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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要巨噬细胞（胞吞作用）对凋亡细胞 (AC) 的清除缺陷会导致许多疾病鉴于调节胞吞作用的基因的治疗潜力，我们试图我们以公正的方式鉴定了该途径的新调节因子，建立了全基因组汇集的 CRISPR。 Cas9 过度表达的小鼠骨髓来源的巨噬细胞的胞吞作用的敲除筛选。最强屏幕点击率的验证发现 Wdfy3（WD 重复和包含 3 的 FYVE 域）作为以前从未涉及胞吞作用或吞噬作用的调节的新型调节剂。研究重点是Wdfy3，对其分子机制和作用进行深入研究被吞噬的 AC 的降解需要招募一部分自噬，但不是全部自噬。 LC3 脂化和随后的吞噬体-溶酶体融合的机制仍然存在重要意义。确定哪些自噬基因参与吞噬货物的降解的知识差距。非选择性规范自噬（“自噬”）不需要的部分对其特别感兴趣 Wdfy3 的治疗靶向激活调节选择性自噬以清除聚集的潜力。蛋白质，但对于饥饿期间的非选择性规范自噬来说是可有可无的，使其成为这样的候选者。我们的数据表明，Wdfy3 敲低可减少小鼠和人类巨噬细胞中的胞吞作用；动脉粥样硬化期间斑块巨噬细胞中被吞噬的 AC 局部表达增加；在人类斑块中，WDFY3 表达与 M2 样巨噬细胞标记物相关。在上下文中，我们认为 Wdfy3 是巨噬细胞通过调节吞噬体形成进行胞吞作用所必需的成熟和 Wdfy3 介导的胞吞作用可预防高脂血症小鼠的动脉粥样硬化目标 1 将通过解决 (A) 胞吞作用的哪个阶段来确定 Wdfy3 的分子机制。受 Wdfy3 调节；(B) Wdfy3 如何招募并参与吞噬体形成及其下游事件；(C) Wdfy3 的过度表达是否以及如何增强胞吞作用；(A) 是否会增强胞吞作用。缺乏骨髓 Wdfy3 的小鼠在地塞米松诱导的胸腺细胞凋亡中具有缺陷的胞吞作用在晚期动脉粥样硬化中，WDFY3 的骨髓过度表达是否会增强体内 (B) 人 iPSC 衍生的巨噬细胞是否具有 WDFY3 null 突变表现出胞吞作用缺陷和炎症增强，目标 3 将检查是否存在其他缺陷。屏幕热门中的自噬基因也是胞吞作用及其依赖性的关键调节因子 Wdfy3。这项研究将 (1) 揭示 Wdfy3 和细胞调节的新的胞吞作用的基本机制。 Wdfy3 介导的胞吞作用作为动脉粥样硬化促胞吞治疗靶点的潜力，以及 (2) 为初级复杂功能表型的全基因组筛选提供了广泛适用的平台巨噬细胞用于公正的新发现。