Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.
ISSN: 1350-9047

inserm-00871432
http://www.hal.inserm.fr/inserm-00871432
DOI : 10.1038/cdd.2012.90
PUBMED : 22814620

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.
ISSN: 0166-445X

hal-00877351
https://hal.archives-ouvertes.fr/hal-00877351
DOI : 10.1016/j.aquatox.2013.05.001
PUBMED : 23721851

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : International audience
Bone Morphogenetic Protein 9 (BMP9) has been recently found to be the physiological ligand for the activin receptor-like kinase 1 (ALK1), and to be a major circulating vascular quiescence factor. Moreover, a soluble chimeric ALK1 protein (ALK1-Fc) has recently been developed and showed powerful anti-tumor growth and anti-angiogenic effects. However, not much is known concerning BMP9. This prompted us to investigate the human endogenous sources of this cytokine and to further characterize its circulating form(s) and its function. Analysis of BMP9 expression reveals that BMP9 is produced by hepatocytes and intrahepatic biliary epithelial cells. Gel filtration analysis combined with ELISA and biological assays demonstrate that BMP9 circulates in plasma (1) as an unprocessed inactive form that can be further activated by furin a serine endoprotease, and (2) as a mature and fully active form (composed of the mature form associated with its prodomain). Analysis of BMP9 circulating levels during mouse development demonstrates that BMP9 peaks during the first 3 weeks after birth and then decreases to 2 ng/mL in adulthood. We also show that circulating BMP9 physiologically induces a constitutive Smad1/5/8 phosphorylation in endothelial cells. Taken together, our results argue for the role of BMP9 as a hepatocyte-derived factor, circulating in inactive (40%) and active (60%) forms, the latter constantly activating endothelial cells to maintain them in a resting state.
ISSN: 1420-682X

hal-00681969
https://hal.archives-ouvertes.fr/hal-00681969
DOI : 10.1007/s00018-011-0751-1
PUBMED : 21710321

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
The catecholamine epinephrine is known to repress expression of hepatic drug metabolizing enzymes such as cytochromes P-450. The present study was designed to determine whether epinephrine may also target expression of main hepatic drug transporters, that play a major role in liver detoxification and are commonly coordinately regulated with drug detoxifying enzymes. Treatment of primary human hepatocytes with 10 mu M epinephrine for 24 h repressed mRNA expression of various transporters, such as the sinusoidal influx transporters NTCP, OATP1B1, OATP2B1, OAT2, OAT7 and OCT1 and the efflux transporters MRP2, MRP3 and BSEP, whereas it induced that of MDR1, but failed to alter that of BCRP. Most of these changes in transporter mRNA levels were also found in epinephrine-exposed human highly-differentiated hepatoma HepaRG cells, which additionally exhibited reduced protein expression of OATP2B1 and MRP3, increased expression of P-glycoprotein and decreased transport activity of NTCP, OATPs and OCT1. Epinephrine effects towards transporter mRNA expression in human hepatocytes were next shown to be correlated to those of the selective beta 2-adrenoreceptor (ADR) agonist fenoterol, of the adenylate cyclase activator forskolin and of the cAMP analogue 8-bromo-cAMP. In addition, the non-selective beta-ADR antagonist carazolol and the selective beta 2-ADR antagonist ICI-118,551, unlike the alpha-ADR antagonist phentolamine, suppressed epinephrine -mediated repressions of transporter mRNA expression. Taken together, these data indicate that epinephrine regulates in vitro expression of main hepatic drug transporters in a beta 2-ADR/adenylate cyclase/cAMP-dependent manner. Hepatic drug transport appears therefore as a target of the beta 2-adrenergic system, which may have to deserve attention for drugs interacting with beta 2-ADRs.
ISSN: 0928-0987

hal-01578583
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578583
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578583/file/Mayati%20et%20al.%20-%20%CE%B22-adrenergic%20receptor-mediated%20in%20vitro%20regulatio.pdf
DOI : 10.1016/j.ejps.2017.06.010
PUBMED : 28603032

Éditeur(s) : HAL CCSD
Résumé : International audience
Acute viral hepatitis results from an inefficient innate immune response to clear the virus and a delayed immune adaptive response. Murine hepatitis virus (MHV) infection represents a unique animal model to identify new escape mechanisms in liver of innate immune responses. The objective of this study was to identify early disorders in TLRs, helicases, cytokines and chemokines favoring the development of a fulminant hepatitis. Groups of C57BL/6 WT and TLR2-/- mice were infected with highly hepatotropic MHV3 and/or weakly hepatotropic MHV-A59 viruses. Histopathological analysis of liver and mRNA expression levels of viral nucleoprotein, viral sensors, interferons, cytokines and chemokines assessed by RT-qPCR were done in the first 3 days of infection. The results showed that liver damages, viral replication and mRNA levels of TLR-2, TLR-3, RIG-1, MDA-5, IL-33, IFN-b, CXCL1, CXCL9, CXCL-10, CXCL-11, CCL3, CCL5, IL-6 and TNF-a increased higher or appeared sooner in MHV3-infected WT than in MHV-A59 and TLR2-/- mice. To address the role of hepatocytes in TLR2-dependent viral replication and innate immune factors, in vitro viral infections were performed on FL83B cells. The results showed that viral replication and mRNA levels of TLR-2 and IL-6 occurred sooner than those of other innate immune parameters. Moreover, blockade of TLR-2 by siRNA decreased IFN-b, TNF-a, CXCL-1, CXCL-10 and CCL-2 expression in infected hepatocytes and also inhibited the viral replication of MHV-A59, and at a lesser extent of MHV3, suggesting that TLR-2 signaling promotes simultaneously the viral replication and the production of innate immune factors in hepatocytes, exacerbating viral hepatitis.
Frontiers of Immunology
Milan, Italy

hal-01118155
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01118155
DOI : 10.3389/conf.fimmu.2013.02.00648

Éditeur(s) : HAL CCSD American Society for Biochemistry and Molecular Biology
Résumé : International audience
2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine formed during the combustion of tobacco. AαC undergoes bioactivation to form electrophilic N-oxidized metabolites that react with DNA to form adducts, which can lead to mutations. Many genotoxicants and toxic electrophiles react with human serum albumin (albumin); however, the chemistry of reactivity of AαC with proteins has not been studied. The genotoxic metabolites, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), 2-nitroso-9H-pyrido[2,3-b]indole (NO-AαC), N-acetyloxy-2-amino-9H-pyrido[2,3-b]indole (N-acetoxy-AαC), and their [(13)C6]AαC-labeled homologues were reacted with albumin. Sites of adduction of AαC to albumin were identified by data-dependent scanning and targeted bottom-up proteomics approaches employing ion trap and Orbitrap MS. AαC-albumin adducts were formed at Cys(34), Tyr(140), and Tyr(150) residues when albumin was reacted with HONH-AαC or NO-AαC. Sulfenamide, sulfinamide, and sulfonamide adduct formation occurred at Cys(34) (AαC-Cys(34)). N-Acetoxy-AαC also formed an adduct at Tyr(332). Albumin-AαC adducts were characterized in human plasma treated with N-oxidized metabolites of AαC and human hepatocytes exposed to AαC. High levels of N-(deoxyguanosin-8-yl)-AαC (dG-C8-AαC) DNA adducts were formed in hepatocytes. The Cys(34) was the sole amino acid of albumin to form adducts with AαC. Albumin also served as an antioxidant and scavenged reactive oxygen species generated by metabolites of AαC in hepatocytes; there was a strong decrease in reduced Cys(34), whereas the levels of Cys(34) sulfinic acid (Cys-SO2H), Cys(34)-sulfonic acid (Cys-SO3H), and Met(329) sulfoxide were greatly increased. Cys(34) adduction products and Cys-SO2H, Cys-SO3H, and Met(329) sulfoxide may be potential biomarkers to assess exposure and oxidative stress associated with AαC and other arylamine toxicants present in tobacco smoke
ISSN: 0021-9258

hal-01169796
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01169796
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01169796/file/Manuscript_KP_JBC_REV_050515_final_TC.pdf
DOI : 10.1074/jbc.M115.646539
PUBMED : 25953894

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-α) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-α-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1K45A) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1LPC-KO), to characterize the role of RIPK1 and TNF-α in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1LPC-KO mice challenged with ConA, TNF-α triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-κB activation, as well as TNF-dependent, but canonical NF-κB-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases. © The Author(s) 2016.
ISSN: 2041-4889

hal-01414916
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01414916
DOI : 10.1038/cddis.2016.362
PUBMED : 27831558

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
ISSN: 0887-2333

hal-00866007
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00866007
DOI : 10.1016/j.tiv.2013.07.003
PUBMED : 23850984

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
Heterocyclic aromatic amines (HAA) are formed in cooked meat, poultry and fish but also arise in tobacco smoke and exhaust gases. HAA are potential human carcinogens, which require metabolic activation to exert their genotoxicity. Human tissues can bioactivate HAA to produce reactive intermediates that bind to DNA. HAA DNA adduct formation occurs in human hepatocytes; however, the potential of HAA to form DNA adducts has not been investigated in human T lymphocytes. In this study, we investigated the ability of human T lymphocytes activated with PMA/Ionomycin or CD3/CD28 to express functional CYP1 activity and bioactivate three major HAA: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) to form DNA adducts. Adducts were measured by ultraperformance liquid chromatography-electrospray ionization/multistage scan mass spectrometry. The highest level of DNA adducts occurred for AαC (16 adducts per 109 nucleotides), followed by PhIP (9 adducts per 109 nucleotides). In contrast, DNA adducts formed from MeIQx and the structurally related aromatic amine 4-aminobiphenyl, a known human carcinogen, were below the limit of detection (< 3 adducts per 109 nucleotides). Moreover, we demonstrate that AαC is a potent inducer of CYP1A1 and CYP1B1 activity through a transcriptional mechanism involving the AhR pathway. Overall, our results highlight the capacity of activated human T lymphocytes to more efficiently bioactivate AαC to form DNA adducts than other prominent HAA or 4-ABP. Environ. Mol. Mutagen. 57:656-667, 2016.
ISSN: 0893-6692

hal-01400486
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01400486
DOI : 10.1002/em.22059
PUBMED : 27801952

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening.
ISSN: 0730-2312

hal-01196600
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196600
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196600/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196600/file/Increasing%203D%20Matrix%20Rigidity_accepted.pdf
DOI : 10.1002/jcb.25356

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
All-trans retinoic acid (atRA) is the active form of vitamin A, known to activate retinoid receptors, especially the heterodimer retinoid X receptor (RXR):retinoic acid receptor (RAR) that otherwise may play a role in regulation of some drug transporters. The present study was designed to characterize the nature of human hepatic transporters that may be targeted by atRA and the heterodimer RXR:RAR. Exposure of human hepatoma HepaRG cells and primary human hepatocytes to 5μM atRA down-regulated mRNA levels of various sinusoidal solute carrier (SLC) influx transporters, including organic anion transporting polypeptide (OATP) 2B1, OATP1B1, organic cation transporter (OCT) 1 and organic anion transporter (OAT) 2, and induced those of the canalicular breast cancer resistance protein (BCRP). The retinoid concomitantly reduced protein expression of OATP2B1 and OATP1B1 and activity of OATPs and OCT1 and induced BCRP protein expression in HepaRG cells. Some transporters such as OATP1B3 and the bile salt export pump (BSEP) were however down-regulated by atRA in primary human hepatocytes, but induced in HepaRG cells, thus pointing out discrepancies between these two liver cell models in terms of detoxifying protein regulation. atRA-mediated repressions of OATP2B1, OATP1B1, OAT2 and OCT1 mRNA expression were finally shown to be counteracted by knocking-down expression of RARα and RXRα through siRNA transfection in HepaRG cells. atRA thus differentially regulated human hepatic drug transporters, mainly in a RXR:RAR-dependent manner, therefore establishing retinoids and retinoid receptors as modulators of liver drug transporter expression.
ISSN: 0928-0987

inserm-00804935
http://www.hal.inserm.fr/inserm-00804935
DOI : 10.1016/j.ejps.2013.01.005
PUBMED : 23352986

Éditeur(s) : HAL CCSD American Society for Biochemistry and Molecular Biology
Résumé : International audience
2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3- b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by 1H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N 2-(β- D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N 2-Gl) and N 2-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b] indole (AαC-HON 2-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN 2-O-Gl). AαC-HN 2-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20-50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (V max/K m) at forming AαC-N 2-Gl (0.67 μl*min -1*mg of protein -1), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl*min -1*mg of protein -1), whereas UGT1A1 was most efficient at forming AαC-HON 2-Gl (5.0 μl*min -1*mg of protein -1). Human hepatocytes produced AαC-N 2-Gl and AαC-HN 2-O-Gl in abundant quantities, but AαC-HON 2-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
ISSN: 0021-9258

hal-00696860
https://hal-anses.archives-ouvertes.fr/hal-00696860
DOI : 10.1074/jbc.M111.320093
PUBMED : 22393056

Éditeur(s) : HAL CCSD American Chemical Society
Résumé : International audience
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats, is metabolically activated to electrophilic intermediates that form covalent adducts with DNA and protein. We previously identified an adduct of PhIP formed at the Cys34 residue of human serum albumin following reaction of albumin with the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP). The major adducted peptide recovered from a tryptic/chymotryptic digest was identified as the missed-cleavage peptide LQQC*[SO2PhIP]PFEDHVK, a [cysteine-S-yl-PhIP]-S-dioxide linked adduct. In this investigation, we have characterized the albumin adduction products of N-sulfooxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed in vivo. Targeted and data-dependent scanning methods showed that N-sulfooxy-PhIP adducted to the Cys34 of albumin in human plasma to form LQQC*[SO2PhIP]PFEDHVK at levels that were 8-10-fold greater than the adduct levels formed with N-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP) or HONH-PhIP. We also discovered that N-sulfooxy-PhIP forms an adduct at the sole tryptophan (Trp214) residue of albumin in the sequence AW*[PhIP]AVAR. However, stable adducts of PhIP with albumin were not detected in human hepatocytes. Instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-PhIP), a solvolysis product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis, suggesting a labile sulfenamide linkage had formed between an N-oxidized intermediate of PhIP and Cys34 of albumin. A stable adduct was formed at the Tyr411 residue of albumin in hepatocytes and identified as a deaminated product of PhIP, Y*[desaminoPhIP]TK, where the 4-HO-tyrosine group bound to the C-2 imidazole atom of PhIP.
ISSN: 0893-228X

hal-01146777
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01146777
DOI : 10.1021/acs.chemrestox.5b00075
PUBMED : 25815793

Éditeur(s) : HAL CCSD BMJ Publishing Group
Résumé : International audience
BACKGROUND: Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated. OBJECTIVE: To assess whether human HSCs are susceptible to HCV infection. DESIGN: We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs. RESULTS: In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication. CONCLUSIONS: Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process.
ISSN: 0017-5749

hal-01063909
https://hal.archives-ouvertes.fr/hal-01063909
DOI : 10.1136/gutjnl-2013-305634
PUBMED : 25063678

Éditeur(s) : HAL CCSD American Physiological Society
Résumé : International audience
The IL-33/ST2 axis plays a protective role in T-cell-mediated hepatitis, but little is known about the functional impact of endogenous IL-33 on liver immunopathology. We used IL-33-deficient mice to investigate the functional effect of endogenous IL-33 in concanavalin A (Con A)-hepatitis. IL-33(-/-) mice displayed more severe Con A liver injury than wild-type (WT) mice, consistent with a hepatoprotective effect of IL-33. The more severe hepatic injury in IL-33(-/-) mice was associated with significantly higher levels of TNF-alpha and IL-1 beta and a larger number of NK cells infiltrating the liver. The expression of Th2 cytokines (IL-4, IL-10) and IL-17 was not significantly varied between WT and IL-33(-/-) mice following Con A-hepatitis. The percentage of CD25(+) NK cells was significantly higher in the livers of IL-33(-/-) mice than in WT mice in association with upregulated expression of CXCR3 in the liver. Regulatory T cells (Treg cells) strongly infiltrated the liver in both WT and IL-33(-/-) mice, but Con A treatment increased their membrane expression of ST2 and CD25 only in WT mice. In vitro, IL-33 had a significant survival effect, increasing the total number of splenocytes, including B cells, CD4(+) and CD8(+) T cells, and the frequency of ST2(+) Treg cells. In conclusion, IL-33 acts as a potent immune modulator protecting the liver through activation of ST2(+) Treg cells and control of NK cells.
ISSN: 0193-1857

hal-01366372
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01366372
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01366372/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01366372/file/Ablation%20of%20interaction%20between%20IL-33%20and%201%20ST2.pdf
DOI : 10.1152/ajpgi.00097.2016

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
: Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures.
ISSN: 0278-6915

hal-00860153
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00860153
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00860153/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-00860153/file/Sergent-et-al-incl-figures-accepted.pdf
DOI : 10.1016/j.fct.2013.07.061

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : International audience
Interleukin-27 (IL-27) belongs to the IL-6/IL-12 family of cytokines, associated with different inflammatory diseases and orchestrates its biological activity via common heterodimeric receptor composed of WSX-1 (IL-27Rα) and gp130. The present study was aimed to investigate the regulation of CXCL9, CXCL10, and CXCL11 chemokines in hepatic cells (human LX-2 cell line derived from normal human stellate cells (HSC), primary human hepatocytes, HSC, and HepG2 cells) and concanavalin A (ConA)-induced liver inflammation. We demonstrated that IL-27, but not IL-6, induced/up-regulated CXCR3 ligand genes (CXCL9, CXCL10, and CXCL11; out of 26 selected genes) in a STAT1-dependent manner in hepatic cells in vitro both at transcript and protein levels. In ConA-induced T cell-mediated hepatic model, we showed that soluble IL-27/IFNγ was elevated following ConA hepatitis in association with increased CXCL9, CXCL10, and CXCL11 expression in the liver. The exogenous IL-27 administration induced CXCR3 ligands in mouse liver at 4 h with any significant effect on recruitment of CXCR3(+) immune cells in the liver. The neutralization of IL-27 during ConA hepatitis differentially modulated (transcript vs protein expression) CXCR3 ligands and IFNγ during ConA-induced hepatitis with down-regulated expression of CXCL9 and CXCL10 at transcript level. The IFNγ, complementary regulated the expression of CXCR3 ligands as their up-regulation during ConA hepatitis, was abolished in IFNγ KO mice. In summary, IL-27 up-regulated the CXCL9, CXCL10, and CXCL11 chemokine expression in hepatic cells. IL-27 regulated CXCR3 ligand expression in IFNγ-dependent manner during acute hepatitis suggesting a complementary role of IL-27 and IFNγ to moderate liver inflammation via regulation of CXCR3 ligands. KEY MESSAGE: IL-27 up-regulated CXCR3 ligand expression in human hepatic cells in vitro. IL-27 up-regulated CXCR3 ligand expression and secretion in ConA hepatitis in vivo. CXCR3 ligand expression was down-regulated by blocking IL-27 or IFNγ deficiency. IL-27 modulated liver injury by regulation of CXCR3 ligands in IFNγ-dependent manner.
ISSN: 0946-2716

hal-01214006
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01214006
DOI : 10.1007/s00109-015-1319-6
PUBMED : 26199110

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
Under physiological conditions, the liver sinusoidal endothelial cells (LSECs) mediate hepatic immune tolerance toward self or foreign antigens through constitutive expression of anti-inflammatory mediators. However, upon viral infection or Toll-like receptor 2 (TLR2) activation, LSECs can achieve proinflammatory functions, but their role in hepatic inflammation during acute viral hepatitis is unknown. Using the highly virulent mouse hepatitis virus type 3 (MHV3) and the attenuated variants 51.6-MHV3 and YAC-MHV3, exhibiting lower tropism for LSECs, we investigated in vivo and in vitro the consequence of LSEC infection on their proinflammatory profiles and the aggravation of acute hepatitis process. In vivo infection with virulent MHV3, in comparison to attenuated strains, resulted in fulminant hepatitis associated with higher hepatic viral load, tissue necrosis, and levels of inflammatory mediators and earlier recruitment of inflammatory cells. Such hepatic inflammatory disorders correlated with disturbed production of interleukin-10 (IL-10) and vascular factors by LSECs. We next showed in vitro that infection of LSECs by the virulent MHV3 strain altered their production of anti-inflammatory cytokines and promoted higher release of proinflammatory and procoagulant factors and earlier cell damage than infection by attenuated strains. This higher replication and proinflammatory activation in LSECs by the virulent MHV3 strain was associated with a specific activation of TLR2 signaling by the virus. We provide evidence that TLR2 activation of LSCEs by MHV3 is an aggravating factor of hepatic inflammation and correlates with the severity of hepatitis. Taken together, these results indicate that preservation of the immunotolerant properties of LSECs during acute viral hepatitis is imperative in order to limit hepatic inflammation and damage. IMPORTANCE Viral hepatitis B and C infections are serious health problems affecting over 350 million and 170 million people worldwide, respectively. It has been suggested that a balance between protection and liver damage mediated by the host's immune response during the acute phase of infection would be determinant in hepatitis outcome. Thus, it appears crucial to identify the factors that predispose in exacerbating liver inflammation to limit hepatocyte injury. Liver sinusoidal endothelial cells (LSECs) can express both anti- and proinflammatory functions, but their role in acute viral hepatitis has never been investigated. Using mouse hepatitis virus (MHV) infections as animal models of viral hepatitis, we report for the first time that in vitro and in vivo infection of LSECs by the pathogenic MHV3 serotype leads to a reversion of their intrinsic anti-inflammatory phenotype toward a proinflammatory profile as well to as disorders in vascular factors, correlating with the severity of hepatitis. These results highlight a new virus-promoted mechanism of exacerbation of liver inflammatory response during acute hepatitis.
ISSN: 0022-538X

hal-01390974
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390974
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390974/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390974/file/Mouse%20Hepatitis%20Virus%20Infection_accepted.pdf
DOI : 10.1128/JVI.01069-16

Éditeur(s) : HAL CCSD American Physiological Society
Résumé : International audience
Excessive or persistent inflammation and hepatocyte death are the key triggers of liver diseases. The poly(ADP-ribose) polymerase (PARP) proteins induce cell death and inflammation. Chemical inhibition of PARP activity protects against liver injury during concanavalin A (ConA)-induced hepatitis. In this mice model, ConA activates immune cells, which promote inflammation and induce hepatocyte death, mediated by the activated invariant natural killer T (iNKT) lymphocyte population. We analyzed immune cell populations in the liver and several lymphoid organs, such as the spleen, thymus, and bone marrow in Parp2-deficient mice to better define the role of PARP proteins in liver immunity and inflammation at steady state and during ConA-induced hepatitis. We show that 1) the genetic inactivation of Parp2, but not Parp1, protected mice from ConA hepatitis without deregulating cytokine expression and leucocyte recruitment; 2) cellularity was lower in the thymus, but not in spleen, liver, or bone marrow of Parp2-/- mice; 3) spleen and liver iNKT lymphocytes, as well as thymic T and NKT lymphocytes were reduced in Parp2 knockout mice. In conclusion, our results suggest that the defect of T-lymphocyte maturation in Parp2 knockout mice leads to a systemic reduction of iNKT cells, reducing hepatocyte death during ConA-mediated liver damage, thus protecting the mice from hepatitis.NEW & NOTEWORTHY The genetic inactivation of Parp2, but not Parp1, protects mice from concanavalin A hepatitis. Immune cell populations are lower in the thymus, but not in the spleen, liver, or bone marrow of Parp2-deficient mice compared with wild-type mice. Spleen and liver invariant natural killer T (NKT) lymphocytes, as well as thymic T and NKT lymphocytes, are reduced in Parp2-deficient mice
ISSN: 0193-1857

hal-01630440
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01630440
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01630440/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01630440/file/102513_1_merged_1499199857-1.pdf
DOI : 10.1152/ajpgi.00436.2016

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Development of hepatocellular carcinoma (HCC) is a complex and progressive disease that involves cycles of liver cell death, inflammation, and tissue regeneration/remodelling. Chemokines and chemokine receptors play numerous and integral roles in the disease progression of HCC. Here we investigated the novel chemokine receptor CXCR7/RDC1 in HCC progression, its two known ligands CXCL12 and CXCL11, as well as the other CXCL12 receptor, CXCR4. Our results show that in a cohort of 408 human HCCs, CXCR7 and CXCL11 were significantly higher in tumours compared to normal liver controls (5- and 10-fold, respectively). Immunohistochemical (IHC) staining on human HCC sections confirmed that both CXCL11 and CXCR7 were much higher in cancer tissues. Furthermore, IHC staining revealed that CXCR7 protein was only expressed in endothelial cells whereas CXCL11 exhibited a much broader tissue expression. At the cellular level we observed that in vitro, human microvascular endothelial cells (HMEC-1) up-regulated CXCR7 under hypoxic and acidic pH conditions, which are well known characteristics of the HCC tumour micro-environment. As for its ligand, we observed that IFNγ robustly induced CXCL11 in hepatic stellate cells, hepatocytes, and HMEC-1s. In addition, in the mouse Diethylnitrosamine model of hepatocarcinogenesis we observed a very strong induction of CXCR7 and CXCL11 transcripts, confirming that CXCR7/CXCL11 up-regulation is conserved between human and mice liver cancer. Altogether, our results strongly support the hypothesis that the CXCL11/CXCR7 pathway is involved HCC progression.
ISSN: 0959-8049

hal-00682463
https://hal.archives-ouvertes.fr/hal-00682463
DOI : 10.1016/j.ejca.2011.06.044
PUBMED : 21778049