Masanori Isogawa

Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo

ABSTRACT Toll-like receptors (TLR) play a key role in innate immunity. To examine the ability of diverse TLRs to modulate hepatitis B virus (HBV) replication, HBV transgenic mice received a single intravenous injection of ligands specific for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. All of the ligands except for TLR2 inhibited HBV replication in the liver noncytopathically within 24 h in a α/β interferon-dependent manner. The ability of these TLR ligands to induce antiviral cytokines at the site of HBV replication suggests that TLR activation could represent a powerful and novel therapeutic strategy for the treatment of chronic HBV infection.

Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment

A paucity of versatile small animal models of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has been an impediment to both furthering understanding of virus biology and testing antiviral therapies. We recently described a regulatable system for repopulating the liver of immunodeficient mice (specifically mice lacking fumaryl acetoacetate hydrolase [Fah], recombination activating gene 2 [Rag2], and the gamma-chain of the receptor for IL-2 [Il-2rgamma]) with human hepatocytes. Here we have shown that a high transplantation dose (3 x 106 to 5 x 106 human hepatocytes/mouse) generates a higher rate of liver chimerism than was previously obtained in these mice, up to 95% human hepatocyte chimerism. Mice with a high level of human liver chimerism propagated both HBV and HCV, and the HCV-infected mice were responsive to antiviral treatment. This human liver chimeric mouse model will expand the experimental possibilities for studying HBV and HCV infection, and possibly other human hepatotropic pathogens, and prove useful for antiviral drug testing.

Pathogenesis of hepatitis B virus infection.

The adaptive immune response is thought to be responsible for viral clearance and disease pathogenesis during hepatitis B virus infection. It is generally acknowledged that the humoral antibody response contributes to the clearance of circulating virus particles and the prevention of viral spread within the host while the cellular immune response eliminates infected cells. The T cell response to the hepatitis B virus (HBV) is vigorous, polyclonal and multispecific in acutely infected patients who successfully clear the virus and relatively weak and narrowly focussed in chronically infected patients, suggesting that clearance of HBV is T cell dependent. The pathogenetic and antiviral potential of the cytotoxic T lymphocyte (CTL) response to HBV has been proven by the induction of a severe necroinflammatory liver disease following the adoptive transfer of HBsAg specific CTL into HBV transgenic mice. Remarkably, the CTLs also purge HBV replicative intermediates from the liver by secreting type 1 inflammatory cytokines thereby limiting virus spread to uninfected cells and reducing the degree of immunopathology required to terminate the infection. Persistent HBV infection is characterized by a weak adaptive immune response, thought to be due to inefficient CD4+ T cell priming early in the infection and subsequent development of a quantitatively and qualitatively ineffective CD8+ T cell response. Other factors that could contribute to viral persistence are immunological tolerance, mutational epitope inactivation, T cell receptor antagonism, incomplete down-regulation of viral replication and infection of immunologically privileged tissues. However, these pathways become apparent only in the setting of an ineffective immune response, which is, therefore, the fundamental underlying cause. Persistent infection is characterized by chronic liver cell injury, regeneration, inflammation, widespread DNA damage and insertional deregulation of cellular growth control genes, which, collectively, lead to cirrhosis of the liver and hepatocellular carcinoma.

Persistent Hepatitis C Virus Infection In Vitro: Coevolution of Virus and Host

ABSTRACT The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.

Platelets mediate cytotoxic T lymphocyte–induced liver damage

We found that platelet depletion reduces intrahepatic accumulation of virus-specific cytotoxic T lymphocytes (CTLs) and organ damage in mouse models of acute viral hepatitis. Transfusion of normal but not activation-blocked platelets in platelet-depleted mice restored accumulation of CTLs and severity of disease. In contrast, anticoagulant treatment that prevented intrahepatic fibrin deposition without reducing platelet counts did not avert liver injury. Thus, activated platelets contribute to CTL-mediated liver immunopathology independently of procoagulant function.

Plasmacytoid dendritic cells sense hepatitis C virus–infected cells, produce interferon, and inhibit infection

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that infects >170 million people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite its ability to block the innate host response in infected hepatocyte cell lines in vitro, HCV induces a strong type 1 interferon (IFN) response in the infected liver. The source of IFN in vivo and how it is induced are currently undefined. Here we report that HCV-infected cells trigger a robust IFN response in plasmacytoid dendritic cells (pDCs) by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 signaling, and we show that the activated pDC supernatant inhibits HCV infection in an IFN receptor-dependent manner. Importantly, the same events are triggered by HCV subgenomic replicon cells but not by free virus particles, suggesting the existence of a novel cell-cell RNA transfer process whereby HCV-infected cells can activate pDCs to produce IFN without infecting them. These results may explain how HCV induces IFN production in the liver, and they reveal a heretofore unsuspected aspect of the innate host response to viruses that can subvert the classical sensing machinery in the cells they infect, and do not infect or directly activate pDCs.

PD-1:PD-L1 Interactions Contribute to the Functional Suppression of Virus-Specific CD8+ T Lymphocytes in the Liver

Mechanisms contributing to the development of chronic viral infections, including chronic hepatitis B virus (HBV) infections, are not well understood. We have shown recently that production of IFN-γ, an important antiviral cytokine, by HBV-specific CTLs is rapidly induced when they enter the liver of HBV transgenic mice, and then rapidly suppressed, despite the continued presence of Ag. Suppression of IFN-γ production by the CTLs coincides with the up-regulation of programmed cell death (PD)-1, a cell surface signaling molecule known to inhibit T cell function. To determine whether PD-1 plays a role in the functional suppression of IFN-γ secretion by CTLs, we treated HBV transgenic mice with blocking Abs specific for PD ligand (PD-L)1, the most widely expressed PD-1 ligand, and adoptively transferred HBV-specific CTLs. Treatment with anti-PD-L1 Abs resulted in a delay in the suppression of IFN-γ-producing CTLs and a concomitant increase in the absolute number of IFN-γ-producing CTLs in the liver. These results indicate that PD-1:PD-L1 interactions contribute to the suppression of IFN-γ secretion observed following Ag recognition in the liver. Blockade of inhibitory pathways such as PD-1:PD-L1 may reverse viral persistence and chronic infection in cases in which the CTL response is suppressed by this mechanism.

Immune effectors required for hepatitis B virus clearance

To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4+ and CD8+ T cells, NK cells, Fas, IFN-gamma (IFN-γ), IFN-alpha/beta receptor (IFN-α/βR1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8+ T cell response in these animals depended on the presence of CD4+ T cells. These results are consistent with a model in which CD4+ T cells serve as master regulators of the adaptive immune response to HBV; CD8+ T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-γ, TNFR1, and IFN-α/βR play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.

MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs

We recently showed that antigen-nonspecific inflammatory cells are recruited into the liver when hepatitis B virus (HBV)-specific CTLs are injected into HBV transgenic mice, and that this process amplifies the severity of liver disease. We also showed that the severity of CTL-induced liver disease is ameliorated by the depletion of Gr-1(+) cells (Gr-1 is an antigen highly expressed by neutrophils), which, secondarily, abolishes the intrahepatic recruitment of all antigen-nonspecific Gr-1(-) mononuclear cells (NK and NKT cells, T and B lymphocytes, monocytes, macrophages, dendritic cells) despite the strong induction of chemokine gene expression. Those results suggested that in addition to chemokine expression, CTL-induced functions are necessary for mononuclear cell recruitment to occur. We now report that MMPs known to be produced by Gr-1(+) cells are rapidly induced in the livers of CTL-injected mice. The inhibition of MMP activity reduced the intrahepatic recruitment of antigen-nonspecific mononuclear cells and much of the attending liver disease without affecting the migration or antiviral potential of antigen-specific CTLs. The notion that the inhibition of MMP activity is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Immunogenicity and Tolerogenicity of Hepatitis B Virus Structural and Nonstructural Proteins: Implications for Immunotherapy of Persistent Viral Infections

ABSTRACT Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8+ T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8+ cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.