Ci Real

Nucleic acid-based polymers effective against hepatitis B Virus infection in patients don’t harbor immunostimulatory properties in primary isolated liver cells

Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2′-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.

Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen

The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection

The interferon‐stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15‐regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV‐infected (n = 54) and uninfected (n = 10) or HBV‐infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll‐like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type‐6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV‐infected patients. In contrast to interferon‐stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock‐down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV.

Hepatic expression of oncogenes Bmi1 and Dkk1 is up-regulated in hepatitis B virus surface antigen-transgenic mice and can be induced by treatment with HBV particles or lipopolysaccharides in vitro : Inflammation and tumor progression

Previous studies have shown that hepatocellular carcinoma (HCC) develops more frequently in hepatitis B virus surface antigen (HBsAg)‐transgenic mice (Alb/HBs) than in wild‐type (WT) mice. However, the mechanism of this HCC model has not been well documented. Toll‐like receptor 4 (Tlr4) signaling probably links innate immunity and HCC progression. This study was designed to investigate the role of innate immunity in hepatocarcinogenesis in Alb/HBs mice. Immunohistochemical analysis of liver specimens from Alb/HBs mice (16 per group) showed that the oncogenes Bmi1 (16/16, 100%) and Dkk1 (13/16, 81.25%) were highly expressed in Alb/HBs mice, whereas the other oncogenes evaluated were expressed in smaller percentages of mice (Afp, 9/16, 56.2%; Ctnnb1, 5/16, 31.3%; Epcam, 0/16; 0%). Comparable results were obtained by quantitative PCR analysis. Hepatic gene expression of Tlr2, Tlr4, Il6 and Tnf was additionally elevated in Alb/HBs mice. Stimulation of primary murine hepatocytes with cell culture‐derived HBV particles or LPS increased the expression of oncogenes (Bmi1, Dkk1) and inflammatory factors (Tnf, Il6, Tlr4). Proliferation and colony formation of hepatoma cells were enhanced by treatment with HBV and LPS and were impaired by the suppression of Bmi1 and Dkk1 by small interfering RNAs. Substantial induction of BMI1 and DKK1 was found in liver biopsy samples from patients with HBV‐related HCC but not in HCC samples without HBV infection background. These findings suggest that innate immunity may link inflammation and tumor progression during chronic HBV infection, involving the oncogenes BMI1 and DKK1.

398 VIRAL REPLICATION IN HBV-TRANSGENIC MICE LACKING THE HBsAg SIGNIFICANTLY INDUCES INTERFERON RESPONSES

Methods: Explanted and transplanted liver from 21 patients (5 with HBV-related, 6 with HBV/HDV-related and 10 with HCVrelated chronic liver disease) were analysed. All but one donor were HBsAg negative, 6 were anti-HBc positive, 14 were negative for all serum markers. From transplanted livers, biopsy specimens obtained before LT, in post-perfusion period and at end of LT were available. HBVDNA was tested in all liver tissue specimens by nested-PCR amplifications specific for 4 different HBV genomic regions. Total HBVDNA, HBV cccDNA and HDV RNA were quantified by specific real time-PCR approaches. Results: At the end of surgery, two of the 5 HBsAg-positive patients (both donors were anti-HBc positive), 1 of the 6 HBV/HDV coinfected patients (the donor was HBsAg-positive) and 2 of the 10 HCV-infected patients (one donor was anti-HBc positive and the other anti-HBc negative) showed the presence of quantifiable amounts of HBVDNA in the liver (range: 6x10-1x10 copies/cells). Sequencing analysis of the isolated HBV genomes showed that they were donor viral strains. HBV cccDNA could be detected and quantified (range: 2x10-2x10 copies/cell) in all the HBV-positive patients. None of the HBV/HDV-infected patients showed HDV RNA in the transplanted liver. Conclusions: 1. HBV genomes infecting the transplanted liver cause the reinfection of the recipients in the course of LT; 2. HDV does not re-infect the transplanted liver at the time of LT; 3. occult HBV infection (namely, presence of intrahepatic HBVDNA in HBsAg negative individuals) may be present in trasplanted liver and may persist during LT independently of the anti-HBc status of the donor.