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INTRODUCTION

Systemic sclerosis (SSc) is a complex autoimmune connective tissue disease characterized by microvascular damage, immune system reactivity and progressive fibrosis of skin and internal organs. Interstitial lung disease is the leading cause of death for SSc patients (SSc-ILD), and the process of lung fibrosis involves also circulating monocytes and alveolar macrophages.

METHODS

Current study aimed to identify monocyte/macrophage phenotypes in lung and peripheral blood of SSc-ILD patients by immunostaining and flow cytometry, respectively. Single immunostaining was performed using primary antibodies against CD68 (pan-macrophage marker), CD80, CD86, TLR4 (M1 markers), CD163, CD204, and CD206 (M2 markers). Flow cytometry analysis included the evaluation of CD45, CD14, CD16 (monocyte lineage), CD1c (dendritic lineage), together with M1 and M2 activation markers on circulating monocytes. Protein synthesis of TLR4 and M2 markers was also investigated in cultured monocytes-derived macrophages (MDMs) from SSc-ILD patients by Western Blotting.

RESULTS

Lung samples were obtained from 9 SSc-ILD patients (50 ± 9 years old) and 5 control non-SSc patients without lung fibrosis (58 ± 23 years old). Alveolar macrophages (CD68 cells) showed a significantly higher positivity of M1 and M2 markers in SSc-ILD lung samples than in controls (p<0.05 for CD80, p<0.01 for CD86, p<0.001 for CD68, p<0.0001 for TLR4, CD163, CD204 and CD206). In CD68 positive areas of SSc-ILD samples, a significantly higher percentage of TLR4, CD163, CD204, and CD206 positive cells was observed compared to CD80 and CD86 positive cells (p<0.001 in both cases), suggesting the possible presence of hybrid TLR4M2 macrophages (CD68CD80CD86TLR4CD163CD204CD206cells) in SSc-ILD samples. A second cohort of 26 SSc-ILD patients (63 ± 14 years old) and 14 SSc patients without ILD (63 ± 19 years old) was recruited for flow cytometry analysis of circulating monocytes. Again, a significantly higher percentage of hybrid TLR4M2 monocytes (CD1cCD80TLR4CD163CD204CD206cells) was found in SSc-ILD positive than SSc-ILD negative patients (p<0.05). Moreover, the protein synthesis of TLR4 and M2 markers was also found higher in cultured MDMs obtained from SSc-ILD patients than in MDMs from SSc patients without ILD and this increase was significantly higher for CD163 (p<0.05) and CD206 (p<0.01).

CONCLUSIONS

The presence of hybrid TLR4M2 markers on both circulating monocytes and resident lung macrophages in SSc-ILD patients, is reported for the first time. Therefore, the detection of circulating hybrid TLR4M2 monocytes in SSc-ILD might represent a further potential biomarker of progressive organ fibrosis, to be searched in blood samples of SSc patients.

引言

系统性硬化症（SSc）是一种复杂的自身免疫性结缔组织疾病，其特征为微血管损伤、免疫系统反应性以及皮肤和内脏器官的进行性纤维化。间质性肺疾病是SSc患者（SSc-ILD）的主要死亡原因，肺纤维化过程还涉及循环单核细胞和肺泡巨噬细胞。

方法

本研究旨在分别通过免疫染色和流式细胞术鉴定SSc-ILD患者肺组织和外周血中的单核细胞/巨噬细胞表型。使用抗CD68（泛巨噬细胞标志物）、CD80、CD86、TLR4（M1标志物）、CD163、CD204和CD206（M2标志物）的一抗进行单免疫染色。流式细胞术分析包括评估CD45、CD14、CD16（单核细胞系）、CD1c（树突状细胞系）以及循环单核细胞上的M1和M2激活标志物。还通过蛋白质印迹法研究了SSc-ILD患者培养的单核细胞衍生巨噬细胞（MDM）中TLR4和M2标志物的蛋白质合成。

结果

从9例SSc-ILD患者（50±9岁）和5例无肺纤维化的对照非SSc患者（58±23岁）获取肺组织样本。肺泡巨噬细胞（CD68阳性细胞）在SSc-ILD肺组织样本中M1和M2标志物的阳性率显著高于对照组（CD80，p<0.05；CD86，p<0.01；CD68，p<0.001；TLR4、CD163、CD204和CD206，p<0.0001）。在SSc-ILD样本的CD68阳性区域，与CD80和CD86阳性细胞相比，观察到TLR4、CD163、CD204和CD206阳性细胞的百分比显著更高（两种情况均为p<0.001），提示SSc-ILD样本中可能存在混合性TLR4M2巨噬细胞（CD68CD80CD86TLR4CD163CD204CD206阳性细胞）。招募了第二组26例SSc-ILD患者（63±14岁）和14例无ILD的SSc患者（63±19岁）进行循环单核细胞的流式细胞术分析。同样，SSc-ILD阳性患者中混合性TLR4M2单核细胞（CD1cCD80TLR4CD163CD204CD206阳性细胞）的百分比显著高于SSc-ILD阴性患者（p<0.05）。此外，还发现从SSc-ILD患者获得的培养MDM中TLR4和M2标志物的蛋白质合成高于无ILD的SSc患者的MDM，且CD163（p<0.05）和CD206（p<0.01）的增加更为显著。