Active Extracellular Vesicles - defining a novel, extracellular metabolic compartment and its role in the control of inflammation.

Inflammation is a normal defence mechanism used by the immune system to help protect from infection and to repair damage caused by trauma. However, the benefits of inflammation are only realised when the response is turned off and the infected or traumatised tissue is returned to normal. This process is known as 'resolution' and relies on immune cells (that have done their protective job) being removed. To allow their removal, these unwanted cells die and, as they do so, they release small membrane bags (called extracellular vesicles: EV). We have shown that these EV are key to attracting 'undertaker' cells ('macrophages') that remove the dying cells by eating them and, crucially, they produce additional signals that drive the tissue to repair.

Relatively little is known of how these EV function. We have identified a molecule that promotes recruitment of macrophages but the manner in which these EV promote 'repair' responses in macrophages remains unknown. Now, our preliminary work has shown that these EV carry a family of active enzymes which will help to produce small lipid molecules that may control inflammation and repair responses. The work proposed here will define the action of these active enzymes that are delivered from dying cells to macrophages in EV. As such, these 'active' EV represent a novel extracellular metabolically-active compartment capable of transmitting inflammation-controlling signals to surrounding cells such as macrophages.

Why is this important? A failure to control inflammatory responses can lead to chronic inflammation and many of the inflammatory diseases associated with ageing. So, by understanding the mechanisms of communication between cells promoting inflammation, and those cells promoting resolution and repair, we will have a clearer insight to possible therapeutic approaches for diseases that are driven by inflammation (e.g. cardiovascular disease, cancer, neurodegeneration). Furthermore, we will study EV from a range of other cells that are known to help promote repair. For example, a type of stem cell, an 'MSC', has held great promise for regenerative medicine but recently the EV released from these cells have been proposed to be active to stimulate a 'repair environment'. Consequently, we will study in detail the inflammation-controlling metabolic activity of EV from MSC. This raises the possibility that we may be able to define the crucial factors required for EV repair activity, opening up novel stem cell-based or cell-free therapies for regenerative medicine.

In order to test our core hypothesis, we will analyse, from a range of dying and viable cells, EV composition for the presence of enzymes, their substrates and their products. We will test the EVs' ability to promote repair in vitro and in vivo and we will inhibit the key enzymes to assess the essential nature of this activity. Furthermore, we will seek to 'tune' or refine cell culture conditions to maximise EV production and function.

This work will, for the first time, detail an inflammation-controlling, metabolically-active extracellular compartment. It will define, at a molecular level, how dying cells communicate with other cells to ensure inflammation is controlled. This is important because ineffective control of inflammation leads to disease. Thus, exploitation of our work will target those conditions where inflammation helps drive disease and will enable novel strategies to promote self-repair.

For inflammation to be successful, it must address the immune challenge, be turned off and tissue must return to its pre-inflamed state. This 'resolution' phase relies on inflammatory cell death (apoptosis) and removal by macrophages. We have shown in vivo that apoptotic cell-derived extracellular vesicles (ACdEV) attract macrophages and this can modify disease. Macrophages must also become 'repair' ('M2') macrophages and the mechanism of action of EV in this process is an important question in the field. Our recent work provides a novel view on this process.

We have identified EV as a novel, extracellular, metabolically-active compartment that carries active enzymes, substrates and products producing inflammation-controlling lipid mediators (e.g. lipoxins, resolvins). Here we will define the role and importance of this 'active EV' compartment in controlling inflammation in vitro & in vivo, generating results that will have a significant impact on the field.

Why is this important? Following inflammation, 'disease or repair' is a key choice that is defined by macrophages. To understand this decision point will enable exploitation to address key issues in regenerative medicine and disease. Mesenchymal stem cells hold great promise in regenerative medicine yet there is increasing evidence that MSC benefit comes from their immune modulating secretome (including EV). Here we will also define the extent to which MSC EV are a metabolically-active, inflammation-modifying compartment. This raises the possibility that, by altering culture conditions to provide cells with increased levels of enzyme substrate (i.e. PUFA), we may produce EV with increased potential for regenerative medicine benefits.

Our work will define a novel mechanism to the control of inflammation and resolution, answering important questions in the field. This novel, fundamental insight to inflammation will have a significant impact on the field of inflammation and regenerative medicine.

Intercellular communication underlies effective functioning of the immune system. Extracellular vesicles (EV) are novel mediators of this communication and are known to impact on a number of physiological and pathological processes. Our work will focus on EV from apoptotic cells that we have shown carry enzymes, substrates and products and we will define the importance of these 'active EV' in controlling inflammation in vitro and in vivo. We will also define the role of EV from MSC, as these are proposed also to modulate inflammation and repair responses.

This work is of great importance as cell death and control of inflammation is vital to healthy ageing and the manner in which EV communicate with the innate immune system is fundamental to the control of inflammation 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 strategic research priority of Bioscience for Health. Through a better understanding of inflammation control, we will seek to manipulate these normal physiological processes for therapeutic gain to address the need for improved health-span.

WHO WILL BENEFIT? This work is of very broad appeal. In the short-term, beneficiaries will be academic basic scientists and clinicians in a wide range of research and therapeutic areas (e.g. cell biology, 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 diseases. For example, EV are of particular interest in cardiovascular disease and cancer. By defining the fundamental communication factors released from dying cells we will shed new light on these important pathologies where recruitment of monocytes to dying cells, and immune modulation, 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 novel mechanisms by which EV control inflammation. It will detail molecular mechanisms that recruit phagocytes and control their inflammatory responses. Such improved knowledge of mechanisms that help resolve inflammation will provide valuable innovative approaches for modulating inappropriate inflammatory control in pathology (e.g tumours and atherosclerotic plaques). In these cases, macrophages infiltrate yet fail to resolve the pathological lesion. Consequently, the ability to modify phagocyte recruitment and response may be of therapeutic benefit. Furthermore, defining new anti-inflammatory control mechanisms within EV will allow production of more potent EV (e.g. from MSC) for regenerative medicine applications.

The assembled team has all the skills for the fundamental studies and the production of novel potential EV 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 bioprocessing for the production of 'designer EV' that may be developed in house at Aston through the marriage of the basic science (Devitt/Milic) and the bioprocess engineers (Dr Hanga and Profs Wall & Hewitt) within Aston. The assembled world-leading team of investigators and collaborators will drive the translation of our cutting edge, basic bioscience.