Efferocytosis meets endocytosis

胞吞作用遇上内吞作用

• 批准号: 10673780
• 负责人: Bishuang Cai
• 金额: $ 42.25万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673780
• 关键词: ATP binding cassette transporter 1; Acceleration; Anabolism; Apoptosis; Apoptotic; Arterial Fatty Streak; Binding; Biochemistry; C-terminal; CRISPR screen; Carrier Proteins; Cell surface; Cells; Cellular biology; Cholesterol; Cholesterol Homeostasis; Cytoskeleton; Data Set; Digestion; Disease; Docosahexaenoic Acids; Endocytosis; Epitopes; Event; Homeostasis; Inflammation; Inflammation Mediators; Inflammatory Response; Intracellular Transport; Learning; Macrophage; Maintenance; Mediating; Mediator; Membrane; Metabolism; Necrosis; Phagocytes; Phagocytosis; Physiological; Process; Resolution; Role; Signal Transduction; Tertiary Protein Structure; Tissues; cholesterol control; chronic inflammatory disease; functional genomics; genome-wide; immunogenic; mouse genetics; novel; novel therapeutic intervention; receptor; single-cell RNA sequencing; tissue repair; trafficking; uptake; vesicle transport

ABSTRACT Apoptosis happens continuously along with the active clearance of apoptotic cells (ACs) by phagocytes or efferocytes, termed “efferocytosis”, to maintain tissue homeostasis. When efferocytosis becomes defective, uncleared ACs undergo post-apoptotic necrosis and release immunogenic epitopes and pro-inflammatory mediators, which leads to chronic inflammatory diseases. Our recent studies revealed a novel role of efferocytosis in accelerating tissue repair as it promotes inflammation resolution by inducing the biosynthesis of specialized pro-resolving mediators (SPMs) that stop inflammatory responses. Therefore, understanding how efferocytosis is successfully carried out is of paramount importance. Much has been learned about the mechanisms of AC recognition and uptake, but how efferocytes degrade ACs and process the metabolic cargo, e.g., cholesterol released from AC digestion, is incompletely understood. Moreover, although efferocytosis and endocytosis share common features, such as involving cytoskeleton rearrangement and intracellular transport of vesicular membrane-bound cargoes, whether efferocytes hijack the endocytic machinery to process AC- derived cargo remains uncertain. In our unpublished results, we found that resolvin D1 (RvD1), a docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments (efferosomes) and LC3-II lipidation, key features in LC3-associated phagocytosis (LAP)-mediated corpse degradation. As our recent study showed that the activation of MerTK, the efferocytosis receptor, was required for RvD1 biosynthesis, these results indicate a novel role of MerTK-RvD1 signaling in LAP-mediated AC degradation. To study whether the key endocytic regulators—the C-terminal Eps15 Homology Domain (EHD) proteins comprising EHD1, EHD2, EHD3, and EHD4—are involved in efferocytosis-related events, we analyzed a single-cell RNA-sequencing (scRNA-seq) dataset from atherosclerotic lesions where a lot of cells undergo apoptosis and found that EHD proteins had heterogeneous expression with high expression of EHD1 and EHD4 in macrophages, the professional efferocytes. We further found that EHD1 enhanced the cell surface levels of the cholesterol efflux transport protein ABCA1 in macrophages during efferocytosis, which indicates that EHD1- mediated endocytic trafficking of ABCA1 may play a role in removing the excess free cholesterol released from digested ACs. Here, we propose to combine approaches in cell biology, biochemistry, mouse genetics, and functional genomics to determine the function and mechanisms of MerTK-RvD1 signaling in LAP and EHD proteins in efferocytosis-related events including maintaining cellular cholesterol homeostasis and controlling endocytic trafficking of MerTK. We will also perform unbiased genome-wide CRISPR screening to identify novel regulators of MerTK levels on the cell surface where macrophages receive ACs. Taken together, understanding these aspects of efferocytosis will shed light on key physiological and pathophysiological processes and suggest novel therapeutic strategies for diseases driven by defective efferocytosis.

摘要 细胞凋亡沿着吞噬细胞对凋亡细胞（AC）的主动清除， 巨噬细胞，称为“巨噬细胞”，以维持组织稳态。当红细胞增多症出现缺陷时， 未清除的AC经历凋亡后坏死并释放免疫原性表位和促炎性表位。 介质，导致慢性炎症性疾病。我们最近的研究揭示了一个新的作用， 红细胞增多症在加速组织修复中的作用，因为它通过诱导 专门的促消退介质（SPM），阻止炎症反应。因此，了解如何 红细胞增多症的成功进行是至关重要的。我们已经了解了很多关于 AC识别和摄取的机制，但巨噬细胞如何降解AC和处理代谢货物， 例如，在一个实施例中，从AC消化释放的胆固醇是不完全理解的。此外，虽然红细胞增多症和 内吞作用具有共同的特征，例如涉及细胞骨架重排和细胞内转运 囊泡膜结合的货物，无论是巨噬细胞劫持的内吞机制，以处理AC- 衍生货物仍不确定。在我们未发表的研究结果中，我们发现resolvin D1（RvD 1）， 二十二碳六烯酸（DHA）衍生的SPM，增强了含AC隔室的酸化 （脂质体）和LC 3-II脂质化，LC 3相关吞噬（CYP）介导的尸体中的关键特征 降解由于我们最近的研究表明，需要激活MerTK（红细胞增多症受体）， 对于RvD 1生物合成，这些结果表明MerTK-RvD 1信号传导在LAP介导的AC中的新作用 降解为了研究内吞的关键调节因子--C末端Eps 15同源结构域（EHD）是否与细胞内吞有关， 蛋白质包括EHD 1，EHD 2，EHD 3，和EHD 4-参与细胞凋亡相关的事件，我们分析了 来自动脉粥样硬化病变的单细胞RNA测序（scRNA-seq）数据集，其中许多细胞经历了 EHD蛋白表达呈异质性，EHD 1和EHD 4在细胞凋亡中呈高表达 在巨噬细胞中，专业的巨噬细胞。我们进一步发现，EHD 1增强了细胞表面的 胆固醇流出转运蛋白ABCA 1在巨噬细胞中的表达，这表明EHD 1- ABCA 1介导的内吞运输可能在清除从细胞中释放的过量游离胆固醇中起作用。 消化的AC在这里，我们建议联合收割机细胞生物学、生物化学、小鼠遗传学和 功能基因组学，以确定MerTK-RvD 1信号转导在EHD和EHD中的功能和机制 蛋白质在细胞凋亡相关事件中的作用，包括维持细胞胆固醇稳态和控制 MerTK的内吞运输。我们还将进行无偏见的全基因组CRISPR筛选，以确定新的 在巨噬细胞接受AC的细胞表面上的MerTK水平的调节剂。综合考虑，理解 红细胞增多症的这些方面将阐明关键的生理和病理生理过程，并提示 用于由缺陷性红细胞增多症驱动的疾病的新治疗策略。