Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma ...(HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.
The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. ...Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1β and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1β and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1β and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.
HepaRG cells are liver bipotent progenitors acquiring hepatocytes features when differentiated in the presence of dimethylsulfoxide (DMSO). Differentiated HepaRG (dHepaRG) are considered the best ...surrogate model to primary human hepatocytes (PHH) and are susceptible to several hepatotropic viruses, including Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) infection. Despite these advantages, HepaRG cells are not widely used for the study of these two viruses because of their long differentiation process and their rather low and variable infection rates. Here, we tested the use of a cocktail of five chemicals (5C) combined or not with DMSO to accelerate the cells' differentiation process. We found that NTCP-mediated HDV entry and replication are similar in HepaRG cells cultivated for only 1 week with 5C and DMSO or differentiated with the regular 4-week protocol. However, even though the NTCP-mediated HBV entry process seemed similar, cccDNA and subsequent HBV replication markers were lower in HepaRG cells cultivated for 1 week with 5C and DMSO compared to the regular differentiation protocol. In conclusion, we set up a new procedure allowing fast differentiation and efficient HDV-infection of HepaRG cells and identified differential culture conditions that may allow to decipher the mechanism behind the establishment of the HBV minichromosome.
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ...ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.
HDV superinfection of chronically HBV-infected patients is the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis/cirrhosis and increased risk of liver ...failure, hepatocellular carcinoma, and death. While HDV infection is not susceptible to available direct anti-HBV drugs, suboptimal responses are obtained with interferon-α-based therapies, and the number of investigational drugs remains limited. We therefore analyzed the effect of several innate immune stimulators on HDV replication in infected hepatocytes.
We used in vitro models of HDV and HBV infection based on primary human hepatocytes (PHHs) and the non-transformed HepaRG cell line that are relevant to explore new innate immune therapies.
We describe here, for the first time, anti-HDV effects of Pam3CSK4 and BS1, agonists of Toll-like receptor (TLR)-1/2, and the lymphotoxin-β receptor (LTβR), respectively. Both types of agonists induced dose-dependent reductions of total intracellular HDV genome and antigenome RNA and of HDV protein levels, without toxicity in cells monoinfected with HDV or co/superinfected with HBV. Moreover, both molecules negatively affected HDV progeny release and strongly decreased their specific infectivity. The latter effect is particularly important since HDV is thought to persist in humans through constant propagation.
Immune-modulators inducing NF-κB pathways in hepatocytes can inhibit HDV replication and should be further evaluated as a possible therapeutic approach in chronically HBV/HDV-infected patients.
Hepatitis delta virus causes the most severe form of viral hepatitis. Despite positive recent developments, effective treatments remain a major clinical need. Herein, we show that immune-modulators that trigger the NF-κB pathways could be effective for the treatment of hepatitis delta infections.
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•HDV leads to the most severe form of chronic viral hepatitis and treatment options are limited.•We demonstrate, for the first time, that immune-modulators inducing the NF-κB pathways can inhibit HDV in hepatocytes.•Pam3CSK4 (TLR1/2 agonist) and BS1 (LTBR agonist) reduced the intracellular and extracellular HDV RNAs and proteins.•Pam3CSK4 and BS1 also strongly reduced specific infectivity of remaining HDV virion progeny.•Pam3CSK4 and BS1 will thereby probably counteract viral spreading in vivo.
The development of new anti–hepatitis B virus (HBV) therapies, especially immunotherapeutic approaches, has been limited by the lack of a primate model more accessible than chimpanzees. We have ...previously demonstrated that sylvanus and cynomolgus macaques are susceptible to in vivo HBV infection after intrahepatic HBV DNA inoculation. In this study, we evaluated the susceptibility of primary macaque hepatocytes (PMHs) to HBV infection with a highly efficient HBV genome–mediated transfer system via a recombinant baculovirus (Bac‐HBV). Freshly prepared PMHs, isolated from macaque liver tissue by collagenase perfusion, were transduced with Bac‐HBV, and intermediates of replication were followed for 9 days post‐transduction. Evidence of HBV replication (hepatitis B surface antigen secretion, viral DNA, RNA, and covalently closed circular DNA) was detected from day 1 to day 9 post‐transduction. HBV markers were dose‐dependent and still detectable at a multiplicity of infection of 10. Importantly, transduced PMHs secreted all typical forms of HBV particles, as evidenced by a cesium chloride gradient as well as transmission electron microscopy. Furthermore, the Toll‐like receptor 9 (TLR9) ligand was used to stimulate freshly prepared macaque peripheral blood mononuclear cells to generate TLR9‐induced cytokines. We then demonstrated the antiviral effects of both TLR9‐induced cytokines and nucleoside analogue (lamivudine) on HBV replication in transduced PMHs. Conclusion: Baculovirus‐mediated genome transfer initiated a full HBV replication cycle in PMHs; thus highlighted both the baculovirus efficiency in crossing the species barrier and macaque susceptibility to HBV infection. Moreover, our results demonstrate the relevance of thus system for antiviral compound evaluations with either nucleoside analogues or inhibitory cytokines. Cynomolgus macaques are readily available, are immunologically closely related to humans, and may therefore represent a promising model for the development of new immunotherapeutic strategies. (HEPATOLOGY 2010)
Aims/hypothesis
Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca
2+
) transfer between ...both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin’s action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in
CypD
(also known as
Ppif
)-knockout (KO) mice.
Methods
We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER–mitochondria interactions, inter-organelle Ca
2+
exchange, organelle homeostasis and insulin action.
Results
Pharmacological and genetic inhibition of CYPD concomitantly reduced ER–mitochondria interactions, inhibited inter-organelle Ca
2+
exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca
2+
transfer from ER to mitochondria was blunted in isolated hepatocytes of
CypD
-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca
2+
transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of
CypD
-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells.
Conclusions/interpretation
Disruption of IP3R-mediated Ca
2+
signalling in the liver of
CypD
-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER–mitochondria Ca
2+
exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.
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