Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

BACKGROUND: Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. METHODS: Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. RESULTS: 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to in vitro induction of cellular senescence, which had no effect. CONCLUSION: Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence

Telomere length, telomere number and nuclear area according to the subcellular location of HBcAg.

<p>Telomere length, telomere number and nuclear area according to the subcellular location of HBcAg.</p

Nuclear HBeAg staining in a representative patient with chronic HBV infection.

<p>HBeAg stains bright green, nuclei stain blue with DAPI and telomeres stain pink.</p

Q-FISH of liver tissue in chronic HBV infection.

<p>Hepatocytes with cytoplasm stained green with antibody to Hepar-1, nuclei stained blue with DAPI and telomeres shown in pink. Note vacuolated nuclei with peripheral telomeres as seen in senescence (13).</p

The mean number of telomeres in normal and HBV-infected liver tissue according to viral load.

<p>The mean number of telomeres in normal and HBV-infected liver tissue according to viral load.</p

(a) Nuclear and cytoplasmic HBcAg staining detected by Q-FISH in liver from a representative patient with chronic HBV infection.

<p>HBcAg stains bright green, nuclei stain blue with DAPI and telomeres stain pink. (b) Nuclear HBcAg staining detected by Q-FISH in liver from a representative patient with chronic HBV infection. HBcAg stains bright green, nuclei stain blue with DAPI and telomeres stain pink.</p

Hepatocytes are arrested in G1 in chronic HBV infection.

<p>Immunoperoxidase staining using cell cycle phase-specific antibodies in a representative case of chronic HBV infection A—E. Immunofluorescence showing the relation between HBcAg expression and Mcm-2 (F) and cyclin A (G). A: Mcm-2 identifies cells that have entered the cycle. B: Cyclin D1, expression is maximal during G1. C: Cyclin A is expressed maximally during S phase. D: Cytoplasmic expression of Cyclin B1 during G2, nuclear and cytoplasmic expression. E: Phosphorylated histone 3 protein expressed in mitosis. F: Immunofluorescence for Mcm-2 (green) and HBcAg (red), showing frequent co-localisation (yellow). G: Immunofluorescence for cyclin A (green) and HBcAg (red). Co-localisation was never seen.</p