巨噬细胞中E3泛素连接酶Cbl-b在心肌炎发生发展中的作用研究

Myocarditis is an acute inflammatory disease of the heart that may cause 5% to 20% of sudden death in young adults, and is the most common cause of dilated cardiomyopathy (DCM) in human. The injury of myocardium is due to the infection of viruses, and also the immunoreaction. . Casitas-B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, negatively regulates T cell responses, and play a crucial in T cell tolerance. Cbl-b knockout mice are highly susceptible to experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA) which are believed to be mediated by Th17. And experimental autoimmune myocarditis (EAM), an animal model of myocarditis, is also mediated by Th17 cells. However, although mice lacking Cbl-b develop severe myocarditis, surprisingly, Cbl-b deficiency in T cells is not the primary driving force for severe disease. Further analysis indicates that Cbl-b does not seem to inhibit Th17 cell differentiation. These data suggest that the severe myocarditis observed in Cbl-b knockout mice may not due to the loss of Cbl-b in T cells but rather in antigen presenting cells (APCs). In support of this notion, Dectin family degradation is compromised in macrophages, which then suppresses Th17 responses and susceptibility to EAM. In this proposal, we will investigate whether Cbl-b deficiency in macrophages results in severe myocarditis and heightened Th17 responses and myocarditis via targeting Dectin family of CLR for ubiquitination in macrophages. A better understanding of the cellular and molecular mechanisms involved in aberrant Th17 responses in EAM and possibly in subset of human myocarditis may provide a useful approach to develop novel strategies against a devastating heart disease.

心肌炎是一种心肌炎症性疾病，是青少年心源性猝死的重要原因之一，与扩张性心肌病的发生密切相关。除病毒感染等直接损害心肌外，免疫反应损伤也在其发病过程中起重要作用。目前认为，心肌炎是一种CD4+ T细胞相关疾病，可能与Th17细胞介导的自身免疫反应有关。Cbl-b是调节免疫与耐受的关键调节因子之一，并可调节CD4+ T细胞的分化。以往研究中，我们发现Cbl-b可影响小鼠心肌Th17细胞应答及自身免疫性心肌炎的发生，但在体外，Cbl-b不直接调节Th17的分化，而调节Dectin-1通路诱导的巨噬细胞IL-6和TNF-α分泌。.本研究拟通过分析心肌炎患者及对照组Cbl-b表达及其临床资料，明确Cbl-b与心肌炎的关系。并通过动物、细胞和分子生物实验明确Cbl-b经何种途经调节巨噬细胞分泌细胞因子、进而影响Th17细胞或通过其他途经对心肌炎产生影响，从而对心肌炎的诊断和临床治疗新的靶点。

心肌炎是一种心肌炎症性疾病，免疫反应损伤也在其发病过程中起重要作用，Cbl-b是调节免疫与耐受的关键调节因子之一，可影响小鼠心肌Th17细胞应答及自身免疫性心肌炎的发生。本研究通过构造小鼠实验性自身免疫性心肌炎模型，利用离体T细胞及抗原提呈细胞探讨了Cbl-b如何对心肌炎产生影响。重要结果及关键数据：1. Cbl-b影响小鼠心肌炎，并可能与Th17活化有关：1）使用MyHC-a诱导自身免疫性心肌炎（EAM）模型，Cbl-b-/-小鼠较对照组心肌炎症更重（炎症评分3.1vs1.8），心肌浸润细胞中Th17更多（10.4%vs7.1%）；2）将Cbl-b-/-及Cbl-b+/+骨髓细胞分别注射至经照射后的Cbl-b+/+及Cbl-b-/-小鼠中，诱导EAM模型，Cbl-b-/-骨髓细胞组心肌炎症更重（炎症评分2.5vs1.4），心肌浸润细胞中Th17更多（8.39%vs4.95%）。2. T细胞中Cbl-b缺乏不影响小鼠心肌炎：将Cbl-b+/+及Cbl-b-/-T细胞注射至Rag-1-/-小鼠中，诱导EAM模型，两组心肌炎症及心肌浸润细胞中Th17表达无差别。3. Cbl-b调节巨噬细胞分泌细胞因子，并可能通过该途径影响Th17分化：1）刺激巨噬细胞及树突状细胞，Cbl-b-/-巨噬细胞分泌IL-6和TNF-a增加（p＜0.05），树突状细胞中二者无差别；2）将刺激后的巨噬细胞与CD4+ Naïve T cell共同培养后体外诱导Th17细胞分化增殖，Cbl-b-/-巨噬细胞使Th17分化增加（zymosan组：3.4%vs5.2%；curdlan组：7.8%vs11.1%）。4. Cbl-b-/-巨噬细胞调节EAM：使用经MyHC-a处理的巨噬细胞诱导小鼠EAM，Cbl-b-/-巨噬细胞组小鼠心肌炎症更重（炎症评分2.8vs1.2），心肌浸润细胞中Th17细胞（27.4%vs11%）、巨噬细胞增多，Th1、Th2及树突状细胞含量两组相似。5. Cbl-b作用于Dectin-1及Dectin-2，通过Dectin/Syk途径调节巨噬细胞分泌IL-6及TNF-，从而调节真菌免疫。科学意义：该研究发现Cbl-b通过影响巨噬细胞分泌细胞因子影响小鼠心肌炎；同时Cbl-b经该途径影响真菌免疫。对于心肌炎的诊断治疗提供了新的思路，并拓展了真菌感染的诊疗方向。