Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

Les voies respiratoires sont exposées à une panoplie de pathogènes. Lors d’une infection virale respiratoire les cellules qui recouvrent ces voies participent activement à la défense immunitaire contre ces derniers en limitant la propagation du virus et en engendrant une réponse proinflammatoire. Un évènement clef dans ces processus est l’activation des facteurs de transcription, notamment le « Nuclear Factor » (NF)-κB et l’« Interferon Regulatory Factor -3 » (IRF-3), qui régulent l’expression des cytokines antivirales et proinflammatoires. Des données récentes démontrent que les dérivés actifs de l’oxygène (ROS), produits suite à une infection virale, ont la capacité de réguler les voies de signalisation enclenchées par NF-κB et IRF-3. Une source importante de ROS est la famille de NADPH oxydases (NOX), qui contient les membres NOX1-5 et DUOX1 et 2. L’objectif de notre étude était d’identifier la NOX qui régule les mécanismes antiviraux et proinflammatoires suite à l’infection avec le virus respiratoire syncytial (RSV), qui cause des complications respiratoires majeures, et le virus Sendai (SeV), un modèle viral non-pathogène. Nos travaux ont permis d’identifier que NOX2 est une molécule clef dans la réponse proinflammatoire suite à l’infection virale. Plus spécifiquement, NOX2 est important pour l’activation de NF-κB et la sécrétion des cytokines régulées par ce dernier. De plus, nous avons observé une forte augmentation de la présence de DUOX2 dans les cellules de voies respiratoires humaines infectées par SeV. Une étude plus approfondie nous a permis de caractériser qu’une synergie entre deux cytokines secrétées lors de l’infection, soit l’interféron (IFN)β et le TNFα est responsable de l’induction de DUOX2. Nous avons aussi découvert que DUOX2 confère une activité antivirale et est nécessaire pour maintenir les taux des cytokines antivirales tardives IFNβ et IFNλ. Lors d’une infection avec RSV, l’induction de DUOX2 n’est pas détectable. Nous avons mis en évidence que RSV interfère avec l’expression de DUOX2 ce qui pourrait suggérer sa pathogénicité. En conclusion, nos travaux démontrent pour la première fois une implication spécifique des NADPH oxydase NOX2 et DUOX suite aux infections virales respiratoires. / The mucosal linings of the airways are constantly exposed to an array of microbial pathogens. During the course of respiratory viral infection, Airway epithelial cells (AEC) actively participate in the innate antiviral immune response by limiting the spread of respiratory viruses and by fostering a proinflammatory environment that attracts and activates players of the immune system. A key step in the establishment of the antiviral and proinflammatory state is the activation of Transcription Factors (TFs), such as Nuclear Factor (NF)-κB and Interferon Regulatory Factor 3 (IRF-3), which regulate the expression of antiviral and proinflammatory cytokines. For the efficient functioning of these events, the signaling pathways involved underlie strict regulatory mechanisms. Recent data suggest that Reactive Oxygen Species (ROS), which are produced upon viral infection, are able to regulate these intracellular signaling pathways. One important source of ROS is the NADPH oxidase (NOX) family of enzymes, which is composed of NOX1-5 and Dual Oxidase (DUOX) 1 and DUOX2. The aim of our study was to identify the NADPH oxidase(s) that regulate(s) antiviral and proinflammatory mechanisms following infection of AEC with Respiratory syncytial virus (RSV), which causes major human lower respiratory tract complications, and Sendai virus (SeV), a non pathogenic virus. During the course of our studies we identified that NOX2 is a key molecule in the early proinflammatory response to RSV and SeV infection. We demonstrate that NOX2 is necessary for the activation of NF-κB. Consequently, NOX2 impacts on the proinflammatory cytokine secretion upon AEC infection. Further, we observed that expression of the ROS-generating NADPH oxidase DUOX2 is strongly increased following infection of AEC with SeV. We identified that DUOX2 induction requires the synergistic stimulation by IFNβ and TNFα. Importantly, DUOX2 exhibited ROS-dependent antiviral action. We identified that DUOX2 was necessary for sustaining the levels of late antiviral cytokines IFNβ and IFNλ. When AEC were infected with RSV, DUOX2 expression was barely detectable. Our data reveal that RSV has developed an evasion mechanism to counteract DUOX2 induction likely contributing to RSV pathogenicity. In conclusion, our work demonstrates for the first time the specific implication of NOX2 and DUOX2 in the antiviral and proinflammatory response to respiratory virus infection.

Profound lymphopenia has been observed during many acute viral infections, and our laboratory has previously documented a type 1 IFN-dependent loss of most memory (CD44hi) and some naïve (CD44lo) CD8 T cells immediately preceding the development of the antiviral T cell response at days 2-4 following lymphocytic choriomeningitis virus (LCMV) infection. In this thesis, I will examine additional mechanisms involved in the early attrition of CD8 T cells and evaluate whether antigen-specific and non-specific CD8 T cells are equally susceptible. Lastly, I will examine whether the early attrition of CD8 T cells contributes to the generation of an effective immune response. Poly(I:C), a potent inducer of type 1 IFN, was previously shown to cause the attrition and apoptosis of CD8α+CD44hi cells in normal mice, but not in type 1 IFN receptor–deficient mice (IFN1-R KO). I questioned whether additional molecule(s) might contribute to the type 1 IFN-induced apoptosis of CD8α+CD44hi cells. I used a PCR array to determine the expression of 84 apoptosis-related genes at 6 hours post-poly(I:C) treatment, relative to an untreated control. There was an 11-fold increase in CD40 RNA expression in CD8α+CD44hi cells isolated from poly(I:C)-treated mice. CD40 protein expression was also increased on CD8α+CD44hi cells, peaking between 9 and 12 hours following poly(I:C) treatment, before declining thereafter. This increase in CD40 protein expression directly correlated with an increase in Annexin V reactivity, an indicator of early apoptosis. Nevertheless, CD40 was not required for the loss of CD8α+CD44hi cells, as both wildtype and CD40-deficient mice were equally susceptible to the poly(I:C)-induced attrition. Upon further characterization, I found this population of CD40+CD8α+CD44hi cells to be CD11c+B220-Thy1.2- MHCIIhi, which is consistent with a “lymphoid” CD8α+ DC phenotype. Kinetic analysis revealed a type 1 IFN-dependent increase in this CD8α+ DC population at 12 hours post-poly(I:C) treatment. This increase was only observed in the spleen, as no increase in percentage was observed in the peritoneal cavity (PEC), lungs, inguinal lymph nodes (iLN), or peripheral blood. Collectively, these results suggest that the type 1 IFN-dependent increase in splenic CD8α+DCs accounts for the observed increase in Annexin V reactive cells following poly(I:C) treatment. These findings required a re-evaluation of the type 1 IFN-induced attrition of CD8+CD44hi T cells with an anti-CD8β antibody, which is a more exclusive marker for T cells than the anti-CD8α antibody. Kinetic analysis revealed a significant decrease in splenic CD8β+CD44hi T cells at 12 hours post-poly(I:C) treatment. This reduction in splenic CD8β+CD44hi T cells was not due to trafficking to other organs, as the PECs, lungs, iLN, lungs, and peripheral blood all exhibited significant, although varying, decreases in the percentage of CD8β+CD44hi T cells at 12 hour following poly(I:C) treatment. These data support the notion that the type 1 IFN-induced attrition of CD8β+CD44hiT cells was a “global” phenomenon and could not be completely due to migration out of the spleen. The attrition of CD8β+CD44hi T cells was also dependent upon type 1 IFN at 3 days post-LCMV infection, as there was no significant reduction of this population in IFN1-R KO mice. The loss of wildtype CD8β+CD44hi T cells correlated with an increased activation of caspases 3 and 8, which are enzymes that play essential roles in apoptosis and inflammation. A significant loss of CD4+CD44hi T cells, which also correlated with an increased activation of caspases 3 and 8, was observed at 3 days post-LCMV infection. Collectively, these results suggest that attrition of both CD4+CD44hi and CD8β+CD44hiT cell populations is type 1 IFN-dependent and associated with the activation of caspases following LCMV infection. At 3 days post-LCMV infection, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had a higher frequency of cells with fragmented DNA, a hallmark characteristic of the late stages of apoptosis, as revealed by terminal transferase dUTP nick end labeling (TUNEL), relative to uninfected controls. This suggests that the loss of both populations was due to apoptosis. Therefore, I questioned whether the LCMV-induced apoptosis of both CD4+CD44hi and CD8β+CD44hi T cell populations occurred through a mitochondrial-induced pathway involving the pro-apoptotic molecule Bim. The attrition of both CD4+CD44hi and CD8β+CD44hi T cells was significantly higher in wildtype mice compared to Bim KO mice at 3 days post-LCMV infection. Moreover, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had higher frequency of TUNEL+ cells, relative to Bim KO populations. These results suggest that the apoptosis of CD8β+CD44hi and CD4+CD44hiT cells, following LCMV infection, might occur through a mitochondrial-induced pathway involving Bim. Studies have shown “lymphoid” CD8α+ DCs to be involved in the phagocytosis of apoptotic lymphocytes. Therefore, I evaluated whether host CD8α+ DCs are capable of phagocytosing apoptotic lymphocytes by adoptively transferring CFSE-labeled wildtype donor splenocytes (Ly5.1) into congenic wildtype hosts (Ly5.2), followed by inoculation with poly(I:C). There was an increased frequency of donor cells (Ly5.1, CFSE+) within the host CD8α+CD11c+ gate at 9 and 12 hours post-poly(I:C) treatment. The results suggest that type 1 IFN-activated CD8α+DCs might aid in the rapid clearance of apoptotic cells during the type 1 IFN-induced attrition associated with viral infections. I next questioned whether TCR engagement by antigen would render CD8 T cells resistant to attrition. I tested whether a high concentration of antigen (GP33 peptide) would protect LCMV-specific naïve TCR transgenic P14 cells specific for the GP33 epitope of LCMV and GP33-specific LCMV-immune cells from depletion. Both naïve P14 and memory GP33-specific donor CD8 T cells decreased substantially 16 hours after inoculation poly(I:C), regardless of whether a high concentration of GP33 peptide was administered to host mice beforehand. The increased activation status of naïve antigen-specific cells via peptide inoculation did not confer resistance to type 1 IFN-induced depletion. Donor naïve P14 and LCMV-specific memory cells were also depleted from day 2 LCMV-infected (Clone 13) hosts by 16 hours post-transfer. These results indicate that antigen engagement does not protect CD8 T cells from the type 1 IFN-induced attrition associated with viral infections. Computer models indicated that early depletion of memory T cells may allow for the generation for a more diverse T cell response to infection by reducing the immunodomination caused by cross-reactive T cells. To test this in a biological system, I questioned whether the reduced apoptosis of the crossreactive memory CD8 population (NP205), in aged LCMV-immune mice (18-22 months), following heterologous virus challenge (PV), would allow it to dominate the immune response. At day 8 post-PV infection, the cross-reactive memory CD8 T cell response (NP205) was more immunodominating in aged LCMV-immune mice relative to younger LCMV-immune mice. This was indicated by the increased ratio of the cross-reactive NP205 response to the newly arising noncross-reactive, PV-specific NP38 response in older LCMV-mice relative to younger LCMV immune-mice, at day 8 post-PV infection. These data suggest that the early attrition of T cells allows for the generation of a more diverse T cell response to infection by reducing the immunodomination caused by crossreactive T cells. Collectively, these findings offer further insight into the early attrition of T cells associated with viral infections.

Dans les patients infectés par HBV, les thérapies avec les analogues de nucléos(t)ides (NAs) ou l'interféron α (IFNα) restent inefficaces pour éradiquer l'infection, à cause d'une forme persistante d'HBV, appelée l'ADN circulaire covalent clos (ADNccc), organisé comme un mini-chromosome. Notre but a été de revisiter l'activité anti-HBV d'un panel de cytokines in vitro en utilisant des hépatocytes non transformés, afin d'identifier de nouvelles options immunothérapeutiques. Parmi toutes les molécules testées, l'IFNβ, l'IFNγ, les IFNλ, le TNFα, l'IL-6, l'IL-1β et le ténofovir (ce dernier utilisé comme contrôle positif) ont montré un effet suppresseur sur la réplication d'HBV aussi fort et parfois plus fort que l'IFNα. La cytokine ayant l'effet le plus élevé sur l'ADN total d'HBV (EC50 ≈ 25 pg/mL), sans cytotoxicité, était l'interleukine-1β (IL-1β), qui est naturellement produite par les cellules de Kupffer (KC), les macrophages du foie. De façon importante, les ARNs totaux d'HBV et l'antigène sécrété HBeAg, mais pas HBsAg, ni l'ADNccc, sont fortement diminués par l'IL-1β. Nous avons donc émis l'hypothèse selon laquelle des promoteurs viraux spécifiques su l'ADNccc pourraient être inhibés, même si l'ADNccc n'est pas dégradé. Ensuite, nous avons étudié le mécanisme de l'activité antivirale de l'IL-1β. Nous avons montré que tous les promoteurs d'HBV sembleraient être inhibés par l'IL-1β. En parallèle, nous avons vérifié que l'IL-1β pouvait activer le promoteur de NF-κB, dont la fonction de transcription a été confirmée. Grâce à cette étude, l'IL-1β a été montré comme ayant un effet antiviral très efficace contre HBV in vitro, par l'intermédiaire de la fixation de NF-κB sur l'ADNccc / In HBV-infected patients, therapies with nucleos(t)ide analogues (NAs) or interferon α (IFNα) remain ineffective in eradicating the infection, because of a persistent form of HBV DNA, namely the covalently closed circular DNA (cccDNA), which is organized as a minichromosome. Our aim was to revisit the anti-HBV activity of a panel of IFNs and pro-inflammatory cytokines in vitro using nontransformed cultured hepatocytes of HBV infection, to identify new immunotherapeutic options. Amongst all molecules tested, IFNβ, IFNγ, IFNλ, TNFα, IL-6, IL-1β and tenofovir showed a suppressive effect on HBV replication at least as strong as, but sometimes stronger than IFNα. The cytokine showing the highest effect on intracellular total HBV DNA without any cytotoxicity, was interleukin-1β (IL-1β), which is naturally produced by Kupffer cells (KC), representing the macrophages of the liver. Importantly, total HBV RNAs and secreted HBeAg, but nor HBsAg, neither cccDNA, were strongly decreased. Thus, we hypothesized that even if cccDNA was not degraded, specific viral promoters on cccDNA could be silenced. Then, we investigated the mechanism of IL-1β antiviral activity. We have shown that all HBV promoters were early inhibited by IL-1β. In the meantime, we have verified that IL-1β can induce nuclear Translocation and expression of NF-κB. We also checked NF-κB functionality. Thanks to this study, IL-1β has been found to have very potent antiviral effect against HBV in vitro, through the binding of NF-κB on cccDNA

Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

L’immunité innée antivirale repose en grande partie sur la production des interférons de type I (IFN-α/β) par les cellules infectées et les cellules immunitaires. Cette synthèse résulte de la reconnaissance de motifs viraux caractéristiques par des récepteurs cellulaires, parmi lesquels les RIG-I-Like Récepteurs (RLR) et le Toll-Like Récepteur 3 (TLR3) détectent l’ARN viral respectivement au niveau du cytosol et des endosomes. La signalisation induite par les RLRs et TLR3 conduit à l’activation d’IRF3 et de NF-κB, deux facteurs de transcription impliqués respectivement dans la production d’IFN-α/β et de cytokines pro-inflammatoires. TBK1 (TANK-Binding Kinase 1) joue un rôle essentiel dans l’immunité innée antivirale, de par la phosphorylation du facteur de transcription IRF3, nécessaire à la production des IFNs de type I. Bien que de nombreuses études aient montré le rôle crucial de cette kinase dans la signalisation antivirale, le processus entrainant son activation est encore mal déterminé à ce jour. Lors de cette étude nous avons démontré que suite à la stimulation du TLR3 et des RLRs, la forme active, ubiquitinylée et phosphorylée, de TBK1 se relocalise au niveau de l’appareil de golgi, grâce à son ubiquitination sur les résidus K30 et K401. Ce mécanisme implique la reconnaissance des chaines d’ubiquitines associées à TBK1 par l’Optineurine (OPTN), permettant la formation d’un complexe autorisant le rapprochement des molécules de TBK1 puis la trans-autophosphorylation au niveau de l’appareil de Golgi. Au cours de ce travail nous avons également découvert qu’OPTN est la cible d’une protéine virale, la protéine NS3 du BTV (Bluetongue Virus), qui neutralise son activité et diminue ainsi l’activation de TBK1 et la signalisation responsable de la sécrétion de cytokines antivirales. / Type-I interferons (IFN-α/β) production and release is a major event in innate antiviral immunity. IFN production depends on the interaction between viral structures and their corresponding cellular sensors. RIG-I-Like Receptors (RLRs) and Toll-Like Receptor 3 (TLR3) sense dsRNAs in the cytosol and endosomes respectively. Stimulation of these receptors by their ligands promotes a signal transduction leading to the activation of the transcription factors NF-κB and IRF3, and consequently to the production of proinflammatory cytokines and Type I Interferons (IFN-I). TBK1 (TANK-Binding Kinase 1), plays a crucial role in antiviral innate immunity, by phosphorylating the transcription factor IRF3, required for the production of type I IFNs. Although many studies have shown the critical role of this kinase in antiviral signaling, the molecular mechanism of its activation are largely unknown. We report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RLRs or TLR3, due to TBK1 ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. We also found that a viral protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling.

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L’activation des lymphocytes T nécessite un double signal. Le premier est antigénique et permet la reconnaissance d’un peptide spécifique présenté à la surface de cellules présentatrices d’antigène (APC). Le second signal est co-stimulateur et implique l’interaction avec des molécules activatrices exprimées par les APC et la présence de cytokines proinflammatoires. Les cellules dendritiques (DC) sont les uniques APC capables de délivrer ce double signal et d'activer les lymphocytes T naïfs, initiant ainsi les réponses immunes primaires. L’immaturité du système immunitaire du nouveau-né est responsable d’une plus grande susceptibilité aux maladies infectieuses ainsi qu’une faible réponse vaccinale. Des déficiences tant au niveau de l’immunité innée que de l’immunité acquise participe à une faible défense face aux agressions. A la naissance, les DC expriment des niveaux faibles de molécules co stimulatrices et présentent un défaut majeur de synthèse d'IL 12, cytokine cruciale pour l’établissement de réponses de type Th1. Le but de ce travail est d’évaluer la capacité des DC du nouveau-né humain à activer les lymphocytes T CD8+.<p>Dans une première approche, nous avons utilisé un modèle unique d’induction de réponse primaire in vitro qui permet d'étudier l'activation de lymphocytes T CD8+ spécifiques de l’antigène Melan-A, une protéine du soi exprimée par les mélanocytes. Ces lymphocytes existent à des fréquences particulièrement élevées chez les individus sains HLA-A2 et présentent les caractéristiques de lymphocytes T naïfs. Dans ce modèle, nous avons d’abord analysé les capacités immunostimulatrices de différentes populations de DC différenciées in vitro. Nous avons observé que les DC différenciées par la culture de monocytes purifiés en présence d'IL-3 et d’IFN-beta sont capables d’initier une réponse fonctionnelle des lymphocytes T CD8+, analogue à celle induite par les DC différenciées en présence de GM-CSF et d’IL-4. Ce même modèle nous a permis de démontrer que, en dépit de leur défaut de production d’IL 12, les DC du nouveau-né sont capables d'induire efficacement une réponse lymphocytaire T CD8+ cytotoxique.<p><p>Afin dévaluer la relevance in vivo de nos observations, nous avons étudié le phénotype et la fonction des DC circulantes chez des nouveau-nés infectés par le cytomégalovirus (CMV). L’infection par le CMV au cours de la vie fœtale représente une situation clinique où le nouveau-né développe une réponse mature et fonctionnelle des lymphocytes T CD8+, alors que celle des lymphocytes T CD4+ est déficiente. Ces expériences ont montré que le phénotype, la fonction et la réponse à différents stimuli des APC présentes en périphérie ne sont pas affectés par l’infection congénitale par le CMV. Ces résultats suggèrent que l’observation des DC circulantes des nouveau-nés infectés par le CMV ne permet pas d’analyser l’influence du virus sur la fonction des DC néonatales. Dans ce but, nous avons reproduit un modèle d’infection in vitro de DC par une souche primaire du CMV. L’utilisation de micropuces à ADN nous a permis de comparer l’expression de gènes différentiellement induits par l’infection des DC d’adultes et de nouveau-nés. Nous avons ainsi révélé une proportion importante de gènes différentiellement induits, parmi lesquels celui de l’IFN-beta. Nous avons confirmé ce défaut au niveau protéique et mis en évidence une production d’IL 12 déficiente en réponse à l’infection par CMV.<p>L’ensemble de nos résultats indique que malgré leur immaturité, les DC du nouveau-né sont capables, dans certaines circonstances, d’induire une réponse lymphocytaire T CD8+ cytotoxique. Cependant, le défaut de production de certaines cytokines co-stimulatrices pourrait être impliqué dans la faible réponse des lymphocytes T CD4+ à l’infection par CMV. Ces observations ont d’importantes implications pour la compréhension de l’induction de réponses cytotoxiques au cours d’infections virales et pour l’élaboration de stratégies vaccinales en début de vie.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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