HBV replication is significantly reduced by IL-6
© Kuo et al; licensee BioMed Central Ltd. 2009
Received: 16 February 2009
Accepted: 20 April 2009
Published: 20 April 2009
Interleukin-6 (IL-6) is a pleiotropic cytokine with pivotal functions in the regulation of the biological responses of several target cells including hepatocytes. The level of serum IL-6 has been reported to be elevated in patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma and represents the best marker of HBV-related clinical progression as compared with several other cytokines. In this study, we found that IL-6 was able to effectively suppress hepatitis B virus (HBV) replication and prevent the accumulation of HBV covalently closed circular DNA (cccDNA) in a human hepatoma cell line. We also demonstrated that the suppression of HBV replication by IL-6 requires concurrently a moderate reduction of viral transcripts/core proteins and a marked decrease in viral genome-containing nucleocapsids. Studies on the stability of existing viral capsids suggest that the IL-6 effect on the reduction of genome-containing nucleocapsids is mediated through the prevention of the formation of genome-containing nucleocapsids, which is similar to the effect of interferons. However, IFN-α/β and IFN-γ did not participate in the IL-6-induced suppression of HBV replication. Taken together, our results will provide important information to better understand the role of IL-6 in the course of HBV infection.
Hepatitis B virus (HBV) is a hepatotropic, non-cytopathic DNA virus (3.2 kb partially double-stranded DNA) that causes acute and chronic hepatitis. More than 350 million people worldwide suffer from chronic hepatitis B (CHB) infection, which is associated with a high risk of developing cirrhosis and hepatocellular carcinoma [1, 2]. The interactions between HBV replication and immune responses against HBV infection play an important role in determining the outcome of virus infection [3, 4]. Previous studies using chimpanzees and transgenic mice models have indicated that HBV clearance occurs prior to the destruction of infected cells [5, 6]. These results suggest that cytokines are likely to be involved in both the regulation of the immune responses and the direct inhibition of HBV replication. Several cytokines have recently been shown to effectively suppress HBV replication in a noncytopathic manner in HBV transgenic mice and in a cell culture system. Interleukin-12 (IL-12), IL-18 and intrahepatic induction of alpha/beta interferon (IFN-α/β) are able to effectively inhibit HBV replication in the liver of transgenic mice [7–9]. IFN-α/β, gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) suppress HBV replication in immortalized murine hepatocytes and human hepatoma cells by preventing the formation of viral capsids or disrupting capsid integrity [10, 11]. Furthermore, IL-4 and transforming growth factor beta-1 (TGF-β1) have been demonstrated to suppress HBV replication in hepatoma cells through the transcriptional regulation of HBV RNA [12, 13]. These studies suggest that inflammatory cytokines play an important role in the antiviral response against HBV infection.
IL-6 is one of the major inflammatory cytokines, and in several types of target cells it affects a variety of biological responses including changes in cell differentiation, growth, apoptosis and the induction of acute-phase responses [14, 15]. In response to liver injury, IL-6 expression is induced in various cell types including endothelial cells, hepatocytes and Kupffer cells . IL-6 plays an important role in promoting hepatic survival by stimulating liver regeneration, and protects the liver from damage caused by immune responses, alcohol and viral infection. The level of serum IL-6 has been reported to be elevated in patients with CHB, cirrhosis and hepatocellular carcinoma, relative to normal subjects [17–19]. IL-6 activity has been shown to be significantly enhanced during acute exacerbation of CHB, which is accompanied by clearance of HBV e antigen (HBeAg). Interestingly, the level of serum IL-6 were reported to be inversely correlated to the transaminase level in patients and represents the best marker of HBV-related clinical progression as compared with IL-10, IL-12 and IFN-γ . Recent experiments have also indicated that gender may influence MyD88-dependent IL-6 production by Kupffer cells, and this may contribute to gender disparity in hepatocarcinogenesis . Using a human-mouse radiation chimera model, Galun et al. found that IL-6 could facilitate HBV infection and suggested that IL-6 might be a potential mediator for HBV entrance into hepatocytes . However, the effect and the mechanisms of action of IL-6 on HBV replication have not been studied in detail.
In this study, we found that IL-6 can effectively suppress HBV replication in an HBV-producing cell line, 1.3ES2 . The suppression of HBV replication requires a moderate reduction of viral transcripts/core proteins and a marked decrease in the formation of HBV genome-containing nucleocapsids. Our studies provide important information to reveal the role of IL-6 in the course of HBV infection.
Materials and methods
HepG2 and 1.3ES2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, as described previously . After culture for 4 days, the confluent 1.3ES2 cells were serum-deprived for 2 days and treated with human IL-6 (R&D Systems Inc., Minneapolis, USA) for various periods to assess the antiviral effect of IL-6. The culture medium was refreshed every 2 days during the experiments. For the neutralization experiment, sheep polyclonal anti-IFN-β antibody (PBL Biomedical Laboratories, New Jersey, USA) was added to the culture medium to block the endogenous IFN-β activity. Human IFN-β (PBL Biomedical Laboratories, New Jersey, USA) was added as a control for the neutralization experiment.
Southern blot analysis
Total DNA (20 μg) was digested with HindIII and separated on 0.7% agarose gels. The gels were treated as described previously  and the DNA samples were transferred onto nylon membranes (Amersham, Freiburg, Germany). After ultraviolet crosslinking and prehybridization, the membranes were hybridized with an HBV-specific probe generated using a random-primed labeling kit (Amersham, Freiburg, Germany).
Analysis of intracellular HBV genome-containing nucleocapsid
Analysis of intracellular HBV genome-containing nucleocapsids was performed as described previously [13, 24]. Briefly, cell lysates were separated using a 0.8% native agarose gel and transferred onto nylon membranes. Capsid-associated nucleic acids were released from the nucleocapsids in situ by denaturing with 0.2 N NaOH/1.5 M NaCl followed by neutralization with 0.2 N Tris-HCl/1.5 M NaCl. The membranes were then hybridized with an HBV-specific probe.
Northern blot analysis
Total RNA was isolated using TRIzol solution (Invitrogen, California, USA) according to the manufacturer's instructions, and 15 μg of isolated RNA was separated using a 1.2% formaldehyde-agarose gel and transferred onto nylon membrane. The membranes were then hybridized with an HBV-specific probe.
Western blot analysis
Total protein (100 μg) was separated using a 15% SDS-polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes (Millipore, Massachusette, USA). The membranes were incubated with anti-core antibody (DakoCytomation, Glostrup, Denmark) or anti-actin antibody (Sigma, St. Louis, MO). The immunoblot signals were examined using enhanced chemiluminescence reagent (Millipore, Massachusette, USA).
Analysis of extracellular HBV nucleic acids
The extracellular pool of HBV nucleic acids was extracted from cell culture medium using a High Pure Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany). The level of viral genomes was then measured by real-time PCR method using a LightCycler® TaqMan® Master Kit (Roche Applied Science, Mannheim, Germany) with a pair of HBV-specific primer (forward primer: 5'-GCTCCAGTTCAGGAACAGTAAAC-3' and reverse primer: 5'-AATCCTCGAGAAGATTGACGAT-3') and the Taqman probe #34. The thermo-cycling parameters were 94°C for 10 min; 50 cycles of 94°C for 10 sec, 60°C for 30 sec, and 72°C for 1 min; and 40°C for 30 sec.
Detection of HBsAg and HBeAg
The levels of HBV surface antigen (HBsAg) and HBeAg in the culture medium were assessed using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer's protocol (Evernew Biotech Inc, Taipei, Taiwan). Each experiment was performed in triplicate and independently repeated three times.
Confluent 1.3ES2 cells were labeled with [35S] methionine-cysteine protein labeling mix (Perkin-Elmer, Connecticut, USA) for 6 h and then chased by replacing with unlabeled medium in the absence or presence of IL-6 (20 ng/ml or 40 ng/ml) for 24 h or 48 h. Cell lysates were harvested and the nucleocapsids were separated from free core protein by centrifugation through a Centricon-100 filter with a retention cutoff of 100 kDa (Millipore, Massachusette, USA). Total core proteins and nucleocapsids were then immunoprecipitated using an anti-core antibody and separated by 12% SDS-polyacrylamide gel electrophoresis. The signal of labeled core protein was visualized by autoradiography.
HBV cccDNAs were isolated as previously described . After complete lysis of the cells with Hirt solution, 750 μl of 5 N NaCl was added to the cell lysate and the mixture incubated on ice overnight. The supernatant was collected after centrifugation and extracted with phenol/chloroform. HBV cccDNA was then precipitated by ethanol and examined by Southern blot analysis.
Primer pairs for RT-PCR
The cDNA templates were amplified by the following primer pairs: (1) IFN-α2b forward primer: 5'-TCCTAGACAAATTCTACACTGAAC-3', IFN-α2b reverse primer: 5'-GCTCTGACAACCTCCC-3'. (2) IFN-β forward primer: 5'-AAACTCATGAGCAGTCTGCA-3', IFN-β reverse primer: 5'-AGGAGATCTTCAGTTTCGGAGG-3'. (3) IFN-γ forward primer: 5'-TCAGCTCTGCATCGTTTTGG-3', IFN-γ reverse primer: 5'-GTTCCATTATCCGCTACATCTGAA-3'. (4) β-actin forward primer: 5'-TGAACTGGCTGACTGCTGTG-3', β-actin reverse primer: 5'-CATCCTTGGCCTCAGCATAG-3'. (5) GBP-1 forward primer: 5'-ACAAGGGAACAGCCTGGACATGG-3', GBP-1 reverse primer: 5'-GCCCACAATTGCCACCACCA-3'. (6) MxA forward primer: 5'-ACCACAGAGGCTCTCAGCAT-3', MxA reverse primer: 5'-CTCAGCTGGTCCTGGATCTC-3'. (7) GAPDH forward primer: 5'-GCTGAGAACGGGAAGC-3', GAPDH reverse primer: 5'-GGTGAAGACGCCAGTG-3'.
IL-6 suppressed HBV replication in the HBV-producing cell line 1.3ES2
IL-6 suppressed HBV replication through moderate reduction of viral transcripts and marked decrease in the genome-containing nucleocapsid levels
IL-6 did not disrupt the HBV nucleocapsids
The antiviral effect of IL-6 was not mediated through induction of interferons
IL-6 prevents the accumulation of HBV cccDNA
In this study, we found that IL-6 suppressed HBV replication in an HBV-replicating cell line. The inhibitory effect was not a result of cell apoptosis since IL-6 treatment increased cell proliferation. We also demonstrated that IL-6 exerted its inhibitory effect through a combination of two different mechanisms which included a moderate reduction of HBV transcripts/core protein coordinately and a marked decrease in the level of HBV genome-containing nucleocapsids. Furthermore, IL-6 also prevents the accumulation of HBV cccDNA. IL-6 has recently been found to suppress HCV RNA replication and protein expression in liver cells . This result coincides with our findings and supports the suggestion that IL-6 is an antiviral cytokine during the progression of chronic hepatitis.
The pathogenesis of HBV-induced liver diseases involves complicated mechanisms related to viral replication and the body's immune responses against HBV infection , including HBV-specific cell-mediated immunity (CMI) and inflammatory cytokines. The CMI responses are essential for the resolution of HBV infection, but several studies have indicated that cytokines are involved in the noncytopathic suppression of virus replication . Of significance to the present study is that the levels of many cytokines (including IL-18, IFNs, TNF-α, IL-4 and TGF-β1) are elevated during hepatitis progression, and have been demonstrated to exhibit antiviral activity in both transgenic mice and cell culture systems [8–10, 12, 13, 24]. Interestingly, the levels of serum IL-6 were reported to represent the best marker of HBV-related clinical progression as compared with IL-10, IL-12 and IFN-γ . The inhibitory mechanisms of the cytokines can be generally divided into two major classes. The first class, which includes IL-4 and TGF-β1, suppresses viral gene expression and subsequently blocks HBV replication. IL-4 suppresses HBV replication through down-regulation of C/EBPα, while TGF-β1 reduces HBV replication through transcriptional inhibition of pregenomic RNA. Cytokines of the second class exert their antiviral effect by either destabilizing viral genome-containing capsids or preventing their assembly. Types I and II IFN activate hepatocellular processes, which prevent the formation of viral genome-containing capsids and subsequently inhibit HBV replication , whereas the inhibitory effect of TNF-α involves the destabilization of viral nucleocapsids . In this study, we demonstrated that IL-6 exerted its antiviral effect through a moderate reduction of viral transcripts similar to IL-4 and TGF-β1 and a more dramatic reduction of viral genome-containing nucleocapsids. These results indicate that the inhibitory mechanism of IL-6 in HBV replication is a combination of both classes of cytokines.
The IL-6 effect on the reduction of HBV genome-containing nucleocapsids may be similar to interferons and mediated through the prevention of the formation of genome-containing nucleocapsids since there is no evidence of the disruption of capsid integrity after IL-6 treatment. The expression levels of IFNs and IFN target genes were not induced during the IL-6 treatment. These results indicate that IL-6 induced suppression of HBV replication is not mediated through the induction of IFNs.
The formation of HBV genome-containing nucleocapsids involves very complicated processes. These processes include dimerization of core protein and multimer association, binding of RNA polymerase to ε structure of pregenomic RNA (pgRNA) and packaging of pgRNA, viral polymerase and cellular factors such as heat shock proteins into capsids [28, 29]. The IL-6 effect on the prevention of the formation of viral genome-containing nucleocapsids could occur at any step of these processes. Similar to the inhibitory effect of IFNs, the exact mechanism involved in the antiviral effect of IL-6 remains to be further investigated.
This work was supported by an intramural research grand PA-096-PP-08 from the National Health Research Institutes, Taiwan. We thank Dr. Ralph Kirby for English editing the manuscript.
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