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Berichtszeitraum: 2015-05-01 bis 2017-04-30

Chronic liver disease (CLD) is the replacement of liver tissue by scar as a consequence of persistent hepatic injury. The main causes of liver injury include alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD) and viral hepatitis. CLD is a progressive life threatening condition and responsible for 170,000 deaths per annum in Europe. NAFLD is characterized by an accumulation of hepatic fat (triglycerides); it is considered as the hepatic manifestation of the metabolic syndrome which comprises insulin resistance, obesity, and hyperlipidemia. The substantial increase of NAFLD incidence tracks obesity epidemic as a result of unhealthy diet and physical inactivity. NAFLD is an umbrella term, which encompasses various levels of hepatic disease from fat accumulation to cirrhosis. Fatty liver (simple steatosis) is an extremely common condition. 10-30% of individuals with steatosis develop some level of hepatic inflammation; namely non-alcoholic steatohepatitis (NASH). Approximately a third of patients with persistent inflammation deposit extracellular matrix proteins in the liver; known as hepatic fibrosis. Progressive scarring of the liver leads to cirrhosis. Therapeutic strategies for NAFLD are limited. Lifestyle modifications (dietary alterations and exercise) are recommended in all stages of NAFLD. NAFLD pathogenesis is not completely elucidated; numerous factors including carbohydrate-lipid metabolism, inflammation, innate immunity and fibrogenesis are involved at various stages of the clinical spectrum. In NASH, inflammation typically contains immune cells. However, it remains unknown how hepatic injury evolves to inflammation; the current theory also proposes the involvement of non-hepatic factors such as adipocyte (fat cells)-driven mediators, gut microbiota and other gut-driven factors. Progression from hepatic lipid deposition to inflammation and cirrhosis follows a substantially variable course. It has been estimated that less than 10% of patients develop CLD. Individual variability for disease progression is partly controlled by environmental factors and genetic background. Moreover, we previously demonstrated that heritable epigenetic modifications could modify susceptibility to CLD. More specifically, liver injury in the ancestors generated adaptive response and attenuated liver disease in the offspring. The associated epigenetic reprogramming, altered CpG methylation and histone marks, was also demonstrated in offspring livers. Currently it is not known if epigenetic signatures could dictate immune cell response and alter disease susceptibility in NAFLD. In this project we tested the hypothesis that lymphocyte subsets are epigenetically programmed into inflammatory state in NASH. We investigated the epigenetic mechanisms controlling hepatic inflammation, particularly NK and NKT immune cells, in NASH.
We used experimental animal models and established a cohort to explore epigenetic mechanisms in NASH. Fluorescence activated cell sorting was used to purify subpopulations of lymphocytes. In order to increase the relevance and translational aspect of the project, significant proportion of evidence was produced from the clinical cohort. Patients were recruited particularly to the research prospectively and allocated into two groups as having simple steatosis or NASH. Blood samples from healthy volunteers (20 participants) were collected for NK and NKT cell isolation.
Main results: Exploring histone modifications regulating inflammatory characteristics of immune cells in fatty liver disease. Lymphocytes are recruited to injured liver by inflammatory signals such as chemokines. Additionally, secreted mediators (cytokines and adipokines) from hepatocytes, adipocytes and gut can potentially direct inflammatory cells through the site of injury. To examine the hypothesis “lymphocytes are epigenetically programmed into inflammatory state in NASH”, firstly transcriptional features of lymphocytes in NAFLD were examined. Isolated NK and NKT cells from animal models and NAFLD patients were analysed by qRT-PCR. Expression profiling targeted inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-10, IL-13, IL-17, significant increases were detected in TNF-α and IL-1β. Additional array studies array studies demonstrated novel inflammatory markers on Natural killer cells which possibly play a role in gaining of inflammatory characteristics presented by peripheral and intrahepatic NK and NKT cells. We also detected major gene expression differences between peripheral (blood) and intrahepatic (liver) NK/NKT cells in NASH. To determine the importance of histone marks in controlling inflammatory features of lymphocytes: We examined the role of histone modifications controlling inflammatory characteristics of NK and NKT cells. It was hypothesized that several histone modifications are altered in either pre-NASH state or progression. Chip-Sequencing were employed to identify stable histone-DNA interactions which revealed that immune cells (NK or NKT cells) demonstrate unique histone alterations in progression from steatosis to non-alcoholic steatohepatitis. Elucidating the role of DNA methylation in the regulation of NK and NKT cells in NASH
To discover how CpG methylation regulates inflammatory phenotype at transcriptional level; DNA methylation and transcriptional repression were investigated in NK/NKT cells. Arrays were employed to investigate methylated cytosines. Validation was performed by loci-specific bisulfite sequencing and pyrosequencing. We found new hepatic and circulating NK cell metylation markers which was strongly linked with NK cell phenotype in steatohepatitis.
We disseminated the results to wide audience. Our research activity and results were disseminated to the public from beginning of the fellowship. Patient and support group meetings were organised at KU Hospital. Talks were delivered by Dr Zeybel and guest speakers. Annual High-school student visits were arranged to inspire young persons. Students spent 3 days in the lab and be familiarized with epigenetics, NAFLD and Marie Curie Actions. We organised an annual an “Epigenetics Workshop” opportunity for postgraduate students from various disciplines. Epigenetic experts were invited to share their experiences. Dr Zeybel designed a project website to publicize the new developments in the laboratory, to promote epigenetic research and share innovations in liver research. The proposed project attracted interests from a wide-range of people from basic scientists, epigeneticists, public health agencies and clinicians to pharmaceutical companies. The potential use of epigenetic signatures as a biomarker of disease, predicting disease progression and treatment responses were discussed by pharmaceutical companies. The research outcomes disseminated through publications and oral presentations in top-tier conferences and peer-reviewed international journals. We published our discoveries in high-impact peer-reviewed journals (Zeybel et al, Molecular Therapy, 2017, Hardy et al, Gut, 2017, Zeybel et al, Clinical Epigenetics, 2016). Dr Zeybel supervised PhD and medical students. In this project, we have worked to discover epigenetic pathways that will be used as a biomarker to diagnose disease activity. This will rely on the collection of blood samples rather than collecting a liver biopsy.