RESUMEN
UNLABELLED: Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well-characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV-infected cells. V protein-transduced HFLC supported enhanced (10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV-inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein-transduced cultures. CONCLUSION: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population.
Asunto(s)
Hepatocitos/virología , Proteínas Virales/biosíntesis , Hepacivirus/fisiología , Hepatitis C/etiología , Hepatitis C/virología , Hepatocitos/metabolismo , Humanos , Interferones/biosíntesis , Interleucinas/antagonistas & inhibidores , Interleucinas/biosíntesis , Lentivirus/genética , Hígado/embriología , Cultivo Primario de Células , Transducción Genética , Replicación Viral/efectos de los fármacosRESUMEN
Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.
Asunto(s)
Modelos Animales de Enfermedad , Hepacivirus/fisiología , Hepatitis C/genética , Hepatitis C/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Adenoviridae/genética , Adenoviridae/fisiología , Animales , Anticuerpos Bloqueadores/inmunología , Antígenos CD/genética , Antígenos CD/metabolismo , Células Cultivadas , Claudina-1 , Genotipo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatocitos/citología , Humanos , Inmunización Pasiva , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Receptores Virales/genética , Receptores Virales/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Tetraspanina 28 , Transfección , Tropismo ViralRESUMEN
Human hematolymphoid mice have become valuable tools for the study of human hematopoiesis and uniquely human pathogens in vivo. Recent improvements in xenorecipient strains allow for long-term reconstitution with a human immune system. However, certain hematopoietic lineages, for example, the myeloid lineage, are underrepresented, possibly because of the limited cross-reactivity of murine and human cytokines. Therefore, we created a nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-γ-null (NOD-SCID IL2Rγ(null)) mouse strain that expressed human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3, termed NSG-SGM3. Transplantation of CD34(+) human hematopoietic stem cells into NSG-SGM3 mice led to robust human hematopoietic reconstitution in blood, spleen, bone marrow, and liver. Human myeloid cell frequencies, specifically, myeloid dendritic cells, were elevated in the bone marrow of humanized NSG-SGM3 mice compared with nontransgenic NSG recipients. Most significant, however, was the increase in the CD4(+)FoxP3(+) regulatory T-cell population in all compartments analyzed. These CD4(+)FoxP3(+) regulatory T cells were functional, as evidenced by their ability to suppress T-cell proliferation. In conclusion, humanized NSG-SGM3 mice might serve as a useful model to study human regulatory T-cell development in vivo, but this unexpected lineage skewing also highlights the importance of adequate spatiotemporal expression of human cytokines for future xenorecipient strain development.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Interleucina-3/metabolismo , Factor de Células Madre/metabolismo , Linfocitos T Reguladores/citología , Animales , Linaje de la Célula , Proliferación Celular , Hematopoyesis/inmunología , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Fenotipo , Linfocitos T Reguladores/inmunología , Timo/citologíaRESUMEN
BACKGROUND & AIMS: Studies of hepatitis C virus (HCV) infection, immunopathogenesis, and resulting liver diseases have been hampered by the lack of a small animal model. We developed humanized mice with human immune system and liver tissues to improve the studies of hepatitis C virus pathogenesis and treatment. METHODS: To promote engraftment of human hepatocytes, we expressed a fusion protein of the FK506 binding protein (FKBP) and caspase 8 under control of the albumin promoter (AFC8), which induces liver cell death, in Balb/C Rag2(-/-) γC-null mice. Cotransplantation of human CD34(+) human hematopoietic stem cells (HSC) and hepatocyte progenitors into the transgenic mice led to efficient engraftment of human leukocytes and hepatocytes. We then infected these humanized mice (AFC8-hu HSC/Hep) with primary HCV isolates and studied HCV-induced immune responses and liver diseases. RESULTS: AFC8-hu HSC/Hep mice supported HCV infection in the liver and generated a human immune T-cell response against HCV. HCV infection induced liver inflammation, hepatitis, and fibrosis, which correlated with activation of stellate cells and expression of human fibrogenic genes. CONCLUSIONS: AFC8-hu HSC/Hep mice are a useful model of HCV infection, the immune response, and liver disease because they contain human immune system and liver cells. These mice become infected with HCV, generate a specific immune response against the virus, and develop liver diseases that include hepatitis and fibrosis. This model might also be used to develop therapeutics for HCV infection.