ANGII/AT1R调节心肌浸润Ly6C(hi)单核细胞极化参与小鼠EAM发生的机制研究

The development of experimental autoimmune myocarditis (EAM) experienced three stages "injury-inflammatory-remodeling". Our previous work demonstrated that inhibition Mφ polarization or blockade angiotensin II (ANGII) could attenuate EAM progression. Our present data showed that ANGII induced Ly6C(hi) monocytes differentiation into M1 phenotype macrophage (Mφ); furthermore, cytokines produced by M1 phenotype Mφ could activate cardiac fibroblast. Therefore, we hypothesized that ANGII secreted by myocardial tissue promoted Ly6C(hi) monocytes differentiation into M1 phenotype Mφ, led to myocardial injury and involved in repair of injury myocardium. To confirm the above hypothesis, the project was carried out, in vitro, to clarify the mechanisms of ANGII modulating Ly6C(hi) monocytes differentiation into M1 phenotype Mφ; in vivo, EAM models were induced WT, Tyk2(-/-), monocytes/Mφ depletion BALB/c mice, respectively. And the heart and serum were collected and analyzed after the mice sacrifice. Additionally, ANGII reperfusion and ANGII receptor antagonists were also employed to confirm the above hypothesis. Following the project, we will expect to furthering confirm the process of myocarditis development and clarify the theory for intervention therapy of inflammatory diseases.

EAM发生经历“损伤-炎症-修复”三个阶段，既往工作证实抑制M1型Mφ极化或ANGII活性均能缓解EAM小鼠心肌损伤。预实验表明ANGII可诱导Ly6C(hi)向M1型Mφ分化/极化；且M1型Mφ分泌的细胞因子可激活成纤维细胞。由此提出“EAM时上调的ANGII诱导Ly6C(hi)单核细胞分化/极化为M1型Mφ导致心肌损伤、启动受损组织修复”的假说。为证实以上假说，本研究拟从体内外两方面进行：体外阐明ANGII调控Ly6C(hi)单核细胞分化机制以及M1型Mφ对成纤维细胞活化、增殖作用；体内通过对WT、Tyk2(-/-)、单核/Mφ选择性敲除小鼠模型诱导及ANGII再灌注与ANGII受体拮抗剂应用，阐明ANGII调控Ly6C(hi)单核细胞向M1型Mφ分化/极化参与EAM发生的机制。通过上述研究有望进一步阐明EAM发生过程，为心肌炎相关疾病的免疫干预治疗奠定基础。

本项目是在前一个基金基础之上，继续深入的研究，旨在阐明血管紧张素II（ANG II）-AT1R轴调控受损心肌组织浸润的CD11b+Ly6Chi单核细胞向炎性巨噬细胞分化/极化的机制及其在心肌炎症损伤中的作用，通过本项目的实施以期为炎性心肌损伤的免疫干预治疗寻找到新的靶点。围绕研究目标完成了以下几方面内容：① 证实ANG II能够通过调控CCR2/5招募CD11b+Ly6Chi单核细胞浸润至心肌损伤部位并通过Erk1/2、P38/ Stat3通路调控心脏浸润的该群细胞向炎性巨噬细胞细胞分化/极化参与EAM炎症损伤。②阐明了心肌损伤时浸润后分化/极化的炎性巨噬细胞的命运，课题组在阐明CD11b+ Ly6Chi单核细胞分化/极化的基础上，进一步证实该群细胞可以被活化的心肌成纤维细胞调控诱导其向M2型转化，重新成为组织定居的巨噬细胞。③ 心肌炎症损伤时B10细胞的存在造成了炎症的迁延不愈。心肌炎症损伤时受损心肌组织和脾脏中存在B10细胞，该细胞能够抑制心肌的纤维化和Th17细胞的分化，心肌组织中释放的PGE2造成了该群细胞的扩增。此外也证实，核HMGB1除了作为一个重要的结构因子之外，可能还对基因转录起了重要的调控作用，如单核/巨噬细胞的HMGB1能够控制该细胞的极化。这一系列工作的完成为全面认识心肌炎症损伤以及发现潜在的治疗靶点奠定基础。