Background & Aims The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such ...interactions during the very early step of infection has not been adequately investigated. Methods We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24 h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. Results We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. Conclusions Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the “stealthy” character of HBV.
Portal vein ligation (PVL) induces liver growth prior to resection. Associating liver partition and portal vein ligation (PVL plus transection=ALPPS) or the addition of the prolyl-hydroxylase ...inhibitor dimethyloxalylglycine (DMOG) to PVL both accelerate growth via stabilization of HIF-α subunits. This study aims at clarifying the crosstalk of hepatocytes (HC), hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) in accelerated liver growth. In vivo, liver volume, HC proliferation, vascular density and HSC activation were assessed in PVL, ALPPS, PVL+DMOG and DMOG alone. Proliferation of HC, HSC and LSEC was determined under DMOG in vitro. Conditioned media experiments of DMOG-exposed cells were performed. ALPPS and PVL+DMOG accelerated liver growth and HC proliferation in comparison to PVL. DMOG alone did not induce HC proliferation, but led to increased vascular density, which was also observed in ALPPS and PVL+DMOG. Activated HSC were detected in ALPPS, PVL+DMOG and DMOG, again not in PVL. In vitro, DMOG had no proliferative effect on HC, but conditioned supernatant of DMOG-treated HSC induced VEGF-dependent proliferation of LSEC. Transcriptome analysis confirmed activation of proangiogenic factors in hypoxic HSC. Hypoxia signaling in HSC induces VEGF-dependent angiogenesis. HSC play a crucial role in the cellular crosstalk of rapid liver regeneration.
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.
Background & Aims:
Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with ...chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells.
Methods:
Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages.
Results:
HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules.
Conclusions:
HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.
Immune-mediated induction of cytidine deaminase APOBEC3B (A3B) expression leads to HBV covalently closed circular DNA (cccDNA) decay. Here, we aimed to decipher the signalling pathway(s) and ...regulatory mechanism(s) involved in A3B induction and related HBV control.
Differentiated HepaRG cells (dHepaRG) knocked-down for NF-κB signalling components, transfected with siRNA or micro RNAs (miRNA), and primary human hepatocytes ± HBV or HBVΔX or HBV-RFP, were treated with lymphotoxin beta receptor (LTβR)-agonist (BS1). The biological outcomes were analysed by reverse transcriptase-qPCR, immunoblotting, luciferase activity, chromatin immune precipitation, electrophoretic mobility-shift assay, targeted-bisulfite-, miRNA-, RNA-, genome-sequencing, and mass-spectrometry.
We found that canonical and non-canonical NF-κB signalling pathways are mandatory for A3B induction and anti-HBV effects. The degree of immune-mediated A3B production is independent of A3B promoter demethylation but is controlled post-transcriptionally by the miRNA 138-5p expression (hsa-miR-138-5p), promoting A3B mRNA decay. Hsa-miR-138-5p over-expression reduced A3B levels and its antiviral effects. Of note, established infection inhibited BS1-induced A3B expression through epigenetic modulation of A3B promoter. Twelve days of treatment with a LTβR-specific agonist BS1 is sufficient to reduce the cccDNA pool by 80% without inducing significant damages to a subset of cancer-related host genes. Interestingly, the A3B-mediated effect on HBV is independent of the transcriptional activity of cccDNA as well as on rcDNA synthesis.
Altogether, A3B represents the only described enzyme to target both transcriptionally active and inactive cccDNA. Thus, inhibiting hsa-miR-138-5p expression should be considered in the combinatorial design of new therapies against HBV, especially in the context of immune-mediated A3B induction.
Immune-mediated induction of cytidine deaminase APOBEC3B is transcriptionally regulated by NF-κB signalling and post-transcriptionally downregulated by hsa-miR-138-5p expression, leading to cccDNA decay. Timely controlled APOBEC3B-mediated cccDNA decay occurs independently of cccDNA transcriptional activity and without damage to a subset of cancer-related genes. Thus, APOBEC3B-mediated cccDNA decay could offer an efficient therapeutic alternative to target hepatitis B virus chronic infection.
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•Impairment of NF-κB signalling prevents APOBEC3B induction and cccDNA decay.•APOBEC3B is post-transcriptionally regulated by the hsa-miR-138-5p.•Over-expression of the hsa-miR-138-5p inhibits APOBEC3B expression and cccDNA decay.•A3B timely induces cccDNA decay without damage to cancer-related genes.•APOBEC3B-mediated cccDNA decay is independent of cccDNA transcriptional activity.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and finding a single reliable biomarker to follow liver degradation is a challenging task. To document the ...relationship between liver failure, hypoxia, and HCC, copper isotope variations (δ65Cu) were evaluated in the serum of HCC-negative and HCC-positive patients as a biomarker of hepatic failure.
We analyzed Cu isotope variations in serum samples from 293 patients with potentially degraded liver functions presenting hepatitis B virus, hepatitis C virus, nonalcoholic steatohepatitis, and alcohol uptake (OH) etiologies and 105 controls. Ninety-five of the patients were diagnosed with HCC.
On average, the δ65Cu values of the serum of patients with F3-F4 fibrosis score or HCC-positive are low. The Cu isotope data are strikingly bimodal with well-defined δ65Cu modes which imperfectly reflect etiology. The population with normal values (ca −0.3‰) is progressively replaced by a population with atypical δ65Cu values (ca −0.8‰), which reflects the progressive degradation of hepatic functions.
The clear bimodality does not correspond to a progressive shift of the δ65Cu values but to a replacement of one population by another. This bimodality sheds light on the persisting difficulties epitomized by α-fetoprotein in finding high-sensitivity and high-specificity HCC biomarkers. It is interpreted as a switch in the resistance of hepatic tissues to the oxidative stress that eventually leads to HCC oncogenesis.
Background and Aims
The liver is an innervated organ that develops a variety of chronic liver disease (CLD). Axon guidance cues (AGCs), of which ephrins, netrins, semaphorins and slits are the main ...representative, are secreted or membrane‐bound proteins that can attract or repel axons through interactions with their growth cones that contain receptors recognizing these messengers. While fundamentally implicated in the physiological development of the nervous system, the expression of AGCs can also be reinduced under acute or chronic conditions, such as CLD, that necessitate redeployment of neural networks.
Methods
This review considers the ad hoc literature through the neglected canonical neural function of these proteins that is also applicable to the diseased liver (and not solely their observed parenchymal impact).
Results
AGCs impact fibrosis regulation, immune functions, viral/host interactions, angiogenesis, and cell growth, both at the CLD and HCC levels. Special attention has been paid to distinguishing correlative and causal data in such datasets in order to streamline data interpretation. While hepatic mechanistic insights are to date limited, bioinformatic evidence for the identification of AGCs mRNAs positive cells, protein expression, quantitative regulation, and prognostic data have been provided. Liver‐pertinent clinical studies based on the US Clinical Trials database are listed. Future research directions derived from AGC targeting are proposed.
Conclusion
This review highlights frequent implication of AGCs in CLD, linking traits of liver disorders and the local autonomic nervous system. Such data should contribute to diversifying current parameters of patient stratification and our understanding of CLD.
Immunoblotting normalization issues have been recently overcome by whole lane staining. Herein, we are taking advantage of these recent advances and of the fluorophore status of the Ponceau S stain ...in order to combine the advantages of whole lane staining and fluorescence. By Ponceau S excitation at 488 nm, we identify the so-called ‘fluorescent Ponceau’ method as more linear, more sensitive and more repeatable than the others in protein lysates of distant biochemical profiles (cells, human and mouse tissues). This essentially cost-free method at the single experiment level provides accessible and robust means for post-blot normalization of many types of analytes.
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•‘Housekeeping protein’ signals robustness is much problematic in western blotting.•Ponceau Red is a fluorescent dye, amenable to fluorescence-based quantification.•Herein, a novel fluorescent Ponceau-based normalization technique is detailed.•It outcompetes regular Ponceau and pertinent commercial products.•Criteria used were sensitivity, linearity, and repeatability.
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