背景:系统性硬化症(SSc)是一种自身免疫性结缔组织疾病,其特征是血管病变和皮肤和一些内脏器官的进行性纤维化,包括肺.巨噬细胞是参与皮肤和肺部免疫炎症损伤的主要细胞,和选择性活化的(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。Nintedanib是一种靶向多种纤维化介质的酪氨酸激酶抑制剂,已被批准用于治疗SSc相关的间质性肺病(ILD)。该研究旨在评估尼达尼布在下调从SSc-ILD患者获得的培养的单核细胞衍生的巨噬细胞(MDMs)中的促纤维化M2表型中的作用。
方法:14例SSc患者,符合2013年ACR/EULAR标准的SSC,10例受ILD影响的SSc患者(SSc-ILDpts),4例未受ILD影响的SSc患者(SScptsno-ILD),和5名自愿健康受试者(HSs),被招募在临床风湿病学系-热那亚大学,在获得伦理委员会批准和患者知情同意后。从外周血中分离出单核细胞,区分为MDM,然后保持在生长培养基中,没有任何处理(未处理的细胞),或用尼达尼布(0.1和1µM)处理3、16和24小时。巨噬细胞清道夫受体(CD204,CD163)的基因表达,甘露糖受体-1(CD206),Mer酪氨酸激酶(MerTK),识别M2巨噬细胞,通过定量实时聚合酶链反应评估TGFβ1和IL10。通过Western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。
结果:培养的未经处理的SSc-ILDMDMs显示CD206的蛋白质合成显着增加(p<0.05),CD204和MerTK(p<0.01),与HS-MDMs相比,MerTK和TGFβ1的基因表达显着上调(p<0.05;p<0.01)。此外,与没有ILD的MDM相比,在SSc-ILDpts的未经处理的MDM中,CD206和MerTK的蛋白质合成和TGFβ1的基因表达显着升高(p<0.05;p<0.01)。在培养的SSc-ILDMDMs中,nintedanib0.1和1µM显著下调CD204,CD206,CD163的基因表达和蛋白质合成(p<0.05),和MerTK(p<0.01)与处理24小时后未处理的细胞相比。限于MerTK和IL10,两种尼达尼布浓度在处理16小时后已经显著下调其基因表达(p<0.05)。在培养的SSc-ILDMDMs中,尼达尼布0.1和1µM显着降低了治疗24小时后活性TGFβ1的释放(p<0.05vs.未处理的细胞)。
结论:在来自SSc-ILDpts的培养MDM中,nintedanib似乎通过显著降低M2细胞表面标志物的基因表达和蛋白质合成来下调促纤维化M2表型,随着TGFβ1释放的显著减少,尤其是MerTK,与肺纤维化有关的酪氨酸激酶受体。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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).
CONCLUSIONS: 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.