Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients. Fourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients’ informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFβ1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test. Cultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells). In cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFβ1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

中文翻译：

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尼达尼布下调从受间质性肺疾病影响的系统性硬化症患者中培养的单核细胞衍生巨噬细胞中的促纤维化 M2 表型

系统性硬化症（SSc）是一种自身免疫性结缔组织疾病，其特征是血管病变以及皮肤和包括肺在内的多个内脏器官的进行性纤维化。巨噬细胞是参与皮肤和肺部免疫炎症损伤的主要细胞，替代性活化（M2）巨噬细胞似乎通过释放促纤维化细胞因子（IL10）和生长因子（TGFβ1）而具有促纤维化作用。尼达尼布是一种针对多种纤维化介质的酪氨酸激酶抑制剂，被批准用于治疗 SSc 相关的间质性肺疾病 (ILD)。该研究旨在评估尼达尼布在下调从 SSc-ILD 患者获得的培养单核细胞源性巨噬细胞 (MDM) 中促纤维化 M2 表型方面的作用。14 名 SSc 患者，满足 2013 年 ACR/EULAR SSc 标准，10 名受 ILD 影响的 SSc 患者 (SSc-ILD 患者)，4 名未受 ILD 影响的 SSc 患者 (SSc 无 ILD 患者)，以及 5 名自愿健康受试者 (HS) ，在获得伦理委员会批准和患者知情同意后，在热那亚大学临床风湿病科招募。从外周血中分离单核细胞，分化为 MDM，然后在不进行任何处理的情况下维持在生长培养基中（未处理的细胞），或用尼达尼布（0.1 和 1μM）处理 3、16 和 24 小时。通过定量实时聚合酶链反应评估巨噬细胞清道夫受体（CD204、CD163）、甘露糖受体-1（CD206）、Mer酪氨酸激酶（MerTK）、识别M2巨噬细胞以及TGFβ1和IL10的基因表达。通过蛋白质印迹研究蛋白质合成，并通过 ELISA 评估活性 TGFβ1 的水平。使用非参数Wilcoxon 检验进行统计分析。培养的未经处理的 SSc-ILD MDM 显示 CD206 (p < 0.05)、CD204 和 MerTK (p < 0.01) 的蛋白质合成显着增加，同时 MerTK 和 TGFβ1 的基因表达显着上调 (p < 0.05；p < 0.01）与 HS-MDM 相比。此外，与没有 ILD 的 MDM 相比，来自 SSc-ILD 患者的培养的未经处理的 MDM 中 CD206 和 MerTK 的蛋白质合成以及 TGFβ1 的基因表达显着较高（p < 0.05；p < 0.01）。在培养的 SSc-ILD MDM 中，与未处理的细胞相比，处理 24 小时后，0.1 和 1μM 尼达尼布显着下调 CD204、CD206、CD163 (p < 0.05) 和 MerTK (p < 0.01) 的基因表达和蛋白质合成。仅限于 MerTK 和 IL10，两种尼达尼布浓度在治疗 16 小时后已显着下调其基因表达（p < 0.05）。在培养的 SSc-ILD MDM 中，治疗 24 小时后，0.1 和 1μM 尼达尼布显着减少活性 TGFβ1 的释放（与未处理细胞相比，p < 0.05）。在 SSc-ILD 患者培养的 MDM 中，