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.

Long-term stimulation of Toll-like receptor 3 in primary human hepatocytes leads to sensitization for antiviral responses induced by poly I:C treatment.

Chronic hepatitis C infection is associated with increased expression of interferon-sensitive genes (ISGs) in the liver, which is, paradoxically, correlated with the nonresponse to interferon (IFN)-based therapies. In the present study PHHs were isolated from HCV-infected or uninfected patients and stimulated with the TLR1-9 ligands for 6-24 h. Expression of cytokines and ISGs was determined by ELISA and qRT-PCR. A comparative analysis was performed for TLR3 signalling, which was also correlated with single nucleotide polymorphisms (SNPs) related to HCV pathogenesis. TLR-activated PHHs produced pro-inflammatory and anti-inflammatory cytokines, whereas IFNs were exclusively induced by TLR3 stimulation. Here, IL-29 and IL-28A were significantly highly expressed than IFN-α and IFN-ß. TLR3-induced IFN response was enhanced in hepatocytes isolated from patients with HCV infection. This hyper-responsiveness could be mimicked in naïve PHHs consistently stimulated with low dose of poly I:C, but not Guardiquimod. The higher responsiveness in PHH isolated from HCV-infected patients could be partially explained by higher frequencies of unfavourable SNP alleles of different SNPs associated with HCV progression and treatment outcome. These data suggest that durable activation of TLR3 but not TLR7, by low doses of viral replicative intermediates, increases the sensitivity to viral invasion. These findings shed new light on the relevance of TLR3 in the pathogenesis of HCV and may provide a possible explanation for the increased ISG expression during chronic HCV infection, the so-called IFN paradox.

Toll-like receptor-mediated immune responses are attenuated in the presence of high levels of hepatitis B virus surface antigen.

It has been recently shown that Toll-like receptor (TLR) signalling in murine nonparenchymal liver cells (NPCs) is suppressed in the presence of Hepatitis B virus surface antigen (HBsAg). It is not clear, however, whether this is also relevant for the adaptive immune responses and how this effect is mediated. Peripheral blood mononuclear cells (PBMCs) from Hepatitis B virus (HBV) patients and controls were stimulated by TLR ligands in the absence or presence of autologous serum. Interestingly, TLR-mediated cytokine expression (Interleukin-6 and -10) as well as TLR3-induced interferon (IFN) expression in PBMCs of HBV patients was significantly higher than in the healthy volunteers, showing a negative correlation with the levels of HBsAg. In addition, TLR3-mediated IFN-γ production was inhibited in the presence of HBV-containing serum. To mechanistically analyse this observation, murine Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs) were stimulated with TLR3 ligands in the presence or absence of HBsAg. Mixed lymphocyte reactions were performed to study T-cell activation induced by TLR-stimulated NPCs. Gene expression of cytokines and TLR3 was analysed by quantitative rt-PCR, and activation of transcription factors was assessed by Western blot or reporter gene assays. TLR-induced expression of interferon γ, interferon sensitive genes and proinflammatory cytokines in murine KCs and LSECs was efficiently suppressed in the presence of HBsAg, whereas the expression of anti-inflammatory cytokines was enhanced. Activation of NFκB, IRF-3 and MAPKs in these liver cells was potently suppressed by HBsAg. T-cell activation mediated through TLR3-stimulated KCs or LSECs was suppressed by HBsAg which could be reverted by anti-IL-10 antibodies. These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to maintain a persistent infection.

MicroRNA-155 controls Toll-like receptor 3- and hepatitis C virus-induced immune responses in the liver.

The hepatitis C virus (HCV) establishes persistent infections despite strong activation of the innate immune system through TLR3 and other sensors. Therefore, we analysed regulatory mechanisms of TLR3-induced immune responses in nonparenchymal liver cells (NPCs). Effects of Interleukin-10 (IL-10), transforming growth factor beta (TGF-ß) and immunoregulatory miR-155 on poly I:C-activated murine (C57BL/6) Kupffer cells (KC) and sinusoidal endothelial cells (LSEC) were assessed in vitro. NPCs were assayed for inflammatory and antiviral cytokines and T-cell (Balb/c)-activating factors. Gene expression of miR-155, IL-10, TGF-ß and interferon sensitive genes (ISGs) in biopsies of patients with HCV was determined by qrt-PCR. TLR3-induced antiviral activity in murine NPCs was potently suppressed by IL-10 and TGF-ß which correlated with decreased TLR3 expression and inhibition of NF-κB and IRF-3 activation. T-cell activation, induced by TLR3-activated NPCs, was also suppressed by IL-10 and TGF-ß, which was associated with a down-regulation of CD80 and CD86. Pretreatment with IL-10 or TGF-ß suppressed TLR3-induced miR-155 expression, which itself positively regulated poly I:C-mediated immune responses, thus counteracting IL-10 or TGF-ß-induced immunosuppression. In addition, hepatic expression of miR-155 was elevated in chronically infected patients with HCV, was associated with an IL-28B SNP (rs12979860) and was inversely correlated with HCV serum load and ISG expression levels. As miR-155 is a key regulator of anti-inflammatory mechanisms that control innate and adaptive hepatic immune responses during HCV infection, miR-155 based therapies may represent a novel mechanism to control HCV in the future.