MICA:The Potential of Multipoint Adult Progenitor Cells as a Novel Therapeutic Strategy in Alcoholic Hepatitis

Liver disease accounts for one in ten of all deaths in people between the ages of 40 and 49 and costs the NHS £1 billion every year. Alcohol is a major cause of liver disease. One way in which alcoholic liver disease can present is alcoholic hepatitis, which is a very dramatic condition with a high mortality, despite current treatments. Therefore, we urgently need to find new treatments for this condition. Understanding the way in which alcohol results in alcoholic hepatitis can help us to identify potential targets for developing new treatments.

Due to previous research, we know which types of cells are responsible for causing liver damage in alcoholic hepatitis. If we can find an effective way of reducing the numbers of these harmful cells getting into the liver, this can be possibly be used to treat alcoholic hepatitis. The most common cause of death in patients with alcoholic hepatitis is infection. Strengthening our body's response against infection is another way of reducing the number of deaths from alcoholic hepatitis.

Several researchers have suggested that certain cells found in the bone marrow, called multipotent adult progenitor cells (MAPC), can help to reduce inflammation. In test-tube studies, animal studies and clinical trials, these cells have been shown to reduce the inflammation associated with a number of conditions, including heart attack and stroke. It has also been shown that bone marrow cells can improve the ability of cells to fight infection. After being injected in over 100 humans so far, it appears that MAPC injections are safe.

However, it is unclear exactly how the MAPC work, or whether they could be used in treatment of alcoholic hepatitis. Testing the effectiveness and safety of treatments in mice before they are used in humans aims to minimise the risk of harm to patients. In our department, we have developed a method to induce alcoholic hepatitis in mice. Using human blood samples from alcoholic hepatitis patients, I can see if the findings in mice are comparable to those in humans, and therefore whether we can use the mouse experiments to accurately predict what will happen in humans.

To assess the usefulness of MAPC in alcoholic hepatitis, I am then going to treat mice with these cells, and assess whether they reduce liver injury. Then I am going to do further experiments to investigate how these cells achieve their effect. For example, I am going to investigate whether MAPC prevent inflammatory cells from entering the liver, and determine exactly which molecules and cells are implicated in this. I will also be testing whether MAPC affect the ability to cells to clear infection. Using human cells in my laboratory work to supplement mouse experiments will help to ensure that my work will be applicable to humans. If we can define the exact mechanisms of action of MAPC, we can refine our treatments to maximise the intended benefits and minimise side effects. These studies will work together to help us to understand if MAPC can be used to treat alcoholic hepatitis and to find out the underlying mechanism.

This project aims to determine whether alcoholic hepatitis (AH) may be amenable to treatment by multipotent adult progenitor cell (MAPC) infusions, and to investigate the underlying mechanisms. I hypothesise that MAPC can reduce liver injury in AH by reducing hepatic sinusoidal endothelial cell (HSEC) activation and ingress of inflammatory effector cells, whilst enhancing neutrophil phagocytic activity. Our department has established a murine model of AH using NRF2-/- mice, fed a Lieber-DeCarli (LdC) diet and alcohol.

Objectives:

(1) I seek to validate our AH murine model by characterising the evolving immunological and regenerative response during injury and recovery. During injury and repair, I will assess liver injury, analyse leukocyte composition using flow cytometry and use DNA labelling techniques to quantify hepatocyte proliferation. Human blood samples will be used for clinical correlation.

(2) I will determine the effect of MAPC on the inflammatory and regenerative response in this mouse model. Infusions will vary in dose (0.125x10^6 to 1x10^6) and will be given either during or after injury (day 13 or 17), to determine optimal dosing and timing of delivery.

(3) I will determine the effect of MAPC on HSEC activation, through study of patterns of adhesion molecule expression. PCR and immunostaining will quantify levels of ICAM, VCAM and VAP1 in human HSEC. Confocal microscopy will be used in mice.

(4) To determine the molecular mechanisms of MAPC action, I will identify potential molecules secreted by MAPC that may mediate their effects on HSEC activation or affect oxidative stress. This will be tested by modulating candidate factors using neutralising antibodies/ siRNA. I will also test effects of MAPC on neutrophil phagocytosis and T-cell proliferation in co-culture studies in vitro.

Defining efficacy and mechanism of action of MAPC in this pre-clinical model of AH will develop a better understanding of potential new treatments.

(1) Academic beneficiaries:

My project will be the first to investigate the effectiveness of multipotent adult progenitor cells (MAPC) in a pre-clinical model of alcoholic hepatitis (AH). My project will gather new information on how MAPC work to reduce inflammation, advancing what is known in the literature. It will also be the first to investigate the effect of MAPC on neutrophil phagocytosis. Our work is likely to be of interest to those specialising in stromal cells, regenerative medicine, cellular therapies, liver disease and sepsis. It will most benefit groups who are currently using MAPC and those doing research into AH. We will freely share our experimental protocols with other researchers, allowing them to use and build on our ideas for other projects. We seek to develop a community of researchers keen on translational research who can support each other. Furthermore, the development of new animal models facilitates pre-clinical research in general, which may attract R&D funding in the UK, within the next five years.

(2) Commercial sector beneficiaries

Our industrial partner, Athersys (a UK-based SME), has extensive experience with MAPC ('Multistem') and will be providing MAPC for this project. Depending on our results, Athersys may disseminate our results to potential benefactors, which will benefit our research team. Greater knowledge of the mechanisms of action of Multistem and its use in AH could increase the use of the cells for a variety of indications, bringing economic benefits for Athersys.

(3) The wider public

This project has the potential to generate data, which can be ultimately translated to provide new treatment options for patients with AH, likely within the next 5-10 years. We hope that this novel treatment will help to reduce the morbidity and mortality associated with severe AH. Mechanistic studies of Multistem could potentially benefit patients with other diseases for which Multistem is being considered, such as stroke, myocardial infarction and graft versus host disease. It may also provide the basis for using MAPC in other conditions not yet studied, such as sepsis. Through public engagement activities, the public will learn about laboratory research, the concept of translational research and AH. I hope that sharing my research experiences will inspire members of the public to get involved in research. Researchers can benefit from public engagement. For example, the public may offer ideas in terms of areas of research needs, or offer input on how trials should be conducted, or how trial information should be communicated to patients. Thus patient opinions will be sought before we design any clinical trial that potentially results from our work. This can be achieved over the next three years. In the longer term, we hope that better treatment for AH will reduce the duration of hospital stay for this group of patients,allowing the NHS resources to be allocated elsewhere in a way which best benefits the wider public, likely in the next 10
years.

(4) Staff working on the project

The staff working on this project will develop key research skills. I will develop specific laboratory skills, including flow cytometry, immunohistochemistry, PCR and confocal microscopy. I will gain experience in animal handling and use of animals to model human disease. I will work closely with laboratory technicians and my project supervisors, thus developing our team-working skills. As I become proficient in these techniques I will offer my support to junior colleagues, who will also benefit, over the next three years. In data collection and analysis, I will develop my skills in use of statistical software. Our public engagement activities will help to build our departments' profile, and develop our own teaching and communication skills. In publishing the results, it will help our department maintain its reputation as a centre of excellence in liver research, and help to attract further funding.