Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection

Background & Aims: The limited availability of hepatitis Delta virus (HDV) infection models has hindered studies of interactions between HDV and infected hepatocytes. The aim was to investigate the antiviral state of HDV infected human hepatocytes in the setting of co-infection with hepatitis B virus (HBV) compared to HBV mono-infection using human liver chimeric mice. Methods: Viral loads, human interferon stimulated genes (hISGs) and cytokines were determined in humanized uPA/SCID/beige (USB) mice by qRT-PCR, ELISA and immunofluorescence. Results: Upon HBV/HDV inoculation, all mice developed viremia, which was accompanied by a significant induction of hISGs (i.e. hISG15, hSTATs, hHLA-E) compared to uninfected mice, while HBV mono-infection led to weaker hISG elevations. In the setting of chronic infection enhancement of innate defense mechanisms was significantly more prominent in HBV/HDV infected mice. Also the induction of human-specific cytokines (hIP10, hTGF-beta, hIFN-beta and hIFN-lambda) was detected in HBV/HDV co-infected animals, while levels remained lower or below detection in uninfected and HBV mono-infected mice. Moreover, despite the average increase of hSTAT levels determined in HBV/HDV infected livers, we observed a weaker hSTAT accumulation in nuclei of hepatocytes displaying very high HDAg levels, suggesting that HDAg may in part limit hSTAT signaling. Conclusions: Establishment of HDV infection provoked a clear enhancement of the antiviral state of the human hepatocytes in chimeric mice. Elevated pre-treatment ISG and interferon levels may directly contribute to inflammation and liver damage, providing a rationale for the more severe course of HDV-associated liver disease. Such antiviral state induction might also contribute to the lower levels of HBV activity frequently found in co-infected hepatocytes. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

A humanized mouse model of liver fibrosis following expansion of transplanted hepatic stellate cells

Hepatic stellate cells (HSCs) are major contributors to liver fibrosis, as hepatic injuries may cause their transdifferentiation into myofibroblast-like cells capable of producing excessive extracellular matrix proteins. Also, HSCs can modulate engraftment of transplanted hepatocytes and contribute to liver regeneration. Therefore, understanding the biology of human HSCs (hHSCs) is important, but effective methods have not been available to address their fate in vivo. To investigate whether HSCs could engraft and repopulate the liver, we transplanted GFP-transduced immortalized hHSCs into immunodeficient NOD/SCID mice. Biodistribution analysis with radiolabeled hHSCs showed that after intrasplenic injection, the majority of transplanted cells rapidly translocated to the liver. GFP-immunohistochemistry demonstrated that transplanted hHSCs engrafted alongside hepatic sinusoids. Prior permeabilization of the sinusoidal endothelial layer with monocrotaline enhanced engraftment of hHSCs. Transplanted hHSCs remained engrafted without relevant proliferation in the healthy liver. However, after CCl4 or bile duct ligation-induced liver damage, transplanted hHSCs expanded and contributed to extracellular matrix production, formation of bridging cell-septae and cirrhosis-like hepatic pseudolobules. CCl4-induced injury recruited hHSCs mainly to zone 3, whereas after bile duct ligation, hHSCs were mainly in zone 1 of the liver lobule. Transplanted hHSCs neither transdifferentiated into other cell types nor formed tumors in these settings. In conclusion, a humanized mouse model was generated by transplanting hHSCs, which proliferated during hepatic injury and inflammation, and contributed to liver fibrosis. The ability to repopulate the liver with transplanted hHSCs will be particularly significant for mechanistic studies of cell-cell interactions and fibrogenesis within the liver

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics

Specific and Nonhepatotoxic Degradation of Nuclear Hepatitis B Virus cccDNA.

peer reviewedCurrent antivirals can control but not eliminate hepatitis-B-virus (HBV), because HBV establishes a stable nuclear cccDNA. Interferon-alpha treatment can clear HBV but is limited by systemic side effects. Here, we describe how interferon-alpha can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-beta-receptor activation as a therapeutic alternative. Interferon-alpha and lymphotoxin-beta-receptor activation up-regulated APOBEC3A and 3B cytidine-deaminases, respectively, in HBV-infected cells, primary hepatocytes and human liver-needle biopsies. HBV-core protein mediated the interaction with nuclear cccDNA resulting in cytidine-deamination, apurinic/apyrimidinic site formation and finally cccDNA degradation that prevented HBV-reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases - e.g., by lymphotoxin-beta-receptor activation - allows development of new therapeutics that combined with existing antivirals may cure hepatitis B

Inhibition of HCV replication by statins in genotype 1b replicon cells.

<p>LucUbiNeo-ET replicon cells were incubated in the presence of fluvastatin (FLV), pravastatin (PRV), simvastatin (SMV), rosuvastatin (ROV) or atorvastatin (ATV) at the indicated concentrations for 24, 48 or 72 hours. (<b>A</b>) Expression of the LDL-receptor (LDLR) was measured by real time RT-qPCR after 48 h of statin incubation (<b>B–F</b>) Dose- and time-dependent inhibition of HCV replication caused by statins was measured by luciferase reporter assay. Statin mediated effects on cell proliferation were measured after 72 hours of incubation (<b>G</b>) and confirmed by Western Blot analysis (<b>H</b>).</p

Matrix conditions predict anti-viral properties of statins.

<p>LucUbiNeo-ET replicon cells growing on PAA gel supports (1–12 kPa: 1 kPa = soft; 12 kPa = stiff) were incubated with FLV for 72 hours. Effects on HCV replication were measured by luciferase assay (<b>A</b>). For the same settings cell viability was measured by MTT assay (<b>B</b>). LucUbiNeo-ET replicon cells were incubated with increasing concentrations of the Rho kinase inhibitor HA1100 alone or in combination with FLV at 5 µM for 72 hours. HCV replication was measured by luciferase reporter assay (<b>C</b>). Viability of LucUbiNeo-ET replicon cells was monitored for the same settings by MTT assay (<b>D</b>). Huh-5-15 replicon cells were incubated with FLV with or without coincubation with HA1100 for 6 hours. HCV replication (<b>E</b>) as well as HO-1 expression (<b>F</b>) was measured by RT-qPCR.</p

Statin-induced HO-1 expression contributes to inhibition of HCV replication and endogenous interferon response.

<p>HO-1-induction was measured by RT-qPCR in Huh-5-15 replicon cells after 6 hours of statin incubation (<b>A</b>). Western Blot analysis was used to visualize the HO-1 protein level was after 8 hours of incubation using 10 or 25 microM of FLV (<b>B</b>). LucUbiNeo-ET replicon cells stably expressing shRNA against HO-1 (shHO1) or a control gene (shGFP) were used to detect baseline or FLV-induced expression of HO-1 by RT-qPCR after 6 hours of incubation (<b>C</b>). ShGFP or shHO1 cells were incubated with statin for 72 hours. HCV replication was measured by luciferase reporter assay (<b>D</b>). LucUbiNeo-ET replicon cells were incubated in the presence of 10 microM of statins for 24 hours. Gene expression levels (interferon alpha 2; interferon alpha 17; interferon response genes) were analyzed by RT-qPCR (<b>E</b>). LucUbiNeo-ET cells were incubated with FLV, IFN alpha (Intron A) and telaprevir (TVR) alone or in combination for 24 hours. Single and combined effects on HCV replication were detected by luciferase assay (<b>F</b>).</p