Publications by authors named "Nathalie Isorce"

11 Publications

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The antioxidant response favors Leishmania parasites survival, limits inflammation and reprograms the host cell metabolism.

PLoS Pathog 2021 Mar 25;17(3):e1009422. Epub 2021 Mar 25.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

The oxidative burst generated by the host immune system can restrict intracellular parasite entry and growth. While this burst leads to the induction of antioxidative enzymes, the molecular mechanisms and the consequences of this counter-response on the life of intracellular human parasites are largely unknown. The transcription factor NF-E2-related factor (NRF2) could be a key mediator of antioxidant signaling during infection due to the entry of parasites. Here, we showed that NRF2 was strongly upregulated in infection with the human Leishmania protozoan parasites, its activation was dependent on a NADPH oxidase 2 (NOX2) and SRC family of protein tyrosine kinases (SFKs) signaling pathway and it reprogrammed host cell metabolism. In inflammatory leishmaniasis caused by a viral endosymbiont inducing TNF-α in chronic leishmaniasis, NRF2 activation promoted parasite persistence but limited TNF-α production and tissue destruction. These data provided evidence of the dual role of NRF2 in protecting both the invading pathogen from reactive oxygen species and the host from an excess of the TNF-α destructive pro-inflammatory cytokine.
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http://dx.doi.org/10.1371/journal.ppat.1009422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993605PMC
March 2021

Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in parasites.

Bio Protoc 2020 May 5;10(9):e3598. Epub 2020 May 5.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique presented here is sequence-independent and is well detailed in the following protocol, taking the example of RNA virus (LRV) in ) species. To summarise, the protocol is divided into four major steps: RNA extraction from the parasites, RNA purification, enzymatic digestions with DNase I and Nuclease S1, and visualization by gel electrophoresis. This method can be used to detect other viral dsRNA in other parasites. It provides an additional tool, complementary to other techniques previously cited and it is easy and quite fast to achieve.
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http://dx.doi.org/10.21769/BioProtoc.3598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842782PMC
May 2020

Importance of polyphosphate in the life cycle.

Microb Cell 2018 Jun 22;5(8):371-384. Epub 2018 Jun 22.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these poly-phosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 () is responsible for polyP synthesis in parasites. During culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient ( ) proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In mouse infections, knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of did not alter the course of infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, promastigotes highly consumed polyP, and both knockdown or deletion of diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.
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http://dx.doi.org/10.15698/mic2018.08.642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116282PMC
June 2018

parasites block the activation of the inflammasome by inhibiting maturation of IL-1β.

Microb Cell 2018 Jan 14;5(3):137-149. Epub 2018 Jan 14.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

The various symptomatic outcomes of cutaneous leishmaniasis relates to the type and potency of its underlying inflammatory responses. Presence of the cytoplasmic RNA virus-1 (LRV1) within , worsens lesional inflammation and parasite burden, as the viral dsRNA genome acts as a potent innate immunogen stimulating Toll-Like-Receptor-3 (TLR3). Here we investigated other innate pattern recognition receptors capable of reacting to dsRNA and potentially contributing to LRV1-mediated inflammatory pathology. We included the cytoplasmic dsRNA sensors, namely, the RIG-like receptors (RLRs) and the inflammasome-dependent and -independent Nod-like-receptors (NLRs). Our study found no role for RLRs or inflammasome-dependent NLRs in the pathology of infection irrespective of its LRV1-status. Further, neither LRV1-bearing (+) nor LRV1-negative () activated the inflammasome . Interestingly, similarly to , infection induced the up-regulation of the A20 protein, known to be involved in the evasion of inflammasome activation. Moreover, we observed that + promoted the transcription of inflammasome-independent NLRC2 (also called NOD2) and NLRC5. However, only NLRC2 showed some contribution to LRV1-dependent pathology. These data confirmed that the endosomal TLR3 pathway is the dominant route of LRV1-dependent signalling, thus excluding the cytosolic and inflammasome pathways. We postulate that avoidance of the inflammasome pathways is likely an important mechanism of virulence in infection irrespective of the LRV1-status.
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http://dx.doi.org/10.15698/mic2018.03.619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826701PMC
January 2018

Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication.

Hepatology 2017 12 6;66(6):1750-1765. Epub 2017 Nov 6.

INSERM U1052, Cancer Research Center of Lyon, Lyon, France.

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified cellular serine/threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)-mediated knockdown, and overexpression of constitutively active PLK1 (PLK1 ). In addition, a humanized liver Fah /Rag2 /Il2rg (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1 increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein.

Conclusion: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV-mediated carcinogenesis. (Hepatology 2017;66:1750-1765).
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http://dx.doi.org/10.1002/hep.29236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658273PMC
December 2017

Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens.

J Immunol 2016 07 25;197(1):356-67. Epub 2016 May 25.

Centre International de Recherche en Infectiologie, INSERM U1111, Ecole Normale Supérieure, Université de Lyon, CNRS-UMR5308, Hospices Civils de Lyon, Lyon 69000, France;

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.
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http://dx.doi.org/10.4049/jimmunol.1502301DOI Listing
July 2016

Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.

Antiviral Res 2016 06 10;130:36-45. Epub 2016 Mar 10.

INSERM U1052, Cancer Research Centre of Lyon (CRCL), 69424 Lyon Cedex 03, France; University of Lyon, Université Claude Bernard (UCBL), UMR_S1052, 69008 Lyon, France; LabEx DEVweCAN, 69008 Lyon, France. Electronic address:

In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were type-I IFNs, IFNγ, type-III IFNs, TNFα, IL-6, IL-1β, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1β, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1β and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1β was found to have a very potent antiviral effect against HBV in vitro. HBV was previously shown to promptly inhibit IL-1β production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1β may help to further inhibit HBV replication in vivo.
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http://dx.doi.org/10.1016/j.antiviral.2016.03.008DOI Listing
June 2016

Cleaved c-FLIP mediates the antiviral effect of TNF-α against hepatitis B virus by dysregulating hepatocyte nuclear factors.

J Hepatol 2016 Feb 25;64(2):268-277. Epub 2015 Sep 25.

Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Republic of Korea; KU Open Innovation Center, Konkuk University, Seoul, Republic of Korea; Research Institute of Medical Sciences, Konkuk University, Seoul, Republic of Korea. Electronic address:

Background & Aims: Cytokines are key molecules implicated in the defense against virus infection. Tumor necrosis factor-alpha (TNF-α) is well known to block the replication of hepatitis B virus (HBV). However, the molecular mechanism and the downstream effector molecules remain largely unknown.

Methods: In this study, we investigated the antiviral effect and mechanism of p22-FLIP (FLICE-inhibitory protein) by ectopic expression in vitro and in vivo. In addition, to provide the biological relevance of our study, we examined that the p22-FLIP is involved in TNF-α-mediated suppression of HBV in primary human hepatocytes.

Results: We found that p22-FLIP, a newly discovered c-FLIP cleavage product, inhibited HBV replication at the transcriptional level in both hepatoma cells and primary human hepatocytes, and that c-FLIP conversion to p22-FLIP was stimulated by the TNF-α/NF-κB pathway. p22-FLIP inhibited HBV replication through the upregulation of HNF3β but downregulation of HNF4α, thus inhibiting both HBV enhancer elements. Finally, p22-FLIP potently inhibited HBV DNA replication in a mouse model of HBV replication.

Conclusions: Taken together, these findings suggest that the anti-apoptotic p22-FLIP serves a novel function of inhibiting HBV transcription, and mediates the antiviral effect of TNF-α against HBV replication.
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http://dx.doi.org/10.1016/j.jhep.2015.09.012DOI Listing
February 2016

Immune-modulators to combat hepatitis B virus infection: From IFN-α to novel investigational immunotherapeutic strategies.

Antiviral Res 2015 Oct 12;122:69-81. Epub 2015 Aug 12.

INSERM, U1052, CNRS UMR_5286, Cancer Research Centre of Lyon (CRCL), Lyon, France; University of Lyon, Université Claude Bernard (UCBL), Lyon, France; Labex DEVweCAN, Lyon, France. Electronic address:

Chronic hepatitis B virus (HBV) infection remains a major challenge for clinicians, as there are only two types of approved therapies: interferon-alpha (IFN-α) or its pegylated form, Peg-IFN-α and nucleoside analogs (e.g. tenofovir, entecavir...). The first are used as finite-duration treatments of around 48-52 weeks, while the second must be taken life-long to prevent rebound. Other immune-modulators, including other types of recombinant IFNs and cytokines/chemokines, could be developed for treating chronic hepatitis B. Alternatively, strategies aimed either at restoring or favoring the endogenous production of IFNs, cytokines and/or chemokines, or at alleviating HBV-mediated inhibitory processes could also be envisaged. In this article, we review current investigational, preclinical and clinical efforts to implement immune-modulatory components in the therapy of chronic hepatitis B. This review forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B".
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http://dx.doi.org/10.1016/j.antiviral.2015.08.008DOI Listing
October 2015

Early inhibition of hepatocyte innate responses by hepatitis B virus.

J Hepatol 2015 Dec 26;63(6):1314-22. Epub 2015 Jul 26.

INSERM U1052, Cancer Research Center of Lyon (CRCL), Lyon 69008, France; University of Lyon, UMR_S1052, UCBL, 69008 Lyon, France; Hospices Civils de Lyon (HCL), 69002 Lyon, France; Institut Universitaire de France (IUF), 75005 Paris, France. Electronic address:

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 24h 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.
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http://dx.doi.org/10.1016/j.jhep.2015.07.014DOI Listing
December 2015

Hippocampal GABAergic neurons are susceptible to amyloid-β toxicity in vitro and are decreased in number in the Alzheimer's disease TgCRND8 mouse model.

J Alzheimers Dis 2012 ;29(2):293-308

Institut de Neurobiologie de la Méditerranée (INMED), U901, Institut National de la Santé et de la Recherche Médicale (INSERM), Marseille, France.

The relevance of γ-amino-butyric acid (GABA)-ergic dysfunctions in the pathology of Alzheimer's disease (AD) remains a matter of debate. In the present study, we characterized the toxicity of amyloid-β (Aβ) on hippocampal GABAergic neurons both in vivo and in vitro. In the TgCRND8 mouse model of AD, we found a significant decrease in the number of hippocampal neurons immunoreactive for glutamate decarboxylase 67 (GAD67), the enzyme synthesizing GABA. This decrease, which was specific for hippocampal CA1-3 fields, was observed at 6 months of age, long after the overproduction of soluble Aβ42 (between 2 and 4 months) and accumulation of insoluble Aβ into amyloid plaques (between 4 and 6 months). In vitro, neurotoxicity was observed in primary hippocampal cultures 72 h following the addition of Aβ42 solutions containing a mixture of soluble oligomers. Taken together, our results suggest that when cultured and exposed to Aβ in vitro, GABAergic neurons are susceptible to Aβ42 neurotoxicity. However, in TgCRND8 mice, the number of GABAergic neurons is not altered up to 6 months, in spite of the massive Aβ load. Combined with the previously reported increased sensitivity to seizures observed in younger (1.5-2 month-old) TgCRND8 mice, it is likely that Aβ toxicity leads to GABAergic neuron dysfunction prior to their losses at a later stage.
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http://dx.doi.org/10.3233/JAD-2011-110830DOI Listing
July 2012