核受体NR4A1介导单核/巨噬细胞免疫表型转换在动脉粥样硬化中的作用

Atherosclerosis is a chronic inflammatory condition that develops from deregulation of the immune system and metabolic pathways. Activated inflammation is a major determinant in the rupture of atherosclerotic lesions. It is now emerging that a central cell player in the innate immune response during atherosclerosis are cells of the monocyte/macrophage. In response to stimulation, they demonstrate the extent of heterogeneity and exert their different function as well. Pro-inflammatory monocytes, recognized by high levels of a marker known as Ly6C, and classically activated (M1) macrophages could produce a wide variety of pro-inflammatory cytokines; while surveillance or resident monocytes with low levels of Ly6C and alternatively activated （M2）macrophages participate in anti-inflammatory responses and promote inflammatory resolution and tissue repair. It has been proposed that monocyte/macrophage polarization is not irrevocable, and that fully differentiated cells can switch between the different phenotypes, a phenomenon referred as cells plasticity. This means that cells promote an inflammatory response at first may have anti-inflammatory properties later. However, very little is known about the transcription factors and the intracellular pathways involved in monocyte/macrophage activation and phenotypic switching. It is important to explore the key mediators in the regulation of the phenotype and role of monocyte/macrophage in inflammation. Thus, they could control the stages of inflammatory response and prevent the progression of atherosclerosis. In our previous study, we found that orphan nuclear receptor NR4A1 is induced by oxLDL via p38 MAPK signal pathway and subsequently protects against inflammation by the inhibition of pro-inflammatory COX-2 pathway in activated macrophage. Our preliminary data also show that NR4A1 could reduce oxLDL-induced intracellular lipid loading by inhibiting lipid influx and enhancing lipid efflux. These studies suggest that NR4A1 may be a key regulator of inflammation in immune cells. In this program, we will detect if the switch between both phenotypes of monocyte/macrophage, the function on the stages of inflammation and eventually the progression of atherosclerosis, are regulated by nuclear receptor NR4A1. Thus we explore the feasibility and effectiveness of nuclear receptor in the therapeutic option for atherosclerosis via the switch between the different phenotypes of monocyte/macrophages.

动脉粥样硬化是危险因素及免疫机制共同参与的慢性炎症过程，激活的炎症是斑块破裂的主要因素。单核/巨噬细胞是血管炎症反应的主要效应细胞。对于刺激，可呈现出不同表现型，执行不同的功能。Ly6C+炎症型单核细胞、活化型（M1型）巨噬细胞亚群，分泌大量促炎因子，导致炎症加剧和疾病进展；而Ly6C-监控型单核细胞、替代型活化（M2型）巨噬细胞亚群，促进炎症消退和组织的修复。免疫细胞具有可塑性，不同表型间可相互转换，然而其调控因子和相关信号通路知之甚少。探讨单核/巨噬细胞免疫表型的调控作用，是控制炎症反应转归及动脉粥样硬化斑块稳定性的关键。我们前期研究发现在激活的巨噬细胞中，核受体NR4A1参与了脂质负荷、炎症反应的调控。此次课题中，我们拟探讨NR4A1在炎症反应中对单核巨噬细胞免疫表型分化的作用，进而对动脉粥样硬化斑块稳定性的调控作用及机制，以探索通过介导免疫细胞表现型转换来治疗动脉粥样硬化的可行性。

动脉粥样硬化是危险因素及免疫机制共同参与的慢性炎症过程，激活的炎症是斑块破裂的主要因素。单核/巨噬细胞是血管炎症反应的主要效应细胞。对于刺激，可呈现出不同表现型，执行不同功能。该课题中，我们探讨了核受体NR4A1(Nur77)对炎症型巨噬细胞生物学功能，免疫表型的调控作用和机制及动脉粥样硬化斑块的稳定性。我们发现，oxLDL能激活细胞内p38 MAPK信号通路，诱导核受体Nur77呈剂量和时间依赖性上调表达，过表达Nur77抑制了oxLDL诱导的巨噬细胞凋亡，进一步机制研究表明Nur77不影响细胞周期增殖相关基因PCNA，p21的表达，而抑制了细胞凋亡相关基因Caspase3的表达，抑制了巨噬细胞的凋亡。Nur77通过其转录活性抑制了炎症型巨噬细胞向树突状细胞分化，包括细胞形态学改变，细胞表面标志物的改变，抑制了oxLDL诱导的细胞内吞作用，T细胞增殖，及炎症因子TNFα和IL12的分泌，而缺失片段TAD和DBD的突变体则没有抑制炎症因子的作用。我们还发现，自噬参与了巨噬细胞炎症反应的调控，发现他汀类药物阿托伐他汀呈剂量和浓度依赖性诱导巨噬细胞自噬，而抑制了LPS诱导的巨噬细胞炎症因子IL-1β和TNFα分泌。给予自噬抑制剂3MA或LY294002,或小干扰RNA(ATG5siRNA)抑制自噬后，则对炎症因子IL-1β和TNFα分泌无明显抑制作用，机制表明阿托伐他汀通过AKT/mTOR信号通路诱导巨噬细胞自噬，抑制炎症因子的分泌，发挥抗炎作用。我们还建立了不稳定斑块的动物模型，易损斑块内可见大量巨噬细胞浸润，自噬相关蛋白（LC3-Ⅱ、p62/SQSTM1）的表达及NLRP3炎症小体的激活。在不稳定斑块研究中,我们还发现，ApoE基因敲除小鼠，阿托伐他汀显著减少了巨噬细胞入侵，内皮下脂质沉积，减少内膜胶原的成分和MMPs的表达，改善斑块的稳定性，进一步机制研究表明，阿托伐他汀抑制了炎症因子MCP-1及CX3CL1的表达及其受体CCR2及CX3CR1的表达。该课题通过对核受体NR4A1(Nur77)对炎症型巨噬细胞生物学功能，免疫表型的调控作用及动脉粥样硬化斑块不稳定性的研究，发现了核受体可以促进活化型巨噬细胞向替代型巨噬细胞转换，抑制细胞的凋亡，炎症反应，及发现了他汀类药物抗炎及斑块稳定的新机制，为动脉粥样硬化的防治提供了新靶点及理论依据。