Joerg F. Schlaak

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.)

Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression

ABSTRACT Alpha/beta interferon (IFN-α/β) is a key mediator of innate antiviral responses but has little effect on the established replication of dengue viruses, which are mosquito-borne flaviviruses of immense global health importance. Understanding how the IFN system is inhibited in dengue virus-infected cells would provide critical insights into disease pathogenesis. In a recent study analyzing the ability of individual dengue virus-encoded proteins to antagonize the IFN response, nonstructural (NS) protein 4B and possibly NS2A and NS4A were identified as candidate IFN antagonists. In monkey cells, NS4B appeared to inhibit both the IFN-α/β and IFN-γ signal transduction pathways, which are distinct but overlapping (J. L. Munoz-Jordan, G. G. Sanchez-Burgos, M. Laurent-Rolle, and A. Garcia-Sastre, Proc. Natl. Acad. Sci. USA 100:14333-14338, 2003). For this study, we examined the effects of dengue virus on the human IFN system, using cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and that expressed all of the dengue virus nonstructural proteins together. We show here that in replicon-containing cells dengue virus RNA replication and the replication of encephalomyocarditis virus, an IFN-sensitive virus, are resistant to the antiviral effects of IFN-α. The presence of dengue virus replicons reduces global IFN-α-stimulated gene expression and specifically inhibits IFN-α but not IFN-γ signal transduction. In cells containing replicons or infected with dengue virus, we found reduced levels of signal transducer and activator of transcription 2 (STAT2), which is a key component of IFN-α but not IFN-γ signaling. Collectively, these data show that dengue virus is capable of subverting the human IFN response by down-regulating STAT2 expression.

Mutational switch of an IL-6 response to an interferon-γ-like response

Signaling through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) is central to the responses to the majority of cytokines and some growth factors, including the interferons (IFNs) and the IL-6 family of cytokines. The biological responses to stimulation through the widely distributed IL-6 and IFN-γ receptors are, however, completely different. Remarkably, it is shown here that, in mouse embryo fibroblasts lacking STAT3, IL-6 mediates an IFN-γ-like response including prolonged activation of STAT1, the induction of multiple IFN-γ-inducible genes, the expression of class II MHC antigens, and an antiviral state. Normal cells exposed to IL-6 thus require a STAT3-dependent function(s) to down-regulate STAT1 activity and prevent an IFN-γ-like response. The data encourage the view that the very disparate IFN-γ and IL-6 JAK/receptor complexes mediate a common set of generic or “core” signals which are subject to STAT3-dependent modulation to provide IL-6 specificity. The switching of one cytokine response to one closely mimicking another as a result of the loss of a single signaling component has profound implications, for example, for the interpretation of the phenotypes of knockout mice and for the clinical use of inhibitors of signaling.

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.)

Modulation of Hepatitis B Virus Replication and Hepatocyte Differentiation by MicroRNA-1

MicroRNAs (miRNAs) are highly conserved small noncoding RNAs participating in regulation of various cellular processes. Viruses have been shown to utilize cellular miRNAs to increase their replication in host cells. Until now, the role of miRNAs in hepatitis B virus (HBV) replication has remained largely unknown. In this study, a number of miRNA mimics were transfected into hepatoma cell lines with HBV replication. It was noted that microRNA‐1 (miR‐1) transfection resulted in a marked increase of HBV replication, accompanied with up‐regulated HBV transcription, antigen expression, and progeny secretion. However, bioinformatics and luciferase reporter analysis suggested that miR‐1 may not target the HBV genome directly but regulate the expression of host genes to enhance HBV replication. Further studies showed that miR‐1 was able to enhance the HBV core promoter transcription activity by augmenting farnesoid X receptor α expression. In addition, miR‐1 arrested the cell cycle at the G1 phase and inhibited cell proliferation by targeting histone deacetylase 4 and E2F transcription factor 5. Analysis of the cellular gene expression profile indicated that miR‐1 transfected hepatoma cells developed a differentiated phenotype of hepatocytes. Conclusion: MiR‐1 regulates the expression of several host genes to enhance HBV replication and reverse cancer cell phenotype, which is apparently beneficial for HBV replication. Our findings provide a novel perspective on the role of miRNAs in host‐virus interactions in HBV infection. (HEPATOLOGY 2011;)

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.

Quality of Life and Psychiatric Complications After Adult Living Donor Liver Transplantation

We investigated the psychosocial effects of a right hepatectomy on donors for adult living donor liver transplantation (ALDLT). Questionnaires were sent to 66 actual donors, who had undergone ALDLT between August 1998 and September 2003, as well as to 139 potential donors, who had been examined as possible candidates for ALDLT; the latter had been excluded and had not undergone surgery. All actual donors reported full recovery within an average period of 14.41 (standard deviation = 8.86) weeks; all had returned to their preoperative employment. In preparation for ALDLT, they had received significantly more support from their families in the decision‐making process than the potential donors had (t = 2.02; degree of freedom = 79; P = 0.047); they also felt better informed about donation than the potential donors (t = 2.04; df = 64; P = 0.045). Psychiatric problems occurred in 6 (14%) female donors in the perioperative period, mostly in connection with unrealistic outcome expectations. Donors with severe postoperative complications (n = 3) demonstrated higher scores of psychiatric symptoms (chi‐square = 6.39; df = 2; P = 0.041). When we compared potential and actual donors, a significant difference in emotional quality of life was not demonstrated (t = 0.41; df = 76; P = 0.684), and it corresponded to that of the normative sample. For donors, perceived emotional quality of life did not depend on the course of recovery of the recipients. Six to 9 months after donation, potential donors reported a significantly higher physical quality of life than actual donors (t = 2.20; df = 56; P = 0.032). In conclusion, female donors, donors with their own major complications, or donors with unrealistic outcome expectations should be provided with adequate psychosocial care. With regard to the psychosocial outcome, ALDLT is a safe intervention for the donor. Liver Transpl 12:1782–1790, 2006.