LMP2/7调控巨噬细胞M2极化参与尘肺纤维化形成的机制研究

Alveolar macrophages are highly plastic cells，which can polarized into M1 and M2 activation states depending on its environments, and it play an essential role in pathogenesis of pulmonary fibrosis. Our previous studies showed that the novel role of immunoproteasome subunits LMP2/7 function for regulating alternative activation of macrophages （M2） by limiting IL-4Rα expression and signaling. Additionally, our preliminary results showed that LMP2/7 protein level were upregulated in lung tissues from mice with silica induced silicosis. Moreover, we found that LMP2/7 were upregulated in BALF（Bronchoalveolar lavage fluid）isolated macrophage. Therefore, we hypothesize that LMP2/LMP7 contribute to the development of silicosis by regulating macrophage M2 polarization. In this project, we will utilize the LMP2 and LMP7 knockout mice to study the role of LMP2/7 in macrophage polarization and pulmonary fibrosis. At first, we will deeply investigate the role of LMP2/LMP7 in macrophage polarization by establishing the model of M2 macrophage polarization, and then we will try to figure out if LMP2/LMP7 can regulate the pro-fibrotic effects of macrophage on alveolar epithelial cell and fibroblast by using coculture model, and finally we will investigate the actual role of LMP2/7 in pathogenesis of pulmonary fibrosis by using a silica induced silicosis mice model. Our study might provide a new mechanistic interpretation and potential therapeutic target for pneumoconiosis.

巨噬细胞有极强可塑性，随微环境改变向M1和M2型极化，其中M2极化与肺纤维化发病密切相关。我们前期发现免疫蛋白酶体亚基LMP2/7可通过影响IL-4/IL-13受体IL-4Rα蛋白水平来调控巨噬细胞M2极化，此外还发现LMP2/7在尘肺病动物模型肺组织以及灌洗液分离巨噬细胞中表达上调。因此我们推测，LMP2/7可通过调控巨噬细胞M2极化参与尘肺纤维化病理过程。本项目拟通过收集尘肺病临床样本、构建LMP2/7敲除小鼠尘肺模型，以及构建M2极化巨噬细胞与肺上皮以及成纤维细胞共培养模型、综合利用细胞分子生物学技术，在临床、整体动物和细胞分子三个层面，明确LMP2/7是否通过调控巨噬细胞M2极化，进而影响肺上皮间质转化以及成纤维细胞增殖、分化和胶原分泌等纤维化过程，来参与尘肺病肺纤维化，并阐明其中细胞分子机制，为尘肺纤维化发病机制提供新理论依据，并为将来治疗尘肺纤维化提供潜在的新靶点。

巨噬细胞有极强可塑性，随微环境改变向M1和M2型极化，极化与肺纤维化发病密切相关。我们前期发现免疫蛋白酶体亚基LMP2/7可通过影响IL-4/IL-13受体IL-4Rα蛋白水平来调控巨噬细胞极化，此外还发现LMP2/7在尘肺病动物模型肺组织以及灌洗液分离巨噬细胞中表达上调。因此我们研究了LMP2/7是否可通过调控巨噬细胞极化参与尘肺纤维化病理过程。本研究首先利用基因芯片及qPCR分析了LMP2敲除对肺泡巨噬细胞M0及M1功能影响，发现敲除LMP2显著增强M1极化，且抑制M0和M1细胞的抗原呈递相关基因H2-M2的表达。进一步利用LMP7抑制剂ONX0194长时处理细胞，利用RNA-seq、WesternBlot及qPCR发现ONX0194 对M1和M2都起显著抑制作用，其机制可能是通过激活自噬和内质网应激通路。最后我们利用二氧化硅尘肺病动物模型系统评估了ONX0914对尘肺纤维化的治疗潜力，发现ONX0914对二氧化硅诱导的小鼠尘肺纤维化无治疗效果。本研究系统性了探索了免疫蛋白酶体在巨噬细胞中的生物学功能，从干预天然免疫细胞角度，为开发免疫蛋白酶体作为治疗尘肺纤维化靶点提供了理论指导。