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

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).

Apoptosis removes unwanted cells in the body. During this process, apoptotic cell-derived extracellular vesicles (acdEV) are released but the full function and structure of acdEV have never been defined. We have shown that acdEV recruit phagocytes and exert anti-inflammatory effects to promote safe, controlled and efficient removal of dying cells. This is important as it ensures successful apoptotic cell clearance and avoidance of inflammation and autoimmunity.

Our previous work highlights important questions in the field of cell death that will be answered in this project. What is the molecular composition of the acdEV? How does this composition relate to acdEV function in ensuring efficient removal of apoptotic cells? This work programme will define those key molecular components that mediate the beneficial immune-modulatory functions of acdEV.

We will identify acdEV (1) PROTEIN components and, using surface biotinylation, acdEV surface proteins - proteins that may mediate the interaction of acdEV with cells of the immune system; (2) LIPID MEDIATORS that may modulate inflammation. Such mediators have been shown to promote resolution of inflammation. Here we will look at the lipidome of acdEV for the first time; and (3) MICRORNA content. We will assess the miRNA ability to mediate intercellular communication and exert anti-inflammatory effects through gene silencing. Using over- or under-expression systems and the use of specific inhibitors we will confirm the role of individual molecular players in the ability of the acdEV to interact with the immune system through in vitro and in vivo functional studies.

This unique work will generate the first comprehensive structure-function analysis of acdEV. It will define those components that interact with the immune system to promote apoptotic cell removal and resolution of inflammation. Thus this work will provide essential detail to enable the manipulation of apoptotic cell clearance for therapy.

Intercellular communication underlies effective functioning of multicellular organisms and extracellular vesicles (EV) are novel mediators of this communication. EV are known to impact on a number of physiological and pathological processes. Our work will focus a particular class of EV (apoptotic cell-derived EV; acdEV) that have never been studied in a detailed and comprehensive manner. This work is of great importance as cell death is vital to healthy ageing and the manner in which cell corpses interact with the immune system is fundamental to the control of inflammation, immune responses and the prevention of important age-associated pathologies (e.g. cardiovascular disease, cancer, inflammation and autoimmunity). The fundamental research outlined in this proposal is thus directly relevant to the BBSRC strategy of Bioscience for Health. Through a better understanding of the mechanisms by which dying cells interact with and modulate the immune system, we will seek to manipulate these normal physiological processes for therapeutic gain.
WHO WILL BENEFIT? This work is of very broad appeal. In the short-term, beneficiaries will be academic e.g. basic scientists and clinicians in a wide range of research and therapeutic areas (e.g. cell death, immunology, inflammation, cardiovascular disease, EV and cellular communication). These will naturally include the immediate workers on this project and other local research groups and students. Our work will significantly advance the field through improved fundamental detail of cellular communication and control of inflammation. Consequently this research will enhance our basic understanding of both physiological and pathological processes in a wide range of disease. For example, EV are of particular interest to the field of cardiovascular disease. By defining the fundamental communication factors released from dying cells we will shed new light on this important pathology where recruitment of monocytes to dying cells in the atherosclerotic plaque drives disease.
In the longer term, beneficiaries will include parties interested in detailed mechanisms for control of inflammation and cell communication (e.g. Pharmaceutical companies, clinicians and patients suffering inflammatory diseases). Ultimately, this work (through its relevance to ageing) will also be of interest to the general public.
HOW WILL THEY BENEFIT? This work will highlight mechanisms by which apoptotic cells interact with the immune system - fundamental for control of inflammation. It will detail molecular mechanisms that recruit phagocytes to identify and silently remove unwanted cells, by defining 'find me' signals and immune-modulatory signals. Such improved knowledge of mechanisms that recruit phagocytes to sites of cell death will provide valuable innovative approaches for modulating inappropriate phagocyte recruitment to sites of cell death. Such sites include tumours and atherosclerotic plaques. In these cases macrophages infiltrate yet fail to resolve the pathological lesion. Consequently the ability to inhibit phagocyte recruitment may be of therapeutic benefit. Furthermore defining new anti-inflammatory factors within acdEV will allow production of synthetic EV to be tested for efficiency as therapeutics.
The assembled team has all the skills for the fundamental studies and the production of novel potential therapeutics. Human therapy is a key long-term goal. Therefore, in the nearer term, output from this research will lever additional funding for focus on development of innovative therapies that may be developed in house at Aston through the marriage of the basic science (Devitt, Griffiths) and the liposome research team (Prof Perrie) within Aston Pharmacy School. The assembled world-leading team of investigators and collaborators will drive the translation of our cutting edge, basic bioscience.