Control of human viral infections by natural killer cells.

Natural killer (NK) cells are effector cells of the innate immune system and are important in the control of viral infections. Their relevance is reflected by the multiple mechanisms evolved by viruses to evade NK cell-mediated immune responses. Over recent years, our understanding of the interplay between NK cell immunity and viral pathogenesis has improved significantly. Here, we review the role of NK cells in the control of four important viral infections in humans: cytomegalovirus, influenza virus, HIV-1, and hepatitis C virus.

Superoxide Dismutase 1 Protects Hepatocytes from Type I Interferon-Driven Oxidative Damage.

Tissue damage caused by viral hepatitis is a major cause of morbidity and mortality worldwide. Using a mouse model of viral hepatitis, we identified virus-induced early transcriptional changes in the redox pathways in the liver, including downregulation of superoxide dismutase 1 (Sod1). Sod1(-/-) mice exhibited increased inflammation and aggravated liver damage upon viral infection, which was independent of T and NK cells and could be ameliorated by antioxidant treatment. Type I interferon (IFN-I) led to a downregulation of Sod1 and caused oxidative liver damage in Sod1(-/-) and wild-type mice. Genetic and pharmacological ablation of the IFN-I signaling pathway protected against virus-induced liver damage. These results delineate IFN-I mediated oxidative stress as a key mediator of virus-induced liver damage and describe a mechanism of innate-immunity-driven pathology, linking IFN-I signaling with antioxidant host defense and infection-associated tissue damage. VIDEO ABSTRACT.

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

BACKGROUND AND AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH AND RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

The specificity protein 3 (SP3) gene in ducks (Anas platyrhynchos): cloning, characterization and expression during viral infection.

Specificity Protein 3 (SP3) is a newly identified regulator of tumor growth and invasiveness in humans. In this study, we identified and characterized the function of duck SP3 (duSP3). The full-length cDNA sequence of the duSP3 gene was cloned via rapid amplification of cDNA ends. It contained 2468 nucleotides, including a 111 base pair (bp) 5'-untranslated region (UTR), 215 bp 3'-UTR, and 2142 bp open reading frame (ORF), which encoded a 713 amino acid (AA) strongly conserved with Avian SP3. Tissue specificity analysis demonstrated that duSP3 was constitutively expressed in the eight tissues tested: liver, spleen, lung, heart, kidney, thymus, breast, and leg; and low expression levels were observed in all tissues, except the spleen and thymus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that duSP3 expression rapidly increased in vitro after stimulation with both the hepatitis virus (DHV-1) and polyriboinosinic polyribocytidylic acid (poly(I:C)). However, the expression under these treatments varied in kidney and liver tissues; in the liver, duSP3 increased significantly at 36 h after the DHV-1 treatment and peaked at 72 h after poly(I:C) stimulation. These results suggested that SP3 may play a positive role in immune responses against viral infections in ducks.

Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury.

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.

Outcome of Antiviral Immunity in the Liver Is Shaped by the Level of Antigen Expressed in Infected Hepatocytes.

The liver bears unique immune properties that support both immune tolerance and immunity, but the mechanisms responsible for clearance versus persistence of virus-infected hepatocytes remain unclear. Here, we dissect the factors determining the outcome of antiviral immunity using recombinant adenoviruses that reflect the hepatropism and hepatrophism of hepatitis viruses. We generated replication-deficient adenoviruses with equimolar expression of ovalbumin, luciferase, and green fluorescent protein driven by a strong ubiquitous cytomegalovirus (CMV) promoter (Ad-CMV-GOL) or by 100-fold weaker, yet hepatocyte-specific, transthyretin (TTR) promoter (Ad-TTR-GOL). Using in vivo bioluminescence to quantitatively and dynamically image luciferase activity, we demonstrated that Ad-TTR-GOL infection always persists, whereas Ad-CMV-GOL infection is always cleared, independent of the number of infected hepatocytes. Failure to clear Ad-TTR-GOL infection involved mechanisms acting during initiation as well as execution of antigen-specific immunity. First, hepatocyte-restricted antigen expression led to delayed and curtailed T-cell expansion-10,000-fold after Ad-CMV-GOL versus 150-fold after Ad-TTR-GOL-infection. Second, CD8 T-cells primed toward antigens selectively expressed by hepatocytes showed high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression levels similar to that seen in chronic hepatitis B. Third, Ad-TTR-GOL but not Ad-CMV-GOL-infected hepatocytes escaped being killed by effector T-cells while still inducing high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression, indicating different thresholds of T-cell receptor signaling relevant for triggering effector functions compared with exhaustion. Conclusion: Our study identifies deficits in the generation of CD8 T-cell immunity toward hepatocyte-expressed antigens and escape of infected hepatocytes expressing low viral antigen levels from effector T-cell killing as independent factors promoting viral persistence. This highlights the importance of addressing both the restauration of CD8 T-cell dysfunction and overcoming local hurdles of effector T-cell function to eliminate virus-infected hepatocytes.

CD8+ lymphocytes but not B lymphocytes are required for protection against chronic hepatitis E virus infection in chickens.

Hepatitis E is an important global disease, causing outbreaks of acute hepatitis in many developing countries and sporadic cases in industrialized countries. Hepatitis E virus (HEV) infection typically causes self-limiting acute hepatitis but can also progress to chronic disease in immunocompromised individuals. The immune response necessary for the prevention of chronic infection is T cell-dependent; however, the arm of cellular immunity responsible for this protection is not currently known. To investigate the contribution of humoral immunity in control of HEV infection and prevention of chronicity, we experimentally infected 20 wild-type (WT) and 18 immunoglobulin knockout (JH-KO) chickens with a chicken strain of HEV (avian HEV). Four weeks postinfection (wpi) with avian HEV, JH-KO chickens were unable to elicit anti-HEV antibody but had statistically significantly lower liver lesion scores than the WT chickens. At 16 wpi, viral RNA in fecal material and liver, and severe liver lesions were undetectable in both groups. To determine the role of cytotoxic lymphocytes in the prevention of chronicity, we infected 20 WT and 20 cyclosporine and CD8+ antibody-treated chickens with the same strain of avian HEV. The CD8 + lymphocyte-depleted, HEV-infected chickens had higher incidences of prolonged fecal viral shedding and statistically significantly higher liver lesion scores than the untreated, HEV-infected birds at 16 wpi. The results indicate that CD8 + lymphocytes are required for viral clearance and reduction of liver lesions in HEV infection while antibodies are not necessary for viral clearance but may contribute to the development of liver lesions in acute HEV infection.

Macrophage scavenger receptor 1 contributes to pathogenesis of fulminant hepatitis via neutrophil-mediated complement activation.

BACKGROUND & AIMS: The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis. METHODS: Msr1-deficient (Msr1-/-) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and complement component 5a (C5a) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion. RESULTS: Our results showed that Msr1-/- mice were resistant to MHV-induced hepatitis. Treatment with the C5a receptor antagonist (C5aRa) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1-/- mice, suggesting that C5a-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection. CONCLUSIONS: Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH. LAY SUMMARY: Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH.

Targeting hepatic macrophages to treat liver diseases.

Our view on liver macrophages in the context of health and disease has been reformed by the recognition of a remarkable heterogeneity of phagocytes in the liver. Liver macrophages consist of ontogenically distinct populations termed Kupffer cells and monocyte-derived macrophages. Kupffer cells are self-renewing, resident and principally non-migratory phagocytes, serving as sentinels for liver homeostasis. Liver injury triggers Kupffer cell activation, leading to inflammatory cytokine and chemokine release. This fosters the infiltration of monocytes into the liver, which give rise to large numbers of inflammatory monocyte-derived macrophages. Liver macrophages are very plastic and adapt their phenotype according to signals derived from the hepatic microenvironment (e.g. danger signals, fatty acids, phagocytosis of cellular debris), which explains their manifold and even opposing functions during disease. These central functions include the perpetuation of inflammation and hepatocyte injury, activation of hepatic stellate cells with subsequent fibrogenesis, and support of tumor development by angiogenesis and T cell suppression. If liver injury ceases, specific molecular signals trigger hepatic macrophages to switch their phenotype towards reparative phagocytes that promote tissue repair and regression of fibrosis. Novel strategies to treat liver disease aim at targeting macrophages. These interventions modulate Kupffer cell activation (e.g. via gut-liver axis or inflammasome formation), monocyte recruitment (e.g. via inhibiting chemokine pathways like CCR2 or CCL2) or macrophage polarization and differentiation (e.g. by nanoparticles). Evidence from mouse models and early clinical studies in patients with non-alcoholic steatohepatitis and fibrosis support the notion that pathogenic macrophage subsets can be successfully translated into novel treatment options for patients with liver disease. LAY SUMMARY: Macrophages (Greek for "big eaters") are a frequent non-parenchymal cell type of the liver that ensures homeostasis, antimicrobial defense and proper metabolism. However, liver macrophages consist of different subtypes regarding their ontogeny (developmental origin), differentiation and function. Understanding this heterogeneity and the critical regulation of inflammation, fibrosis and cancer by macrophage subsets opens promising new options for treating liver diseases.

Acute Liver Damage Associated with Innate Immune Activation in a Small Nonhuman Primate Model of Hepacivirus Infection.

UNLABELLED: Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection. IMPORTANCE: Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study.

Crosstalk of liver immune cells and cell death mechanisms in different murine models of liver injury and its clinical relevance.

BACKGROUND: Liver inflammation or hepatitis is a result of pluripotent interactions of cell death molecules, cytokines, chemokines and the resident immune cells collectively called as microenvironment. The interplay of these inflammatory mediators and switching of immune responses during hepatotoxic, viral, drug-induced and immune cell-mediated hepatitis decide the fate of liver pathology. The present review aimed to describe the mechanisms of liver injury, its relevance to human liver pathology and insights for the future therapeutic interventions. DATA SOURCES: The data of mouse hepatic models and relevant human liver diseases presented in this review are systematically collected from PubMed, ScienceDirect and the Web of Science databases published in English. RESULTS: The hepatotoxic liver injury in mice induced by the metabolites of CCl4, acetaminophen or alcohol represent necrotic cell death with activation of cytochrome pathway, formation of reactive oxygen species (ROS) and mitochondrial damage. The Fas or TNF-alpha induced apoptotic liver injury was dependent on activation of caspases, release of cytochrome c and apoptosome formation. The ConA-hepatitis demonstrated the involvement of TRAIL-dependent necrotic/necroptotic cell death with activation of RIPK1/3. The alpha-GalCer-induced liver injury was mediated by TNF-alpha. The LPS-induced hepatitis involved TNF-alpha, Fas/FasL, and perforin/granzyme cell death pathways. The MHV3 or Poly(I:C) induced liver injury was mediated by natural killer cells and TNF-alpha signaling. The necrotic ischemia-reperfusion liver injury was mediated by hypoxia, ROS, and pro-inflammatory cytokines; however, necroptotic cell death was found in partial hepatectomy. The crucial role of immune cells and cell death mediators in viral hepatitis (HBV, HCV), drug-induced liver injury, non-alcoholic fatty liver disease and alcoholic liver disease in human were discussed. CONCLUSIONS: The mouse animal models of hepatitis provide a parallel approach for the study of human liver pathology. Blocking or stimulating the pathways associated with liver cell death could unveil the novel therapeutic strategies in the management of liver diseases.

Toll-like receptor-2 exacerbates murine acute viral hepatitis.

Viral replication in the liver is generally detected by cellular endosomal Toll-like receptors (TLRs) and cytosolic helicase sensors that trigger antiviral inflammatory responses. Recent evidence suggests that surface TLR2 may also contribute to viral detection through recognition of viral coat proteins but its role in the outcome of acute viral infection remains elusive. In this study, we examined in vivo the role of TLR2 in acute infections induced by the highly hepatotrophic mouse hepatitis virus (MHV) type 3 and weakly hepatotrophic MHV-A59 serotype. To address this, C57BL/6 (wild-type; WT) and TLR2 knockout (KO) groups of mice were intraperitoneally infected with MHV3 or MHV-A59. MHV3 infection provoked a fulminant hepatitis in WT mice, characterized by early mortality and high alanine and aspartate transaminase levels, histopathological lesions and viral replication whereas infection of TLR2 KO mice was markedly less severe. MHV-A59 provoked a comparable mild and subclinical hepatitis in WT and TLR2 KO mice. MHV3-induced fulminant hepatitis in WT mice correlated with higher hepatic expression of interferon-ß, interleukin-6, tumour necrosis factor-α, CXCL1, CCL2, CXCL10 and alarmin (interleukin-33) than in MHV-A59-infected WT mice and in MHV3-infected TLR2 KO mice. Intrahepatic recruited neutrophils, natural killer cells, natural killer T cells or macrophages rapidly decreased in MHV3-infected WT mice whereas they were sustained in MHV-A59-infected WT mice and MHV3-infected TLR2 KO. MHV3 in vitro infection of macrophagic cells induced rapid and higher viral replication and/or interleukin-6 induction in comparison to MHV-A59, and depended on viral activation of TLR2 and p38 mitogen-activated protein kinase. Taken together, these results support a new aggravating inflammatory role for TLR2 in MHV3-induced acute fulminant hepatitis.

Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs.

UNLABELLED: Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure. IMPORTANCE: More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

T-cell immunity and hepatitis C virus reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen in a chimpanzee.

UNLABELLED: Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T-cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)-free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct-acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T-cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T-cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape-prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection. CONCLUSIONS: The intrahepatic HCV-specific CD8+ T-cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T-cell responses and reduce the risk of a second persistent infection.

T-cell Ig and ITIM domain regulates natural killer cell activation in murine acute viral hepatitis.

UNLABELLED: Uncontrolled natural killer (NK) cell activation during the early response to acute viral infection can lead to severe immunopathology, and the mechanisms NK cells use to achieve self-tolerance in such contexts are currently unclear. Here, NK cells up-regulated a coinhibitory receptor, T-cell Ig and ITIM domain (TIGIT), during challenge with the viral double-stranded RNA (dsRNA) analog poly I:C. Blocking TIGIT by antibody treatment in vivo or a genetic deficiency in Tigit enhanced NK cell activation and aggravated liver injury in a poly I:C/D-GalN-induced model of acute fulminant hepatitis, suggesting that TIGIT is normally required for protecting against NK cell-mediated liver injury. Furthermore, adoptively transferring Tigit(-/-) NK cells into NK cell-deficient Nfil3(-/-) mice also resulted in elevated liver injury. Reconstituting Kupffer cell-depleted mice with poliovirus receptor (PVR/CD155, a TIGIT ligand)-silenced Kupffer cells led to aggravated liver injury in a TIGIT-dependent manner. Blocking TIGIT in an NK-Kupffer cell coculture in vitro enhanced NK cell activation and interferon-gamma (IFN-γ) production in a PVR-dependent manner. We also found that TIGIT was up-regulated selectively on NK cells and protected against liver injury in an acute adenovirus infection model in both an NK cell- and Kupffer cell-dependent manner. Knocking down PVR in Kupffer cells resulted in aggravated liver injury in response to adenovirus infection in a TIGIT-dependent manner. CONCLUSION: TIGIT negatively regulates NK-Kupffer cell crosstalk and alleviates liver injury in response to poly I:C/D-GalN challenge or acute adenovirus infection, suggesting a novel mechanism of NK cell self-tolerance in liver homeostasis during acute viral infection.

C5a/C5aR pathway is essential for the pathogenesis of murine viral fulminant hepatitis by way of potentiating Fgl2/fibroleukin expression.

UNLABELLED: Viral fulminant hepatitis (FH) remains a serious clinical problem with very high mortality. Lacking understanding of FH pathogenesis has in essence hindered efficient clinical treatment. Inferring from a correlation observed between the genetic differences in the complement component 5 (C5) and the susceptibility of mouse strains to murine hepatitis virus strain-3 (MHV-3) infections, we propose that excessive complement activation plays a critical role in the development of FH. We show that MHV-3 infection causes massive complement activation, along with a rapid increase in serum C5a levels and quick development of FH in susceptible strains. Mice deficient in the C5a receptor (C5aR) or the susceptible strains treated with C5aR antagonists (C5aRa) exhibit significant attenuation of the disease, accompanied by a remarkable reduction of hepatic fibrinogen-like protein 2 (Fgl2), a hallmark protein that causes necrosis of infected livers. In accordance, biopsy of FH patients shows a dramatic increase of Fgl2 expression, which correlates with C5aR up-regulation in the liver. In vitro C5a administration accelerates MHV-3-induced Fgl2 secretion by macrophages. Furthermore, inhibiting ERK1/2 and p38 efficiently blocks C5a-mediated Fgl2 production during viral infections. CONCLUSION: These data provide evidence that mouse susceptibility to MHV-3-induced FH may rely on C5a/C5aR interactions, for which ERK1/2 and p38 pathways participate in up-regulating Fgl2 expression. Inhibition of C5a/C5aR interactions is expected to be beneficial in the clinical treatment of FH patients.

The prevalence of duck hepatitis A virus types 1 and 3 on Korean duck farms.

This study reports the prevalence of duck hepatitis A virus (DHAV) types 1 and 3 on Korean duck farms. By RT-nested PCR assays specific for DHAV-1 or DHAV-3, DHAV-1 was detected in 9 of 157 liver samples (5.7 %) from 2 of 30 farms (6.7 %), and DHAV-3 was positive in 104 of 157 liver samples (66.2 %) from 23 of 30 farms (76.7 %). Dual infections with DHAV-1 and DHAV-3 were detected in 23 of 157 samples (14.6 %) from 5 of 30 farms (16.7 %). The data indicate that DHAV-3 infections are prevalent and that DHAV-1 reemerged in Korea, resulting in dual infections on several farms. Our data will help to establish a vaccination policy against DHAV-1 and DHAV-3 in Korea.

CD62L is critical for maturation and accumulation of murine hepatic NK cells in response to viral infection.

NK cells play critical roles in the first line of defense against viruses and other pathogens. However, the factors that control NK cell recruitment into local sites to exert effector functions during viral infection remain poorly understood. In this study, we found that murine NK cells in various organs could be divided into CD62L(-) and CD62L(+) subsets, the latter of which were less abundant in the liver and exhibited a relatively mature NK cell phenotype and a stronger cytotoxic function. Moreover, NK cells acquired CD62L expression after birth, and the frequency of CD62L(+) NK cells gradually increased during postnatal development. In models of polyinosinic-polycytidylic acid administration and adenovirus infection in vivo, CD62L(+) NK cell frequency and absolute numbers in the liver rapidly and markedly increased as a result of the augmented differentiation of CD62L(-) to CD62L(+) NK cells and recruitment of peripheral mature NK cells to the liver. However, blocking CD62L prior to administering viral stimuli in vivo abolished viral stimulation-induced NK cell accumulation and maturation in the liver. Collectively, these data suggest that CD62L marks a mature NK cell subset, as well as affects the magnitude of the local NK cell response to viral infection.

Characterization of human antiviral adaptive immune responses during hepatotropic virus infection in HLA-transgenic human immune system mice.

Humanized mice have emerged as a promising model to study human immunity in vivo. Although they are susceptible to many pathogens exhibiting an almost exclusive human tropism, human immune responses to infection remain functionally impaired. It has recently been demonstrated that the expression of HLA molecules improves human immunity to lymphotropic virus infections in humanized mice. However, little is known about the extent of functional human immune responses in nonlymphoid tissues, such as in the liver, and the role of HLA expression in this context. Therefore, we analyzed human antiviral immunity in humanized mice during a hepatotropic adenovirus infection. We compared immune responses of conventional humanized NOD SCID IL-2Rγ-deficient (NSG) mice to those of a novel NOD SCID IL-2Rγ-deficient strain transgenic for both HLA-A*0201 and a chimeric HLA-DR*0101 molecule. Using a firefly luciferase-expressing adenovirus and in vivo bioluminescence imaging, we demonstrate a human T cell-dependent partial clearance of adenovirus-infected cells from the liver of HLA-transgenic humanized mice. This correlated with liver infiltration and activation of T cells, as well as the detection of Ag-specific humoral and cellular immune responses. When infected with a hepatitis C virus NS3-expressing adenovirus, HLA-transgenic humanized mice mounted an HLA-A*0201-restricted hepatitis C virus NS3-specific CD8(+) T cell response. In conclusion, our study provides evidence for the generation of partial functional antiviral immune responses against a hepatotropic pathogen in humanized HLA-transgenic mice. The adenovirus reporter system used in our study may serve as simple in vivo method to evaluate future strategies for improving human intrahepatic immune responses in humanized mice.