The role of the innate immune system in chronic hepatitis B infection

The global burden of Hepatitis B virus infection remains high despite the availability of a highly effective vaccine and potent antiviral agents. It is estimated 350-400 million people are chronically infected, and consequently at risk of developing the complications of cirrhosis, liver failure and hepatocellular carcinoma. The host immune system appears to be a major determinant of the impact of HBV infection in a given individual, influencing the risk of viral persistence following acute infection, the different phases of chronic infection, and also mediating much of the virus associated liver injury. In addition, immunomodulatory therapy comprises one of the major classes of treatment for chronic hepatitis B (CHB). Much of the early focus centred on the adaptive immune system, however recently there has been increasing interest in the role of innate immune mechanisms. Equipped with a broad range of pattern recognition receptors, effector cells and a variety of secreted immunomodulatory and antiviral cytokines, this arm of the host immune system appears ideally positioned to both mediate the immediate antiviral response and direct the adaptive immune response. However, despite the recognition of its potential importance, the mechanisms that underlie innate immune function in CHB remain poorly defined. Interferon-α is a key mediator of the innate immune system and was the first recognised effective treatment for CHB. Pegylated Interferon-α (Peg-IFN) is now the preferred formulation, but despite offering the advantage of a fixed treatment course and potential for durable responses is used far less commonly than nucleos(t)ide analogues due to lower rates of viral suppression and a less favourable adverse event profile. Such concerns are further heightened by a lack of published data relating to its use in a real world setting where outcomes are often poorer than in controlled studies. The first aim of this thesis was to perform a real world study of CHB patients treated with Peg-IFN to determine its viability as a therapeutic option in day to day clinical practice. In a large multicenter cohort, the observed rate of HBeAg to Anti-HBe seroconversion was 32%, similar to that of multiple large controlled trials. In these HBeAg positive patients the HBV DNA suppression rates below both the detectable threshold of 351 IU/ml and the widely used cutoff of 2000 IU/ml were 22% and 16% respectively. In the HBeAg negative patients, the corresponding rates of viral suppression were 37% and 50% which compare favourably to the published literature. In a subset of patients followed for 2 years after completion of therapy, the durability of the viral suppresion ranged from 50-75%. Concerns regarding the tolerability of Peg-IFN in a real world setting did not bear out in this study, with over 90% of patients completing the 48 week treatment course and only 2 patients experiencing significant AEs. Plasmacytoid dendritic cells (pDC) are the most potent cellular producers of IFN-α in the peripheral blood, and together with myeloid dendritic cells (mDC) are considered sentinels of the innate immune system. Like much of this arm of host immunity, the activity and function of both subsets is mediated predominantly through the family of toll like receptors (TLR). The second aim of this thesis was to examine TLR mediated function of DCs in the setting of CHB. Myeloid DC production of the TNF-α in response to TLR2 stimulation was found to be reduced in patients with HBeAg positive CHB compared to HBeAg negative patients and controls, a finding not previously described. This is consistent with previous studies that suggest the HBeAg appears to target TLR2 in CHB as part of a strategy by the virus to avoid immune detection and clearance. No changes were noted in the mDC response to TLR4 signalling despite evidence that this receptor may be a mediator of tissue damage in other models of liver disease. Plasmacytoid DC IFN-α production in response to stimulation with Influenza virus which acts through TLR7 was significantly impaired across all stages of CHB independent of viral load, HBeAg status or ALT. Similar findings have been described in relation to pDC TLR9 signalling, and together suggest a major functional impairment involving key components of the innate immune system in CHB. Natural killer cells are another key mediator of the innate immune system and have a capacity for both antiviral activity as well as tissue damage. The precise role of NK cells in CHB remains unclear, particularly in relation to TLR signalling. In a series of experiments, NK cells were shown to produce IFN-γ in response to PBMC stimulation with agonists of TLRs 3, 4, 7/8 and 9. The cytokine response to TLR9 was significantly impaired in CHB patients compared to controls. Further experiments suggested this impairment involved indirect pathways mediated through IFN-α. In contrast, the NK cell capacity to upregulate the activation marker CD69 in response to TLR9 stimulation was not affected in CHB, raising the possibility that other aspects of NK cell function such as the capacity to mediate tissue injury remain intact. In an effort to understand the wider impact of TLR dysfunction in CHB, the IFN-γ responses of naïve and memory CD4+ and CD8+ T cells and NKT cells to TLR agonists were also examined. All cellular subsets produced cytokine in response to PBMC stimulation with TLR 3,4,7/8 and 9 agonists, but no differences were noted in these responses between CHB patients and controls. Studies involving HBV specific T cells may be required to further understand the impact of TLR dysfunction on the adaptive immune system In conclusion, this thesis provides a number of insights into the significance of the innate immune system in CHB. TLR mediated changes in DCs and NK cells favour viral persistence rather than clearance through impaired type 1 IFN production. The functional dichotomy observed in NK cells in response to TLR 9 supports a model where activated NK cells may still have the potential to mediate tissue damage despite impaired antiviral activity. Such insights have the potential to further contribute to the ongoing development of immunotherapies that are likely to hold the most promise in achieving the goal of sustained viral clearance.