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

Lead Research Organisation: Aston University
Department Name: Sch of Life and Health Sciences

Abstract

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).

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We have discovered that detailed molecular structure of extracellular vesicles from dying white blood cells. Within this work we have identified a family of important inflammation controlling enzymes. Our current work funded through additional BBSRC funding, will characterise the function of these enzymes and their role in regenerative medicine.
Exploitation Route The experimental data that is currently being prepared for publication will provide novel approaches for researchers within the field of regenerative medicine.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description A mass spectrometry centre for the analysis of glycerolipids, glycerophospholipids and sphingolipids, and their lipid oxidation products
Amount £637,476 (GBP)
Funding ID BB/S01943X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 06/2020
 
Description A novel therapy for lymphoma & atherosclerosis - identification of commercialisation approaches
Amount £10,717 (GBP)
Funding ID BB/S000011/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 11/2018
 
Description Active Extracellular Vesicles - defining a novel, extracellular metabolic compartment and its role in the control of inflammation.
Amount £777,973 (GBP)
Funding ID BB/S00324X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 08/2021
 
Description Towards defining the functional surface of extracellular vesicles in dementia
Amount £70,341 (GBP)
Funding ID ARUK-EG2018A-006 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 02/2019
 
Description Understanding the impact of ageing on 'active Extracellular Vesicles' from Mesenchymal Stromal Cells (MSC) and consequences on repair responses: towards novel therapies in wound healing.
Amount £49,990 (GBP)
Funding ID AIS2110\33 
Organisation The Dunhill Medical Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2022 
End 10/2022
 
Title Data underpinning "CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups" 
Description  
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
URL http://researchdata.aston.ac.uk/id/eprint/454
 
Description Invited conference talk - Cambridge SelectBio (Extracellular Vesicles: Biology and Therapeutic Potential) - PI Prof Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 2016 SelectBio: Extracellular Vesicles Biology & Therapeutic Potential (Cambridge). Invited talk.

This conference presentation at the SelectBio conference formed the basis of closer working relationships with other members of the scientific field of Extracellular Vesicles.
Invited conference talk - Rotterdam 2016 ISEV (International Society for Extracellular Vesicles) - PI Prof Devitt
Year(s) Of Engagement Activity 2016
URL https://selectbiosciences.com/conferences/biographies.aspx?speaker=1235882&conf=eve2016
 
Description BBSRC Immunology Working Group Member 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The initial meetings are designed to help direct BBSRC future funding for Immunology research. I was invited to become a member based on my research profile which is supported by the research funding on these grants from the NC3R and BBSRC.
Year(s) Of Engagement Activity 2016,2017
 
Description Biochem Soc meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a Biochemical Society 'hot topic' meeting that was co-organised by Dr Devitt, Prof Griffiths and Dr Brown (Aston University). The speakers and audience were international. The topic was "Metabolic Drivers of Immunity". This was directly relevant to the vesicle research work within the Devitt research group. The international networking was excellent and plans for International collaborations were made.
Year(s) Of Engagement Activity 2015
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/HT010/view/Conference/Default.aspx
 
Description Brazil EV Workshop 2016 - Ivana Milic 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an international research workshop aimed at Early Career Researchers. Dr Ivana Milic attended to network with other international ECR in this Newton Fund/British Council/FAPESP funded workshop "Extracellular Vesicles in health and disease".
Year(s) Of Engagement Activity 2016
URL http://www.ukev.org.uk/Brazil/About.html
 
Description Engagement focused website, blog or social media channel - Twitter (@AC_d_EV) 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This is a Twitter account for Extracellular Vesicle research from Aston - covering both our BBSRC responsive mode grants and our iCASE.
Year(s) Of Engagement Activity 2016,2017
URL https://twitter.com/AC_d_EV
 
Description Invitation to talk at the UK Immunomodulation and Safety Hub workshop: UK Regenerative Medicine Platform (London 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an excellent workshop that was out of discipline. The invite came as a result of the organisers seeing previous research talks from the PI (Devitt) at other meetings.
Year(s) Of Engagement Activity 2016
URL https://www.liverpool.ac.uk/translational-medicine/events/ukrmp-workshop/
 
Description Invited Chair of a Discussion session at ISEV 2016 - PI Prof Devitt 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference at the International Congress of the International Society for Extracellular Vesicles is the world-leading platform for scientific discussions and presentation relating to Extracellular Vesicle research. The invite for PI (Prof Devitt) came as a result of the previous work presented at ISEV in 2014.
Year(s) Of Engagement Activity 2016
URL http://www.isev.org/page/isev2016
 
Description Invited Talk -British Society for Immunology Congress 2017 -Andrew Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invited presentation. A strong networking opportunity.
Year(s) Of Engagement Activity 2017
URL https://www.immunology.org/events/bsi-congress
 
Description Invited conference talk - Rotterdam 2016 ISEV (International Society for Extracellular Vesicles) - PDRA Dr Ivana Milic 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference presentation at the International Congress of the International Society for Extracellular Vesicles formed the basis of close working relationships with other members of the scientific field of Extracellular Vesicles. Our research group was singled out for special note in the Conference Summit up talk. Our PDRA won top prize for the Oral Talk. The conference enabled closer working relationships between international and UK national research groups. It cemented an application to host a Royal Society meeting.

The PDRA have also been invited back to speak again in 2017.
Year(s) Of Engagement Activity 2016
URL http://www.isev.org/page/isev2016
 
Description Invited conference talk - Rotterdam 2016 ISEV (International Society for Extracellular Vesicles) - PI Prof Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference presentation at the International Congress of the International Society for Extracellular Vesicles formed the basis of close working relationships with other members of the scientific field of Extracellular Vesicles. Our research group was singled out for special note in the Conference Summit up talk. Our PDRA won top prize for the Oral Talk. The conference enabled closer working relationships between international and UK national research groups. It cemented an application to host a Royal Society meeting.

I have also been invited back to speak again in 2017.
Year(s) Of Engagement Activity 2016
URL http://www.isev.org/page/isev2016
 
Description Invited research presentation at UK EV symposium Oxford 2016 - Prof Andrew Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an invited research presentation at the UK premier Extracellular Vesicle conference 2016. A valuable networking opportunity with data presented by Prof Andrew Devitt from multiple BBSRC-funded work.
Year(s) Of Engagement Activity 2016
URL http://www.ukev.org.uk/UKEV2016.html
 
Description Invited research presentation at a Biochemical Society meeting - Prof Andrew Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an invited talk at a research conference that brought together world-leading lipid researchers. This was a valuable networking event and enabled future research plans to be made with international researchers.
Year(s) Of Engagement Activity 2017
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA191/view/Conference/Default.aspx
 
Description Invited research presentation at a Biochemical Society meeting - presentation by PDRA Dr Ivana Milic 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact This was an invited talk at a research conference that brought together world-leading lipid researchers. This was a valuable networking event and enabled future research plans to be made with international researchers.
Year(s) Of Engagement Activity 2017
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA191/view/Conference/Default.aspx
 
Description Research in Progress Seminar 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Seminar presentation with Q&A session
Year(s) Of Engagement Activity 2016
 
Description Royal Society meeting - Extracellular vesicles and the tumour microenvironment 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a highly successful Royal Society meeting. It is in the process of resulting in a Meeting Report document for general distribution. As a result of this meeting, new research collaborations are forming.
Year(s) Of Engagement Activity 2017
URL https://royalsociety.org/science-events-and-lectures/2017/0
 
Description School Visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talk to year 12 students about our research and careers in science.
Year(s) Of Engagement Activity 2017
 
Description School Visit (Coventry) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact A talk about my research and career to school students, followed by a Q&A session. The school reported increased engagement with the careers office re science courses, particularly Biology.
Year(s) Of Engagement Activity 2015
 
Description Sixth Form Talk (Coventry) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact ~ 60 Sixth Form students engaged in a morning of talks and interactive sessions regarding my research and HE more generally. Still awaiting feedback.
Year(s) Of Engagement Activity 2016
 
Description Twitter (@ProfDevitt) 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This is a Twitter account for all research from the Devitt research group at Aston. It reaches to students (UG/PG), business, public and practitioners.
Year(s) Of Engagement Activity 2014,2015,2016,2017
URL https://twitter.com/ProfDevitt
 
Description Twitter (EV) 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This is a new Twitter account to announce updates to the programme of work on the various Extracellular Vesicle grants that are running within the Devitt group at Aston. Through tweets and retweets we will hit a wide audience of the public, scientists and students.
Year(s) Of Engagement Activity 2016
URL https://twitter.com/AC_d_EV
 
Description UKEV 2016 & 2018 Poster -Ivana Milic 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A valuable networking opportunity at the UK premier Extracellular Vesicle conference.
2016 A poster presentation of our BBSRC EV data detailing lipidomic analyses. Presented by Dr Ivana Milic. Included important data from the BSBRC iCASE award with data from Mr Allan Cameron.
2018 Proteomic data from our muscle cell work
Year(s) Of Engagement Activity 2016,2018
URL http://www.ukev.org.uk/UKEV2016.html
 
Description UKEV 2017 Talk -Ivana Milic 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invited talk at a world-leading conference for the study of EV (the International Society for Extracellular Vesicles).
Year(s) Of Engagement Activity 2017
URL http://www.isev.org/page/isev2017