Novel Regulation and Targeting of Macrophages Metabolism in Neuroinflammatory Disorders

摘要 髓系细胞在多发性硬化症(MS)的中枢神经系统脱髓鞘和轴突破坏中起关键作用 实验性自身免疫性脑脊髓炎(EAE)。该病的早期特征是 存在致病的活化巨噬细胞(M1型)，而恢复期与 交替激活的巨噬细胞(M2型)，释放抗炎细胞因子，分解 致病性炎症。活化的M1巨噬细胞依赖糖酵解促进生物合成途径 产生炎症介质。然而，抗炎的M2巨噬细胞主要依靠线粒体 呼吸。一磷酸腺苷激活的蛋白激酶(AMPK)调节能量代谢，因此 控制糖酵解和线粒体呼吸之间的平衡。我们之前报道过AMPKα1 基因敲除(KO)小鼠出现严重的EAE，表明AMPK的激活是保护性的，但其分子机制 AMPK是如何调控EAE疾病进展的尚不清楚。AMPKα1-KO巨噬细胞表现出高度- 炎症表型和代谢率较低。AMPKα1-KO巨噬细胞也表现为糖酵解- 三羧酸(TCA)循环重构，导致内源性激素水平失衡 代谢物，琥珀酸和衣康酸，调节促炎和抗炎巨噬细胞功能， 分别进行了分析。它们的水平受琥珀酸脱氢酶(SDH)和免疫应答基因的严格控制 1(IRG1)。我们推测，AMPKα1的缺失重塑了糖酵解-三氯乙酸途径，导致 琥珀酸和衣康酸水平不平衡，这促进了M1表型而不是M2表型。 这反过来促进Th17细胞并抑制T调节细胞，从而导致高炎性中枢神经系统免疫 反应和中枢神经系统组织损伤。为了验证我们的假设，我们生成了单核细胞特异性AMPKα1 KO 和巨噬细胞特异性的、成分活跃的AMPKα1T172D转基因小鼠。在目标1中，我们将研究如何 巨噬细胞AMPKα1功能丧失或获得调节M1/M2巨噬细胞极化 因此，Th17和Tregs的分化和疾病结局。目标2下的研究将阐明 AMPKα1缺失导致糖酵解-三氯乙酸代谢失衡的机制 巨噬细胞中的琥珀酸和衣康酸代谢物，进而决定巨噬细胞的表型。这个 拟议的研究有望通过阐明代谢调节机制产生积极影响 在疾病中负责巨噬细胞的可塑性并研究AMPKα1作为潜在的治疗靶点 对于女士来说，我们创新的小鼠遗传模型和精确的代谢组学方法将使我们能够识别 炎性细胞中AMPKα1特异的细胞代谢通路的明显重新连接。归根结底，这 可以利用这一过程来量身定制新的治疗策略，以解决或限制自身免疫性炎症 中枢神经系统。

ABSTRACT Myeloid cells play a critical role in CNS demyelination and axonal destruction of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The early phase of the disease is characterized by the presence of pathogenic activated macrophages (M1 type), while the recovery phase is associated with alternatively-activated macrophages (M2 type) which release anti-inflammatory cytokines that resolve the pathogenic inflammation. Activated M1 macrophages depend on glycolysis to boost biosynthetic pathways to produce inflammatory mediators. However, anti-inflammatory M2 macrophages rely primarily on mitochondrial respiration. Adenosine monophosphate-activated protein kinase (AMPK) regulates energy metabolism, and thus controls the balance between glycolysis and mitochondrial respiration. We reported previously that AMPKα1 knockout (KO) mice develop severe EAE indicating AMPK activation is protective, yet the molecular mechanism by which AMPK regulates EAE disease progression is not known. AMPKα1-KO macrophages exhibit a hyper- inflammatory phenotype and have a lower rate of metabolism. AMPKα1-KO macrophages also show glycolysis- tricarboxylic acid (TCA) cycle remodeling, which results in an imbalance in the levels of the endogenous metabolites, succinate and itaconate, which regulate pro- and anti-inflammatory macrophage functions, respectively. Their levels are tightly controlled by succinate dehydrogenase (SDH) and immune responsive gene 1 (IRG1), respectively. We hypothesize that the loss of AMPKα1 remodels the glycolytic-TCA pathway causing an imbalance in the levels of succinate and itaconate, which promotes an M1 phenotype over an M2 phenotype. This, in turn, promotes Th17 cells and suppresses T regulatory cells leading to a hyperinflammatory CNS immune response and CNS tissue damage. To test our hypothesis, we have generated monocyte-specific AMPKα1 KO and macrophage-specific, constitutively active AMPKα1T172D transgenic mice. In Aim 1, we will examine how the loss or gain of function of AMPKα1 in macrophages regulates M1 versus M2 macrophage polarization and consequently, Th17 and Tregs differentiation and disease outcomes. Studies under Aim 2 will elucidate the mechanism by which the loss of AMPKα1 reprograms glycolysis-TCA metabolism leading to an imbalance of succinate and itaconate metabolites in macrophages, which in turn, determine the macrophage phenotype. The proposed study is expected to have a positive impact by elucidating the metabolic regulatory mechanism responsible for macrophage plasticity during disease and investigating AMPKα1 as a potential therapeutic target for MS. Our innovative genetic mouse models and precise metabolomics approach will allow us to identify the apparent rewiring of cellular metabolic pathways specific to AMPKα1 in hyperinflammatory cells. Ultimately, this process could be exploited to tailor novel therapeutic strategies to resolve or limit autoimmune inflammation in the CNS.