Discovering Wdfy3 as a novel regulator of macrophage efferocytosis by genome-wide CRISPR screen

通过全基因组 CRISPR 筛选发现 Wdfy3 作为巨噬细胞胞吞作用的新型调节因子

• 批准号: 10606558
• 负责人: Hanrui Zhang
• 金额: $ 59.35万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606558
• 关键词: Acute; Address; Apoptosis; Apoptotic; Arterial Fatty Streak; Atherosclerosis; Autophagocytosis; Binding; Biological Process; Bone Marrow; CRISPR screen; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Dependence; Dexamethasone; Disease; Eating; Event; Genes; Goals; Guide RNA; Human; Impairment; In Vitro; Individual; Inflammation; Knock-in; Knock-out; Knowledge; Lysosomes; Macrophage; Mediating; Molecular; Mus; Myelogenous; Pathway interactions; Performance; Phagocytes; Phagocytosis; Phagosomes; Phenotype; Proteins; Regulation; Role; Small Interfering RNA; Starvation; Testing; Therapeutic; Thymus Gland; Validation; WD Repeat; genome wide screen; genome-wide; in vivo; induced pluripotent stem cell; innovation; insight; interest; knock-down; mouse model; mutation carrier; novel; null mutation; nutrient deprivation; overexpression; protein aggregation; recruit; seal; therapeutic target

PROJECT SUMMARY Defective clearance of apoptotic cells (AC) by macrophages (efferocytosis) contribute to many diseases including atherosclerosis. Given the therapeutic potential of genes that regulate efferocytosis, we sought to identify novel regulators of this pathway in an unbiased manner. We established a genome-wide pooled CRISPR knockout screen for efferocytosis in Cas9-overexpressing murine bone marrow-derived macrophages. Individual validation of the strongest screen hits has uncovered Wdfy3 (WD repeat and FYVE domain containing 3) as a novel regulator previously never implicated in the regulation of efferocytosis or phagocytosis. The goal of this study is to focus on Wdfy3 for in-depth mechanistic studies of its molecular mechanisms and roles in atherosclerosis. Degradation of engulfed AC requires the recruitment of a subset, but not all of the autophagy machinery for LC3 lipidation and subsequent phagosome-lysosome fusion. There remains a significant knowledge gap to identify which autophagy genes participate in the degradation of phagocytosed cargos. Those that are not required for non-selective canonical autophagy (“self-eating”) are of particular interest for their potential of therapeutic targeting for activation. Wdfy3 regulates selective autophagy for clearance of aggregated proteins, but is dispensable for non-selective canonical autophagy during starvation, making it such a candidate. Our data show that Wdfy3 knockdown reduces efferocytosis in murine and human macrophages; WDFY3 is co- localized with engulfed AC; Wdfy3 expression is increased in plaque macrophages during atherosclerosis regression; in human plaque, WDFY3 expression is correlated with M2-like macrophage markers. Within this context, we hypothesize that Wdfy3 is required for macrophage efferocytosis by regulating phagosome formation and maturation and Wdfy3-mediated efferocytosis protects against atherosclerosis in hyperlipidemic mice models. Aim 1 will determine the molecular mechanisms of Wdfy3 by addressing (A) which stage of efferocytosis is regulated by Wdfy3; (B) How Wdfy3 is recruited and participates in phagosome formation and downstream events; (C) whether and how overexpression of Wdfy3 enhances efferocytosis. Aim 2 will determine (A) whether mice lacking myeloid Wdfy3 have defective efferocytosis in dexamethasone-induced thymic apoptosis and in advanced atherosclerosis; and whether myeloid overexpression of WDFY3 will enhance in vivo efferocytosis and alleviate atherosclerosis; (B) whether human iPSC-derived macrophages with WDFY3 null mutations demonstrate defective efferocytosis and enhanced inflammation. Aim 3 will examine whether other autophagy genes among the top screen hits are also key regulators of efferocytosis and their dependence on Wdfy3. This study will (1) reveal novel fundamental mechanisms of efferocytosis regulated by Wdfy3 and the potential of Wdfy3-mediated efferocytosis as a target for pro-efferocytotic therapy in atherosclerosis, and (2) provide a broadly-applicable platform for genome-wide screen of complex functional phenotypes in primary macrophages for unbiased novel discoveries.

项目摘要 通过巨噬细胞（for经菌病）对凋亡细胞（AC）的缺陷清除有助于许多疾病 包括动脉粥样硬化。鉴于调节传染性胞症的基因的治疗潜力，我们试图 以公正的方式识别该途径的新型调节剂。我们建立了一个全基因组汇总的CRISPR 敲除筛查筛分过表达CAS9的鼠骨髓衍生的巨噬细胞中的筛查。个人 强烈屏幕命中的验证已发现WDFY3（WD重复和包含3的FYVE域）作为一个 新型调节剂以前从不隐含在肿瘤病或吞噬作用的调节中。目标的目标 研究将重点放在WDFY3上，以进行其分子机制的深入机械研究，并在 动脉粥样硬化。吞没的AC的退化需要招募子集，但不是所有自噬 LC3脂质的机械以及随后的吞噬体散糖体融合。仍然很重要 知识差距以识别哪些自噬基因参与吞噬碳的降解。那些 非选择性的规范自噬（“自我吃”）不需要的是特别感兴趣的 热靶向激活的潜力。 WDFY3调节选择性自噬以清除聚合 蛋白质，但对于饥饿期间的非选择性规范自噬是可分配的，使其成为候选者。 我们的数据表明，WDFY3敲低可减少鼠和人类巨噬细胞中的吞噬作用。 WDFY3是共同的 与吞没的交流电本地化；在动脉粥样硬化期间，WDFY3的表达增加了 回归；在人斑块中，WDFY3表达与M2样巨噬细胞标记相关。在此 上下文，我们假设WDFY3是通过控制吞噬体形成的巨噬细胞吞噬作用所必需的 成熟和WDFY3介导的肿瘤病可预防高脂血症小鼠的动脉粥样硬化 型号。 AIM 1将通过解决（a）哪个阶段的肿瘤性阶段来确定WDFY3的分子机制 由WDFY3调节； （b）如何招募WDFY3并参与吞噬体形成和下游 事件； （c）WDFY3的过度表达以及如何增强吞噬作用。 AIM 2将确定（a）是否 缺乏髓样WDFY3的小鼠在地塞米松诱导的胸腺凋亡和 在晚期动脉粥样硬化中； WDFY3的髓样过表达是否会增强体内 肿瘤病和减轻动脉粥样硬化； （b）人类IPSC衍生的巨噬细胞是否具有WDFY3 NULL 突变表现出缺陷的肿瘤病和炎症增强。 AIM 3会检查其他是否 顶部屏幕击中中的自噬基因也是胚细胞增多症的关键调节剂及其对 WDFY3。这项研究将（1）揭示由WDFY3和THE调节的新型肿瘤病的基本机制 WDFY3介导的胞吞作用的潜力是动脉粥样硬化中促肌细胞疗法的靶标，（2） 为主要功能表型的全基因组屏幕提供广泛适用的平台 无偏见的新发现的巨噬细胞。