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

人体内受损、感染、老化或不需要的细胞会通过一种被称为“细胞凋亡”的高度受控的过程死亡。细胞凋亡在一系列正常组织功能、胚胎发育、清除感染细胞、控制对感染的免疫反应以及包括炎症疾病、癌症、神经退行性疾病和心血管疾病在内的一系列与年龄相关的疾病中都是重要的。在死亡水平较高的地点，专业的“清道夫”细胞(被称为巨噬细胞)被招募。这些吞噬细胞被吸引来从垂死的细胞释放“找到我”的信号，然后吞噬细胞受体将“吃我”的信号绑定到垂死的细胞表面。吞噬细胞迅速掩埋死亡细胞是至关重要的，否则可能会发生死亡细胞内容物的泄漏。这进而导致炎症(如动脉粥样硬化)和自身免疫性疾病(如系统性红斑狼疮)的灾难性后果。我们通过定义‘吃我’信号(一种称为ICAM-3的蛋白质)和一个重要的调节进食的受体(CD14)，对细胞死亡领域产生了重大影响。然而，尽管人们对介导吞噬死亡细胞的分子知道得很多，但对“找到我”信号的身份和功能却知之甚少。我们是研究这些信号的性质的一小群科学家之一。当细胞死亡时，细胞释放出小的膜‘袋’(‘凋亡性细胞衍生的细胞外小泡’或‘acdEV’)，这些小袋从死亡细胞的表面起泡并被掐掉。这些acdEV的全部功能尚不清楚，对其组成也知之甚少。我们最近已经证明，垂死的白细胞在acdEV上释放ICAM-3，以创建一条吸引人的“路径”，将吞噬细胞引导到细胞死亡部位，在那里它们可以安全和可控的方式清除细胞身体。然而，人们对acdEV的其他功能知之甚少。我们的工作为细胞死亡领域提出了重要的问题。(1)acdEV的组成是什么？(2)这种组成与功能有什么关系？在这项工作方案中，我们将解决我们知识中的这些根本差距。我们将分析不同类型的死亡细胞(免疫细胞和非免疫系统细胞)释放的acdEV，并确定这些颗粒的组成和功能。我们将对(A)这些acdEV内和表面所含的蛋白质进行详细分析，以确定acdEV的蛋白质组成；(B)acdEV的脂类成分；以及(C)acdEV内所含的小核酸分子(‘microRNA’)。我们将通过增加或减少acdEV的分子存在来关注单个的acdEV组分，并在体外和活体的一系列检测中评估它们与免疫系统相互作用和调节的功能能力。这项工作将首次详细说明acdEV的分子组成并将其与acdEV的功能联系起来，从而回答重要的问题并显著推进该领域的发展。它将在分子水平上定义死亡细胞如何与其他细胞沟通，以确保它们被迅速移除而不会导致炎症。这一点很重要，因为死亡细胞的缺陷清除会导致疾病。因此，我们的工作将针对那些无效的死亡细胞清除导致破坏性炎症反应的条件(例如，在动脉粥样硬化中)。

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).