulated with IL-5 have a higher level of TIMP-1 and TIMP-2. A-B showing the mRNA levels of both TIMP-1 and TIMP-2 in both Normal and asthma derived lung fibroblasts. 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2^−ΔΔct. Data shown are the mean ± SD of five separate experiments; two-tailed paired student’s t-Test statistical analysis was performed *P < 0.05. A TIMP1, and (B) TIMP2. C-D Western immunoblots and densitometric analysis of expression in normal and asthmatic derived lung fibroblasts post 48 h of IL-5 stimulation. Data shown are the mean ± SD after normalization to the untreated control and are representative of five different experiments; Tubulin was used as loading control, significance was assessed using two-tailed paired t-Test, *p < 0.05. C TIMP-1, and (D) TIMP-2. E MMP-9/TIMP-1 ratios in normal and asthmatic fibroblasts in the presence of IL-5, significance was assessed using two tailed paired student’s t-Test, *p < 0.05. F MMP-2/TIMP-2 ratio in normal and asthmatic fibroblasts in the presence of IL-5, significance was assessed using two tailed paired student'st-Test

Fig. 4

IL-5 promotes the expression of profibrotic and proinflammatory cytokine in asthmatic fibroblasts. A-D Expression of cytokines mRNA upon IL-5 stimulation (A) mRNA levels of TGF-β, (B) mRNA levels of IL11, (C) mRNA levels of IL6, and (D) mRNA levels of TNF-α in normal and asthma derived lung fibroblasts post 1 h of IL-5 stimulation. For all, 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2.^−ΔΔct. Data shown are the mean of the fold change between IL-5 stimulated compared to control ± SD of three separate experiment; paired two-tailed student’s t-Test statistical analysis was done *p < 0.05. E–F Flow cytometric analysis of intracellular expression of cytokines. Histogram show expression in unstimulated (blue histogram) or IL-5-stimulated (red histogram) cells. Graphs show the mean expression in IL-5 stimulated cells normalized to control ± SD of five separate experiments for (E) IL-6, (F) TGF-β, and (G) TNF-α. Significance was assessed using two tailed paired student’s t-Test, *p < 0.05. H Showing the secreted levels of TGF-β from normal and asthmatic derived fibroblasts supernatant measured by ELISA, (I) Showing the secreted levels of IL-6 from normal and asthmatic derived fibroblasts supernatant measured by ELISA. Data shown are the mean of the fold change between IL-5 stimulated compared to control ± SD of six separate experiment; paired two-tailed student’s t-Test statistical analysis was done *p < 0.05, **p < 0.01

Fig. 5

IL-5 inducing the expression of IL-5Ra in lung derived fibroblasts. A mRNA levels of IL5RA in both normal and asthmatic derived lung fibroblasts. 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2.^−ΔΔct. Data shown are the mean ± SD of four separate experiment; two-tailed paired student’s t-Test statistical analysis was done * p < 0.05, ** p < 0.01, *** p < 0.001. B Cultured normal and asthmatic fibroblasts, captured at 40X magnification, display immunofluorescence staining for IL-5Rα (green), vimentin (red), and DAPI (blue) in both cell types, along with mouse and rabbit IgG isotype controls. C Evaluation of the mean fluorescence intensity (MFI) of IL-5Rα expression in lung fibroblasts, quantified following 24 h of IL-5 stimulation. Statistical significance was determined using two-tailed paired Student's t-test based on data from three independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 6

Lung derived fibroblasts stimulated with IL-5 exhibit a distinct transcriptional profiles and enhances cellular survival. A Leading edge analysis showed a significant enrichment in the regulation of programmed cell death. The heatmap demonstrates the clustering of upregulated and down regulated genes, particularly NR4A1, FZD9 and FOXO1; upregulated genes presented in red, down regulated genes presented in green. B Representative figure of Annexin V staining of both normal and asthma derived fibroblasts post 48 h of IL-5 stimulation, (C) bar graphs representing the percentage of both early and late apoptotic cells post IL-5 stimulation, data shown is the mean ± SD of four separate experiment, (D) representative immunoblots of Caspase-3 in asthma derived lung fibroblasts pre and post IL-5 stimulation, (E) Densitometric analysis of the immunoblots, proteins were normalized against GAPDH, data shown are the mean ± SD of four separate experiment; unpaired two-tailed student’s t-Test statistical analysis was done *p < 0.05, **p < 0.01. F The upregulation of the mRNA level of NR4A1 post IL-5 stimulation in asthmatic derived fibroblasts, 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2.^−ΔΔct. Data are the mean ± SD of four separate experiments; significance was determined using two-tailed paired student t-Test ** p < 0.01