Effector CD8(+) T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.

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 ␣/␤ 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.We have previously shown that hepatitis B virus (HBV)-specific CD8 ϩ cytotoxic T lymphocytes and CD4 ϩ helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting gamma interferon (IFN-␥) when they recognize viral antigen (6,8). This antiviral effect could be also induced in response to IFN-␣/␤ that was produced in the liver during lymphocytic choriomeningitis virus, murine cytomegalovirus, and adenovirus infections (2, 7). More recently, we have demonstrated that NKT cells, NK cells, and antigenpresenting cells inhibit HBV replication when they are activated by alpha-galactosylceramide (16), interleukin-12 (IL-12) (3), , and an agonistic anti-CD40 antibody injection (17), respectively. Collectively, these results suggest that HBV replication can be controlled by innate immune response if it is activated in the liver.Toll-like receptors (TLRs) are essential for the recognition of invading pathogens and serve as an important link between innate and adaptive immunity. TLRs can discriminate various microbial components, such as triacylated lipopeptides (recognized by TLR1/TLR2 heterodimer) (30), diacylated lipopeptides (recognized by TLR2/TLR6 heterodimer) (23), doublestranded RNA (dsRNA; recognized by TLR3) (1), lipopolysaccharide (LPS; recognized by TLR4) (27), flagellin from bacterial flagella (recognized by TLR5) (11), singlestranded RNA (ssRNA; recognized by TLR7/8) (4, 12), and bacterial DNA containing the unmethylated CpG motif (recognized by TLR9) (13). While the TLRs have been shown to play a crucial role in the innate recognition of bacterial and fungal pathogens, recent studies also suggest the importance of TLRs in antiviral immunity in vivo (14,19,20).Although we have reported that poly(I · C) inhibits HBV replication by inducing IFN-␣/␤ (21), little is known about the ability of other TLRs to control of HBV. Accumulating evidence suggests that each TLR transduces its signals by distinct but overlapping signaling pathways. For instance, TLR3 and TLR4 appear to signal mainly through a MyD88-independent, TRIF-dependent pathway, while TLR2, TLR5, TLR7, and TLR9 signaling appears to be MyD88 dependent and TRIF independent (14). Thus, to examine the potential antiviral effect of these different TLR signaling pathways, groups of three or more age-, sex-, and serum HBeAg-matched transgenic mice from lineage 1.3.32 (9) were injected intravenousl...

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 γ-chain of the receptor for IL-2 [Il-2rγ]) with human hepatocytes. Here we have shown that a high transplantation dose (3 × 10 6 to 5 × 10 6 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.

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.

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.innate immune response | TLR7 | Toll-like receptor | hepatocyte H epatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1). Type 1 interferons (IFNα/β) play critical roles in the defense against virus infection. The HCV NS3/4A protease strongly inhibits type 1 IFN induction in infected cells by cleaving a key intermediate in the double-stranded RNA (dsRNA) signaling pathway (2-4). Nonetheless, HCV strongly induces IFN-stimulated gene (ISG) expression in the infected liver (5-7). The discrepancy between these observations suggests that type 1 IFNs may be produced by liver cells other than infected hepatocytes. Plasmacytoid dendritic cells (pDCs) are a highly specialized subset of dendritic cells that produce type 1 IFNs in response to microbial stimuli (8,9) and are abundant in the HCV-infected liver (10). Although HCV has been reported to suppress pDC numbers and function (11-13), their role in the control of HCV infection has not been examined. Here we show that pDCs produce large amounts of type 1 IFN via Toll-like receptor 7 (TLR7) signaling that is induced by direct cell-to-cell contact with HCV-infected cells. Importantly, these events require viral RNA replication but not virion formation in the stimulator cells. These results could explain how IFN is produced during natural HCV infection, and they reveal a host response mechanism to HCV and possibly other viruses that do not infect or directly activate pDCs.