Neutrophils recruitment during sepsis: Critical points and crossroads

The mechanisms that initiate an inflammatory systemic response to a bacterial infection lead to a high mortality and constitute the first cause of death in Critical Care Units (ICU`s). Sepsis is a poorly understood disease and despite life support techniques and the administration of antibiotics, not much more can be done to improve its diagnosis and treatment. The present article has as main objective to discuss the role of neutrophils recruitment in sepsis, dissecting the molecular mechanisms implicated in this complex process and its importance to the pathogenesis of this outstanding cause of death.

Gene expression reprogramming protects macrophage from septic-induced cell death

Sepsis induces a systemic inflammatory response leading to tissue damage and cell death. LPS tolerance affects inflammatory response. To comprehend potential new mechanisms of immune regulation in endotoxemia, we examined macrophage mRNA expression by macroarray affected by LPS tolerance. LPS tolerance was induced with subcutaneous administration of 1 mg/kg/day of LPS over 5 days. Macrophages were isolated from the spleen and the expression of 1200 genes was quantitatively analyzed by the macroarray technique. The tolerant group displayed relevant changes in the expression of 84 mRNA when compared to naive mice. A functional group of genes related to cell death regulation was identified. PARP-1, caspase 3, FASL and TRAIL genes were confirmed by RT-PCR to present lower expression in tolerant mice. In addition, reduced expression of the pro-inflammatory genes TNF-alpha and IFN-gamma in the tolerant group was demonstrated. Following this, animals were challenged with polymicrobial sepsis. Flow cytometry analysis showed reduced necrosis and apoptosis in macrophages from the tolerant group compared to the naive group. Finally, a survival study showed a significant reduction in mortality in the tolerant group. Thus, in the current study we provide evidence for the selective reprogramming of the gene expression of cell death pathways during LPS tolerance and link these changes to protection from cell death and enhanced survival rates. (C) 2010 Elsevier Ltd. All rights reserved.

Endotoxin tolerance: Selective alterations in gene expression and protection against lymphocyte death

Extensive lymphocyte apoptosis may be an important cause of immune suppression in sepsis. Here we investigated the effect of LPS tolerance on lymphocyte apoptosis in an experimental model of polymicrobial infection. Tolerance was induced by the injection of lipopolysaccharide (1.0 mg/kg/subcutaneously) once a day for 5 days. Macroarray analysis of mRNA isolated from T-(CD4) lymphocytes was used to identify genes that are differentially expressed during LPS tolerance. In addition, assessment of the expression of apoptosis-associated lymphocyte gene products and apoptotic events was performed on the 8th day; 6 h after the terminal challenge with polymicrobial infection or high-dose LPS administration. Survival studies with polymicrobial infection were also conducted. LPS tolerance induced a broad reprogramming of cell death pathways, including a suppression of receptor-mediated and mitochondrial apoptotic pathways, inflammatory caspases, alternate apoptotic pathways, as well as reduced expression of genes involved in necrosis. These alterations led to a marked resistance of lymphocytes against cell death during the subsequent period of sepsis. In addition, LPS tolerance produced an increased differentiation of T-lymphocytes to T(H)1 and T(H)2, with a T(H)1 differentiation predominance. Thus, in the current study we provide an evidence for a marked reprogramming of gene expression of multiple cell death pathways during LPS tolerance. These alterations may play a significant role in the observed protection of the animals from a subsequent lethal polymicrobial sepsis challenge. (C) 2009 Elsevier GmbH. All rights reserved.

Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and XRCC4 DNA repair genes in systemic lupus erythematosus

Impaired DNA repair efficiency in systematic lupus erythematosus (SLE) patients has been reported ill some studies, mainly regarding the repair of oxidative damage, but little is known about repair kinetics towards primarily single-stranded DNA breaks. In the present study, we aimed to investigate: (a) the efficiency of SLE peripheral blood leucocytes in repairing DNA damage induced by ionizing radiation and (b) the association of DNA repair gene (XRCC1 Arg399Gln, XRCC3 Thr241Met and XRCC4 Ile401Thr) polymorphisms in SLE patients, considering the whole group, or stratified sub-groups according to clinical and laboratory features. A total of 163 SLE patients and 125 healthy control were studied. The kinetics of DNA strand break repair was evaluated by the comet assay, and genotyping for DNA repair genes was performed by PCR-RFLP. Compared with controls. SLE leucocytes exhibited decreased efficiency of DNA repair evaluated at 30 min following irradiation. A significant association with DNA repair gene polymorphisms was not observed for the whole group of SLE patients; however, the XRCC1Arg399Gln polymorphism was associated with the presence of anti-dsDNA antibody. The concomitance of two DNA repair polymorphic sites was associated with the presence of neuropsychiatric manifestations and antiphospholipid antibody syndrome. Taken together, these results indicated that SLE leucocytes repair less efficiently the radiation-induced DNA damage, and DNA repair polymorphic sites may predispose to the development of particular clinical and laboratory features. Lupus (2008) 17, 988-995.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Caspase substrates and neurodegenerative diseases.

Apoptosis is induced by the cleavage of a subset of cellular proteins by proteases of the caspase family. Numerous (hundreds) caspase substrates have been described but only for a few of them is the function of their cleavage by caspases well understood. In this review, apoptosis and caspases will first be introduced. The main focus will then be directed to the caspase substrates, the actual "workers" doing the job of mediating and regulating the apoptotic process. The caspase substrates whose functions upon cleavage have been carefully investigated and those that are potentially involved in neurodegenerative diseases will be discussed in detail.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

PARP inhibition attenuates histopathological lesion in ischemia/reperfusion renal mouse model after cold prolonged ischemia.

We test the hypothesis that PARP inhibition can decrease acute tubular necrosis (ATN) and other renal lesions related to prolonged cold ischemia/reperfusion (IR) in kidneys preserved at 4°C in University of Wisconsin (UW) solution. Material and Methods. We used 30 male Parp1(+/+) wild-type and 15 male Parp1(0/0) knockout C57BL/6 mice. Fifteen of these wild-type mice were pretreated with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) at a concentration of 15 mg/kg body weight, used as PARP inhibitor. Subgroups of mice were established (A: IR 45 min/6 h; B: IR + 48 h in UW solution; and C: IR + 48 h in UW solution plus DPQ). We processed samples for morphological, immunohistochemical, ultrastructural, and western-blotting studies. Results. Prolonged cold ischemia time in UW solution increased PARP-1 expression and kidney injury. Preconditioning with PARP inhibitor DPQ plus DPQ supplementation in UW solution decreased PARP-1 nuclear expression in renal tubules and renal damage. Parp1(0/0) knockout mice were more resistant to IR-induced renal lesion. In conclusion, PARP inhibition attenuates ATN and other IR-related renal lesions in mouse kidneys under prolonged cold storage in UW solution. If confirmed, these data suggest that pharmacological manipulation of PARP activity may have salutary effects in cold-stored organs at transplantation.

Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.

Xanthine oxidase inhibitor allopurinol attenuates the development of diabetic cardiomyopathy.

In this study, we investigated the effect of the xanthine oxidase (XO) inhibitor, allopurinol (ALP), on cardiac dysfunction, oxidative-nitrosative stress, apoptosis, poly(ADP-ribose) polymerase (PARP) activity and fibrosis associated with diabetic cardiomyopathy in mice. Diabetes was induced in C57/BL6 mice by injection of streptozotocin. Control and diabetic animals were treated with ALP or placebo. Left ventricular systolic and diastolic functions were measured by pressure-volume system 10 weeks after established diabetes. Myocardial XO, p22(phox), p40(phox), p47(phox), gp91(phox), iNOS, eNOS mRNA and/or protein levels, ROS and nitrotyrosine (NT) formation, caspase3/7 and PARP activity, chromatin fragmentation and various markers of fibrosis (collagen-1, TGF-beta, CTGF, fibronectin) were measured using molecular biology and biochemistry methods or immunohistochemistry. Diabetes was characterized by increased myocardial, liver and serum XO activity (but not expression), increased myocardial ROS generation, p22(phox), p40(phox), p47(phox), p91(phox) mRNA expression, iNOS (but not eNOS) expression, NT generation, caspase 3/7 and PARP activity/expression, chromatin fragmentation and fibrosis (enhanced accumulation of collagen, TGF-beta, CTGF and fibronectin), and declined systolic and diastolic myocardial performance. ALP attenuated the diabetes-induced increased myocardial, liver and serum XO activity, myocardial ROS, NT generation, iNOS expression, apoptosis, PARP activity and fibrosis, which were accompanied by improved systolic (measured by the evaluation of both load-dependent and independent indices of myocardial contractility) and diastolic performance of the hearts of treated diabetic animals. Thus, XO inhibition with ALP improves type 1 diabetes-induced cardiac dysfunction by decreasing oxidative/nitrosative stress and fibrosis, which may have important clinical implications for the treatment and prevention of diabetic cardiomyopathy and vascular dysfunction.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Persistent infection of arenaviruses : molecular mechanisms and pathogenesis

Arenaviruses are enveloped negative single strand RNA viruses that include a number of important human pathogens. The most prevalent human pathogen among the arenaviruses is the Old World arenavirus Lassa virus (LASV) which is endemic in West Africa from Senegal to Cameroon. LASV is the etiologic agent of a severe viral hemorrhagic fever named Lassa fever whose mortality rate can reach 30% in hospitalized patients. One of the hallmarks of fatal arenavirus infection in humans is the absence of an effective innate and adaptive immune response. In nature, arenaviruses are carried by rodents which represent the natural reservoirs as well as the vectors for transmission. In their natural rodent reservoir, arenaviruses have the ability to establish persistent infection without any overt signs and symptoms of pathology. We believe that the modulation of the host cell's innate immunity by arenaviruses is a key determinant for persistence in the natural host and for the pathogenesis in man. In this thesis, we studied the interaction of arenaviruses with two main branches of the host's innate anti-viral defense, the type I interferon (IFN) system and virus-induced mitochondrial apoptosis. The arenavirus nucleoprotein (NP) is responsible for the anti-IFN activity of arenaviruses. Specifically, NP blocks the activation and the nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) which leads to type I IFN production. LASV and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) NPs contain a 3'-5'exoribonuclease domain in the C terminal part that has been linked to the anti-IFN activity of NP. In the first project, we sought to identify cellular component(s) of the type I IFN induction pathway targeted by the viral NP. Our study revealed that LCMV NP prevents the activation of IRF3 by blocking phosphorylation of the transcription factor. We found that LCMV NP specifically targets the IRF-activating kinase IKKs, and this specific binding is conserved within the Arenaviridae. We could also demonstrate that LCMV NP associates with the kinase domain of IKKs involving NP's C-terminal region. Lastly, we showed that the binding of LCMV NP inhibits the kinase activity of IKKs. This study allowed the discovery of a new cellular interacting partner of arenavirus NP. This newly described association may play a role in the anti-IFN activity of arenaviruses but potentially also in other aspects of arenavirus infection. For the second project, we investigated the ability of arenaviruses to avoid and/or suppress mitochondrial apoptosis. As persistent viruses, arenaviruses evolved a "hit and stay" survival strategy where the apoptosis of the host cell would be deleterious. We found that LCMV does not induce mitochondrial apoptosis at any time during infection. Specifically, no caspase activity, no cytochrome c release from the mitochondria as well as no cleavage of poly (ADP-ribose) polymerase (PARP) were detected during LCMV infection. Interestingly, we found that virus-induced mitochondrial apoptosis remains fully functional in LCMV infected cells, while the induction of type IIFN is blocked. Since both type IIFN production and virus- induced mitochondrial apoptosis critically depend on the pattern recognition receptor (PRR) RIG-I, we examined the role of RIG-I in apoptosis in LCMV infected cells. Notably, virus- induced mitochondrial apoptosis in LCMV infected cells was found to be independent of RIG- I and MDA5, but still depended on MAVS. Our study uncovered a novel mechanism by which arenaviruses alter the host cell's pro-apoptotic signaling pathway. This might represent a strategy arenaviruses developed to maintain this branch of the innate anti-viral defense in absence of type I IFN response. Taken together, these results allow a better understanding of the interaction of arenaviruses with the host cell's innate immunity, contributing to our knowledge about pathogenic properties of these important viruses. A better comprehension of arenavirus virulence may open new avenues for vaccine development and may suggest new antiviral targets for therapeutic intervention against arenavirus infections. - Les arenavirus sont des virus enveloppés à ARN simple brin qui comportent un grand nombre de pathogènes humains. Le pathogène humain le plus important parmi les arenavirus est le virus de Lassa qui est endémique en Afrique de l'Ouest, du Sénégal au Cameroun. Le virus de Lassa est l'agent étiologique d'une fièvre hémorragique sévère appelée fièvre de Lassa, et dont le taux de mortalité peut atteindre 30% chez les patients hospitalisés. L'une des caractéristiques principales des infections fatales à arenavirus chez l'Homme est l'absence de réponse immunitaire innée et adaptative. Dans la nature, les arenavirus sont hébergés par différentes espèces de rongeur, qui représentent à la fois les réservoirs naturels et les vecteurs de transmission des arenavirus. Dans leur hôte naturel, les arenavirus ont la capacité d'établir une infection persistante sans symptôme manifeste d'une quelconque pathologie. Nous pensons que la modulation de système immunitaire inné de la cellule hôte par les arenavirus est un paramètre clé pour la persistance au sein de l'hôte naturel, ainsi que pour la pathogenèse chez l'Homme. L'objectif de cette thèse était d'étudier l'interaction des arenavirus avec deux branches essentielles de la défense antivirale innée de la cellule hôte, le système interféron (IFN) de type I et l'apoptose. La nucléoprotéine virale (NP) est responsable de l'activité anti-IFN des arenavirus. Plus spécifiquement, la NP bloque 1'activation et la translocation nucléaire du facteur de transcription IRF3 qui conduit à la production des IFNs de type I. La NP du virus de Lassa et celle du virus de la chorioméningite lymphocytaire (LCMV), l'arénavirus prototypique, possèdent dans leur extrémité C-terminale un domaine 3'-5' exoribonucléase qui a été associé à l'activité anti-IFN de ces protéines. Dans un premier projet, nous avons cherché à identifier des composants cellulaires de la cascade de signalisation induisant la production d'IFNs de type I qui pourraient être ciblés par la NP virale. Nos recherches ont révélé que la NP de LCMV empêche 1'activation d'IRF3 en bloquant la phosphorylation du facteur de transcription. Nous avons découvert que la NP de LCMV cible spécifiquement la kinase IKKe, et que cette interaction spécifique est conservée à travers la famille des Arenaviridae. Notre étude a aussi permis de démontrer que la NP de LCMV interagit avec le domaine kinase d'IKKe et que l'extrémité C-terminale de la NP est impliquée. Pour finir, nous avons pu établir que l'association avec la NP de LCMV inhibe l'activité kinase d'IKKe. Cette première étude présente la découverte d'un nouveau facteur cellulaire d'interaction avec la NP des arenavirus. Cette association pourrait jouer un rôle dans l'activité anti-IFN des arénavirus, mais aussi potentiellement dans d'autres aspects des infections à arénavirus. Pour le second projet, nous nous sommes intéressés à la capacité des arénavirus à éviter et/ou supprimer l'apoptose mitochondriale. En tant que virus persistants, les arénavirus ont évolué vers une stratégie de survie "hit and stay" pour laquelle l'apoptose de la cellule hôte serait néfaste. Nous avons observé qu'à aucun moment durant l'infection LCMV n'induit l'apoptose mitochondriale. Spécifiquement, aucune activité de caspase, aucune libération mitochondriale de cytochrome c ainsi qu'aucun clivage de la polymerase poly(ADP-ribose) (PARP) n'a été détecté pendant l'infection à LCMV. Il est intéressant de noter que l'apoptose mitochondriale induite par les virus reste parfaitement fonctionnelle dans les cellules infectées par LCMV, alors que l'induction de la réponse IFN de type I est bloquée dans les mêmes cellules. La production des IFNs de type I et l'apoptose mitochondriale induite par les virus dépendent toutes deux du récepteur de reconnaissance de motifs moléculaires RIG-I. Nous avons, par conséquent, investigué le rôle de RIG-I dans l'apoptose qui a lieu dans les cellules infectées par LCMV lorsqu'on les surinfecte avec un autre virus pro-apoptotique. En particulier, l'apoptose mitochondriale induite par les surinfections s'est révélée indépendante de RIG-I et MDA5, mais dépendante de MAVS dans les cellules précédemment infectées par LCMV. Notre étude démontre ainsi l'existence d'un nouveau mécanisme par lequel les arénavirus altèrent la cascade de signalisation pro-apoptotique de la cellule hôte. Il est possible que les arénavirus aient développé une stratégie permettant de maintenir fonctionnelle cette branche de la défense antivirale innée en l'absence de réponse IFN de type I. En conclusion, ces résultats nous amènent à mieux comprendre l'interaction des arénavirus avec l'immunité innée de la cellule hôte, ce qui contribue aussi à améliorer notre connaissance des propriétés pathogéniques de ces virus. Une meilleure compréhension des facteurs de virulence des arénavirus permet, d'une part, le développement de vaccins et peut, d'autre part, servir de base pour la découverte de nouvelles cibles thérapeutiques utilisées dans le traitement des infections à arénavirus.