Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (1), 3284

Intracellular interleukin-32γ Mediates Antiviral Activity of Cytokines Against Hepatitis B Virus

Affiliations

Intracellular interleukin-32γ Mediates Antiviral Activity of Cytokines Against Hepatitis B Virus

Doo Hyun Kim et al. Nat Commun.

Abstract

Cytokines are involved in early host defense against pathogen infections. In particular, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) have critical functions in non-cytopathic elimination of hepatitis B virus (HBV) in hepatocytes. However, the molecular mechanisms and mediator molecules are largely unknown. Here we show that interleukin-32 (IL-32) is induced by TNF and IFN-γ in hepatocytes, and inhibits the replication of HBV by acting intracellularly to suppress HBV transcription and replication. The gamma isoform of IL-32 (IL-32γ) inhibits viral enhancer activities by downregulating liver-enriched transcription factors. Our data are validated in both an in vivo HBV mouse model and primary human hepatocytes. This study thus suggests that IL-32γ functions as intracellular effector in hepatocytes for suppressing HBV replication to implicate a possible mechanism of non-cytopathic viral clearance.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Induction of IL-32 is involved in cytokine-mediated inhibition of HBV. a Huh7 cells were treated with the indicated concentrations of TNF-α and IFN-γ for 48 h, and the IL-32 level was determined by western blot analysis. Relative cell viability was measured by the XTT method. b Expression of IL-32γ induced by cytokines or transient transfection was observed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm. c Huh7 cells were transfected with the HBV 1.2 and GFP plasmids and treated with TNF-α and IFN-γ. HBV replication was determined by Southern blotting. Expression levels of IL-32 and GFP (transfection control) were determined by western blotting. d HBV 1.2 and siRNAs (20 nM) were co-transfected into Huh7 or HepG2 cells. Next day, cytokines were added with fresh medium. HBV replication and IL-32 expression were determined by Southern and western blotting, respectively. p < 0.05 by Student’s t-test. e Huh7 or HepG2 cells were transfected with the HBV 1.2 plasmid and treated with recombinant human IL-32γ (rhIL-32γ) for 3 days. HBV replication was determined by Southern blotting. f The levels of the IL-32 protein in culture medium and lysates of cytokine-treated Huh7 cells were determined by ELISA. The total amount of secreted (supernatant) or intracellular IL-32 in whole cells grown in a 12-well plate are shown. g The levels of the IL-32 protein in culture medium and lysates of cytokine-treated PHHs and differentiated HepaRG cells were determined by ELISA. Dashed bars represent the level of IL-32 obtained after transfection with pCAGGS (control) or IL-32 plasmids. Data (a, c, d, f, g) were obtained from three independent experiments (mean ± S.D.). p < 0.05 by Student’s t-test
Fig. 2
Fig. 2
Inhibition of HBV replication by IL-32γ through an intracellular event and the effect of IL-32γ on other viruses. a Vectors for HBV 1.2 and IL-32γ were co-transfected into Huh7 cells. At 48 h post-transfection, viral replication, protein expression, and cell viability were analyzed by Southern blotting, western blotting, and XTT assay, respectively. b The levels of the IL-32 protein in culture medium and lysates of IL-32γ-transfected Huh7 cells were measured by ELISA. c Schematic illustration of IL-32 isoforms. Shaded boxes represent the N-terminal and C-terminal extensions in IL-32γ. d Vectors for HBV 1.2, IL-32α, IL-32β, or IL-32γ were transfected in Huh7 cells as indicated. At 48 h post-transfection, viral replication and protein expression were analyzed by Southern and western blotting, respectively. Cells treated with cytokines were used as a control. eh IL-32 expression vector or empty vector was transfected into Huh7 cells. After 16 h, cells were infected with herpes simplex virus 1 (HSV-1_GFP) or influenza A virus (A/WSN/33_GFP) at 1 MOI. Cells were treated with the indicated concentrations cytokines (TNF-α and IFN-γ). Treatment with GCV (1 μg/mL) or IFNs (1000 U/mL IFN-α and 100 U/mL IFN-γ) was used as a positive control for HSV-1 and A/WSN/33, respectively. The GFP signals were monitored at 24 h post-infection. Magnification, ×100; scale bar, 100 μm. Data (a, b, d, g, h) were obtained from three independent experiments (mean ± S.D.)
Fig. 3
Fig. 3
IL-32γ suppresses HBV transcription by downregulating viral enhancer/core promoter activities. a, b At 72 h post-co-transfection of Huh7 cells with HBV 1.2 and IL-32γ vectors, the levels of HBV RNAs and proteins were determined by Northern and western blotting, respectively. The 18S RNA was used as a loading control. c Cartoon of various HBV enhancer reporter mutants used in this study. d Effect of IL-32γ on each enhancer reporter mutant in Huh7 cells. Relative luciferase activity of each enhancer clone was determined at 48 h post-co-transfection with either empty or IL-32γ vector. e HBV 1.2 and siRNAs (20 nM) were co-transfected into Huh7 cells. Next day, the cells were treated with cytokines (TNF-α and IFN-γ) in fresh medium. At 72 h post-transfection, the cells were harvested and the levels of HBV RNAs were determined by Northern blot analysis. f Huh7 cells were co-transfected with HBV 1.2 and empty or IL-32γ vector. Next day, the cells were treated with IFN-γ. At 72 h post-transfection, the cells were harvested and nuclear and cytoplasmic fractions were isolated. Total RNA was extracted and subjected to Northern blot analysis. g Analysis of HBV transcription rate by nuclear run-on assay using the nuclear fractions shown in f. The level of HBV transcription was normalized to that of GAPDH RNA. Data (d) were obtained from three independent experiments (mean ± S.D.)
Fig. 4
Fig. 4
IL-32γ downregulates HNF1α and HNF4α and reduces their binding to enhancers. a Map of liver-enriched transcription factor binding to HBV enhancers. bd Huh7 cells were co-transfected with the HBV 1.2 and IL-32γ vectors or were treated with cytokines after HBV 1.2 transfection. The levels of transcription factors were determined by semi-quantitative RT-PCR (b), real-time PCR (c), or western blot analysis (d. e, f) Effect of IL-32γ and cytokines on the expression of HNF1α and HNF4α was analyzed by confocal fluorescence microscopy. At 24 h after transfection or treatment, immunofluorescence assay was performed using indicated antibodies (magnification, ×400; scale bar, 50 μm.). g, h Chromatin immunoprecipitation (ChIP) assay. Control or IL-32γ vector was transfected into Huh7 cells and ChIP assay was performed using anti-HNF4α (g) or anti-HNF1α (h) antibody. The level of the IL-32γ protein was determined by western blotting. The regions for R1–R3 were shown in above diagram (a). i Electrophoretic mobility shift assay (EMSA). An aliquot of 2 µg nuclear extracts was used. A cold competitor (50-fold) was used as a negative control. The protein complex was confirmed by western blotting using anti-HNF4α antibody. Data (c) was obtained from three independent experiments (mean ± S.D.). p < 0.001, p < 0.01 by Student’s t-test
Fig. 5
Fig. 5
Involvement of ERK1/2 signaling in IL-32γ-mediated inhibition of HBV replication. a Effect of IL-32γ and cytokines on MAPK signaling in Huh7 cells. TNF-α and IFN-γ were added for 30 min before harvest. Activation of MAPK signaling pathways was determined by western blotting with the indicated antibodies. b Effect of ERK1/2 activation inhibitor (10 µM U0126) on the expression of HNFs. ce Effect of HNF4α (c), HNF1α (d), and U0126 (e) on IL-32γ-mediated inhibition of HBV replication. Viral replication and protein expression were determined by Southern and western blot analyses, respectively. U0126 was added for 48 h at a final concentration of 10 µM. Data (ce) were obtained from three independent experiments (mean ± S.D.). p < 0.001, p < 0.05 by Student’s t-test
Fig. 6
Fig. 6
IL-32γ suppresses HBV in a mouse model. a Six-week-old male mice (3 mice per group) were killed 4 days after hydrodynamic injection of the indicated plasmids: control vector, 20 µg; HBV 1.2, 20 µg; β-gal, 5 µg. HBV replication was analyzed by Southern blotting using 50 mg of liver tissues. The level of IL-32γ and β-actin were analyzed by western blotting. b The level of secreted HBsAg in serum was measured by ELISA. c Immunofluorescence analysis of the HBV core, β-gal, and IL-32γ proteins in mouse liver tissues prepared as in a. β-gal protein staining was used as a control for hydrodynamic injection. Magnification, ×200; scale bar, 50 μm. d Immunofluorescence analysis of the co-expression of RFP and GFP proteins in the same hepatocytes. Each plasmid (pEF1α-RFP and pEF1α-GFP; 10 µg) was hydrodynamically injected into mouse liver. Sections were prepared with a tissue cryotome. Green and red fluorescence signals were observed with an inverted fluorescence microscope at indicated magnification (scale bar, 50 μm). Data (a, b) were obtained from three independent experiments (mean ± S.D.). p < 0.001 by Student’s t-test
Fig. 7
Fig. 7
IL-32γ is involved in cytokine-mediated suppression of HBV in PHHs. a The experimental scheme. PHHs were infected with 1000 genome equivalents HBV per cell. At 4 days post-infection (dpi), the HBeAg level was determined by ELISA. b Effect of IL-32 knockdown on cytokine-induced downregulation of HNF1α and HNF4α. PHHs were infected with HBV and shIL-32 lentivirus as described in a. Cytokines were added for 2 days before harvest. c, d Effect of IL-32 knockdown on cytokine-induced inhibition of HBV replication and HBeAg secretion. Aliquots of cell lysates were used for real-time PCR and HBeAg ELISA. e A hypothetical model of IL-32γ-mediated suppression of HBV through downregulation of HNF1α and HNF4α expression. When hepatocytes are infected with HBV, immune cells secrete TNF-α and IFN-γ, which induce the expression of IL-32γ. IL-32γ activates ERK1/2, which in turn downregulates the expression of HNF1α and HNF4α. Finally, the binding of HNFs to the viral enhancers/core promoter is reduced, which consequently inhibits HBV transcription and replication. Data (c, d) were obtained from three independent experiments (mean ± S.D.). p < 0.001 by Student’s t-test

Similar articles

See all similar articles

Cited by 5 articles

References

    1. Guidotti LG, et al. Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proc. Natl Acad. Sci. USA. 1994;91:3764–3768. doi: 10.1073/pnas.91.9.3764. - DOI - PMC - PubMed
    1. Guidotti LG, et al. Viral clearance without destruction of infected cells during acute HBV infection. Science. 1999;284:825–829. doi: 10.1126/science.284.5415.825. - DOI - PubMed
    1. McClary H, Koch R, Chisari FV, Guidotti LG. Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J. Virol. 2000;74:2255–2264. doi: 10.1128/JVI.74.5.2255-2264.2000. - DOI - PMC - PubMed
    1. Ganem D, Prince AM. Hepatitis B virus infection—natural history and clinical consequences. N. Engl. J. Med. 2004;350:1118–1129. doi: 10.1056/NEJMra031087. - DOI - PubMed
    1. Pasquetto V, Wieland SF, Uprichard SL, Tripodi M, Chisari FV. Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures. J. Virol. 2002;76:5646–5653. doi: 10.1128/JVI.76.11.5646-5653.2002. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback