Multimodal profiling of inflammatory and immune cells to determine stage and treatment response in non-alcoholic steatohepatitis and type II diabetes

Non-alcoholic fatty liver disease, or fatty liver for short, is the most common cause of chronic liver disease worldwide. It affects two thirds of people living with type 2 diabetes in Europe and over three quarters of people with obesity. Fatty liver is caused by a build-up of fat in liver cells. For most people, fatty liver is a benign condition, but one in six people will go on to develop the aggressive form of disease, called non-alcoholic steatohepatitis, called NASH for short. NASH is a form of inflammation in the liver and can damage the liver cells. NASH can lead to liver scarring and eventually, in some patients, to cirrhosis, liver failure and even liver cancer. The challenge to doctors and nurses looking after people with fatty liver is to identify which patients might go on to develop the more aggressive disease, so that interventions and treatments could be targeted to those at greatest need.

We know that patients with NASH and particularly those with scarring or fibrosis in the liver are at greater risk of developing advanced disease. However, the only way to diagnose NASH and fibrosis is to do a liver biopsy. This is an invasive and costly test that involves passing a needle through the skin and taking a small sample of liver that can be studied under the microscope. Although it is a very useful test, it is not possible to use liver biopsy to find out which of the millions of patients with fatty liver are at risk, and who has the mild form that is unlikely to progress. Some non-invasive blood tests or scans are available, but they are not accurate enough, on their own, to guide patient care today or to study the many new treatments in development.

The researchers in this project aim to address the problem by learning more about the way in which NASH develops, paying particular attention to inflammation. Inflammation is our body's response to infection and injury. In inflammation, specialised cells, including immune cells are switched on to fight infections and repair damaged tissues. Inflammation in the liver is the main feature of NASH, and immune cells can also be found in fat tissues and the blood of patients with type 2 diabetes and obesity. However, although lots of teams are looking for new tests and treatments for NASH, they are not studying cells of the immune system, despite the fact that these cells are a critical feature of NASH. This project will address this gap in our knowledge.

The researchers in this project have already found that the numbers of certain immune cells, called T cells, in the blood differ between benign fatty liver and NASH and now they want to study this in more detail. They will study patients with type 2 diabetes, obesity and fatty liver who are about to have bariatric, or weight reduction surgery (which can effectively treat fatty liver in some patients). The researchers will take samples of liver, fat tissue and blood and use state-of-the-art technology to find all the genes that are activated in over 2000 individual immune cells from each specimen. They will compare the patterns of genes that are turned on or off in different parts of the body in patients with different stages of fatty liver. This will help them build a picture of how the immune system affects NASH. They will study the immune cells they discover in the laboratory and see whether these cells alter the toxic effects of fats on liver cells. They will then ask the patients who had surgery for further samples one year after the operation to see if the patients' immune systems have returned to normal, using the same cutting-edge techniques, which will indicate that the cells they discovered at the start of the study really are part of the problem in NASH. The results of this study will help the researchers understand more about NASH, design new tests for NASH that mean fewer people will need liver biopsy and may even tell us about new ways in which we can treat NASH in the future.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide, affecting 2/3 of people living with type 2 diabetes in Europe. For most patients, NAFLD is a benign condition, but a minority of patients develop non-alcoholic steatohepatitis (NASH) and liver fibrosis which can progress to cirrhosis, liver failure and liver cancer. Currently the only means of distinguishing patients with NASH and fibrosis from those who have uncomplicated steatosis is to perform a liver biopsy. It is neither convenient nor desirable to use this invasive and costly procedure to risk-stratify patients. Non-invasive markers can help detect established advanced disease, but are not reliable at detecting patients with NASH and fibrosis at risk of progression. One reason for the absence of a reliable biomarker is our incomplete understanding of NASH pathogenesis, which we will address in the current proposal. Our preliminary data using cytometry by time of flight to study peripheral blood mononuclear cells has shown an increase in cytotoxic CD8+ T cells and Th1 cells in patients with NASH and significant fibrosis compared to patients with simple steatosis or healthy controls. We hypothesise that adaptive immune cells, specifically pro-inflammatory T cells, play a pathogenic role in NASH. Single cell transcriptomics and downstream mechanistic studies will give us unique insights into the pathogenesis of NASH and inform biomarker selection and lead to novel treatment targets. We will:
1. Use cutting-edge CITE-Seq single cell RNA sequencing to determine type, activation status and clonality of T cells (and other immune cells), in liver, blood and adipose tissue sampled at the time of bariatric surgery from patients with type 2 diabetes, obesity and fatty liver.
2. Determine function of T cell subsets identified in Objective 1 in in vitro models of lipotoxicity and fibrosis.
3. Determine effect of treatment-induced NASH resolution on T and other immune cells.

Non-alcoholic fatty liver disease (NAFLD respectively) affects 15-33% of the adult population worldwide. Up to one in five patients with NAFLD have the progressive fibroinflammatory form of disease, non-alcoholic steatohepatitis or NASH. The PI's recent work combining the healthcare records of 18 million European adults published in the British Medical Journal and BMC Medicine shows that despite significant under-recording of disease, the incidence and prevalence of NAFLD and NASH diagnoses are rising in line with the rising prevalence of obesity. In the 2016 Health Survey for England, two thirds of adult men and over half of women were overweight or obese. It is estimated that by 2020, NASH will be the leading indication for liver transplantation in the USA. The key independent risk factor for life-threatening outcomes such as cirrhosis and hepatocellular carcinoma is type II diabetes.

The asymptomatic nature of NAFLD and NASH means that many patients are not diagnosed and those who have fatty liver on imaging or abnormal liver tests do not have further testing done to determine whether they have NASH and are therefore at risk of progression. There are currently no drugs licensed for the treatment of inflammatory liver diseases, therefore the cornerstone of treatment is behavior and lifestyle change, but adherence to such changes is limited and effect on long term outcomes has not been demonstrated.

Public health campaigns now focus on reducing the consumption of sugary drinks, increasing exercise awareness and access to facilities, education, and legislation, with particular attention on childhood obesity that affects a third of 10-11 year old children in the East End of London and Essex. However such public health measures will take many years to impact on the burden of established liver disease in the population. Therefore better understanding of the mechanisms of disease are needed to drive better diagnostic and biomarker discovery and to discover novel therapeutic approaches.

The findings from this research will impact upon a range of stakeholders including patients, healthcare professionals and healthcare providers. These are detailed in the attached Pathways to Impact document that outlines planned academic, public engagement and education activities. It is likely that there will be commercial exploitation opportunities for the body that funds the research and the PI's experience with this process will help early identification of such opportunities. The project brings to bear novel experimental and therapeutic approaches, combining antibody-mediated detection with single-cell RNA sequencing (CITE-seq) with novel bioinformatic approaches. The experience of refining and adapting these techniques in human disease will inform future projects in a broad range of disciplines. Furthermore, the strategy to study the immune mechanisms in NASH beyond the T cell subsets identified in our preliminary work will identify alternative potential diagnostic and therapeutic approaches. Whether new treatments are based on the populations of interest we discover or based on the molecules they secrete, strong partnerships with all the stakeholders, including industry, will be required to develop these targets rapidly and to take them back into the clinic.