R. Broering

Hepatitis B virus suppresses toll-like receptor–mediated innate immune responses in murine parenchymal and nonparenchymal liver cells†

We have previously shown that Toll‐like receptor (TLR)‐activated murine nonparenchymal liver cells [(NPC); Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC)] can suppress hepatitis B virus (HBV) replication. Therefore, the aim of this study was to investigate whether HBV has the ability to counteract the TLR‐mediated control of its replication. Freshly purified murine hepatocytes and NPCs obtained from C57BL6 mice were stimulated by TLR 1‐9 ligands in the presence or absence of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV virions, or supernatants from HBV‐producing HBV‐Met cells, and HBV replication was suppressed by anti‐ hepatitis B virus X protein (HBx) small interfering RNA (siRNA) in HBV‐Met cells. Supernatants were collected and tested for antiviral cytokines by viral protection assay. HBV gene expression and replication was analyzed by southern blot. RNA and proteins were analyzed by quantitative reverse transcription polymerase chain reaction (RT‐PCR) or western blot and enzyme‐linked immunosorbent assay, respectively. Pretreatment of hepatocytes and NPCs with HBV‐Met cells supernatants, HBsAg, HBeAg, or HBV virions almost completely abrogated TLR‐induced antiviral activity, which correlated with suppression of interferon beta (IFN‐β) production and subsequent interferon‐stimulated gene induction as well as suppressed activation of interferon regulatory factor 3 (IRF‐3), nuclear factor kappa B (NF‐κB), and extracellular signal‐regulated kinase (ERK) 1/2. In HBV‐infected HBV‐Met cells, TLR stimulation did not induce antiviral cytokines in contrast to primary hepatocytes. TLR‐stimulated expression of proinflammatory cytokines [tumor necrosis factor alpha (TNF‐α), interleukin‐6 (IL‐6)], and activation of IRF‐3 was suppressed after up‐regulation of HBV replication in HBV‐Met cells. Accordingly, suppression of HBV replication by siRNA led to activation or expression of proinflammatory transcription factors and cytokines. Conclusion: Our data indicate that HBV can suppress the TLR‐induced antiviral activity of liver cells. This has major implications for the interaction between HBV and the immune system. (HEPATOLOGY 2009.)

Toll‐like receptor‐mediated control of HBV replication by nonparenchymal liver cells in mice

Hepatitis B virus (HBV) infection is one of the most frequent causes of chronic liver disease worldwide. Because recent studies have suggested that Toll‐like receptor (TLR)‐based therapies may be a promising approach in the treatment of HBV infection, we studied the role of the local innate immune system of the liver as a possible mediator of this effect. Murine nonparenchymal cells, including Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs), were isolated from C57/BL6 wild‐type or MyD88−/− mice and stimulated by agonists of TLR1 to TLR9. Supernatants were harvested and assayed for their antiviral activity against HBV in HBV‐Met cells. No direct antiviral effect of TLR agonists could be observed. In controls (myeloid dendritic cells), TLR1, TLR3, TLR4, TLR7, and TLR9 activation lead to production of antiviral cytokines. By contrast, only supernatants from TLR3‐stimulated and TLR4‐stimulated KCs and TLR3‐stimulated LSECs from wild‐type mice were able to potently suppress HBV replication as assessed via Southern blotting. Similar results were found with cells from MyD88−/− mice, indicating that the effect was independent of this signaling pathway. Cellular HBV RNA and hepatitis B surface antigen or hepatitis B e antigen levels in supernatants remained unchanged. Using neutralizing antibodies, we demonstrated that the TLR3‐mediated effect but not the TLR4‐mediated effect is mediated exclusively through interferon‐β. Conclusion: Our data indicate that the innate immune system of the liver can control HBV replication after activation by TLR agonists. This has implications for the development of TLR‐based therapeutic approaches against HBV. (HEPATOLOGY 2007.)

Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific

Little is known of how the Toll‐like receptor (TLR) system can modulate the function of non‐parenchymal liver cells (NPC) as a major component of the innate and adaptive immune system of the liver. To investigate the diversification of TLR signalling pathways in NPC, we isolated Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild‐type C57BL/6 mice and examined their responses to TLR1 to TLR9 agonists. The data show that KC respond to all TLR ligands by producing tumour necrosis factor‐α (TNF‐α) or interleukin‐6 (IL‐6), to TLR3 and TLR4 ligands only by producing interferon‐β (IFN‐β), to TLR1 and TLR8 ligands by significantly up‐regulating major histocompatibility complex (MHC) class II and costimulatory molecules, and to TLR1, ‐2, ‐4 and ‐6 ligands by inducing high levels of T‐cell proliferation and IFN‐γ production in the mixed lymphocyte reaction (MLR). Similarly, LSEC respond to TLR1 to ‐4, ‐6, ‐8 and ‐9 ligands by producing TNF‐α, to TLR3 and ‐4 ligands by producing IL‐6, and to TLR3 ligands by producing IFN‐β. Interestingly, despite significant up‐regulation of MHC class II and co‐stimulatory molecules in response to TLR8 ligands, LSEC stimulated by TLR1, ‐2 or ‐6 could stimulate allogeneic T cells as assessed by MLR. By contrast, myeloid dendritic cells, used as positive control for classical antigen‐presenting cells, respond to TLR1, ‐2, ‐4 and ‐9 ligands by both up‐regulation of CD40 and activation of allogeneic T cells. In conclusion, NPC display a restricted TLR‐mediated activation profile when compared with ‘classical’ antigen‐presenting cells which may, at least in part, explain their tolerogenic function in the liver.

The interferon stimulated gene 15 functions as a proviral factor for the hepatitis C virus and as a regulator of the IFN response

Background Non-response to combination therapy by patients with hepatitis C virus (HCV) has previously been associated with a strong hepatic upregulation of interferon stimulated genes (ISGs) including ISG15. Therefore, the aim of this study was to further elucidate the functional role of this molecule. Methods ISG15 expression was suppressed by siRNAs or enhanced by over-expression in genomic and subgenomic human or murine HCV replicon systems. In addition, ISG15 expression was analysed in liver samples of patients with HCV prior to antiviral therapy and correlated with clinical and virological parameters. Results Short- or long-term knockdown of ISG15 expression suppressed HCV replication comparable to IFNs without evidence for the induction of resistant mutations. Triple therapy consisting of ISG15 knockdown, interferon α (IFNα) and ribavirin led to complete suppression of the HCV NS5A protein, corresponding to 99% suppression of HCV-RNA compared to 75% suppression by IFNα and ribavirin only. Combination treatment of ISG15 knockdown and IFN was associated with enhanced and prolonged expression of selected ISGs. Consistent with these in vitro data, high hepatic ISG15 levels correlated with the unfavourable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN during the initial phase of treatment. Conclusions ISG15 plays an important role in the HCV replication cycle. Therefore, therapies based on the suppression of ISG15 may provide a promising strategy to overcome non-response to standard combination treatment in the future. Furthermore, analysis of ISG15 prior to therapy may be useful to predict short-term and long-term outcome and thus tailor antiviral therapy with pegIFN and ribavirin.

Toll-like receptor-stimulated non-parenchymal liver cells can regulate hepatitis C virus replication.

BACKGROUND/AIMS The aim of this study was to further elucidate the role of the IFN and the Toll-like receptor (TLR) system in the control of HCV replication by non-parenchymal liver cells (NPC). METHODS Murine HCV replicon bearing MH1 cells were incubated with supernatants from TLR1-9-stimulated murine NPC (Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC)) and bone marrow-derived myeloid dendritic cells (mDC). HCV replication and expression of IFN-stimulated genes (ISGs) as well as TLR1-9 mRNA were determined by real-time rtPCR. RESULTS IFNs (-alpha, -beta, -gamma) and TLR3 ligands only (despite the expression of TLR1-7 and TLR9 mRNA) achieved direct suppression of HCV replication by about 80-90% or 60%, respectively. Supernatants from TLR3- and 4-stimulated NPC only, however, led to potent suppression of HCV replication through IFN-beta and induction of ISGs. By contrast, mDCs could be stimulated by TLR2, -3, -4, -7 and -9 to produce antiviral cytokines. CONCLUSIONS TLR3- and TLR4-stimulated NPC are able to regulate HCV replication through production of IFN-beta. This can also, at least partly explain the high level of ISG expression in HCV infected livers. These novel findings are of particular relevance for the control of HCV replication by the innate immune system of the liver.

Toll-like receptor activated human and murine hepatic stellate cells are potent regulators of hepatitis C virus replication.

BACKGROUND/AIMS While hepatic stellate cells (HSC) are known to be key mediators of liver fibrosis, only little is known about their functional role in the innate immune system of the liver. METHODS To address this question, murine HSC were isolated from livers of C57BL/6J mice and human HSC were isolated from liver samples obtained from resections and liver explants. HSC were stimulated with Toll-like receptor (TLR) 1-9 ligands for 20 h. Supernatants were harvested and used in virus protection assays (encephalomyocarditis virus, EMCV) as well as in human and murine hepatitis C virus (HCV) replicon systems. Expression of interferon (IFN), retinoic acid-inducible gene-I (RIG-I), and interferon-stimulated genes (ISGs) was assessed by quantitative reverse transcription polymerase chain reaction. RESULTS While all TLRs were detectable in HSC, in murine HSC only TLR 3 and -4 agonists could induce cytokines that had an antiviral effect upon EMCV and HCV replication. IFN-beta was the main cytokine mediating the antiviral activity of TLR 3-stimulated HSC whereas other cytokines of undefined nature were involved in TLR 4-mediated antiviral effects. In human HSC, only TLR 3 stimulation led to production of antiviral cytokines. The antiviral effect was related to the up-regulation of ISGs and RIG-I in target cells. CONCLUSIONS These data demonstrate that murine and human HSC have as yet unrecognized antiviral properties when activated through the TLR-system and TLR 3/HCV in particular. This sheds new light on their role in the innate immune system of the liver and their participation in the control of HCV replication.

Role of Toll-like receptor 2 in the immune response against hepadnaviral infection

BACKGROUND & AIMS The Toll-like receptor 2 (TLR2) has recently been recognized to play an important role in the pathogenesis of chronic hepatitis B virus (HBV) infection. In the present study, we examined the role of TLR2 in hepadnaviral infection in hepatoma cell lines and the woodchuck model. METHODS The expression of TLR2 and pro-inflammatory cytokines was quantified by real time RT-PCR. TLR2-associated signaling pathways in hepatocytes were examined by Western blot. HBV replication and gene expression were assessed by Southern blot, Northern blot and specific ELISA, respectively. RESULTS TLR2 ligands activated NF-κB, PI3K/Akt, and different arms of MAPK signaling pathways and induced the production of pro-inflammatory cytokines in hepatocytes. TLR2-mediated innate immune responses led to reduction of HBV/woodchuck hepatitis virus (WHV) replication and gene expression in HepG2.2.15 cells and primary woodchuck hepatocytes. Furthermore, the antiviral activity of TLR2 ligands was abolished by pretreatment with U0126 and rapamycin, inhibitors of the MAPK/ERK and PI3K/Akt pathways, respectively. In the woodchuck model, relatively low levels of TLR2 expression were found in peripheral blood mononuclear cells (PBMCs) and in liver tissues from chronic WHV carriers. TLR2 expression in PBMCs was inversely correlated with WHV DNA titers in acute WHV infection and in entecavir-treated chronic WHV carriers. CONCLUSIONS These data suggest that hepatocytes play an active role in TLR2-mediated antiviral responses during hepadnaviral infection. The mutual inhibition of HBV replication and TLR2 signaling represents an important aspect of HBV infection and should be considered in the new therapeutic concept against chronic HBV infection.

Role of Toll-like receptors in liver health and disease

TLRs (Toll-like receptors), as evolutionarily conserved germline-encoded pattern recognition receptors, have a crucial role in early host defence by recognizing so-called PAMPs (pathogen-associated molecular patterns) and may serve as an important link between innate and adaptive immunity. In the liver, TLRs play an important role in the wound healing and regeneration processes, but they are also involved in the pathogenesis and progression of various inflammatory liver diseases, including autoimmune liver disease, alcoholic liver disease, non-alcoholic steatohepatitis, fibrogenesis, and chronic HBV (hepatitis B virus) and HCV (hepatitis C virus) infection. Hepatitis viruses have developed different evading strategies to subvert the innate immune system. Thus recent studies have suggested that TLR-based therapies may represent a promising approach in the treatment in viral hepatitis. The present review focuses on the role of the local innate immune system, and TLRs in particular, in the liver.

Preclinical development of TLR ligands as drugs for the treatment of chronic viral infections

Introduction: Toll-like receptors (TLRs) have been identified as key regulators of innate and adaptive immune responses in viral infection. Recent progress in this field revealed that there are significant interactions between the TLR system and pathogens in chronic viral infections. Therefore, TLR ligands have great potential for the treatment of chronic viral infections. Areas covered: This review provides an overview of the methodology for preclinical testing of TLR ligands for three major viral infections: hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). TLR ligands have shown potent antiviral activity in different cell culture systems as well as animal models for these infections and induce the production of antiviral cytokines, modulated cellular immunological functions and antiviral effects in vivo. Expert opinion: The recent progress in this field demonstrated that activation of a large number of TLR ligands is effective against viral infections in cell culture systems and animal models. Exploring these models, further in-depth elucidation of the molecular and immunological mechanisms of the antiviral activity of TLR ligands will be necessary to develop them into clinical useful drugs.

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection.

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.