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

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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).