Stephen Locarnini

Characterization of the innate immune signalling pathways in hepatocyte cell lines.

Summary.  Hepatitis B virus (HBV) infection is a major cause of liver‐related morbidity and mortality. Toll‐like receptors (TLRs) have recently been recognized to play an important role in the pathogenesis of chronic hepatitis B (CH‐B). Furthermore, manipulation of TLR signalling pathways shows potential as an antiviral therapeutic strategy. Whether hepatocytes themselves possess intact TLR signalling pathways remains controversial. It is critical that cell culture models be developed to allow investigation of the interaction between HBV and the TLR signalling pathways. We have screened three hepatocyte cell lines for the integrity of pro‐inflammatory responses and antiviral cytokines following stimulation with interleukin‐1 (IL‐1) and different TLR ligands. We observed that Huh‐7, HepG2 and PH5CH8 cells selectively responded to IL‐1 and TLR2 ligands, leading to the activation of NF‐κB. In addition, the PH5CH8 cell lines were able to induce type 1 interferon (IFN) via both TLR3 and RIG‐I following stimulation with poly I:C, HepG2 cells mounted an IFN response via RIG‐I only, whereas Huh‐7 cells were unresponsive. We conclude that the hepatocyte cell lines investigated display a repertoire of TLR signalling, albeit limited, suggesting that hepatocytes may themselves play an active role in innate immune responses to viruses such as HBV. Furthermore, particular hepatoma cell lines are suitable for investigating the interaction between HBV and hepatocyte‐expressed pattern recognition receptors.

Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen

ABSTRACT The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

The hepatitis B e antigen suppresses IL-1β-mediated NF-κB activation in hepatocytes

Summary.  Previous clinical studies have demonstrated an association between the hepatitis B e antigen and Toll‐like receptor (TLR) expression and signalling. Therefore, the aim of this study was to develop an in vitro assay to measure the effect of hepatitis B virus proteins, including the precore protein, on signalling mediated by members of the Toll‐like/interleukin 1 (TIR) superfamily, by measuring NF‐κB promoter activity. The basal level of NF‐κB reporter activity was measured in three hepatocyte cell lines (Huh7, HepG2 and PH5CH8) and one kidney cell line (HEK293) using a luciferase assay. All cell lines were virtually refractory to stimulation with lipopolysaccharide; however, PH5CH8 cells had a robust activation of NF‐κB in response to IL‐1β stimulation, with ∼40‐fold higher activation than the unstimulated control, a higher degree of activation than that observed in either Huh7 and HepG2, or HEK293 and HEK293‐TLR2 cells. In PH5CH8 cells transfected with pCI expression constructs and stimulated with IL‐1β, we showed that the precursor form of the precore protein, p25, inhibits NF‐κB activation by up to 30% and the cytosolic form, p22, inhibits NF‐κB activation by 70%. The core protein, p21, which shares significant homology with the precore protein except for a 10‐amino acid extension at the N‐terminus, had no effect on NF‐κB activation. We hypothesize that the inhibition of IL‐1β‐mediated NF‐κB activation by the precore protein may be a mechanism that allows the virus to persist, suggesting a role for the pool of precore protein that remains intracellular.

Effects of interferon alpha therapy on the catalytic domains of the polymerase gene and basal core promoter, precore and core regions of hepatitis B virus

Aims: The aim of the present study was to examine the catalytic domains of the polymerase gene, the basal core promoter and the precore and core regions of the hepatitis B virus (HBV) genome for specific mutations. These may account for the response to interferon alpha (IFN‐α) treatment, which may have prognostic value.

Assessment of chronic hepatitis B: the importance of hepatitis B virus DNA testing

Background:  Chronic hepatitis B (CHB) has an estimated prevalence of 90 000 to 160 000 in Australia. Cirrhosis and hepatocellular carcinoma are important complications of CHB and appropriate evaluation of hepatitis B surface antigen (HBsAg)‐positive individuals is vital to identify treatment candidates.

Chapter 20 – Novel approaches in the management of chronic HBV infection

Publisher Summary The treatment of chronic hepatitis B infection is restricted to alpha interferon (IFN-α) and a small number of nucleoside analogues with infrequent clearance of infection. The failure to eradicate chronic infection and the development of resistance may limit their usefulness in the long term. The main goals in the treatment of chronic hepatitis B are (1) prevention of liver fibrosis that requires long-term suppression of HBV replication and (2) prevention of hepatocellular carcinoma (HCC), where treatment needs to be initiated before HBV DNA integration into host cell DNA has occurred. Antiviral nucleoside analogues are effective and convenient therapeutic agents but do not eradicate chronic infection. The management of chronic hepatitis B should be based on an understanding of the pathogenesis of the disease. There are three key elements: virus, hepatocyte, and immune response. The best therapeutic approach is based on immune modulation with cytokines and therapeutic vaccines, blocking viral replication using antiviral agents and cytokines, and preventing re-infection by neutralizing antibodies. These in vitro and in vivo models provide invaluable information on combining nucleoside analogue therapy for chronic hepatitis B infection and help to guide future clinical trials of combinations of antiviral, antisense, and immune modulating therapeutics