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-kappaB. 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.
Hepatoma cell lines expressed functional IL-1 receptor and TLR2 receptors, which when stimulated led to a signalling cascade that inhibited HBV replication. These data support an active role for hepatocytes in inhibiting HBV replication and provide a rationale for the development of TLR agonists as potentially novel antiviral agents.
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.
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