• chronic hepatitis B
• immune infiltration
• innate immunity
• population‐specific expression analysis

• China Postdoctoral Science Foundation

• hepatitis B virus (HBV)
• immune system
• polymorphism
• signaling
• toll-like receptors (TLR)

• Humans
• Immunity, Innate
• Toll-Like Receptors/metabolism
• Hepatitis B/genetics
• Hepatitis B virus
• Cytokines/metabolism

• antiviral
• autophagy
• hepatitis B virus (HBV)
• mitogen-activated protein kinases (MAPKs)
• nuclear factor-kappa B (NF-κB)
• tannic acid (TA)

• Adaptive immune response
• B cells
• Immunotherapy
• T cells
• Toll-like receptor
• Viral infection

• hepatitis B virus
• prolonged HBV infection
• toll-like receptors

• Chronic hepatitis B
• Direct-acting antivirals
• Functional cure
• Gene silencing
• Immunotherapy
• Therapeutic vaccination

• Antiviral Agents/therapeutic use
• Hepatitis B/drug therapy
• Hepatitis B Surface Antigens
• Hepatitis B virus/genetics
• Hepatitis B, Chronic/drug therapy
• Hepatitis C, Chronic/drug therapy
• Humans

• Animals
• Antibodies, Neutralizing/immunology
• Hepatitis B/immunology
• Hepatitis B/metabolism
• Hepatitis B virus/immunology
• Hepatocytes/immunology
• Hepatocytes/metabolism
• Humans
• Immunity, Innate
• Interleukin-1beta/immunology
• Interleukin-6/immunology
• Lipoproteins/metabolism
• MAP Kinase Signaling System
• Mice
• NF-kappa B/metabolism
• Phosphorylation
• Toll-Like Receptor 2/metabolism
• Transcriptome
• Tumor Necrosis Factor-alpha/immunology
• p38 Mitogen-Activated Protein Kinases/metabolism

Woodchuck infected with woodchuck hepatitis virus (WHV) represents the pathogenically nearest model of hepatitis B and associated hepatocellular carcinoma (HCC). This naturally occurring animal model also is highly valuable for development and preclinical evaluation of new anti-HBV agents and immunotherapies against chronic hepatitis (CH) B and HCC. Studies in this system uncovered a number of molecular and immunological processes which contribute or likely contribute to the immunopathogenesis of liver disease and modulation of the systemic and intrahepatic innate and adaptive immune responses during hepadnaviral infection. Among them, inhibition of presentation of the class I major histocompatibility complex on chronically infected hepatocytes and a role of WHV envelope proteins in this process, as well as augmented hepatocyte cytotoxicity mediated by constitutively expressed components of CD95 (Fas) ligand- and perforin-dependent pathways, capable of eliminating cells brought to contact with hepatocyte surface, including activated T lymphocytes, were uncovered. Other findings pointed to a role of autoimmune response against hepatocyte asialoglycoprotein receptor in augmenting severity of liver damage in hepadnaviral CH. It was also documented that WHV in the first few hours activates intrahepatic innate immunity that transiently decreases hepatic virus load. However, this activation is not translated in a timely manner to induction of virus-specific T cell response which appears to be hindered by defective activation of antigen presenting cells and presentation of viral epitopes to T cells. The early WHV infection also induces generalized polyclonal activation of T cells that precedes emergence of virus-specific T lymphocyte reactivity. The combination of these mechanisms hinder recognition of virus allowing its dissemination in the initial, asymptomatic stages of infection before adaptive cellular response became apparent. This review will highlight a range of diverse mechanisms uncovered in the woodchuck model which affect effectiveness of the anti-viral systemic and intrahepatic immune responses, and modify liver disease outcomes. Further exploration of these and other mechanisms, either already discovered or yet unknown, and their interactions should bring more comprehensive understanding of HBV pathogenesis and help to identify novel targets for therapeutic and preventive interventions. The woodchuck model is uniquely positioned to further contribute to these advances.

• asialoglycoprotein receptor
• hepatocyte as cytotoxic immune effector
• intrahepatic innate and adaptive immune responses
• major histocompatibility complex presentation
• pre-acute infection
• toll-like receptors
• virus-hepatocyte interaction
• woodchuck model of hepatitis B

Although several evidences suggesting the vital roles that innate immunity plays in the persistence and elimination of chronic hepatitis B virus (CHB) infection, the exact mechanism is still complicated. Here, we successfully polarized monocytes derived from healthy human peripheral blood mononuclear cells (PBMCs) into M1/M2 macrophages and detected the effects of hepatitis B core antigen (HBcAg) on the polarization and function of macrophages via the Toll-like receptor (TLR) 2 signaling pathway. The results showed that HBcAg had a negligible impact on M1 polarization, while it effectively impaired M2 polarization and promoted the production of pro-inflammatory cytokines such as IL-6 and TNF-α. Additionally, HBcAg treatment increased TLR2 expression on M2 macrophages and TLR2 blockade abolished the effects of HBcAg on the impaired phenotype and pro-inflammatory cytokine productions of M2 macrophages. Signaling pathway analysis revealed that the nuclear factor κB (NF-κB) pathway, the downstream of TLR2, was upregulated upon HBcAg treatment in both M1 and M2 macrophages. Furthermore, a CD8⁺ T-macrophage coculture system implied that compared with PBS stimulation, HBcAg-stimulated M2 macrophages regained their ability to activate CD8⁺ T cells with higher secretion of IFN-γ. Finally, we found impaired expression of M2-related molecules and increased levels of pro-inflammation cytokines in M2 macrophages from CHB patients upon HBcAg stimulation. In conclusion, these results imply a favorable role of HBcAg in the establishment of a pro-inflammatory microenvironment by macrophages, which may suggest a potential therapeutic strategy of HBcAg-induced macrophage activation in CHB infection.

• HBV core protein
• Toll-like receptor 2
• hepatitis B virus
• inflammatory cytokines
• macrophage polarization

• HBV
• hepatocytes
• in-vitro cell models
• innate immunity

• Cell Line
• Cells, Cultured
• Genome, Viral
• Genomics/methods
• Hepatitis B/immunology
• Hepatitis B/metabolism
• Hepatitis B/virology
• Hepatitis B virus/genetics
• Hepatitis B virus/immunology
• Hepatocytes/immunology
• Hepatocytes/metabolism
• Hepatocytes/virology
• Host-Pathogen Interactions/immunology
• Humans
• Immunity, Innate

• adaptive immunity
• chronic hepatitis B
• functional cure
• hepatitis B virus
• innate immunity

Woodchucks infected with woodchuck hepatitis virus (WHV) represent a highly valuable model of human hepatitis B virus (HBV) infection, chronic hepatitis (CH), and virus induced-primary liver cancer. Toll-like receptors (TLRs) are important mediators of immune responses playing pivotal roles in the pathogenesis of viral diseases; however, their expression profiles in different forms of infection and stages of hepatitis, and in healthy animals remain essentially unknown. In this study, woodchuck TLRs 1–10 exon fragments were identified and TLR genes transcription quantified in livers, primary hepatocytes, peripheral blood mononuclear cells (PBMC), and in selected organs during experimental WHV infection. Among others, liver biopsies from acute hepatitis (AH) and CH showed significantly augmented expression of the majority of TLRs when compared to healthy and woodchucks prior to AH, with resolved AH or primary occult infection. In contrast to the liver tissue, significant upregulation of TLR3, TLR4, and TLR10, but downregulation of TLR7, characterized hepatocytes derived from livers of animals with resolved AH accompanied by secondary occult infection. Hepatocytes from CH showed significantly lower expression or a trend toward suppression of several TLRs when compared to hepatocytes from healthy animals and woodchucks with other forms of infection or hepatitis, suggesting that hepatocyte innate immune response is downregulated during CH. Contrastingly, upregulated transcription of some TLRs characterized PBMC throughout CH. Our study uncovered that TLR expression significantly varies between different forms of hepadnaviral infection and whether infection is accompanied or not by hepatitis. The results showed that the profiles of TLRs’ expression in circulating lymphomononuclear cells do not mirror accurately those of livers and hepatocytes from infected animals. These findings are of importance to the understanding of immune process operating at different sites targeted by virus in the course of hepadnaviral infection and evaluation of future therapies modifying antiviral innate responses in the woodchuck model.

• PBMC
• hepadnaviral infection
• hepatocytes
• liver
• lymphatic organs
• toll-like receptors
• woodchuck model of hepatitis B

Toll-like receptors (TLRs) play a crucial role in activation of innate immunity, which is essential for inducing effective adaptive immune responses. Our previous studies have shown that toll-like receptor 2 (TLR2) is required to induce effective virus-specific T-cell responses against hepatitis B virus (HBV) in vivo. However, the contribution of TLR2 activation to adaptive immunity and HBV clearance remains to be clarified. In this study, we explored the hydrodynamic injection (HI) mouse models for HBV infection and examined how the TLR2 agonist Pam3CSK (P3C) influences HBV control and modulates HBV-specific T-cell response if applied in vivo. We found that TLR2 activation by P3C injection leads to the rapid but transient production of serum proinflammatory factors interleukin-6 and tumor necrosis factor-α and activation of CD8⁺ T cells in vivo. Then, the anti-HBV effect and HBV-specific T-cell immunity were investigated by TLR2 activation in the mouse models for persistent or acute HBV infections using HBV plasmids pAAV-HBV1.2 and pSM2, respectively. Both P3C application at early stage and pre-activation promoted HBV clearance, while only TLR2 pre-activation enhanced HBV-specific T-cell response in the liver. In the mouse model for acute HBV infection, P3C application had no significant effect on HBV clearance though P3C significantly enhanced the HBV-specific T-cell response. Collectively, TLR2 pre-activation enhances HBV-specific T-cell responses and accelerates HBV clearance in HI mouse models. Thus, the modulation of host immune status by TLR2 agonists may be explored for immunotherapeutic strategies to control HBV infection.

• T-cell immunity
• hepatitis B virus
• mouse model
• proinflammatory cytokines
• toll-like receptor 2

• Chronic viral infection
• Toll-like receptor (TLR)
• adaptive immunity
• hepatitis B virus (HBV)
• human immunodeficiency virus (HIV)
• interferon (IFN)
• pro-inflammatory cytokines

Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or “adjuvant” potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.

Immune defense against infection with the hepatitis B virus (HBV) is complex and involves both host innate and adaptive immune systems. It is well accepted that the development of sufficient HBV-specific T cell and B cell responses are required for controlling an HBV infection. However, the contribution of innate immunity to removing HBV has been explored in recent years. Toll-like receptors (TLRs) are recognized as the first line of antiviral immunity because they initiate intracellular signaling pathways to induce antiviral mediators such as interferons (IFNs) and other cytokines. Recent studies show that the activation of TLR-mediated signaling pathways results in a suppression of HBV replication in vitro and in vivo. However, HBV has also evolved strategies to counter TLR responses including the suppression of TLR expression and the blockage of downstream signaling pathways. Antiviral treatment in chronic HBV-infected patients leads to an upregulation of TLR expression and the restoration of its innate antiviral functions. Thus, TLR activation may serve as an additional immunotherapeutic option for treating chronic HBV infection in combination with antiviral treatment.

• adaptive immunity
• hepatitis B virus
• immunotherapy
• innate immunity
• toll-like receptors

• Hepatitis B virus
• Hepatitis activity
• Immune response
• Toll-like receptors

• CD4+ T cells
• T helper cell 17
• chronic hepatitis B
• chronic hepatitis B-related liver failure
• toll-like receptor 2

• Cirrhosis
• Hepatitis B virus (HBV)
• Hepatocellular carcinoma
• Innate immunity
• Pathogen recognition receptors

• Carcinoma, Hepatocellular/immunology
• Carcinoma, Hepatocellular/virology
• Hepatitis B virus/immunology
• Hepatitis B, Chronic/immunology
• Host-Pathogen Interactions
• Humans
• Immunity, Innate/immunology
• Liver Neoplasms/immunology
• Liver Neoplasms/virology
• Receptors, Pattern Recognition/immunology

• clinical phases of chronic hepatitis B infection
• hepatitis B virus
• innate immune responses

The outcome of hepatitis B viral (HBV) infection is determined by the complex interactions between replicating HBV and the immune system. While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively, the contribution of innate immune mechanisms remains to be defined. Here we examined the role of the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signaling pathway in adaptive immune responses and viral clearance by exploring the HBV mouse model. Hydrodynamic injection with a replication-competent HBV genome was performed in wild-type mice (WT) and a panel of mouse strains lacking specific innate immunity component expression. We found higher levels of HBV protein production and replication in Tlr2^−/−, Tlr23479^−/−, 3d/Tlr24^−/−, Myd88/Trif^−/− and Irak4^−/− mice, which was associated with reduced HBV-specific CD8⁺ T-cell responses in these mice. Importantly, HBV clearance was delayed for more than 2 weeks in 3d/Tlr24^−/−, Myd88/Trif^−/− and Irak4^−/− mice compared to WT mice. HBV-specific CD8⁺ T-cell responses were functionally impaired for producing the cytokines IFN-γ, TNF-α and IL-2 in TLR signaling-deficient mice compared to WT mice. In conclusion, the IL-1R/TLR signaling pathway might contribute to controlling HBV infection by augmenting HBV-specific CD8⁺ T-cell responses.

• HBV
• immune therapeutic concepts
• innate immunity
• liver immunity
• viral escape

• FHB-ALF
• TLR2.
• hepatitis B
• pathway enrichment

• Adult
• Alanine Transaminase/metabolism
• Animals
• DNA, Viral/genetics
• Female
• Hepatitis B/complications
• Hepatitis B Core Antigens/blood
• Hepatitis B virus
• Humans
• Liver/virology
• Liver Failure, Acute/genetics
• Liver Failure, Acute/virology
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Middle Aged
• Mutation
• Signal Transduction
• T-Lymphocytes, Regulatory/metabolism
• Toll-Like Receptor 2/genetics
• Young Adult

Toll like receptors (TLRs) play an important role in innate immunity and various studies suggest that TLRs play a crucial role in pathogenesis of hepatitis B virus (HBV) infection. The present study aims in looking into the status of crucial host and viral gene expression on inciting TLR7.

The transcription of TLR7 pathway signaling molecules and HBV DNA viral load were quantified by Real Time-PCR after stimulation of TLR7 with its imiquimod based ligand, R837. Cell cycle analysis was performed using flow-cytometry. Expression of TLR7 and chief cell cycle regulator governing G1/S transition, p53 was also seen in liver biopsysss samples of CHB patients. HBV induced alteration in histone modifications in HepG2 cells and its restoration on TLR7 activation was determined using western blot.

The TLR7 expression remains downregulated in HepG2.2.15 cells and in liver biopsy samples from CHB patients. Interestingly HBV DNA viral load showed an inverse relationship with the TLR7 expression in the biopsy samples. We also evaluated the anti-viral activity of R837, an agonist of TLR7. It was observed that there was a suppression of HBV replication and viral protein production upon TLR7 stimulation. R837 triggers the anti-viral action probably through the Jun N-terminal Kinase (JNK) pathway. We also observed a downregulation of histone H3K9Me3 repression mark upon R837 treatment in HBV replicating HepG2.2.15 cells, mimicking that of un-infected HepG2 cells. Additionally, the G1/S cell cycle arrest introduced by HBV in HepG2.2.15 cells was released upon ligand treatment.

The study thus holds a close insight into the changes in hepatocyte micro-environment on TLR7 stimulation in HBV infection.

The online version of this article (doi:10.1186/s12879-017-2189-z) contains supplementary material, which is available to authorized users.

• Cell-cycle arrest
• Epigenetics
• Hepatitis B virus
• Innate immune response
• TLR7

• hepatitis B virus
• immunotherapy
• viral targets

Toll like receptors plays a significant anti-viral role in different infections. The aim of this study was to look into the role of toll like receptor 4 (TLR4) in hepatitis B virus (HBV) infection.

Real time PCR was used to analyze the transcription of TLR4 signaling molecules, cell cycle regulators and HBV DNA viral load after triggering the HepG2.2.15 cells with TLR4 specific ligand. Nuclear factor (NF)-κB translocation on TLR4 activation was analyzed using microscopic techniques. Protein and cell cycle analysis was done using Western Blot and FACS respectively.

The present study shows that TLR4 activation represses HBV infection. As a result of HBV suppression, there are several changes in host factors which include partial release in G1/S cell cycle arrest and changes in host epigenetic marks. Finally, it was observed that anti-viral action of TLR4 takes place through the NF-κB pathway.

The study shows that TLR4 activation in HBV infection brings about changes in hepatocyte microenvironment and can be used for developing a promising therapeutic target in future.

• Hepatitis B virus
• Toll like receptor 4
• Cell cycle
• Epigenetic marks
• Innate immune response

• Active Transport, Cell Nucleus
• Cellular Microenvironment
• DNA Methylation
• DNA, Viral/genetics
• Dose-Response Relationship, Drug
• Epigenesis, Genetic
• G1 Phase Cell Cycle Checkpoints
• Hep G2 Cells
• Hepacivirus/genetics
• Hepacivirus/pathogenicity
• Hepatitis B, Chronic/genetics
• Hepatitis B, Chronic/metabolism
• Hepatitis B, Chronic/prevention & control
• Hepatitis B, Chronic/virology
• Hepatocytes/drug effects
• Hepatocytes/metabolism
• Hepatocytes/virology
• Host-Pathogen Interactions
• Humans
• Ligands
• Lipopolysaccharides/pharmacology
• NF-kappa B/metabolism
• Signal Transduction
• Time Factors
• Toll-Like Receptor 4/agonists
• Toll-Like Receptor 4/genetics
• Toll-Like Receptor 4/metabolism
• Viral Load

• Antiviral therapy
• Combination therapy
• Gene expression
• HBV cure
• HBsAg
• Immunomodulation
• Novel antiviral strategies
• cccDNA

Cellular microRNAs (miRNAs) are able to influence hepatitis B virus (HBV) replication directly by binding to HBV transcripts or indirectly by targeting cellular factors. Here, we investigate the effect of epigenetically regulated miR-449a on HBV replication and the underlying mechanisms. miR-449a expression was lower in human hepatocellular carcinoma (HCC) cells than in primary hepatocytes and could be induced by trichostatin A. Ectopic miR-449a expression in HCC cells strongly enhanced HBV replication, transcription, progeny virions secretion, and antigen expression in a dose-dependent manner. miR-449a directly targeted cAMP-responsive element binding protein 5 (CREB5), which in turn induced the expression of farnesoid X receptor α (FXRα), a transcription factor that facilitates HBV replication. CREB5 knockdown and overexpression demonstrated that it is a negative regulator of HBV replication. Additionally, miR-449a overexpression inhibited proliferation, caused cell cycle arrest, and promoted HCC cell differentiation. The results indicated that epigenetically regulated miR-449a targets CREB5 to increase FXRα expression, thereby promoting HBV replication and gene expression. Our findings provide a new understanding of the role of miRNAs in HBV replication.

• Direct acting antivirals (DAA)
• Hepatitis B virus
• Host-targeting antivirals (HTA)
• Immunotherapy
• Viral targets

• C/EBP-β
• Toll-like receptors
• chronic
• hepatitis B virus
• miRNA

Hepatitis B virus (HBV) is a hepatotropic DNA virus and its infection results in acute or chronic hepatitis. It is reported that the host innate immune system contributes to viral control and liver pathology, while whether and how HBV can trigger the components of innate immunity remains controversial. In recent years, the data accumulated from HBV-infected patients, cellular and animal models have challenged the concept of a stealth virus for HBV infection. This editorial focuses on the current findings about the innate immune recognition to HBV. Such evaluation could help us to understand HBV immunopathogenesis and develop novel immune therapeutic strategies to combat HBV infection.

• Hepatitis B virus
• Pathogen-recognition receptor
• Hepatocytes
• Interferon
• Innate immunity

• Cytokines/chemokines
• Hepatitis B virus
• Immune-modulators
• Immunotherapy
• Interferon

• Chronic hepatitis B
• Hepatitis B virus
• Pegylated interferon
• Telbivudine
• Toll-like receptor 2

• Adolescent
• Adult
• Antiviral Agents/therapeutic use
• Cross-Sectional Studies
• Female
• Gene Expression Profiling
• Hepatitis B virus/immunology
• Hepatitis B, Chronic/immunology
• Humans
• Interferon-alpha/therapeutic use
• Interleukin-6/metabolism
• Male
• Middle Aged
• Polyethylene Glycols/therapeutic use
• Recombinant Proteins/therapeutic use
• Telbivudine
• Thymidine/analogs & derivatives
• Thymidine/therapeutic use
• Toll-Like Receptor 2/biosynthesis
• Toll-Like Receptor 2/blood
• Tumor Necrosis Factor-alpha/metabolism
• Young Adult

• Drug resistance
• Hepatitis B therapy
• Immunotherapy
• Interferon-α
• Nucleos(t)ide analogues

Hepatitis B virus (HBV) persistent infection is associated with ineffective immune response for the clearance of virus. Immunomodulators represent an important class of therapeutics, which potentially could be beneficial for the treatment of HBV infection. The particulate yeast-derived glucan (PYDG) has been shown to enhance the innate and adaptive immune responses. We therefore, assessed the efficacy of PYDG in enhancing HBV specific immune responses by employing the hydrodynamic injection-based (HDI) HBV transfection mouse model. Mice were intragatric administered PYDG daily for 9 weeks post pAAV/HBV1.2 hydrodynamic injection. PYDG treatment significantly promoted HBV DNA clearance and production of HBsAb compared to control mice. PYDG treatment resulted in recruitment of macrophages, dendritic cells (DCs) and effector T cells to the liver microenvironment, accompanied by a significantly augmented DCs maturation and HBV-specific IFN-γ and TNF-α production by T cell. In addition, enhanced production of Th1 cytokines in liver tissue interstitial fluid (TIF) was associated with PYDG administration. Live imaging showed the accumulation of PYDG in the mouse liver. Our results demonstrate that PYDG treatment significantly enhances HBV-specific Th1 immune responses, accompanied by clearance of HBV DNA, and therefore holds promise for further development of therapeutics against chronic hepatitis B.

• Adaptive Immunity/drug effects
• Adaptive Immunity/immunology
• Animals
• Dendritic Cells/drug effects
• Dendritic Cells/immunology
• Disease Models, Animal
• Glucans/administration & dosage
• Glucans/chemistry
• Glucans/immunology
• Hepatitis B virus/drug effects
• Hepatitis B virus/immunology
• Hepatitis B virus/pathogenicity
• Hepatitis B, Chronic/drug therapy
• Hepatitis B, Chronic/immunology
• Hepatitis B, Chronic/virology
• Humans
• Immunologic Factors/administration & dosage
• Immunologic Factors/chemistry
• Injections
• Liver/drug effects
• Liver/immunology
• Macrophages/drug effects
• Macrophages/immunology
• Mice
• Saccharomyces cerevisiae/chemistry
• T-Lymphocyte Subsets/drug effects
• T-Lymphocyte Subsets/immunology
• Virus Replication/drug effects
• Virus Replication/immunology

• In ovo
• Infectious laryngotracheitis virus
• Lipoteichoic acid
• Lung
• Toll-like receptor 2