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Abstract
BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects.MethodsPrimary normal human lung and IPF-FB were exposed to TGF-β (5 ng/ml), Azithromycin (50 μM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry.ResultsAZT significantly reduced collagen secretion in TGF-β treated IPF-FB compared to TGF-β treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-β treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-β treated IPF-FB.ConclusionWe report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.
Highlights
Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed
AZT has enhanced anti-fibrotic effects on extracellular matrix formation, cytokine production and myofibroblast differentiation in IPF fibroblasts compared to controls We investigated whether Azithromycin (AZT) differently affects the fibrotic response in lung fibroblasts from normal human lung fibroblasts and IPF fibroblasts
For western blot analysis (d: FN; f: α-smooth muscle actin (αSMA)) one-way ANOVA followed by Tukey’s post was used to analyze data. a Col1A1 gene expression was significantly reduced in control and IPF fibroblasts after AZT and transforming growth factor-β (TGF-β) treatment compared to TGF-β alone (**p-value ≤0.01, ***p-value ≤0.001). b FN gene expression was not significantly altered after AZT neither in control nor in IPF fibroblasts compared to TGF-β alone. c Collagen production was measured by ELISA in cell culture supernatant from control (n = 4) and IPF fibroblasts (n = 5)
Summary
Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects. Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease causing dyspnea and cough, which leads to respiratory failure [1]. New drugs slow down disease progression and may prolong survival [3, 4]. New and improved drug strategies are urgently needed
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