程序性死亡因子-1及其配体信号通路在肺纤维化中的免疫调控作用

Pulmonary fibrosis is the result of lung injury followed by an abnormal repair leading to thickening of alveolar walls and scarring of the lungs. Following lung injury, there is an accumulation of inflammatory mediators and immune cells that take part in the pathogenesis of pulmonary fibrosis. Programmed death 1 (PD-1), PD-ligand 1 (PD-L1), and PD-L2 were recognized inhibitory receptors in T cell activation, proliferation and cytokine production, but their roles in pulmonary fibrosis immunity remain poorly understood. In our previous study, the down regulation of PD-1 on T cells in the lungs of idiopathic pulmonary fibrosis (IPF) patients was detected, indicating that PD-1/PD-Ls pathway played roles in the processing of lung fibrosis. We aimed to evaluate the immune regulation of PD-1/PD-Ls pathways on T cells in the patients with IPF and bleomycin-induced mice pulmonary fibrosis. Concentrations of soluble PD-1, PD-L1 and PD-L2 in bronchoalveolar lavage (BAL) supernatant of IPF patients were detected. Expression of PD-1 on CD4+ and CD8+ T cells and expressions of PD-Ls on monocytes of peripheral blood and marcophages from BAL were determined using flow cytometry and real time PCR. The impacts of PD-1/PD-Ls pathways on CD4+ and CD8+ T cells were also explored in IPF. By using a mouse model of lung fibrosis, the inflammation and fibrosis of the lungs were dynamic observed in PD-1-deficient mice on different time points. Furthermore, the role of PD-1/PD-Ls pathway in the early and late stages of bleomycin-induced lung fibrosis in mice was determined by depleting them with anti-PD-1 antibody. We wish to clarify the effects of PD-1/PD-Ls pathway on pulmonary fibrosis immunity.

肺纤维化是肺泡上皮细胞损伤和异常修复过程，伴随肺炎症细胞激活。程序性死亡因子(PD)-1及其配体信号通路在抗原递呈中发挥负性调节作用。我们前期发现，IPF患者肺脏T淋巴细胞PD-1表达下调，提示PD-1/PD-L通路在肺纤维化中发挥免疫调控作用。进而推测特发性肺纤维化（IPF）患者肺脏PD-1/PD-L信号通路受抑制，导致CD4+和CD8+T淋巴细胞过度活化，加重肺脏炎症，使肺纤维化进程加速。为此，我们以IPF患者为研究对象，探讨人类肺纤维化中外周血和肺脏PD-1及其配体表达和来源，不同细胞因子对PD-1/PD-L细胞表达的调节；探讨IPF中PD-1/PD-L通路在T细胞表面活性分子的表达与粘附活性与机制；动态观察PD-1敲基因小鼠模型肺脏炎症和纤维化，研究PD-1/PD-L通路对肺纤维化发生发展的作用；利用肺纤维化模型，证实肺纤维化早期或晚期阻断PD-1通路对肺纤维化形成的作用，深化对肺纤维化机制的认识。

背景：肺间质纤维化是异质性间质性肺疾病共同的病理改变之一。特发性肺纤维化（IPF）、结缔组织病相关性肺纤维化（CTD-IP）、以石棉肺和矽肺为代表的尘肺病是不同病因的慢性纤维化性肺疾病。程序性死亡因子1（PD-1）与其配体PD-L1或PD-L2结合后，可能通过抑制T细胞和B细胞的激活，抑制细胞因子的生成，发挥负性免疫调控作用。调节性T细胞（Tregs）是具有免疫抑制效应的T细胞亚群，我们的研究发现CD4+Foxp3+T细胞3个表型和功能各不相同的亚群在IPF中为失衡状态，外周血aTreg比例与疾病的严重性呈负相关。研究内容：采用流式细胞仪检测不同类型肺纤维化患者外周血和肺脏PD-1和PD-L1，PD-L2的差异性表达；检测矽肺患者外周血中Tregs及其亚群的数目和功能，探讨Tregs在矽肺的表达和可能的免疫调节作用。 结果：1. IPF患者BALF中CD8+T淋巴细胞负性免疫调节表型PD-1的表达与CTD-IP和健康人相比显著地下降，肺脏CD4+T淋巴细胞PD-1的表达也有下降趋势；同时，IPF患者PD-L1和PD-L2的表达与对照组相比下调，提示IPF患者PD-1/PD-L信号通路表达受到抑制。进一步发现，石棉肺和矽肺T淋巴细胞或单核细胞PD-1/PD-Ls通路表达均下调，在石棉肺中PD-1在外周的表达水平与疾病严重程度呈显著正相关。并且，石棉肺或矽肺CD8+PD-1+T细胞的比例与CD8+CD28+T细胞的比例呈显著正相关。 2. 矽肺rTregs比例显著地低于健康人，而aTregs和FrⅢ的比例与健康人相比无显著性差异。CD28是T细胞活化第二信号分子，外周血矽肺CD8+T细胞CD28的表达显著地低于健康人；CD38和HLA-DR是淋巴细胞活化抗原，矽肺外周血 CD8+T细胞CD38表达显著地低于健康人，CD8+T细胞HLADR的表达显著地高于健康人。CD31主要表达在新近从胸腺迁移出的细胞表面，矽肺rTregs的CD31表达水平显著高于aTregs和FrⅢ，提示绝大多数的rTregs有可能新近来源于胸腺。意义：PD-1/PD-Ls通路在不同慢性肺纤维化患者均表达为下调，IPF和矽肺均表现为Tregs失衡，可能参与了肺纤维化过程。