Cbl-b/C-Cbl 双敲单核巨噬细胞在特发性肺纤维化发生发展中的作用及机制研究

Idiopathic pulmonary fibrosis is a chronic, progressive and interstitial lung disease, Accumulating evidence demonstrated that inflammation caused by macrophages play pivotal role in the development of IPF.However, it is still largely unclear which molecule causes the activation of macrophages. We establish the LysM-Cre+ Cbl-b-/-/C-Cblf/f mouse model with ablation of both Cbl-b/C-Cbl E3 ubiquitin ligase in monocytes / macrophages.The 100% dKO mice were died of IPF like disease in about 35 days. Mature macrophages were infiltrated in lung, the alveolar structure were severely destroyed. dKO mice BM can developed more mature macrophages cultured with M-CSF in vitro, suggests that Cbl-b/C-Cbl may inhibit the MCSF1R pathwy to prevent the differentiation of macrophage. Other preliminary results further suggest that Cbl-b/C-Cbl may ubiquitinate PU.1 which are important transcriptional factor of MCSF1R, and directly ubiquitinated MCSF1R, meanwhile, the Cbl-b/C-Cbl double deficent macrophages have lower expression of SHIP1 which are essential signal molecule in MCSF1R pathway. All together imply that Cbl-b/C-Cbl synergisticly function on the transcription and ubinquitination of MCSF1R,and impair the signal pathway of MCSF1R by down-regulating the SHIP1. In this project, we plan to extensively study the effect of Cbl-b/C-Cbl on MCSF1R and its signal pathway, and clarify the mechanism of Cbl-b/C-Cbl in macrophage-induced IPF.

特发性肺纤维化是一种慢性、进行性、纤维化性间质性肺疾病，巨噬细胞导致的炎症在IPF发生发展中的作用越来越得到肯定，但何种原因引起了巨噬细胞的活化尚不甚清楚。在单核/巨噬细胞中我们同时敲除E3泛素化连接酶Cbl-b/C-Cbl后，发现小鼠在35天左右出现典型的以成熟巨噬细胞侵润为主的IPF样症状，且100%的死亡。体外用M-CSF诱导巨噬细胞分化，发现dKO小鼠BM可以得到更多的巨噬细胞；Western-blot、IP等实验初步提示Cbl-b/C-Cbl可能通过泛素化PU.1影响了MCSFR的转录，并直接泛素化MCSF1R，同时Cbl-b/C-Cbl影响其信号通路中的SHIP1 等分子的稳定性，从多种途径协同作用影响巨噬细胞的分化和成熟。本课题拟在已有预试验基础上，详细研究Cbl-b/C-Cbl对MCSFR本身及其信号通路的影响，阐明Cbl-b/C-Cbl缺失的巨噬细胞诱导IPF的作用机制。

作为 E3泛素连接酶，Cbl家族成员参与多种免疫细胞的调控。然而，c-Cbl和 Cbl-b如何共同调控巨噬细胞的功能仍不清楚。为了探索Cbl-b和c-Cbl在巨噬细胞中是否发挥的冗余作用，我们构建了在单核巨噬细胞中特异性敲除Cbl-b和c-Cbl的双敲除小鼠（LysM Cre+ c-Cblf/f Cbl-b-/-，dKO)、c-Cbl条件性敲除小鼠（LysM Cre+ c-Cblf/f，c-Cbl cKO）、Cbl-b 单敲除小鼠（Cbl-b KO）。有趣的是dKO小鼠在出生两周后开始生长缓慢，2个月左右所有dKO小鼠死亡。H&E结果显示现dKO小鼠的肺脏破坏严重，并且伴有大量炎症细胞侵润,天狼猩红染色显示dKO小鼠发生了特发性肺纤维化。FACS结果分析发现dKO小鼠肺脏中侵润的炎症细胞主要为肺泡巨噬细胞，其增殖增强，凋亡减少。.我们发现用M-CSF培养 dKO小鼠的BMDM，其克隆计数明显多于WT组。且M-CSFR的蛋白表达在dKO小鼠的AMs中特异性增多。免疫共沉淀实验表明M-CSFR与Cbl-b和c-Cbl之间均存在相互作用。并且Cbl-b和c-Cbl可以降解M-CSF受体。同时M-CSF受体的mRNA表达也在dKO小鼠的AMs中特异性增多，所以我们推测Cbl-b和c-Cbl也可以在转录水平上调控M-CSF受体的水平。接下来通过全蛋白质谱检测发现转录因子PU.1在dKO小鼠的AMs中特异性富集。在dKO小鼠BMDM中敲低PU.1可以抑制巨噬细胞克隆形成以及M-CSF受体表达，并且Cbl-b/c-Cbl均可泛素化PU.1。这些证据表明Cbl-b和c-Cbl可以通过泛素化 PU.1 从而调控 M-CSFR的转录。 .此外，双敲除（dKO）肺泡巨噬细胞还高表达一些炎症因子。在 AMs中，双敲除导致了一些microRNA的表达量显著的降低，许多研究报道，这些miRNA在炎症和癌变过程中表达异常。接下来我们选择了四种差异最明显的miRNA（miR-135a、miR-143、miR-145a和miR-199a）进行进一步研究。我们将四种miRNA mimics转染DKO小鼠BMDM。结果表明，Cbl-b/c-Cbl的缺失显著促进巨噬细胞分泌促炎因子。而这些作用可通过恢复miR-143的表达而逆转。由此可见，miR-143可能是抗炎治疗DKO小鼠肺部炎症的候选药物之一。