Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells.

Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.

Emerging anti-HDV drugs and HBV cure strategies with anti-HDV activity.

Hepatitis delta virus (HDV) is a satellite RNA virus that requires the presence of hepatitis B virus (HBV) for its replication. HDV/HBV co-infection is often associated with a faster disease progression of chronic hepatitis in comparison to HBV mono-infection. Therefore, the development of novel antiviral therapies targeting HDV represents a high priority and an urgent medical need. In this review, we summarize the ongoing efforts to evaluate promising HDV-specific drugs, such as lonafarnib (LNF), pegylated interferon lambda (PEG-IFN-λ) and their use as a combination therapy. Furthermore, we review the most recent developments in the area of anti-HBV drugs with potential effects against HDV, including therapeutic agents targeting hepatitis B surface antigen (HBsAg) expression, secretion and function. Finally, we consider the important insights that have emerged from the development of these potential antiviral strategies, as well as the intriguing questions that remain to be elucidated in this rapidly changing field.

Hepatitis Delta Virus Antigens Trigger Oxidative Stress, Activate Antioxidant Nrf2/ARE Pathway, and Induce Unfolded Protein Response.

Hepatitis delta virus (HDV) is a viroid-like satellite that may co-infect individuals together with hepatitis B virus (HBV), as well as cause superinfection by infecting patients with chronic hepatitis B (CHB). Being a defective virus, HDV requires HBV structural proteins for virion production. Although the virus encodes just two forms of its single antigen, it enhances the progression of liver disease to cirrhosis in CHB patients and increases the incidence of hepatocellular carcinoma. HDV pathogenesis so far has been attributed to virus-induced humoral and cellular immune responses, while other factors have been neglected. Here, we evaluated the impact of the virus on the redox status of hepatocytes, as oxidative stress is believed to contribute to the pathogenesis of various viruses, including HBV and hepatitis C virus (HCV). We show that the overexpression of large HDV antigen (L-HDAg) or autonomous replication of the viral genome in cells leads to increased production of reactive oxygen species (ROS). It also leads to the upregulated expression of NADPH oxidases 1 and 4, cytochrome P450 2E1, and ER oxidoreductin 1α, which have previously been shown to mediate oxidative stress induced by HCV. Both HDV antigens also activated the Nrf2/ARE pathway, which controls the expression of a spectrum of antioxidant enzymes. Finally, HDV and its large antigen also induced endoplasmic reticulum (ER) stress and the concomitant unfolded protein response (UPR). In conclusion, HDV may enhance oxidative and ER stress induced by HBV, thus aggravating HBV-associated pathologies, including inflammation, liver fibrosis, and the development of cirrhosis and hepatocellular carcinoma.

Inflammasome-independent NLRP3 function enforces ATM activity in response to genotoxic stress.

NLRP3 is a pattern recognition receptor with a well-documented role in inducing inflammasome assembly in response to cellular stress. Deregulation of its activity leads to many inflammatory disorders including gouty arthritis, Alzheimer disease, and cancer. Whereas its role in the context of cancer has been mostly explored in the immune compartment, whether NLRP3 exerts functions unrelated to immunity in cancer development remains unexplored. Here, we demonstrate that NLRP3 interacts with the ATM kinase to control the activation of the DNA damage response, independently of its inflammasome activity. NLRP3 down-regulation in both broncho- and mammary human epithelial cells significantly impairs ATM pathway activation, leading to lower p53 activation, and provides cells with the ability to resist apoptosis induced by acute genotoxic stress. Interestingly, NLRP3 expression is down-regulated in non-small cell lung cancers and breast cancers, and its expression positively correlates with patient overall survival. Our findings identify a novel non-immune function for NLRP3 in maintaining genome integrity and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways to maintain cell integrity.

SARS-CoV-2 Establishes a Productive Infection in Hepatoma and Glioblastoma Multiforme Cell Lines.

Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.

Hepatitis C virus replication requires integrity of mitochondria-associated ER membranes.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

Plasma-Like Culture Medium for the Study of Viruses.

Viral infections attract more and more attention, especially after the emergence of novel zoonotic coronaviruses and the monkeypox virus over the last 2 decades. Research on viruses is based to a great extent on mammalian cell lines that are permissive to the respective viruses. These cell lines are usually cultivated according to the protocols established in the 1950s to 1970s, although it is clear that classical media have a significant imprint on cell growth, phenotype, and especially metabolism. So, recently in the field of biochemistry and metabolomics novel culture media have been developed that resemble human blood plasma. As perturbations in metabolic and redox pathways during infection are considered significant factors of viral pathogenesis, these novel medium formulations should be adapted by the virology field. So far, there are only scarce data available on viral propagation efficiencies in cells cultivated in plasma-like media. But several groups have presented convincing data on the use of such media for cultivation of uninfected cells. The aim of the present review is to summarize the current state of research in the field of plasma-resembling culture media and to point out the influence of media on various cellular processes in uninfected cells that may play important roles in viral replication and pathogenesis in order to sensitize virology research to the use of such media.

An Update on the Metabolic Landscape of Oncogenic Viruses.

Viruses play an important role in cancer development as about 12% of cancer types are linked to viral infections. Viruses that induce cellular transformation are known as oncoviruses. Although the mechanisms of viral oncogenesis differ between viruses, all oncogenic viruses share the ability to establish persistent chronic infections with no obvious symptoms for years. During these prolonged infections, oncogenic viruses manipulate cell signaling pathways that control cell cycle progression, apoptosis, inflammation, and metabolism. Importantly, it seems that most oncoviruses depend on these changes for their persistence and amplification. Metabolic changes induced by oncoviruses share many common features with cancer metabolism. Indeed, viruses, like proliferating cancer cells, require increased biosynthetic precursors for virion production, need to balance cellular redox homeostasis, and need to ensure host cell survival in a given tissue microenvironment. Thus, like for cancer cells, viral replication and persistence of infected cells frequently depend on metabolic changes. Here, we draw parallels between metabolic changes observed in cancers or induced by oncoviruses, with a focus on pathways involved in the regulation of glucose, lipid, and amino acids. We describe whether and how oncoviruses depend on metabolic changes, with the perspective of targeting them for antiviral and onco-therapeutic approaches in the context of viral infections.

2-Deoxyglucose, an Inhibitor of Glycolysis, Enhances the Oncolytic Effect of Coxsackievirus.

Glioblastoma multiforme (GBM) is one of the most common types of brain tumor. Despite intensive research, patients with GBM have a poor prognosis due to a very high rate of relapse and significant side effects of the treatment, with a median survival of 14.6 months. Oncolytic viruses are considered a promising strategy to eliminate GBM and other types of cancer, and several viruses have already been introduced into clinical practice. However, identification of the factors that underly the sensitivity of tumor species to oncolytic viruses or that modulate their clinical efficacy remains an important target. Here, we show that Coxsackievirus B5 (CVB5) demonstrates high oncolytic potential towards GBM primary cell species and cell lines. Moreover, 2-deoxyglucose (2DG), an inhibitor of glycolysis, potentiates the cytopathic effects of CVB5 in most of the cancer cell lines tested. The cells in which the inhibition of glycolysis enhanced oncolysis are characterized by high mitochondrial respiratory activity and glycolytic capacity, as determined by Seahorse analysis. Thus, 2-deoxyglucose and other analogs should be considered as adjuvants for oncolytic therapy of glioblastoma multiforme.

Hepatic inflammation elicits production of proinflammatory netrin-1 through exclusive activation of translation.

BACKGROUND AND AIMS: Netrin-1 displays protumoral properties, though the pathological contexts and processes involved in its induction remain understudied. The liver is a major model of inflammation-associated cancer development, leading to HCC. APPROACH AND RESULTS: A panel of cell biology and biochemistry approaches (reverse transcription quantitative polymerase chain reaction, reporter assays, run-on, polysome fractionation, cross linking immunoprecipitation, filter binding assay, subcellular fractionation, western blotting, immunoprecipitation, stable isotope labeling by amino acids in cell culture) on in vitro-grown primary hepatocytes, human liver cell lines, mouse samples and clinical samples was used. We identify netrin-1 as a hepatic inflammation-inducible factor and decipher its mode of activation through an exhaustive eliminative approach. We show that netrin-1 up-regulation relies on a hitherto unknown mode of induction, namely its exclusive translational activation. This process includes the transfer of NTN1 (netrin-1) mRNA to the endoplasmic reticulum and the direct interaction between the Staufen-1 protein and this transcript as well as netrin-1 mobilization from its cell-bound form. Finally, we explore the impact of a phase 2 clinical trial-tested humanized anti-netrin-1 antibody (NP137) in two distinct, toll-like receptor (TLR) 2/TLR3/TLR6-dependent, hepatic inflammatory mouse settings. We observe a clear anti-inflammatory activity indicating the proinflammatory impact of netrin-1 on several chemokines and Ly6C+ macrophages. CONCLUSIONS: These results identify netrin-1 as an inflammation-inducible factor in the liver through an atypical mechanism as well as its contribution to hepatic inflammation.

Heparanase-1 is upregulated by hepatitis C virus and favors its replication.

BACKGROUND & AIMS: Over time, chronic HCV infection can lead to hepatocellular carcinoma (HCC), a process that involves changes to the liver extracellular matrix (ECM). However, the exact mechanisms by which HCV induces HCC remain unclear. Therefore, we sought to investigate the impact of HCV on the liver ECM, with a focus on heparanase-1 (HPSE). METHODS: HPSE expression was assessed by quantitative reverse-transcription PCR, immunoblotting and immunofluorescence in liver biopsies infected or not with HCV, and in 10-day-infected hepatoma Huh7.5 cells. Cell lines deficient for or overexpressing HPSE were established to study its role during infection. RESULTS: HCV propagation led to significant HPSE induction, in vivo and in vitro. HPSE enhanced infection when exogenously expressed or supplemented as a recombinant protein. Conversely, when HPSE expression was downregulated or its activity blocked, HCV infection dropped, suggesting a role of HPSE in the HCV life cycle. We further studied the underlying mechanisms of such observations and found that HPSE favored HCV release by enhancing CD63 synthesis and exosome secretion, but not by stimulating HCV entry or genome replication. We also showed that virus-induced oxidative stress was involved in HPSE induction, most likely through NF-κB activation. CONCLUSIONS: We report for the first time that HCV infection is favored by HPSE, and upregulates HPSE expression and secretion, which may result in pathogenic alterations of the ECM. LAY SUMMARY: Chronic hepatitis C virus (HCV) infection can lead to hepatocellular carcinoma development in a process that involves derangement of the extracellular matrix (ECM). Herein, we show that heparanase-1, a protein involved in ECM degradation and remodeling, favors HCV infection and is upregulated by HCV infection; this upregulation may result in pathogenic alterations of the ECM.

Cultivation of Cells in a Physiological Plasmax Medium Increases Mitochondrial Respiratory Capacity and Reduces Replication Levels of RNA Viruses.

Changes in metabolic pathways are often associated with the development of various pathologies including cancer, inflammatory diseases, obesity and metabolic syndrome. Identification of the particular metabolic events that are dysregulated may yield strategies for pharmacologic intervention. However, such studies are hampered by the use of classic cell media that do not reflect the metabolite composition that exists in blood plasma and which cause non-physiological adaptations in cultured cells. In recent years two groups presented media that aim to reflect the composition of human plasma, namely human plasma-like medium (HPLM) and Plasmax. Here we describe that, in four different mammalian cell lines, Plasmax enhances mitochondrial respiration. This is associated with the formation of vast mitochondrial networks and enhanced production of reactive oxygen species (ROS). Interestingly, cells cultivated in Plasmax displayed significantly less lysosomes than when any standard media were used. Finally, cells cultivated in Plasmax support replication of various RNA viruses, such as hepatitis C virus (HCV) influenza A virus (IAV), severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and several others, albeit at lower levels and with delayed kinetics. In conclusion, studies of metabolism in the context of viral infections, especially those concerning mitochondria, lysosomes, or redox systems, should be performed in Plasmax medium.

Effect of endothelial cell heterogeneity on nanoparticle uptake.

Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino-modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelium to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine.

Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin.

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.

Hypoxia sensing by hepatic stellate cells leads to VEGF-dependent angiogenesis and may contribute to accelerated liver regeneration.

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.

Metabolic Hallmarks of Hepatic Stellate Cells in Liver Fibrosis.

Liver fibrosis is a regenerative process that occurs after injury. It is characterized by the deposition of connective tissue by specialized fibroblasts and concomitant proliferative responses. Chronic damage that stimulates fibrogenic processes in the long-term may result in the deposition of excess matrix tissue and impairment of liver functions. End-stage fibrosis is referred to as cirrhosis and predisposes strongly to the loss of liver functions (decompensation) and hepatocellular carcinoma. Liver fibrosis is a pathology common to a number of different chronic liver diseases, including alcoholic liver disease, non-alcoholic fatty liver disease, and viral hepatitis. The predominant cell type responsible for fibrogenesis is hepatic stellate cells (HSCs). In response to inflammatory stimuli or hepatocyte death, HSCs undergo trans-differentiation to myofibroblast-like cells. Recent evidence shows that metabolic alterations in HSCs are important for the trans-differentiation process and thus offer new possibilities for therapeutic interventions. The aim of this review is to summarize current knowledge of the metabolic changes that occur during HSC activation with a particular focus on the retinol and lipid metabolism, the central carbon metabolism, and associated redox or stress-related signaling pathways.

Regulation of Mitochondria-Associated Membranes (MAMs) by NO/sGC/PKG Participates in the Control of Hepatic Insulin Response.

Under physiological conditions, nitric oxide (NO) produced by the endothelial NO synthase (eNOS) upregulates hepatic insulin sensitivity. Recently, contact sites between the endoplasmic reticulum and mitochondria named mitochondria-associated membranes (MAMs) emerged as a crucial hub for insulin signaling in the liver. As mitochondria are targets of NO, we explored whether NO regulates hepatic insulin sensitivity by targeting MAMs. In Huh7 cells, primary rat hepatocytes and mouse livers, enhancing NO concentration increased MAMs, whereas inhibiting eNOS decreased them. In vitro, those effects were prevented by inhibiting protein kinase G (PKG) and mimicked by activating soluble guanylate cyclase (sGC) and PKG. In agreement with the regulation of MAMs, increasing NO concentration improved insulin signaling, both in vitro and in vivo, while eNOS inhibition disrupted this response. Finally, inhibition of insulin signaling by wortmannin did not affect the impact of NO on MAMs, while experimental MAM disruption, using either targeted silencing of cyclophilin D or the overexpression of the organelle spacer fetal and adult testis-expressed 1 (FATE-1), significantly blunted the effects of NO on both MAMs and insulin response. Therefore, under physiological conditions, NO participates to the regulation of MAM integrity through the sGC/PKG pathway and concomitantly improves hepatic insulin sensitivity. Altogether, our data suggest that the induction of MAMs participate in the impact of NO on hepatocyte insulin response.

Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation.

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.