BACKGROUND 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 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. METHODS Primary 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. RESULTS AZT 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. CONCLUSION We 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.

中文翻译：

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与对照组相比，阿奇霉素对特发性肺纤维化 (IPF) 患者的肺成纤维细胞具有增强作用[已纠正]。

背景特发性肺纤维化（IPF）是一种无法治愈的慢性致命性肺部疾病，迫切需要新的药物策略。患病细胞和健康细胞之间的行为差​​异是众所周知的，从 IPF 患者和对照中分离的细胞之间的药物反应也可能不同。大环内酯类阿奇霉素 (AZT) 具有抗炎和免疫调节特性。最近已经描述了抗纤维化作用。然而，与对照 FB 直接比较对原代 IPF 成纤维细胞 (FB) 的抗纤维化作用尚不清楚。我们假设 IPF-FB 在抗纤维化作用方面与 AZT 的反应不同。方法 在基因表达分析之前，将原代正常人肺和 IPF-FB 单独或联合暴露于 TGF-β (5 ng/ml)、阿奇霉素 (50 μM)。通过 ELISA 评估前胶原 Iα1 分泌，通过蛋白质印迹评估蛋白表达（αSMA、纤连蛋白、ATP6V1B2、LC3 AB (II/I)、p62、Bcl-xL）。在对照-FB中进行阿奇霉素处理后进行微阵列分析以筛选相关基因和通路。通过流式细胞术分析细胞凋亡和腔内溶酶体pH。结果 与单独使用 TGF-β 治疗相比，AZT 显着减少 TGF-β 治疗的 IPF-FB 中的胶原蛋白分泌，但在对照-FB 中则不然。在 IPF 和对照 FB 中，AZT 治疗后促纤维化基因表达同样降低。P62 和 LC3II/I 蛋白质印迹显示对照和 IPF-FB 中 AZT 后自噬通量受损，对照和 IPF-FB 中 AZT 后 LC3II/I 显着增加，表明自噬抑制增强。与 AZT 后的对照组相比，TGF-β 处理的 IPF-FB 的早期细胞凋亡显着更高。用 AZT 处理的对照 FB 的微阵列分析显示溶酶体途径受损。与对照组相比，IPF-FB 中添加 AZT 后，ATP 酶和溶酶体 pH 调节剂 ATP6V0D2 的增加显着减少。IPF 和对照 FB 中的溶酶体功能均受损，但 TGF-β 处理的 IPF-FB 中 pH 值显着升高。结论 我们报告了 AZT 后不同的治疗反应，与对照 FB 相比，IPF 的抗纤维化和促凋亡作用增强。溶酶体功能可能受损会导致这些影响。总之，IPF 和对照细胞的不同基线细胞表型和行为有助于增强 AZT 治疗后 IPF-FB 的抗纤维化和促凋亡作用，并加强其作为新的潜在抗纤维化化合物的作用，应进一步评估在临床研究中。