Defining the molecular structure-function relationships of extracellular vesicles from dying cells.

身体中受损、感染、老化或不需要的细胞通过一个高度受控的过程死亡，称为“细胞死亡”。细胞凋亡在一系列正常组织功能、胚胎发育、去除感染细胞、控制对感染的免疫反应以及一系列与年龄相关的疾病状况（包括炎性疾病、癌症、神经变性和心血管疾病）中是重要的。在死亡水平高的部位，专业的“清道夫”细胞（称为巨噬细胞的吞噬细胞）被招募。这些吞噬细胞被垂死细胞释放的“找到我”信号所吸引，然后吞噬细胞受体在垂死细胞表面结合“吃我”信号。吞噬细胞迅速掩埋死亡细胞是至关重要的，因为否则会发生死亡细胞内容物的泄漏。这反过来又导致炎症（例如动脉粥样硬化）和自身免疫性疾病（例如系统性红斑狼疮）的灾难性后果。通过定义“吃我”信号（一种称为ICAM-3的蛋白质）和介导进食的重要受体（CD 14），我们对细胞死亡领域产生了重大影响。然而，尽管对介导吞噬细胞去除垂死细胞的分子了解很多，但对“找到我”信号的身份和功能知之甚少。当细胞死亡时，细胞会释放出小的膜“囊”（“凋亡细胞衍生的细胞外囊泡”或“acdEV”），这些囊泡会从垂死细胞的表面起泡并夹断。这些acdEV的全部功能是未知的，并且对其组成知之甚少。我们最近已经表明，垂死的白色血细胞在acdEV上释放ICAM-3，以创建一个有吸引力的“路径”，将吞噬细胞引导到细胞死亡的部位，在那里它们可以以安全和受控的方式清除细胞尸体。然而，很少有人知道其他acdEV功能。我们的工作为细胞死亡领域提出了重要问题。(1)acdEV的组成是什么？(2)这个组合与功能有什么关系？在本工作方案中，我们将处理我们知识中的这些根本性差距。我们将分析不同类型的垂死细胞（免疫和非免疫系统细胞）释放的acdEV，并定义这些颗粒的组成和功能。我们将详细分析（A）这些acdEV内部和表面所含的蛋白质，以确定acdEV的蛋白质组成;（B）acdEV的脂质成分;和（C）acdEV内部所含的小核酸分子（“microRNA”）。我们将通过增加或减少其分子存在来关注单个acdEV成分，并在一系列体外和体内试验中评估其与免疫系统相互作用和调节免疫系统的功能能力。这项工作将首次详细介绍分子组成并将其与acdEV的功能联系起来，从而回答重要问题并显着推进该领域。它将在分子水平上定义垂死细胞如何与其他细胞沟通，以确保它们被迅速清除而不会导致炎症。这一点很重要，因为死亡细胞的清除缺陷会导致疾病。因此，利用我们的工作将针对那些条件下，无效的死细胞清除导致破坏性炎症反应（如动脉粥样硬化）。

Damaged, infected, aged or unwanted cells in the body die via a highly controlled process known as 'apoptosis'. Apoptosis is important in a range of normal tissue functions, embryonic development, removing infected cells, controlling immune responses to infection, and in a range of age-associated disease conditions including inflammatory diseases, cancer, neurodegeneration and cardiovascular disease. At sites where levels of death are high, professional 'scavenger' cells (phagocytes known as macrophages) are recruited. These phagocytes are attracted to 'find me' signals released from dying cells and then phagocyte receptors bind 'eat me' signals on the dying cell surface. It is crucially important for phagocytes to bury dying cells quickly because otherwise leakage of dead cell contents can occur. This in turn leads to catastrophic consequences of inflammation (e.g. in atherosclerosis) and autoimmune disease (e.g. systemic lupus erythematosus). We have made a significant impact on the field of cell death by defining an 'eat me' signal (a protein called ICAM-3) and an important receptor that mediates eating (CD14). However, whilst much is known of molecules that mediate phagocytic removal of dying cells, very little is known of the identity and function of the 'find me' signals. We are one of a small group of scientists addressing the nature of these signals.When dying, cells release small membrane 'bags' ('apoptotic cell-derived extracellular vesicles' or 'acdEV') that bleb and pinch off from the surface of dying cells. The full function of these acdEV is unknown and little is known of their composition. We have shown recently that dying white blood cells release ICAM-3 on acdEV to create an attractive 'path' that leads phagocytes to the site of cell death where they can clear the cell corpses in a safe and controlled manner. However, little is known of other acdEV functions. Our work raised important questions for the field of cell death. (1) What is the composition of acdEV? (2) How does this composition relate to function? In this programme of work we will address these fundamental gaps in our knowledge. We will analyse acdEV release from different types of dying cells (immune and non-immune system cells) and will define the composition and function of these particles. We will undertake a detailed analysis of (A) the PROTEINS contained within and on the surface of these acdEV to define the protein constitution of the acdEV; (B) the LIPID constituents of the acdEV; and (C) the small nucleic acid molecules ('microRNA') contained within the acdEV. We will focus on individual acdEV components by increasing or decreasing their molecular presence and assess their functional ability to interact with and modulate the immune system in a range of assays both in vitro and in vivo.This work will, for the first time, detail the molecular composition and link this to function of acdEV thereby answering important questions and advancing the field significantly. It will define, at a molecular level, how dying cells communicate with other cells to ensure that they are removed rapidly without leading to inflammation. This is important because defective clearance of dying cells leads to disease. Thus exploitation of our work will target those conditions where inefficient dead cell clearance results in damaging inflammatory responses (e.g. in atherosclerosis).