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.

项目总结 巨噬细胞对凋亡细胞(AC)的缺陷清除(胞泡吞噬)导致许多疾病 包括动脉粥样硬化。考虑到调节泡沫化的基因的治疗潜力，我们试图 以不偏不倚的方式确定这一途径的新调控因子。我们建立了全基因组池CRISPR Cas9过表达小鼠骨髓来源的巨噬细胞胞吐作用的基因敲除筛选。个体 对最强屏幕点击量的验证发现Wdfy3(WD重复和FYVE结构域包含3)是一个 新的调节剂以前从未涉及到吞噬或吞噬的调节。这样做的目的是 研究的重点是Wdfy3，对其分子机制和作用进行深入的研究 动脉硬化。吞噬AC的降解需要补充一个子集，但不是所有的自噬 LC3脂化和随后的吞噬小体-溶酶体融合的机制。仍然存在着一个重要的 确定哪些自噬基因参与吞噬的货物的降解的知识差距。那些 非选择性典型自噬(“自噬”)所不需要的基因对于它们的 治疗靶向激活的可能性。Wdfy3调节选择性自噬清除聚集 蛋白质，但对于饥饿期间的非选择性典型自噬来说是可有可无的，使其成为这样一个候选。 我们的数据显示，Wdfy3基因敲除减少了小鼠和人巨噬细胞的吞噬作用；WDFY3是共同的- 动脉粥样硬化斑块巨噬细胞Wdfy3表达增加 回归：在人类斑块中，WDFY3的表达与M2样巨噬细胞标志物相关。在这个范围内 背景，我们假设Wdfy3通过调节吞噬小体的形成是巨噬细胞泡腾所必需的。 成熟和Wdfy3介导的泡腾作用对高脂血症小鼠动脉粥样硬化的保护作用 模特们。目标1将通过解决(A)哪个阶段的胞吐来确定Wdfy3的分子机制 受Wdfy3调控；(B)Wdfy3如何被招募并参与吞噬小体的形成和下游 事件；(C)过表达Wdfy3是否以及如何增强泡沫化作用。目标2将决定(A)是否 缺乏髓系Wdfy3的小鼠在地塞米松诱导的胸腺细胞凋亡和 在晚期动脉粥样硬化中；以及WDFY3的髓系过表达是否会在体内增强 吞噬和缓解动脉粥样硬化；(B)WDFY3缺失的人IPSC来源的巨噬细胞 突变表现为有缺陷的泡泡吞噬和炎症增强。目标3将检查是否有其他 热门屏幕中的自噬基因也是泡沫化的关键调节因素，它们依赖于 Wdfy3.这项研究将(1)揭示Wdfy3和Wdfy3调节胞吐作用的新的基本机制。 Wdfy3介导的泡腾作用作为动脉粥样硬化泡腾治疗的靶点的可能性，以及(2) 提供了一个广泛适用的平台，用于在全基因组范围内筛选初级肺癌的复杂功能表型 巨噬细胞为无偏见的新发现。