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It has been recently shown that Toll-like receptor (TLR) signalling in murine nonparenchymal liver cells (NPCs) is suppressed in the presence of Hepatitis B virus surface antigen (HBsAg). It is not clear, however, whether this is also relevant for the adaptive immune responses and how this effect is mediated. Peripheral blood mononuclear cells (PBMCs) from Hepatitis B virus (HBV) patients and controls were stimulated by TLR ligands in the absence or presence of autologous serum. Interestingly, TLR-mediated cytokine expression (Interleukin-6 and -10) as well as TLR3-induced interferon (IFN) expression in PBMCs of HBV patients was significantly higher than in the healthy volunteers, showing a negative correlation with the levels of HBsAg. In addition, TLR3-mediated IFN-γ production was inhibited in the presence of HBV-containing serum. To mechanistically analyse this observation, murine Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs) were stimulated with TLR3 ligands in the presence or absence of HBsAg. Mixed lymphocyte reactions were performed to study T-cell activation induced by TLR-stimulated NPCs. Gene expression of cytokines and TLR3 was analysed by quantitative rt-PCR, and activation of transcription factors was assessed by Western blot or reporter gene assays. TLR-induced expression of interferon γ, interferon sensitive genes and proinflammatory cytokines in murine KCs and LSECs was efficiently suppressed in the presence of HBsAg, whereas the expression of anti-inflammatory cytokines was enhanced. Activation of NFκB, IRF-3 and MAPKs in these liver cells was potently suppressed by HBsAg. T-cell activation mediated through TLR3-stimulated KCs or LSECs was suppressed by HBsAg which could be reverted by anti-IL-10 antibodies. These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to maintain a persistent infection., (© 2014 John Wiley & Sons Ltd.)

Objectives: The therapeutic application of small interfering RNAs (siRNAs) is limited by the induction of severe off-target effects, especially in the liver. Therefore, we assessed the potential of differently modified siRNAs to induce the hepatic innate immune system in vitro and in vivo., Methods: Primary isolated liver cells were transfected with siRNAs against apolipoprotein B1 (APOB1), luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in lipid nanoparticles (LNPs) and administered intravenously to C57BL/6 mice. Liver tissue was collected 6-48 h after injection and knock-down efficiency or immune responses were determined by quantitative reverse-transcription-linked PCR., Results: Unmodified GAL siRNA transiently induced the expression of TNF-α, IL-6, IL-10, IFN-β and IFN-sensitive gene 15 in vivo, whereas a formulation of 2'-O-methylated-LUC siRNA had no such effects. Formulation of unmodified APOB1-specific siRNA suppressed APOB1 mRNA levels by ~80% in the liver 48h after application. The results were paralleled in vitro, where transfection of liver cells with unmodified siRNAs, but not with chemically modified siRNAs, led to cell-type-specific induction of immune genes. These immune responses were not observed in MYD88-deficient mice or in chloroquine-treated cells in vitro., Conclusions: Our data indicate that siRNAs activate endosomal Toll-like receptors in different liver-derived cell types to various degrees, in vitro. LNP-formulated siRNA selectively leads to hepatic knock-down of target genes in vivo. Here, off-target immune responses are restricted to non-parenchymal liver cells. However, 2'-O-methyl modifications of siRNA largely avoid immune-stimulatory effects, which is a crucial prerequisite for the development of safe and efficient RNA-interference-based therapeutics.

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.

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.

Objective: Only little is known about the mechanisms of action of corticosteroids in the treatment of inflammatory liver diseases. As there is increasing evidence that stimulation of the innate immune system plays an important pathogenetic role in these conditions, we hypothesized that steroids may interfere with the activation of the Toll-like receptor (TLR) system of the liver., Methods: To test this hypothesis, murine non-parenchymal liver cells (Kupffer cells, liver sinusoidal endothelial cells) and primary hepatocytes were stimulated with TLR 1-9 ligands in the presence or absence of dexamethasone. Expression of pro- and anti-inflammatory cytokines was determined by quantitative reverse transcription-PCR or ELISA, respectively. Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation was assessed by western blot analysis., Results: TLR agonists induced the expression of pro- [tumor necrosis factor-α (TNF-α), IL-6, IL-1β, IFN-β] and anti-inflammatory cytokines [IL-10, transforming growth factor-β (TGF-β)], which was differentially modulated by steroid treatment. TNF-α and IL-6 expression was suppressed by dexamethasone, while IL-10 but not TGF-β was enhanced after TLR stimulation. IFN-β production induced by TLR 4 agonists but not TLR 3 agonists was inhibited by dexamethasone. TLR expression itself was down-regulated by steroid treatment in a cell type-specific manner. These effects were associated with suppression of the TLR-mediated activation of NF-κB., Conclusions: TLR signaling is modulated by corticosteroids in a cell type-specific fashion resulting in down-regulation of TLR expression, suppression of pro-inflammatory and up-regulation of anti-inflammatory cytokines. This represents an as yet unknown mechanism of action for corticosteroids that may at least in part explain their therapeutic effects in inflammatory liver diseases.

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-alpha (TNF-alpha) or interleukin-6 (IL-6), to TLR3 and TLR4 ligands only by producing interferon-beta (IFN-beta), 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-gamma production in the mixed lymphocyte reaction (MLR). Similarly, LSEC respond to TLR1 to -4, -6, -8 and -9 ligands by producing TNF-alpha, to TLR3 and -4 ligands by producing IL-6, and to TLR3 ligands by producing IFN-beta. 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.

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.

Unlabelled: 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-beta) production and subsequent interferon-stimulated gene induction as well as suppressed activation of interferon regulatory factor 3 (IRF-3), nuclear factor kappa B (NF-kappaB), 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-alpha), 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.

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.

Unlabelled: 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-beta., 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.

The interferon-stimulated gene 15 ( ISG15) plays an important role in the pathogenesis of hepatitis C virus ( HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected ( n = 54) and uninfected ( n = 10) or HBV-infected controls ( n = 23). Primary human hepatocytes ( PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 ( PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific si RNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/ AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV. [ABSTRACT FROM AUTHOR]

Chronic hepatitis C infection is associated with increased expression of interferon-sensitive genes ( ISGs) in the liver, which is, paradoxically, correlated with the nonresponse to interferon ( IFN)-based therapies. In the present study PHHs were isolated from HCV-infected or uninfected patients and stimulated with the TLR1-9 ligands for 6-24 h. Expression of cytokines and ISGs was determined by ELISA and qRT- PCR. A comparative analysis was performed for TLR3 signalling, which was also correlated with single nucleotide polymorphisms ( SNPs) related to HCV pathogenesis. TLR-activated PHHs produced pro-inflammatory and anti-inflammatory cytokines, whereas IFNs were exclusively induced by TLR3 stimulation. Here, IL-29 and IL-28 A were significantly highly expressed than IFN-α and IFN-β. TLR3-induced IFN response was enhanced in hepatocytes isolated from patients with HCV infection. This hyper-responsiveness could be mimicked in naïve PHHs consistently stimulated with low dose of poly I: C, but not Guardiquimod. The higher responsiveness in PHH isolated from HCV-infected patients could be partially explained by higher frequencies of unfavourable SNP alleles of different SNPs associated with HCV progression and treatment outcome. These data suggest that durable activation of TLR3 but not TLR7, by low doses of viral replicative intermediates, increases the sensitivity to viral invasion. These findings shed new light on the relevance of TLR3 in the pathogenesis of HCV and may provide a possible explanation for the increased ISG expression during chronic HCV infection, the so-called IFN paradox. [ABSTRACT FROM AUTHOR]

The hepatitis C virus ( HCV) establishes persistent infections despite strong activation of the innate immune system through TLR3 and other sensors. Therefore, we analysed regulatory mechanisms of TLR3-induced immune responses in nonparenchymal liver cells ( NPCs). Effects of Interleukin-10 ( IL-10), transforming growth factor beta ( TGF-β) and immunoregulatory mi R-155 on poly I: C-activated murine ( C57 BL/6) Kupffer cells ( KC) and sinusoidal endothelial cells ( LSEC) were assessed in vitro. NPCs were assayed for inflammatory and antiviral cytokines and T-cell ( Balb/c)-activating factors. Gene expression of mi R-155, IL-10, TGF-β and interferon sensitive genes ( ISGs) in biopsies of patients with HCV was determined by qrt- PCR. TLR3-induced antiviral activity in murine NPCs was potently suppressed by IL-10 and TGF-β which correlated with decreased TLR3 expression and inhibition of NF-κ B and IRF-3 activation. T-cell activation, induced by TLR3-activated NPCs, was also suppressed by IL-10 and TGF-β, which was associated with a down-regulation of CD80 and CD86. Pretreatment with IL-10 or TGF-β suppressed TLR3-induced mi R-155 expression, which itself positively regulated poly I: C-mediated immune responses, thus counteracting IL-10 or TGF-β-induced immunosuppression. In addition, hepatic expression of mi R-155 was elevated in chronically infected patients with HCV, was associated with an IL-28 B SNP (rs12979860) and was inversely correlated with HCV serum load and ISG expression levels. As mi R-155 is a key regulator of anti-inflammatory mechanisms that control innate and adaptive hepatic immune responses during HCV infection, mi R-155 based therapies may represent a novel mechanism to control HCV in the future. [ABSTRACT FROM AUTHOR]