iTR35细胞通过IL-35调节纤维细胞分化、迁移、分泌功能及其在特发性肺纤维化发病中的作用研究

Idiopathic Pulmonary Fibrosis (IPF) is a kind of chronic lung disease for which there is limited cure and whose etiopathogensis and pathogenesis are not clear. Its pathogenesis is regulated by immune mechanisms. We have found that Foxp3+ T regular (Treg) cells take part in the pathogenesis of lung fibrosis by promoting the differentiation, migration and secretion of fibrocytes. If iTR35 cells, this new kind of Foxp3- Treg cells take part in the pathogenesis of IPF is not clear. IL-35 is the key cytokine produced by iTR35 cells and Foxp3+ Treg cells. Some scientists found that it might take part in the pathogenesis of tissue fibrosis but it has not been studied in IPF. We found lower level of iTR35 cells and IL-35 in the peripheral blood and serum of IPF patients compared to the healthy control. The differentiation and migration of fibrocytes could be regulated by IL-12 and IL-35 belongs to IL-12 family. So we presume that iTR35 cells and Foxp3+ Treg cells might take part in the pathogenesis of lung fibrosis by regulating the differentiation, migration and secretion of fibrocytes through IL-35. We aim to detect the number and the percentage of iTR35 cells, Foxp3+ Treg cells, fibrocytes and the level of IL-35 in the IPF patients and lung fibrosis mouse model. We will co-culture the iTR35 cells and fibrocytes, Foxp3+ Treg cells and fibrocytes, IL-35 and fibrocytes in order to detect the effect of iTR35 cells, Foxp3+ Treg cells and IL-35 to the differentiation, migration and secretion of fibrocytes. We will also detect the effect of iTR35 cells, Foxp3+ Treg cells and IL-35 to lung fibrosis and fibrocytes in the lung fibrosis mouse model. Our aim is to clarify if iTR35 cells and Foxp3+ Treg cells take part in the pathogenesis of IPF by regulating the differentiation, migration and secretion of fibrocytes through IL-35 and try to find new therapy methods for IPF.

特发性肺纤维化（IPF）的发生受免疫机制调控。申请者曾发现Treg细胞通过调节纤维细胞分化、迁移和分泌功能而参与肺纤维化的发生。iTR35细胞是一种新型Treg细胞，IL-35是iTR35等Treg细胞分泌的发挥免疫调节作用的最重要细胞因子，新近研究发现IL-35参与组织纤维化的发生，但在IPF中的作用未见研究。申请者初步观察到IPF患者外周血iTR35细胞数量和血浆IL-35水平下降。纤维细胞的分化、迁移等受IL-12的调节，而IL-35属于IL-12家族，故推测iTR35等Treg细胞可能通过分泌IL-35调节纤维细胞分化、迁移和分泌功能而参与肺纤维化的发生。本研究拟利用IPF患者和肺纤维化小鼠模型，通过体外共培养和动物模型体内干预等技术，探讨iTR35等Treg细胞是否通过分泌IL-35调节纤维细胞分化、迁移和分泌功能而参与IPF的发生，以期为IPF的治疗寻找新的靶点和方法。

既往研究证实Treg细胞在抑制肺纤维化的发生中起重要作用，IL-35是iTR35等Treg细胞分泌的发挥免疫调节作用的重要因子，与组织纤维化密切相关。外周血纤维细胞被报道在纤维化过程中可以转化为成纤维细胞而参与纤维化的发生。Treg细胞能抑制纤维细胞从而阻止肺纤维化的发生，具体机制并不明确。本研发现特发性肺纤维化（IPF）组的PBMCs中纤维细胞比例较健康人上调，而Foxp3+Treg细胞比例、iTR35细胞比例及IL-35表达水平较健康人下调；IPF组的外周血血浆中IL-35水平较健康人对照下调，IL-10及TGF-β的表达较健康人上调。IPF患者Treg细胞体外培养上清中IL-35表达水平较健康人下调，TGF-β1及IL-10水平较健康人上调。在体外对Treg细胞的IL-35基因进行沉默和过表达干预后并分别用干预后的Treg细胞培养上清以及rh IL-35对PBMCs进行干预培养发现，rh IL-35干预的PBMCs分化为纤维细胞较对照组少、分化程度低且形态不典型，其细胞培养上清中的胶原表达较对照组下调。沉默Treg细胞IL-35表达的上清干预培养的纤维细胞的胶原表达较对照组下调，而过表达Treg细胞IL-35表达的上清干预培养的纤维细胞的胶原表达较对照组上调。BLM致肺纤维化小鼠模型中小鼠BALF中IL-35、IL-10表达下调、TGF-β表达上调；BLM小鼠肺组织中IL-35两个亚基EBI3及P35表达下调，纤维细胞比例上调，Foxp3+Treg细胞比例和iTR35细胞比例下调。抑制Treg细胞、iTR35细胞及IL-35表达后，BLM小鼠肺纤维化程度加重，BALF中IL-35、IL-10表达水平较对照组下调、TGF-β表达水平较对照组上调；BLM小鼠肺组织中EBI3及P35 mRNA及蛋白水平均下调，纤维细胞比例上调，Foxp3+Treg细胞比例和iTR35细胞比例下调。而过表达IL-35的BLM小鼠肺纤维化程度较其对照组改善，BALF中IL-35、IL-10表达水平较对照组上调、TGF-β表达水平较对照组下调；BLM小鼠肺组织中EBI3及P35 mRNA及蛋白水平表达均上调，纤维细胞比例下调，Foxp3+Treg细胞和iTR35细胞比例上调。这说明iTR35细胞和Foxp3+Treg细胞可能通过分泌IL-35调节纤维细胞分化、分泌等功能参与肺纤维化的发生