M. Trippler

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.

Identification of type I and type II interferon‐induced effectors controlling hepatitis C virus replication

Persistent infection with hepatitis C virus (HCV) can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All current therapies of hepatitis C include interferon‐alpha (IFN‐α). Moreover, IFN‐gamma (IFN‐γ), the only type II IFN, strongly inhibits HCV replication in vitro and is the primary mediator of HCV‐specific antiviral T‐cell responses. However, for both cytokines the precise set of effector protein(s) responsible for replication inhibition is not known. The aim of this study was the identification of IFN‐α and IFN‐γ stimulated genes (ISGs) responsible for controlling HCV replication. We devised an RNA interference (RNAi)‐based “gain of function” screen and identified, in addition to known ISGs earlier reported to suppress HCV replication, several new ones with proven antiviral activity. These include IFIT3 (IFN‐induced protein with tetratricopeptide repeats 3), TRIM14 (tripartite motif containing 14), PLSCR1 (phospholipid scramblase 1), and NOS2 (nitric oxide synthase 2, inducible). All ISGs identified in this study were up‐regulated both by IFN‐α and IFN‐γ, demonstrating a substantial overlap of HCV‐specific effectors induced by either cytokine. Nevertheless, some ISGs were more specific for IFN‐α or IFN‐γ, which was most pronounced in case of PLSCR1 and NOS2 that were identified as main effectors of IFN‐γ‐mediated anti‐HCV activity. Combinatorial knockdowns of ISGs suggest additive or synergistic effects demonstrating that with either IFN, inhibition of HCV replication is caused by the combined action of multiple ISGs. Conclusion: Our study identifies a number of novel ISGs contributing to the suppression of HCV replication by type I and type II IFN. We demonstrate a substantial overlap of antiviral programs triggered by either cytokine and show that suppression of HCV replication is mediated by the concerted action of multiple effectors. (HEPATOLOGY 2012;56:2082–2093)

Dissecting the Interferon-Induced Inhibition of Hepatitis C Virus Replication by Using a Novel Host Cell Line

ABSTRACT The Hepatitis C virus (HCV), a member of the family Flaviviridae, is a major cause of chronic liver disease. Patients are currently treated with alpha interferon (IFN-α) that is given alone or in combination with ribavirin. Unfortunately, this treatment is ineffective in eliminating the virus in a large proportion of individuals. IFN-induced antiviral activities have been intensively studied in the HCV replicon system. It was found that both IFN-α and IFN-γ inhibit HCV replicons, but the underlying mechanisms have not yet been identified. Of note is that nearly all of these studies were performed with the human hepatoma cell line Huh-7. Here, we report that genotypes 1b and 2a replicons also replicate in the human hepatoblastoma cell line HuH6. Similar to what has been described for Huh-7 cells, we observed that efficient HCV replication in HuH6 cells depends on the presence of cell culture-adaptive mutations and the permissiveness of the host cell. However, three major differences exist: in HuH6 cells, viral replication is (i) independent from ongoing cell proliferation, (ii) less sensitive to certain antiviral compounds, and (iii) highly resistant to IFN-γ. The latter is not due to a general defect in IFN signaling, as IFN-γ induces the nuclear translocation of signal transducer and activator of transcription 1 (STAT1), the enhanced transcription of several IFN-regulated genes, and the inhibition of unrelated viruses such as influenza A virus and Semliki Forest virus. Taken together, the results establish HuH6 replicon cells as a valuable tool for IFN studies and for the evaluation of antiviral compounds.

Clinical evaluation and applications of the Amplicor HBV Monitor test, a quantitative HBV DNA PCR assay

Viral load has emerged recently as a reliable marker of disease progression and therapeutic efficacy in chronic infections, including AIDS and hepatitis C. The clinical management of type B hepatitis could also be improved by monitoring viremia levels in patients with chronic liver disease undergoing anti-viral treatment. To address this question we evaluated the performance of a newly developed, quantitative PCR assay (Amplicor HBV Monitor test, Roche Diagnostic Systems) in the assessment of viremia changes over time in a group of 45 patients with chronic active hepatitis (CAH) who received interferon treatment. Of the 45 patients, 14 were HBsAg and anti-HBeAg positive and 31 HBsAg, HBeAg positive. Follow-up extended up to 24 months. An average of ten samples per patient were analyzed for levels of ALT, IgM anti-HBc (Abbott Laboratories), HBV DNA by in-house dot-blot hybridization and hybridization-capture assays (HBV-DNA hybrid capture kit, Murex Diagnostics) and by Amplicor HBV Monitor. A sustained biochemical response was observed at the end of treatment in 12 HBeAg-positive and in seven anti-HBeAg positive patients. This was accompanied by the disappearance of HBeAg and of HBV DNA (hybridization assays) in all cases and by the clearance of IgM anti-HBc in 70% of the cases. Viremia (quantitative PCR assay) became undetectable only in 25-30% of cases and was associated with the loss of HBsAg. A good correlation was observed between the time course of IgM anti-HBc, quantitative PCR and dot-blot hybridization although the latter missed 33% of viremic samples. Together, these results indicate that the Amplicor HBV Monitor test is a robust and standardized assay for quantifying HBV viremia levels in the range from 10(2) to 10(7) copies/ml. Compared to other current markers, viral load may provide additional clinical information by predicting long term virologic response and HBsAg clearance in patients with normal ALT at the end of interferon therapy.

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.

Dysregulation of innate immunity in hepatitis C virus genotype 1 IL28B‐unfavorable genotype patients: Impaired viral kinetics and therapeutic response

Recent studies have shown that a single‐nucleotide polymorphism upstream of the interleukin‐28B (IL28B) gene plays a major role in predicting therapeutic response in hepatitis C virus (HCV)‐infected patients treated with pegylated interferon (PEG‐IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics, IFN pharmacokinetics, IFN pharmacodynamics, and gene expression profiles. Two prospective cohorts (human immunodeficiency virus [HIV]/HCV‐coinfected and HCV‐monoinfected) completing treatment with IFN/ribavirin were enrolled. Patients were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort, frequent serum sampling was completed for HCV RNA and IFN levels. DNA microarray of peripheral blood mononuclear cells and individual expression of IFN‐stimulated genes (ISGs) were quantified on IFN therapy. The IL28B‐favorable (CC) genotype was associated with improved therapeutic response compared with unfavorable (CT or TT) genotypes. Patients with a favorable genotype had greater first‐ and second‐phase viral kinetics (P = 0.004 and P = 0.036, respectively), IFN maximum antiviral efficiency (P = 0.007) and infected cell death loss (P = 0.009) compared with unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of natural killer cells, from patients with unfavorable genotypes (P <0.004). Induction of innate immunity genes was also lower in unfavorable genotypes. ISG expression at baseline and induction with IFN was independent of IL28B genotype. Conclusion: Carriers of the IL28B‐favorable genotype were more likely to have superior innate immune response to IFN therapy compared with unfavorable genotypes, suggesting that the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of ISG expression. (HEPATOLOGY 2012)