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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-28 A 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. [ABSTRACT FROM AUTHOR]

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-β) and immunoregulatory mi R-155 on poly I: C-activated murine ( C57 BL/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 mi R-155, IL-10, TGF-β 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-β which correlated with decreased TLR3 expression and inhibition of NF-κ B and IRF-3 activation. T-cell activation, induced by TLR3-activated NPCs, was also suppressed by IL-10 and TGF-β, which was associated with a down-regulation of CD80 and CD86. Pretreatment with IL-10 or TGF-β suppressed TLR3-induced mi R-155 expression, which itself positively regulated poly I: C-mediated immune responses, thus counteracting IL-10 or TGF-β-induced immunosuppression. In addition, hepatic expression of mi R-155 was elevated in chronically infected patients with HCV, was associated with an IL-28 B SNP (rs12979860) and was inversely correlated with HCV serum load and ISG expression levels. As mi R-155 is a key regulator of anti-inflammatory mechanisms that control innate and adaptive hepatic immune responses during HCV infection, mi R-155 based therapies may represent a novel mechanism to control HCV in the future. [ABSTRACT FROM AUTHOR]

Background & Aims: Hepatitis B surface antigen (HBsAg) drives hepatocarcinogenesis. Factors and mechanisms involved in this progression remain poorly defined, hindering the development of effective therapeutic strategies. Therefore, the mechanisms involved in the HBsAg-induced transformation of normal liver into hepatocellular carcinoma (HCC) were investigated., Methods: Hemizygous Tg(Alb1HBV)44Bri/J mice were examined for HBsAg-induced carcinogenic events. Gene set-enrichment analysis identified significant signatures in HBsAg-transgenic mice that correlated with endoplasmic reticulum (ER) stress, unfolded protein response, autophagy and proliferation. These events were investigated by western blotting, immunohistochemical and immunocytochemical staining in 2-, 8- and 12-month-old HBsAg-transgenic mice. The results were verified in HBsAg-overexpressing Hepa1-6 cells and validated in human HBV-related HCC samples., Results: Increased BiP expression in HBsAg-transgenic mice indicated induction of the unfolded protein response. In addition, early-phase autophagy was enhanced (increased BECN1 and LC3B) and late-phase autophagy blocked (increased p62) in HBsAg-transgenic mice. Finally, HBsAg altered lysosomal acidification via ATF4- and ATF6-mediated downregulation of lysosome-associated membrane protein 2 (LAMP2) expression. In patients, HBV-related HCC and adjacent tissues showed increased BiP, p62 and downregulated LAMP2 compared to uninfected controls. In vitro, the use of ER stress inhibitors reversed the HBsAg-related suppression of LAMP2. Furthermore, HBsAg promoted hepatocellular proliferation as indicated by Ki67, cleaved caspase-3 and AFP staining in paraffin-embedded liver sections from HBsAg-transgenic mice. These results were further verified by colony formation assays in HBsAg-expressing Hepa1-6 cells. Interestingly, inhibition of ER stress in HBsAg-overexpressing Hepa1-6 cells suppressed HBsAg-mediated cell proliferation., Conclusions: These data showed that HBsAg directly induces ER stress, impairs autophagy and promotes proliferation, thereby driving hepatocarcinogenesis. In addition, this study expanded the understanding of HBsAg-mediated intracellular events in carcinogenesis., Impact and Implications: Factors and mechanisms involved in hepatocarcinogenesis driven by hepatitis B surface antigen (HBsAg) are poorly defined, hindering the development of effective therapeutic strategies. This study showed that HBsAg-induced endoplasmic reticulum stress suppressed LAMP2, thereby mediating autophagic injury. The present data suggest that restoring LAMP2 function in chronic HBV infection may have both antiviral and anti-cancer effects. This study has provided insights into the role of HBsAg-mediated intracellular events in carcinogenesis and thereby has relevance for future drug development., Competing Interests: Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Authors.)

Hepatitis B virus (HBV)-transgenic mice exhibit competent innate immunity and are therefore an ideal model for considering intrinsic or cell-based mechanisms in HBV pathophysiology. A highly replicative model that has been little used, let alone characterized, is the Tg1.4HBV-s-rec strain derived from cross breeding of HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1-HBV]Bri44) or lack (Tg1.4HBV-s-mut) the hepatitis B surface antigen (HBsAg). Tg1.4HBV-s-rec hepatocytes secreted HBsAg, Hepatitis B extracellular antigen (HBeAg) and produced HBV virions. Transmission electron microscopy visualised viral particles (Tg1.4HBV-s-rec), nuclear capsid formations (Tg1.4HBV-s-mut and Tg1.4HBV-s-rec) and endoplasmic reticulum malformations (Alb/HBs). Viral replication in Tg1.4HBV-s-rec and Tg1.4HBV-s-mut differed in HBsAg expression and interestingly in the distribution of HBV core antigen (HBcAg) and HBV × protein. While in Tg1.4HBV-s-mut hepatocytes, the HBcAg was located in the cytoplasm, in Tg1.4HBV-s-rec hepatocytes, the HBcAg appeared in the nuclei, suggesting a more productive replication. Finally, Tg1.4HBV-s-rec mice showed symptoms of mild hepatitis, with reduced liver function and elevated serum transaminases, which appeared to be related to natural killer T cell activation. In conclusion, the study of Alb/HBs, Tg1.4HBV-s-mut and their F1 progeny provides a powerful tool to elucidate HBV pathophysiology, especially in the early HBeAg-positive phases of chronic infection and chronic hepatitis., (© 2023. The Author(s).)

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background and Aims: Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation., Methods: Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies., Results: Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16 INK4a , p19 ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle., Conclusion: HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches., (© 2023 The Authors. Liver International published by John Wiley & Sons Ltd.)

Background and Aims: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far., Approach and Results: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection., Conclusions: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Immunopathology in hepatitis B virus (HBV) infection is driven by innate and adaptive immunity. Whether the hepatitis B surface antigen (HBsAg) affects hepatic antiviral signalling was investigated in HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1HBV]Bri44), lack (Tg1.4HBV-s-mut3) or secrete (Tg1.4HBV-s-rec (F1, Tg1.4HBV-s-mut × Alb/HBs) the HBsAg. Herein, the responsiveness of TLR3 and RIG-I in primary parenchymal and non-parenchymal liver cells was determined in vitro and in vivo. Cell type-specific and mouse strain-dependent interferon, cytokine and chemokine expression were observed by LEGENDplex™ and validated by quantitative PCR. In vitro, the hepatocytes, liver sinusoidal endothelial cells and Kupffer cells of Tg1.4HBV-s-rec mice showed poly(I:C) susceptibilities similar to the wild-type controls, while in the remaining leucocyte fraction the interferon, cytokine and chemokine induction was reduced. On the contrary, poly(I:C)-injected 1.4TgHBV-s-rec mice showed suppressed interferon, cytokine and chemokine levels in hepatocytes but increased levels in the leucocyte fraction. Thus, we concluded that liver cells of Tg1.4HBV-s-rec mice, which produce HBV particles and release the HBsAg, responded to exogenous TLR3/RIG-I stimuli in vitro but exhibited a tolerogenic environment in vivo.

Previous studies have revealed multiple tissue- or cell-specific or enriched miRNA profiles. However, miRNA profiles enriched in hepatic cell types and their effect on HBV replication have not been well elucidated. In this study, primary human hepatocytes (PHHs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) were prepared from liver specimens of non-HBV-infected patients. Four hepatic cell type-enriched miRNA profiles were identified from purified liver cells miRNA microarray assay. The results revealed that 12 miRNAs, including miR-122-5p and miR-192-3p were PHH-enriched; 9 miRNAs, including miR-142-5p and miR-155-5p were KC-enriched; 6 miRNAs, including miR-126-3p and miR-222-3p were LSEC-enriched; and 14 miRNAs, including miR-214-3p and miR-199a-3p were HSC-enriched. By testing the effect of 11 PHH-enriched miRNAs on HBV production, we observed that miR-192-3p had the greatest pro-virus effect in hepatic cell lines. Moreover, we further found that miR-192-3p promoted HBV replication and gene expression through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in HepG2.2.15 cells. Additionally, the serum and hepatic miR-192-3p expression levels were significantly higher in chronic hepatitis B patients than in healthy controls and serum miR-192-3p positively correlated with the serum levels of HBV DNA and HBsAg. Collectively, we identified miRNA profiles enriched in four hepatic cell types and revealed that PHH-enriched miR-192-3p promoted HBV replication through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in hepatic cell lines. Our study provides a specific perspective for the role of hepatic cell type-enriched miRNA in interaction with viral replication and various liver pathogenesis.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Hepatitis B virus (HBV) infection causes acute and chronic liver diseases, including severe hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Interferon alpha 2a (IFNα-2a) is commonly used for treating chronic HBV infection. However, its efficacy remains relatively low. Yet, the immunological and molecular mechanisms for successful IFNα-2a treatment remain elusive. One issue is whether the application of increasing IFNα doses may modulate cellular processes and HBV replication in hepatic cells. In the present study, we focused on the interaction of IFNα signaling with other cellular signaling pathways and the consequence for HBV replication. The results showed that with the concentration of 6000 U/ml IFNα-2a treatment downregulated the activity of not only the Akt/mTOR signaling but also the AMPK signaling. Additionally, IFNα-2a treatment increased the formation of the autophagosomes by blocking autophagic degradation. Furthermore, IFNα-2a treatment inhibited the Akt/mTOR signaling and initiated autophagy under low and high glucose concentrations. In reverse, inhibition of autophagy using 3-methyladenine (3-MA) and glucose concentrations influenced the expression of IFNα-2a-induced ISG15 and IFITM1. Despite of ISGs induction, HBV replication and gene expression in HepG2.2.15 cells, a cell model with continuous HBV replication, were slightly increased at high doses of IFNα-2a. In conclusion, our study indicates that IFNα-2a treatment may interfere with multiple intracellular signaling pathways, facilitate autophagy initiation, and block autophagic degradation, thereby resulting in slightly enhanced HBV replication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Kemper, Broering, Chen, Yuan, Wang and Lu.)

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

A transcriptome-wide analysis of human liver for demonstrating differences between young and old humans has not yet been performed. However, identifying major age-related alterations in hepatic gene expression may pinpoint ontogenetic shifts with important hepatic and systemic consequences, provide novel pharmacogenetic information, offer clues to efficiently counteract symptoms of old age, and improve the overarching understanding of individual decline. Next-generation sequencing (NGS) data analyzed by the Mann-Whitney nonparametric test and Ensemble Feature Selection (EFS) bioinformatics identified 44 transcripts among 60,617 total and 19,986 protein-encoding transcripts that significantly ( p = 0.0003 to 0.0464) and strikingly (EFS score > 0.3:16 transcripts; EFS score > 0.2:28 transcripts) differ between young and old livers. Most of these age-related transcripts were assigned to the categories 'regulome', 'inflammaging', 'regeneration', and 'pharmacogenes'. NGS results were confirmed by quantitative real-time polymerase chain reaction. Our results have important implications for the areas of ontogeny/aging and the age-dependent increase in major liver diseases. Finally, we present a broadly substantiated and testable hypothesis on a genetically governed 'aging cascade', wherein PPP1R10 acts as a putative ontogenetic master regulator, prominently flanked by IGFALS and DUSP1 . This transcriptome-wide analysis of human liver offers potential clues towards developing safer and improved therapeutic interventions against major liver diseases and increased insights into key mechanisms underlying aging.