Efferocytosis meets endocytosis

胞吞作用遇上内吞作用

基本信息

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
项目类别：
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

项目摘要

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 In our unpublished results, we found that resolvin D1 (RvD1), a docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments (efferosomes)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.

抽象的细胞凋亡随着吞噬细胞或细胞主动清除凋亡细胞（AC）而持续发生。胞吞作用，称为“胞吞作用”，当胞吞作用出现缺陷时，维持组织稳态。未清除的 AC 会发生凋亡后坏死并释放免疫原性表位和促炎物质我们最近的研究揭示了导致慢性炎症疾病的新作用。胞吞作用加速组织修复，因为它通过诱导生物合成促进炎症消退因此，了解如何阻止炎症反应的专门促消解介质 (SPM)。胞吞作用的成功进行是至关重要的。 AC 识别和摄取的机制，但是传细胞如何降解 AC 并处理代谢货物，例如，尽管胞吞作用和 AC 消化释放的胆固醇尚不完全清楚。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运输囊泡膜结合货物，泡细胞是否劫持内吞机制来处理 AC- 在我们未发表的结果中，我们发现 resolvin D1 (RvD1) 是一种衍生货物。二十二碳六烯酸 (DHA) 衍生的 SPM，增强了含有 AC 的隔室的酸化 (efferosomes) 和 LC3-II 脂化，LC3 相关吞噬作用 (LAP) 介导的尸体的关键特征正如我们最近的研究表明，需要激活 MerTK（胞吞作用受体）。对于 RvD1 生物合成，这些结果表明 MerTK-RvD1 信号在 LAP 介导的 AC 中的新作用研究关键的内吞调节因子——C 端 Eps15 同源域 (EHD) 是否会降解。我们分析了包含 EHD1、EHD2、EHD3 和 EHD4 的蛋白质参与胞吞作用相关事件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动脉粥样硬化病变细胞凋亡，发现EHD蛋白具有异质性表达，其中EHD1和EHD4高表达在巨噬细胞中，我们进一步发现 EHD1 增强了细胞表面的水平。胞吞作用期间巨噬细胞中的胆固醇外流转运蛋白 ABCA1，这表明 EHD1- ABCA1 介导的内吞转运可能在去除从细胞中释放的过量游离胆固醇方面发挥作用。在这里，我们建议结合细胞生物学、生物化学、小鼠遗传学和功能基因组学确定 MerTK-RvD1 信号在 LAP 和 EHD 中的功能和机制胞吞作用相关事件中的蛋白质，包括维持细胞胆固醇稳态和控制我们还将进行无偏倚的全基因组 CRISPR 筛选，以鉴定新的 MerTK。巨噬细胞接收 AC 的细胞表面 MerTK 水平的调节剂。胞吞作用的这些方面将揭示关键的生理和病理生理过程，并表明针对由胞吞作用缺陷引起的疾病的新治疗策略。