HBV replication is significantly reduced by IL-6

Abstract

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-alpha/beta and IFN-gamma 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.

Figure 1

IL-6 suppresses HBV replication in a dose-dependent manner. (A) 1.3ES2 cells were treated 6 days after plating (when the cells had reached confluence) with various doses of IL-6 (0, 5, 10, 20, 40 ng/ml). The cell lysates were harvested after 4 days treatment with IL-6 and equal amounts of sample were analyzed by particle blot analysis using native agarose gel electrophoresis. HBV genome-containing nucleocapsids were detected by Southern blot analysis of the disrupted nucleocapsids, using an HBV-specific probe. Expression of β-actin was used as an internal control for sample loading. The signals were quantified by densitometry analysis and expressed as percentage of the control cells to indicate the inhibitory effect of IL-6. (B) Effect of IL-6 on growth curve. The cells were treated with IL-6, as described above. The cell number was determined by the trypan blue exclusion method.

Figure 2

IL-6 suppresses HBV replication in a time-dependent manner. Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 2 days (day2), 4 days (day4) or 6 days (day6). For the analysis of HBV replicative intermediates, equal amounts of total DNA were subjected to HindIII digestion and separated by electrophoresis. The integrated viral genome and viral replicative intermediates were then examined by Southern blot analysis using an HBV-specific probe. Bands corresponding to the integrated genome, the relaxed circular double-stranded DNA (RC) and single-stranded DNA (SS) are indicated individually. The intensity of the integrated HBV genome in each lane reflected the amounts of total DNA and was served as the internal control for sample loading. The signals were quantified by densitometry analysis and expressed as percentage of the respective control cells to indicate the inhibitory effect of IL-6.

Figure 3

IL-6 decreases the levels of HBV transcripts, core protein, and genome-containing nucleocapsids. Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 2 days (day2), 4 days (day4) or 6 days (day6). For RNA analysis (panel for RNA), total RNAs were extracted and equal amounts of samples were subjected to Northern blot analysis to reveal the expression profile of the HBV transcripts, namely those of 3.5-kb and 2.4-kb/2.1-kb respectively. The expression level of 18S rRNA was used as an internal control for sample loading. For Western blot analysis (panel for Proteins), cell lysates were harvested and equal amounts of samples were separated by SDS-polyacrylamide gel electrophoresis. The total core protein was subsequently detected by immunoblot analysis with antibodies against core protein. The expression level of β-actin was used as an internal control for sample loading. For particle blot analysis, equal amounts of cell lysates were assayed for viral capsid by native agarose gel electrophoresis. HBV genome-containing nucleocapsids were then detected by Southern blot analysis of the disrupted nucleocapsids using an HBV-specific probe (panel for Genome-containing nucleocapsids). The signals including 3.5-kb RNA, core proteins and genome-containing nucleocapsids were quantified by densitometry analysis and expressed as the average percentage of respective control cells from three independent experiments to indicate the inhibitory effect of IL-6. Results shown are representative of three independent experiments. *: The reduction in levels of HBV genome-containing nucleocapsids was compared with that of core proteins and found to be highly significant (P < 0.05 as monitored by Pearson χ² test).

Figure 4

IL-6 does not suppress the levels of secreted HBeAg and HBsAg. Confluent cells were treated for 6 days with various doses of IL-6 (0, 5, 10, 20, 40 ng/ml). (A) The level of extracellular viral genome-containing nucleocapsids in the culture medium was measured by real-time PCR method and normalized with total cell number. (B and C) The levels of secreted HBeAg and HBsAg in the culture medium were measured by ELISA. Results shown are representative of three independent experiments.

Figure 5

IL-6 does not disrupt the HBV nucleocapsids. To assess the IL-6 effect on the stability of HBV nucleocapsids, confluent cells were labeled with [³⁵S] methionine-cysteine protein labeling mix for 6 h and then chased 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. Total core protein and nucleocapsids were then immunoprecipitated using an anti-core antibody and separated by SDS-polyacrylamide gel electrophoresis. The labeled core protein was visualized by autoradiography. The signals were quantified by densitometry analysis and expressed as percentage of the respective control cells to indicate the IL-6 effect on capsid stability. Results shown are representative of three independent experiments.

Figure 6

The antiviral effect of IL-6 on HBV replication is not mediated through induction of IFNs. (A) Confluent 1.3ES2 cells were treated with or without 20 ng/ml of IL-6 for 2 day (day2) or 4 days (day4). Total RNA of each sample was extracted for the detection of IFN-α2b, IFN-β, IFN-γ and β-actin by RT-PCR analysis. Cells were transfected with 20 μg poly (I:C) for 6 h and served as the positive control for the induction of IFN-α2b and IFN-β simultaneously. Plasmid containing IFN-γ gene was served as the positive control for the detection of IFN-γ. (B) Confluent 1.3ES2 cells were treated with or without 20 ng/ml of IL-6 for 1 day (day1), 3 days (day3) or 6 days (day6). Total RNA of each sample was extracted for the detection of GBP-1 (target gene for IFN-γ), MxA (target gene for type I IFN) and GAPDH by RT-PCR analysis. Cells were treated with IFN-β or IFN-γ for 18 h and served as the positive control for the induction of GBP-1 and MxA genes. (C) Confluent 1.3ES2 cells were treated with various concentration of IL-6 or IFN-β for 4 days in the absence or presence of anti-IFN-β polyclonal antibody (400 U/ml). The level of HBV genome-containing nucleocapsids was measured as mentioned above. The expression level of β-actin was used as an internal control for sample loading.

Figure 7

IL-6 decreases the formation of HBV cccDNA. Confluent cells (day0) were treated with or without 20 ng/ml of IL-6 for 4 days (day4) or 6 days (day6) and subjected to Hirt extraction to prepare HBV cccDNA. The Hirt extract was thermally denatured at 85°C for 5 min, digested with EcoRI and analyzed by Southern blotting using an HBV-specific probe. Bands corresponding to HBV cccDNA and HBV single-stranded DNA (SS) are indicated. The signals were quantified by densitometry analysis and expressed as percentage of the control cells to indicate the IL-6 effect on the accumulation of HBV cccDNA.