Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines

Abstract

We have previously shown that hepatitis B virus (HBV) replication is inhibited noncytopathically in the livers of transgenic mice following injection of HBV-specific cytotoxic T lymphocytes (CTLs) or infection with unrelated hepatotropic viruses, including lymphocytic choriomeningitis virus (LCMV) and adenovirus. These effects are mediated by gamma interferon (IFNgamma), tumor necrosis factor alpha (TNFalpha), and IFNalpha/beta. In the present study, we crossed HBV transgenic mice with mice genetically deficient for IFNgamma (IFNgammaKO), the TNFalpha receptor (TNFalphaRKO), or the IFNalpha/beta receptor (IFNalpha/betaRKO) in order to determine the relative contribution of each cytokine to the antiviral effects observed in each of these systems. Interestingly, we showed that HBV replicates in unmanipulated IFNgammaKO and IFNalpha/betaRKO mice at levels higher than those observed in control mice, implying that baseline levels of these cytokines control HBV replication in the absence of inflammation. We also showed that IFNgamma mediates most of the antiviral effect of the CTLs while IFNalpha/beta is primarily responsible for the early inhibitory effect of LCMV and adenovirus on HBV replication. In addition, we showed that the hepatic induction of IFNalpha/beta observed after injection of poly(I. C) is sufficient to inhibit HBV replication and that a similar antiviral effect is achieved by systemic administration of very high doses of IFNalpha. We also compared the relative sensitivity of LCMV and adenovirus to control by IFNgamma, TNFalpha, or IFNalpha/beta in these animals. Importantly, IFNalpha/betaRKO mice, and to a lesser extent IFNgammaKO mice, showed higher hepatic levels of LCMV RNA and adenovirus DNA and RNA than control mice, underscoring the importance of both interferons in controlling these other viral infections as well.

FIG. 1

High levels of HBV replication in livers of mice genetically deficient for IFNγ or the IFNα/β receptor. Six age (8 to 10 weeks)-, sex (male)-, and serum HBeAg-matched mice that were either heterozygous (+/−) or homozygous (−/−) for the indicated null mutation were sacrificed, and their livers were harvested. Following extraction, total hepatic RNA and DNA in each group were pooled and analyzed for HBV gene expression and replication by Northern and Southern blot analyses. The membranes were hybridized with ³²P-labeled HBV-, 2′,5′-OAS-, and GAPDH-specific DNA probes. Southern blot analysis was performed with 30 μg of total hepatic DNA. All DNA samples were RNAse treated before being subjected to gel electrophoresis. Bands corresponding to the integrated transgene and to relaxed-circular (RC) and single-stranded (SS) linear HBV DNA replicative forms are indicated. The integrated transgene can be used to normalize the amount of DNA bound to the membrane. The filter was hybridized with a ³²P-labeled HBV-specific DNA probe. Total hepatic RNA (10 μg) from the same mice was also analyzed by RNase protection assay for the expression of IFNγ and TNFα transcripts and for the expression of CD3, CD4, CD8, and F480, as indicated. The mRNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. The mean sALT activity, measured at the time of autopsy, is indicated (bottom) for each group and is expressed in units per liter.

FIG. 2

IFN-γ produced by HBsAg-specific CTLs is sufficient to inhibit HBV replication in the livers of HBV-transgenic mice. Age-, sex-, and serum HBeAg-matched transgenic mice from the same groups described in the legend to Fig. 1 were injected intravenously with 2.5 × 10⁷ CTLs and sacrificed 2 days later (d.2). Total hepatic RNA and DNA were analyzed for HBV gene expression and replication and for mRNAs for 2′,5′-OAS, IFNγ, TNFα, and T-cell (CD8, CD4, and CD3) and macrophage (F480) markers exactly as described in the legend to Fig. 1. Results were compared with those for livers pooled from 10 age-, sex-, and serum HBeAg-matched transgenic saline-injected controls (NaCl). The mean sALT activity, measured at the time of autopsy, is indicated (bottom) for each group and is expressed in units per liter. RC and SS, relaxed-circular and single-stranded linear HBV DNA replicative forms, respectively.

FIG. 3

IFN-dependent inhibition of HBV and LCMV replication. Age-, sex-, and serum HBeAg-matched transgenic mice from the same groups of mice described in the legend to Fig. 1 were infected with LCMV WE clone 2.2 (2 × 10⁶ PFU/mouse) and sacrificed either 24 h (d1) or 7 days (d7) after infection. Total hepatic RNA and DNA were analyzed for HBV gene expression and replication and for mRNAs for 2′,5′-OAS, IFNγ, TNFα, and T-cell (CD8, CD4, and CD3) and macrophage (F480) markers exactly as described in the legend to Fig. 1. Northern blot membranes were also hybridized with a ³²P-labeled LCMV-specific DNA probe (top). Animals indicated as wild type (wt) were heterozygous for the IFNα/β receptor null mutation. Results were compared with those for livers pooled from 10 age-, sex-, and serum HBeAg-matched transgenic saline-injected controls (NaCl) that were sacrificed 1 day after NaCl injection. The mean sALT activity, measured at the time of autopsy, is indicated (bottom) for each group and is expressed in units per liter. Int., integrated; RC and SS, relaxed-circular and single-stranded linear HBV DNA replicative forms, respectively.

FIG. 4

IFN-dependent inhibition of HBV replication and of adenovirus entry and gene expression. Age-, sex-, and serum HBeAg-matched transgenic mice from the groups described in the legend to Fig. 1 were infected with a dose (1.5 × 10⁹ PFU/mouse) of a replication-deficient, lacZ-expressing adenovirus (Ad.CblacZ) that rapidly infects all of the hepatocytes. At 24 h (d1) or 7 days (d7) postinfection, the mice were sacrificed and total hepatic RNA and DNA were analyzed for HBV gene expression and replication and for mRNAs for 2′,5′-OAS, IFNγ, TNFα, and T-cell (CD8, CD4, and CD3) and macrophage (F480) markers exactly as described in the legend to Fig. 1. Northern and Southern blot membranes were also hybridized with a ³²P-labeled lacZ-specific DNA probe (top). Animals indicated as wild type (wt) were heterozygous for the IFNα/β receptor null mutation. Results were compared with those for livers pooled from 10 age-, sex-, and serum HBeAg-matched transgenic saline-injected controls (NaCl) that were sacrificed 1 day after NaCl injection. The mean sALT activity, measured at the time of autopsy, is indicated (bottom) for each group and is expressed in units per liter. Int., integrated; RC and SS, relaxed-circular and single-stranded linear HBV DNA replicative forms.

FIG. 5

Time course of adenovirus infection in IFNα/βRKO mice. Age-, sex-, and serum HBeAg-matched transgenic mice that were heterozygous (+/−) or homozygous (−/−) for the IFNα/β receptor null mutation were infected intravenously with Ad.CBlacZ (1.5 × 10⁹ PFU/mouse) and sacrificed at the indicated time points (day 1 [d. 1] through day 5 [d. 5] plus day 7 [d. 7]). Results were compared with those for livers pooled from 10 uninjected age-, sex-, and serum HBeAg-matched transgenic controls (d.0). Total hepatic RNA and DNA were analyzed for HBV gene expression and replication and for mRNAs for 2′,5′-OAS, IFNγ, TNFα, and T-cell (CD8, CD4, and CD3) and macrophage (F480) markers exactly as described in the legend to Fig. 1. Northern and Southern blot membranes were also hybridized with a ³²P-labeled lacZ-specific DNA probe (top). The mean sALT activity, measured at the time of autopsy, is indicated (bottom) for each group and is expressed in units per liter. RC and SS, relaxed-circular and single-stranded HBV DNA replicative forms.

FIG. 6

IFNα/β mediates the antiviral effect of poly(I · C). Age-, sex-, and serum HBeAg-matched transgenic mice from the same groups of mice described in the legend to Fig. 1 were sacrificed 24 h after receiving single injections of poly(I · C) (200 μg/mouse). Total hepatic RNA and DNA were analyzed for HBV gene expression and replication and for mRNAs for 2′,5′-OAS, IFNγ, TNFα, and T-cell (CD8, CD4, and CD3) and macrophage (F480) markers exactly as described in the legend to Fig. 1. Animals indicated as wild type (wt) were heterozygous for the IFNα/β receptor null mutation. Results were compared with those observed in livers pooled from 10 age-, sex-, and serum HBeAg-matched transgenic saline-injected controls (NaCl). The mean sALT activity, measured at the time of autopsy, is indicated (bottom) for each group and is expressed in units per liter. RC and SS, relaxed-circular and single-stranded HBV DNA replicative forms, respectively.

FIG. 7

High doses of IFNα administered systemically inhibit HBV replication. Age-, sex-, and serum HBeAg-matched transgenic mice (lineage 1.3.46) were injected intravenously with various doses of IFNα, and their livers were harvested 24 h after injection for Southern analysis of HBV DNA replicative forms and Northern analysis of 2′,5′-OAS RNA exactly as described in the legend to Fig. 1. Results were compared with those for livers pooled from 10 age-, sex-, and serum HBeAg-matched transgenic saline-injected controls (NaCl). Results were also compared with those for livers harvested 24 h after injection with poly(I · C) (200 μg/mouse) or infection with LCMV (2 × 10⁶ PFU/mouse) or adenovirus (1.5 × 10⁹ PFU/mouse). RC and SS, relaxed-circular and single-stranded HBV DNA replicative forms, respectively.