RESUMEN
BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. CONCLUSIONS: IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.
Asunto(s)
Hepatitis B/metabolismo , Interferón gamma/farmacología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Células Cultivadas , Técnicas de Cocultivo , Replicación del ADN/efectos de los fármacos , ADN Viral/efectos de los fármacos , ADN Viral/inmunología , Ensayo de Inmunoadsorción Enzimática , Células Hep G2/inmunología , Células Hep G2/metabolismo , Hepacivirus/metabolismo , Hepatitis B/fisiopatología , Hepatitis B Crónica/inmunología , Humanos , Linfocitos T/inmunología , Carga ViralRESUMEN
UNLABELLED: With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1alpha and HNF 4alpha, two transcription factors essential for HBV gene expression and replication. CONCLUSION: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.
Asunto(s)
Regulación de la Expresión Génica , Hepatitis B/inmunología , Interferones/fisiología , Interleucina-6/fisiología , Células Cultivadas , Citocinas/biosíntesis , Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Factor Nuclear 1-alfa del Hepatocito/antagonistas & inhibidores , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Transcripción Genética , Replicación ViralRESUMEN
Adenovirus type 12 (Ad12) propagation in hamster BHK21 cells is blocked prior to viral DNA replication. The amounts of Ad12 DNA in the nuclei or cytoplasm of hamster cells are about 2 orders of magnitude (2 h postinfection [p.i.]) and 4 to 5 orders of magnitude (48 h p.i.) lower than in permissive human cells. Cell line BHK21-hCAR is transgenic for and expresses the human coxsackie- and adenovirus receptor (hCAR) gene. Nuclear uptake of Ad12 DNA in BHK21-hCAR cells is markedly increased compared to that in naïve BHK21 cells. Ad12 elicits a cytopathic effect in BHK21-hCAR cells but not in BHK21 cells. Quantitative PCR or [(3)H]thymidine labeling followed by zone velocity sedimentation fails to detect Ad12 DNA replication in BHK21 or BHK21-hCAR cells. Newly assembled Ad12 virions cannot be detected. Thus, the block in Ad12 DNA replication in hamster cells is not released by enhanced nuclear import of Ad12 DNA.
Asunto(s)
Adenoviridae/fisiología , Núcleo Celular/virología , ADN Viral/metabolismo , Receptores Virales/biosíntesis , Internalización del Virus , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Núcleo Celular/metabolismo , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Cricetinae , Receptores Virales/genética , Replicación Viral/fisiologíaRESUMEN
BACKGROUND & AIMS: The human hepatitis B virus (HBV) is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection. METHODS: We performed gene expression profiling of primary human hepatocytes infected with HBV and proved the results in HBV-replicating cell lines and human liver tissue using real-time polymerase chain reaction and Western blotting. Activation of signal transducer and activator of transcription (STAT3) was examined in HBV-replicating human hepatocytes, HBV-replicating mice, and liver tissue from HBV-infected individuals using Western blotting, STAT3-luciferase reporter assay, and immunohistochemistry. The consequences of STAT3 activation on HBV infection and cell survival were studied by chemical inhibition of STAT3 phosphorylation and small interfering RNA-mediated knockdown of STAT3. RESULTS: Gene expression profiling of HBV-infected primary human hepatocytes detected no interferon response, while genes encoding for acute phase and antiapoptotic proteins were up-regulated. This gene regulation was confirmed in liver tissue samples of patients with chronic HBV infection and in HBV-related hepatocellular carcinoma. Pathway analysis revealed activation of STAT3 to be the major regulator. Interleukin-6-dependent and -independent activation of STAT3 was detected in HBV-replicating hepatocytes in cell culture and in vivo. Prevention of STAT3 activation by inhibition of Janus tyrosine kinases as well as small interfering RNA-mediated knockdown of STAT3-induced apoptosis and reduced HBV replication and gene expression. CONCLUSIONS: HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells. This very likely supports HBV-related carcinogenesis.
RESUMEN
The intramuscular (i.m.) injection of human adenovirus type 12 (Ad12) into newborn Syrian hamsters caused widespread dissemination of up to 15 tumors over the entire peritoneal cavity in 70-90% of the animals within 30-50 days. Subcutaneous (s.c.) injections led to local tumor formation only. Independent of location, tumor histology revealed Homer-Wright rosette-like structures typical for primitive neuroectodermal tumors (PNET). All tumor cells showed markers indicative of neuroectodermal and mesenchymal derivations. Each Ad12-induced tumor cell carried multiple copies of integrated Ad12 genomes at one chromosomal site which was different for each tumor. For Ad12 tumor induction in hamsters, the patterns of Ad12 viral and cellular gene expression were important and were affected by changes in DNA methylation, both in the integrated Ad12 DNA and the cellular genome. By applying the bisulfite protocol, the de novo DNA methylation in the integrated Ad12 genomes was determined. These patterns were complex, characterized by regional initiation and by excluding genome segments in the E1A and E1B promoters. In all tumors, the Ad12 segments E1A, E1B, E2A, parts of E3 and E4 were similarly transcribed, as shown by the RT-PCR and DNA microarray methods. Changes in the transcription of a large number of cellular genes was assessed by using mouse gene microarrays encompassing about 1980 different mouse genes with 87-96% homology to hamster genes. Similarities and differences existed in the transcription of cellular genes of different functional classes among the different Ad12-induced tumors. These alterations in cellular gene transcription may be an important parameter in the oncogenic transformation by Ad12.
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Adenovirus Humanos/patogenicidad , Neoplasias Experimentales/virología , Tumores Neuroectodérmicos/virología , Cavidad Peritoneal/patología , Proteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Animales Recién Nacidos , Cricetinae , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Inyecciones Intramusculares , Mesocricetus , Metilación , Ratones , Neoplasias Experimentales/patología , Tumores Neuroectodérmicos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Análisis de Secuencia de ADN , Transcripción Genética , Proteínas Virales/genética , Integración ViralRESUMEN
Studies of the influence of parasites on host fitness generally conclude that parasites have a strong negative effect on their hosts. In this study, we have investigated experimentally the role of Polymorphus minutus, an acanthocephalan parasite, on the salinity tolerance of the freshwater amphipod Gammarus roeseli, one of its intermediate hosts. Unexpectedly, P. minutus-infected gammarids were more tolerant to salinity stress than uninfected ones. The mean lethal salt concentrations for 50% mortality of hosts tested were 17.3 (infected) and 9.7 g/L (uninfected). The parasitic load (one or two parasites per host) did not affect the result. The size of hosts had no significant influence on the salinity tolerance of either infected or uninfected gammarids. The mobility of all types of gammarid decreased when the salinity exceeded 9.0 g/L, but there was no significant difference between infected and uninfected gammarids. We discuss the higher salinity tolerance of infected amphipods in relation to O(2) consumption and osmoregulation. Finally, we demonstrate that the salinity tolerance is enhanced in the parasitized amphipod but without a significant change in behavior or an osmoregulatory adjustment.
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Acantocéfalos/patogenicidad , Crustáceos/parasitología , Acantocéfalos/enzimología , Adenosina Trifosfatasas/metabolismo , Animales , Tolerancia a Medicamentos , Agua Dulce , Cloruro de Sodio/farmacologíaRESUMEN
BACKGROUND & AIMS: Induction of heme oxygenase-1 (HO-1) has been shown to be beneficial in immune-mediated liver damage. We now investigate the effects of HO-1 induction in models of human hepatitis B virus (HBV) infection. METHODS: Adenoviral transfer of an HBV 1.3 genome into wild-type mice was used as a model for acute hepatitis B. HBV transgenic animals were used as a model for chronic HBV infection. HBV replication was assessed by HBV viremia, antigenemia, and Southern blotting, liver damage was assessed by serum alanine aminotransferase activities and histopathology of liver sections. To investigate HO-1 effects on HBV replication at a molecular level, stably HBV-transfected hepatoma cells were used. HBV gene expression, protein stability, transcription, and replication were determined. HO-1 was induced by either cobalt-protoporphyrin-IX or over expressed by adenoviral gene transfer. RESULTS: In the acute hepatitis B model, liver injury was reduced significantly after HO-1 induction. In addition, HO-1 showed a pronounced antiviral effect, which was confirmed in stably HBV-transfected hepatoma cells and in persistently HBV replicating transgenic mice. We showed that HO-1 induction repressed HBV replication directly in hepatocytes at a posttranscriptional step by reducing stability of HBV core protein and thus blocking refill of nuclear HBV covalently closed circular (ccc)DNA. Small interfering RNA directed against HO-1 proved that this effect depended on the expression level of HO-1. CONCLUSIONS: Besides its hepatoprotective effect, HO-1 showed a pronounced antiviral activity in HBV infection. Therefore, induction of HO-1 might be a novel therapeutic option for inflammatory flares of hepatitis B.
Asunto(s)
Antivirales/farmacología , Hemo-Oxigenasa 1/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B/prevención & control , Hígado/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Protoporfirinas/farmacología , Adenoviridae/genética , Animales , Antivirales/uso terapéutico , Línea Celular Tumoral , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Inducción Enzimática/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Vectores Genéticos , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Hepatitis B/enzimología , Hepatitis B/genética , Hepatitis B/patología , Humanos , Hígado/enzimología , Hígado/patología , Hígado/virología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Protoporfirinas/uso terapéutico , Interferencia de ARN , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , ARN Viral/metabolismo , Transfección , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.