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

项目摘要 巨噬细胞对凋亡细胞（AC）的清除缺陷（巨噬细胞增多症）导致许多疾病 包括动脉粥样硬化。考虑到调节红细胞增多症的基因的治疗潜力，我们试图 以公正的方式鉴定该途径的新调节剂。我们建立了一个全基因组的混合CRISPR 在过表达Cas9的鼠骨髓来源的巨噬细胞中敲除筛选巨噬细胞增多症。个人 最强筛选命中的验证已经揭示了Wdfy 3（WD重复序列和含有3的FYVE结构域）作为一种 一种新的调节剂，以前从未涉及调节吞噬作用或吞噬作用。这个目标 研究的重点是Wdfy 3的分子机制和作用， 动脉粥样硬化吞噬的AC的降解需要募集一个子集，但不是所有的自噬 LC 3脂质化和随后的吞噬体-溶酶体融合的机制。仍然有一个重要的 知识差距，以确定哪些自噬基因参与降解的吞噬货物。那些 非选择性典型自噬（“自食”）不需要的蛋白质， 治疗靶向激活的潜力。Wdfy 3调节选择性自噬以清除聚集的 蛋白质，但在饥饿期间非选择性典型自噬，使其成为这样的候选者。 我们的数据显示，Wdfy 3敲低减少了鼠和人巨噬细胞中的巨噬细胞增多症; Wdfy 3与巨噬细胞共表达。 局限于吞噬的AC;动脉粥样硬化期间斑块巨噬细胞中Wdfy 3表达增加 在人类斑块中，WDFY 3表达与M2样巨噬细胞标志物相关。在这 在此背景下，我们假设Wdfy 3通过调节吞噬体的形成而成为巨噬细胞胞吐作用所需的 成熟和Wdfy 3介导的红细胞增多对高脂血症小鼠动脉粥样硬化的保护作用 模型目的1将确定Wdfy 3的分子机制，通过解决（A）在红细胞增多症的哪个阶段， （B）Wdfy 3如何被募集并参与吞噬体形成和下游 事件;（C）Wdfy 3的过表达是否以及如何增强红细胞增多症。目标2将确定（A）是否 缺乏髓系Wdfy 3的小鼠在地塞米松诱导的胸腺凋亡中具有缺陷性红细胞增多症， 在晚期动脉粥样硬化中;以及WDFY 3的髓样过表达是否会在体内增强 （B）WDFY 3无效的人iPSC衍生的巨噬细胞是否 突变表明缺陷性红细胞增多症和增强的炎症。目标3将审查其他 自噬基因在最热门的筛选中也是红细胞增多症的关键调节因子， Wdfy3.本研究将（1）揭示Wdfy 3调控的红细胞增多症的新的基本机制， Wdfy 3介导的巨噬细胞增多症作为动脉粥样硬化中促巨噬细胞治疗的靶点的潜力，以及（2） 提供了一个广泛适用的平台，用于在基因组范围内筛选原发性肝癌中的复杂功能表型， 无偏见的新发现。

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.