IL-17 Induced Autophagy Regulates Mitochondrial Dysfunction and Fibrosis in Severe Asthmatic Bronchial Fibroblasts

Abstract

The accumulation of fibroblasts, their synthesis of extracellular matrix (ECM) proteins and their innate resistance to apoptosis are characteristics of subepithelial fibrosis observed in severe asthma. Interleukin-17 (IL-17) is an important regulator of airway remodeling in asthma. However, the contribution of IL-17 to the pro-fibrotic phenotype of bronchial fibroblasts is not well-characterized. In this study, we investigated whether IL-17 induced autophagy regulates mitochondrial and pro-fibrotic function in bronchial fibroblasts. The primary cultured bronchial fibroblasts isolated from non-asthmatic (NHBF) and severe asthmatic (DHBF) subjects were treated with IL-17 in order to ascertain its effect on mitochondrial function, mitochondrial quality control, and apoptosis using immunoblotting and flow cytometric analyses. At baseline, DHBF exhibited higher levels of mitophagy and mitochondrial biogenesis compared to NHBF. Immunohistochemical evaluation of bronchial biopsies showed intense PINK1 immunoreactivity in severe asthma than in control. IL-17 intensified the mitochondrial dysfunction and impaired the mitochondrial quality control machinery in NHBF and DHBF. Moreover, IL-17 augmented a pro-fibrotic and anti-apoptotic response in both group of fibroblasts. Inhibition of autophagy using bafilomycin-A1 reduced PINK1 expression in NHBF and restored the IL-17 mediated changes in PINK1 to their basal levels in DHBF. Bafilomycin-A1 also reversed the IL-17 associated fibrotic response in these fibroblasts, suggesting a role for IL-17 induced autophagy in the induction of fibrosis in bronchial fibroblasts. Taken together, our findings suggest that IL-17 induced autophagy promotes mitochondrial dysfunction and fibrosis in bronchial fibroblasts from both non-asthmatic and severe asthmatic subjects. Our study provides insights into the therapeutic potential of targeting autophagy in ameliorating fibrosis, particularly in severe asthmatic individuals.

Keywords: IL-17; autophagy; bronchial fibroblasts; fibrosis; mitochondria; mitochondrial dysfunction; severe asthma.

Figure 1

Enhancement of mitochondrial quality control in severe asthmatic (S-As) fibroblasts. In order to measure the basal expression levels, non-asthmatic fibroblasts (NHBF), and S-As fibroblasts (DHBF) were serum-starved for 24 h and thereafter, cultured in DMEM complete medium for 24 h. The fibroblasts were then subjected to Western Blot or flow cytometric analysis. β-actin or GAPDH was used as loading control as indicated. (A) Representative immunoblots and (B) densitometric analysis of autophagy markers, with LC3B lipidation represented as the ratio of LC3BII to LC3BI, and LAMP2A expression, mitophagy markers, PINK1 and Parkin, and mitochondrial biogenesis markers, SIRT1 and PGC1α. Data representative of three independent experiments. (C) Bar charts indicating NHBF (green) and DHBF (red) fluorescence when stained with MitoTracker Green, (D) MitoTracker Red and (E) Annexin V followed by flow cytometric analysis. 10,000 events were analyzed in each flow cytometry experiment. Data representative of three independent experiments. (F) Densitometric analysis and representative immunoblots depicting expression of anti-apoptotic protein, Survivin, in NHBF and DHBF. Data presented as mean ± SEM relative to NHBF (where indicated). Statistical significance assessed by Mann Whitney test. *p < 0.05, **p < 0.01.

Figure 2

Increased PINK1 expression in severe asthmatic bronchial biopsy tissues. Representative images taken at 40X magnification showing PINK1 immunostaining developed with 3,3′-diaminobenzidine diaminobenzidine (brown). Nuclei were counterstained with hematoxylin (blue). Representative bronchial biopsy sections from (A) healthy control showing weak, (B) severe asthmatic showing moderate to strong PINK1 protein expression. Arrows refer to bronchial epithelium. Arrowheads refer to fibroblasts.

Figure 3

IL-17 increases mitochondrial dysfunction in bronchial fibroblasts. NHBF and DHBF were serum-starved for 24 h and thereafter, cultured in DMEM complete medium without or with IL-17 (25 ng/ml) for up to 96 h. (A) mRNA expression of IL-6 in NHBF and DHBF upon IL-17 stimulation was analyzed by qPCR and expressed as fold expression change relative to the respective untreated control post normalization to housekeeping gene 18 s rRNA. Data representative of n = 3. (B) Representative histograms and bar charts indicating fluorescence in mean fluorescence intensity (MFI) in cells stained with MitoTracker Green, (C) MitoTracker Red, and (D) Annexin V followed by flow cytometric analysis. 10,000 events were analyzed in each flow cytometry experiment. Data representative of three independent experiments. (E) Densitometric analysis and representative immunoblots depicting expression of anti-apoptotic protein, Survivin, in NHBF (left panel) and DHBF (right panel) upon IL-17 stimulation. GAPDH was used as loading control. Data presented as mean ± SEM after normalization to the respective untreated control (Complete Medium). Statistical significance assessed by unpaired t-test with multiple comparisons using the Holm-Sidak method. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4

IL-17 impairs mitochondrial quality control in bronchial fibroblasts. NHBF and DHBF were serum-starved for 24 h and thereafter, cultured in DMEM complete medium without or with IL-17 (25 ng/ml) for 48 h for mRNA analysis and 96 h for protein analysis. (A) mRNA expression of autophagy genes, BECN1, ATG5, LC3B, SQSTM1, and LAMP2 in NHBF and DHBF upon IL-17 stimulation was analyzed by qPCR and expressed as fold expression change relative to the respective untreated control post normalization to housekeeping gene 18 s rRNA. Data representative of n = 3. (B) Representative immunoblots and (C) densitometric analysis of autophagy markers, with LC3B lipidation represented as the ratio of LC3BII to LC3BI and p62 expression, mitophagy markers, PINK1 and Parkin, and mitochondrial biogenesis markers, SIRT1, and PGC1α. Data representative of three independent experiments. β-actin was used as loading control. Data presented as mean ± SEM after normalization to the respective untreated control (Complete Medium). Statistical significance assessed by unpaired t-test with multiple comparisons using the Holm-Sidak method. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Autophagy triggers IL-17 induced mitochondrial dysfunction. NHBF and DHBF were serum-starved for 24 h, pre-treated with Bafilomycin-A1 (10 nM) for 4 h and thereafter, cultured in DMEM complete medium without or with IL-17 (25 ng/ml) for 48 h for mRNA analysis and 96 h for protein analysis. The cells were co-incubated with FCCP (10 μM) in the final 2 h of treatment. (A) mRNA expression of autophagy gene, BECN1, in NHBF (left panel) and DHBF (right panel) was analyzed by qPCR and expressed as fold expression change relative to the respective untreated control post normalization to housekeeping gene 18 s rRNA. Data representative of n = 3. (B) Representative immunoblots and (C) densitometric analysis depicting expression of autophagy markers, LC3B and p62, in NHBF and DHBF. GAPDH was used as loading control. Data representative of two independent experiments. (D) mRNA expression of PINK1 and (E) Parkin in NHBF (left panel) and DHBF (right panel) was analyzed by qPCR and expressed as fold expression change relative to the respective untreated control post normalization to housekeeping gene 18 s rRNA. Data representative of n = 3. Data presented as mean ± SEM after normalization to the respective untreated control. Statistical significance assessed by 1-way ANOVA with Tukey's multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

Autophagy triggers IL-17 induced pro-fibrotic phenotype. NHBF and DHBF were serum-starved for 24 h, pre-treated with Bafilomycin-A1 (10 nM) for 4 h and thereafter, cultured in DMEM complete medium without or with IL-17 (25 ng/ml) for 48 h. (A) mRNA expression of fibrotic genes, COL1A1, COL3A1, COL5A1 and FN1, at baseline in NHBF and DHBF. Data representative of n = 3. mRNA expression of (B) COL1A1, (C) COL3A1, (D) COL5A1, (E) FN1, and (F) ACTA2, in NHBF (left panel) and DHBF (right panel) was analyzed by qPCR and expressed as fold expression change relative to the untreated control post normalization to housekeeping gene 18s rRNA. Data representative of n=3. Data presented as mean ± SEM after normalization to NHBF or the respective untreated control. Statistical significance assessed by (A) unpaired two-tailed t-test or (B–F) 1-way ANOVA with Tukey's multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.