The influence of the liver on NK cell education: a role in Hepatitis C?

Hepatitis C is a common viral infection of the liver. Approximately 20% of individuals are able to clear hepatitis C without treatment, however the remaining 80% are chronically infected. This can lead to liver failure and cancer. In the UK 216,000 people are infected with an estimate of 150 million worldwide. While treatments are available, 30% are still unable to clear the virus. There is no vaccine for Hepatitis C despite much work in this field.

Understanding the mechanisms of viral clearance can impact vaccine design and new therapies. Whether a person is able to resolve hepatitis C depends on external factors, including alcohol use and age, but also on genetics. Patients who clear hepatitis C mount an excellent immune response to the virus. The strength of this defence system is decided by variations (polymorphisms) in genes which determine vital components of the body's immune system.

We are interested in Natural Killer (NK) cells, a key player in the early immune response to hepatitis C. These cells have surface receptors, called Killer cell Immunoglobulin-like Receptors (KIRs), which interact with HLA receptors on liver cells infected by hepatitis C. Specific combinations of KIRs and their HLA pairs determine spontaneous and treatment related outcomes. The most significant genetic variable associated with Hepatitis C Virus (HCV) however is the IL28B gene, which determines components of the late immune response. We have found that the strength of these associations is influenced by the type of HCV virus present. The IL28B cascade is strongly predictive of response in patients infected with HCV type 1, whereas the KIR pathway is important for types 2 and 3.

We propose that different people clear HCV via different mechanisms, i.e. either the IL28B or KIR pathway dominates depending on the patient's genetics and the type of HCV virus present. Recent data from our group suggests the strength of the IL28B system may affect the way NK cells develop. While the protective IL28B gene causes a strong release of chemicals (cytokines), particularly in the presence of HCV type 1, allowing patients to resolve HCV via this mechanism, it may weaken NK cells. Instead of allowing immature NK cells to gain KIR and grow into cytotoxic cells, a strong cytokine response may direct NK cells to develop into a hypofunctional group less able to kill cells infected by HCV. This would override the influence of protective KIR:HLA pairs, making NK cells tolerant of infection. We therefore hypothesise NK cells can be weakened during their maturation by (1) the type of HCV present; (2) the strength of chemical response induced by IL28B. We suspect the site at which this divergence occurs is the liver. Our work in liver transplantation has identified the STAT4 protein as a potential driver of this differentiation.

We are keen to test this hypothesise and obtain clear definition of the above pathways. We have three specific aims. Aim 1: To study the functional maturation of NK cells in the liver and blood of non-HCV infected patients. We will obtain liver specimens from patients undergoing removal of cancer spread from the bowel to the liver and examine a sample of blood from the same patients. Aim 2: To study how STAT4 affects NK cell maturation. Aim 3: To test our model that NK cells take on different roles in HCV types 1 and 3 related to the liver cytokine environment, by taking a sample of blood from 40 patients infected with HCV. This research will be supervised by Professor Khakoo, who has published a number of landmark papers in this field and will be carried out by myself.

In summary our work will link NK cell development and function with clinical outcomes in hepatitis C. Understanding how NK cell subpopulations are generated could be key to developing novel therapies or a vaccination. These insights will be important for other viral infections and liver diseases in which NK cell development is affected.

Introduction
Hepatitis C virus (HCV) is a chronic infection that leads to liver failure and cancer affecting 216,000 people in the UK and 150 million worldwide. Natural Killer (NK) cells are important components of the innate immune response to HCV. Specific combinations of NK cell receptors, including KIRs and their HLA-I ligands determine both spontaneous and treatment related outcomes, as do other polymorphisms within the innate immune system affecting the expression of IL28B. Why certain diseases are able to escape the NK cell response is unclear, however we suspect that changes occur at the level of NK cell maturation in the liver directing them towards a hypofunctional or tolerant subpopulation.

Hypothesis
I propose that NK cells enter the liver as an immature subset that is modulated by the intrahepatic cytokine environment in response to a particular HCV genotype. In this way the HCV virus may determine NK cell response. We will investigate how the liver and HCV infection affect NK cell development and function, focusing on STAT4 as a key molecule in these processes. We also aim to establish how Th1 and Th3 cytokines induced by IL28B/IFNL4 polymorphisms may affect this pathway.

Methodology
Aim 1: Determine the phenotype and function of NK cells in the liver and blood of non-HCV infected patients.
Aim 2: Study how STAT4 affects NK cell maturation, which genes it targets and how this relates to NK cell phenotype
Aim 3: Investigate whether abnormalities in NK cells in HCV are genotype specific and if this relates to the hepatic microenvironment

Medical opportunities
Our work will integrate NK cell development and function with clinical outcomes in HCV. Understanding the influence of the liver on NK cell development could be important for therapy for chronic liver disease and developing a vaccine against HCV infection. Insights will also be relevant to other viral infections in which NK cell development could be affected.

The World Health Organisation estimate 150 million individuals worldwide are potentially infected with hepatitis C, and 216,000 in the UK. The majority develop a chronic infection. Treatment is currently prolonged, expensive and troubled by significant side effects. Treatment failure, and indeed failure to treat patients at all, as commonly occurs due to economic constraints abroad and under-recognition of this largely asymptomatic disease, usually results in cirrhosis or liver cancer. Huge costs follow in terms of hospital bed days, radiological, endoscopic and surgical interventions. Furthermore hepatitis C is a huge burden on the transplant services accounting for 18% of transplants undertaken in the UK in 2011. Therefore this area of study is important and can have a large impact on health.

The work will benefit primarily the academic community. However due to the wide-reaching nature of the research it can benefit academics in hepatology, virology, immunology and infectious diseases. As this is a novel area of study we anticipate that the work will have an international impact. Our work could thus stimulate research into novel aspects of liver immunology, hepatitis and natural killer cell biology leading to an expansion of our knowledge base within the UK and worldwide

The work could also have implications for the pharmaceutical industry, as it could help establish natural killer cells as targets for novel therapeutics or vaccine design, as NK cells are thought to cross talk with T cells. This would have commercial implications for industry in the UK and globally, helping to establish cellular-based therapies as a potential alternative to current anti-viral agents. It could thus benefit patients with hepatitis and other liver diseases, and so may benefit the public sector (NHS) on a national basis. Thus it could contribute to the health of the nation.