Macrophage and Monocyte Metabolic Adaptation in Hemolysis and Sickle Cell Disease

巨噬细胞和单核细胞代谢对溶血和镰刀细胞病的适应 项目总结 溶血是一系列不同病理的统一特征，包括遗传性疾病，如镰状细胞 疾病(SCD)、包括疟疾在内的传染病、败血症等全身性病理以及药物 毒性和自身免疫性疾病。游离血红素的释放会引起氧化损伤，导致免疫细胞 激活、内皮损伤、缺血和终末器官毒性。巨噬细胞等先天免疫细胞 专门研究血红素解毒，虽然现在很明显巨噬细胞改变了它们的细胞代谢 为了执行特定的效应器功能，允许巨噬细胞存活的代谢适应 血红素清除的毒性应激尚不清楚。我们小组的初步数据显示，在对血红素的反应中 负载巨噬细胞将葡萄糖代谢转移到磷酸戊糖途径(PPP)，这允许 NADPH的快速产生和氧化还原动态平衡的维持。这种代谢适应是依赖于 关于血红素加氧酶的活性，以及在血红素分解过程中释放的一氧化碳是 诱导代谢适应PPP的介体。我们还发现PPP的活动是必需的 对于有效的血红素解毒，以及PPP在酶和转录水平上的上调 SCD小鼠模型，而令人惊讶的是，SCD患者的PBMC，PPP酶下调 在疾病恶化期间，与基线时的同一患者相比。综合来看，这些数据 提出巨噬细胞/单核细胞代谢适应在溶血反应中的关键作用，并指出 将新陈代谢作为一种潜在的治疗途径用于治疗由血红素引起的病理。 这项提议验证了这样一种假设，即血红素分解释放的一氧化碳会改变葡萄糖 朝向PPP的代谢及其促进PPP的药理作用 清除血红素，改善溶血引起的损伤。这一假设将通过 完成以下具体目标。具体目标1是确定细胞内的机制，通过它 一氧化碳推动代谢朝向购买力平价：Subaim 1A测试一氧化碳抑制胱硫醚β的假说 合成酶(CBS)导致磷酸果糖激酶(PFK)FB3活性丧失，从而分流葡萄糖代谢 远离糖酵解，转向购买力平价。Subaim 1B然后检查药物抑制是否 PFKFB3可促进血红素清除，减轻低氧引起的溶血损伤 血管闭塞。具体目标2是检查目前治疗SCD的谷氨酰胺如何改变免疫代谢 小鼠和人SCD循环单核细胞的分布。Subaim 2A体内外检测谷氨酰胺 改变细胞代谢，促进PPP，改善溶血引起的病理改变。苏巴伊姆2B 结合转录和生物能量学分析来表征PBMCs的免疫代谢状态 以确定新陈代谢在稳定状态、疾病恶化期间或 作为对治疗的回应。

Macrophage and Monocyte Metabolic Adaptation to Hemolysis and Sickle Cell Disease PROJECT SUMMARY Hemolysis is a unifying feature of a diverse set of pathologies including inherited disorders such as sickle cell disease (SCD), infectious diseases including malaria, systemic pathologies such as sepsis, as well as drug toxicity and autoimmune disease. The release of free heme causes oxidative damage, leading to immune cell activation, endothelial damage, ischemia, and end-organ toxicity. Innate immune cells such as macrophages are specialized in heme detoxification, and while it is now evident that macrophages alter their cellular metabolism in order to carry out specific effector functions, the metabolic adaptations that allow macrophages to survive the toxic stress of heme clearance remain unknown. Preliminary data from our group reveal that in response to heme loading macrophages shift glucose metabolism toward the pentose phosphate pathway (PPP), which allows for the rapid production of NADPH and maintenance of redox homeostasis. This metabolic adaptation is dependent on the activity of heme oxygenase, and that carbon monoxide (CO) released during heme breakdown is the mediator that induces the metabolic adaptation toward the PPP. We also find that activity of the PPP is required for effective heme detoxification, and that the PPP is upregulated at the enzymatic and transcriptional level in a mouse model of SCD, while, surprisingly, PBMCs from patients with SCD, PPP enzymes are downregulated during disease exacerbation when compared with the same patient at baseline. Taken together, these data suggest a critical role for macrophage/monocyte metabolic adaptation in the response to hemolysis, and point toward manipulation of metabolism as a potential therapeutic avenue for treatment of heme-driven pathologies. This proposal tests the hypothesis that carbon monoxide released by heme breakdown shifts glucose metabolism toward the PPP and that pharmacologic manipulation of this pathway to promote the PPP promotes heme clearance and ameliorates hemolysis-induced damage. This hypothesis will be tested through the completion of the following specific aims. Specific Aim 1 is to determine the intracellular mechanism by which CO drives metabolism toward the PPP: Subaim 1A tests the hypothesis that CO inhibition of cystathionine beta synthetase (CBS) results in loss of phosphofructokinase (PFK) FB3 activity, which shunts glucose metabolism away from glycolysis and toward the PPP. Subaim 1B then examines whether pharmacologic inhibition of PFKFB3 can promote heme clearance and reduce hemolysis-induced damage in hypoxia-induced vasoocclusion. Specific Aim 2 is to examine how the current SCD treatment glutamine alters immunometabolic profile of circulating monocytes in mouse and human SCD. Subaim 2A tests in vitro and in vivo whether glutamine alters cellular metabolism to promote the PPP and ameliorate hemolysis-induced pathology. Subaim 2B integrates transcriptomic and bioenergetics analyses to characterize the immunometabolic status of PBMCs from patients with SCD, to determine how metabolism is altered at steady state, during disease exacerbation, or in response to treatment.