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

Abstract

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-β. 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.

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 hepatic induction of PSMA6 during chronic viral infection and its correlation with ISG expression suggest that PSMA6 expression is likely to be controlled by the innate immune system. Functional TLR signalling in human PHH has recently been demonstrated . Here, we determined the expression of PSMA6 and ISG15 in untreated and stimulated PHH at the RNA level.…”

“…Toll‐like receptor agonists were purchased from InvivoGen (Toulouse, France) and used as previously described . Recombinant human IFN‐ α was purchased from Sigma (Heidenheim, Germany).…”

“…Primary human hepatocytes (PHH) were isolated from resected or explanted liver tissue as previously described . The study was approved by the local ethics committee.…”

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

“…The lack of any observable type 1 interferon response (INFA4 and INFB1) with REP 2055, REP 2139 or REP 2165 in any cell type suggests that the cytoplasmic transit of these NAPs does not stimulate cytoplasmic or endosomal PAMP receptors. Antiviral activity of TLR-stimulated parenchymal and non-parenchymal human liver cells seems to be restricted to TLR3 in vitro 3435. Cytosolic pathogen recognition receptors like RIG-I, STING and cGAS can be activated in human hepatocytes as well, leading to antiviral signaling3637.…”

Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy

“…The purities of PHH (95.5 ± 1.7%), KC (94.5 ± 1.2%), LSEC (97.8 ± 1.1%), and HSC (97.1 ± 1.5%) could be reached as determined by immunofluorescence staining with specific markers (21). These cells were stimulated using the Human toll-like receptor (TLR) 1–9 Agonist Kit (Invivogen, Toulouse, France) at recommended concentrations for 6 h. Cells were then collected for RNA extraction and CXCL13 gene expression analysis by real-time RT-PCR was performed using commercially available primer set (Qiagen, Hilden, Germany) and normalized to β-actin as previously described (22). …”