No description available.

Double stranded RNA (dsRNA), the genomic material of some viruses and a replication intermediate in others, is recognized by multiple signaling receptors that initiate the anti-viral response1. Viruses have developed mechanisms to circumvent the anti-viral response by targeting components of the signaling pathway. An example of one such pathway is the TLR3 signaling pathway, which contains a kinase complex that activates interferon regulatory factor 3 (IRF3), leading to production of type I interferons. The kinase complex consists of a scaffold protein, NAK-associated protein 1 (NAP1), and two kinases, TANK-binding kinase 1 (TBK-1) and IκB kinase epsilon (IKKε). A fourty residue sequence in NAP1 was discovered that mediated its interaction with TBK1 and IKKε, termed the kinase binding domain (KBD)1. However, the function of NAP1 in mediating kinase activation is unknown and understanding this is the long-term goal of this project. The goal of this thesis was to test the dependency of NAP1’s dimeric structure on mediating interactions with the kinases. Biochemical characterization of recombinant targets was completed using size-exclusion chromatography (SEC) and NAP1 KBD WT eluted as a dimeric species. CFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 KBD and scaffold binding motif (SBM) of the kinases, TBK-1 and IKKε, were generated to assess interactions using fluorescence resonance energy transfer (FRET). NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis was attempted to identify the residues necessary for NAP1 dimerization and any effect dimerization may have on kinase recognition. This thesis shows data to support that NAP1 KBD forms stable homo-oligomers and directly interacts with a small C-terminal portion of TBK1 and IKKε.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

L'encéphalite herpétique (EH), causée par l'herpès simplex virus-1 (HSV-1), peut résulter de défauts monogéniques de l'immunité médiée par TLR3. L'induction d'interférons (IFNs)-α/β ou -λ via TLR3 est cruciale à la protection après infection primaire avec HSV-1 dans le système nerveux central (SNC). Nous décrivons deux enfants avec l'EH portant différentes mutations hétérozygotes (D50A et G159A) dans TBK1, encodant TANK-Binding Kinase 1, une kinase aux carrefours de multiples voies de signalisation induisant des IFNs. Les deux allèles mutants de TBK1 sont perte-de-fonction par des mécanismes différents: instabilité de la protéine (D50A) ou perte d'activité kinase (G159A). Ces allèles sont associés à un trait autosomal dominant (AD) par des mécanismes différents: haplotype-insuffisance (D50A) ou dominance négative (G159A). Un défaut de réponses à poly(I:C) par TLR3 est observable dans les fibroblastes hétérozygotes pour G159A, et non pour D50A TBK1. Néanmoins, la réplication virale et la mortalité cellulaire après infection par deux virus dépendants de TLR3 (HSV-1 et VSV) étaient élevées dans les fibroblastes des deux patients. Ces phénotypes peuvent être sauvés par IFN-α2b. De plus, la production d'IFNs en réponse à des agonistes et virus indépendants de TLR3 est maintenue dans les PBMCs et fibroblastes des patients. Le phénotype cellulaire restreint, partiel représente ainsi le phénotype clinique de ces patients, limité à l'EH. Ces données identifient la déficience partielle AD de TBK1 comme une nouvelle étiologie génétique de l'EH de l'enfance, et indiquent que TBK1 est essentiel pour le contrôle de HSV-1 dans le SNC, médié par TLR3 et dépendant des IFNs

Orientador: Prof. Dr. Daniel Carneiro Carrettiero / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2015. / Doenca de Alzheimer (DA) e uma doenca neurodegenerativa progressiva provocada por perda de sinapses e neuronios, caracterizado por disfuncao da memoria e comprometimento cognitivo global. O anormalmente hiperfosforilada, insoluvel, a proteina tau filamentosa mostrou-se ser o componente principal de emaranhados neurofibrilares, uma caracteristica patologica da DA. Neuroinflamacao e o indutor secundario resposta patologica a deposicao de A e morte celular neuronal associada com DA. Pesquisas documentaram o envolvimento de receptores Toll-like (TLRs) na neuroinflamacao, contribuindo para a patogenese da DA. Todos os TLR ativam as vias inflamatorias conservadas, ativando adicionalmente o Fator Nuclear-kB (NF-kB), resultando na libertacao e a producao de citocinas pro-inflamatorias. Entre a familia TLR, varios estudos indicaram o envolvimento de TLR3 e TLR4 contribuindo para neuroinflamacao. Alem disso, um corpo de dados indica o envolvimento de BAG2 na degradacao da tau fosforilada. Neste contexto, foi investigado o papel de TLR3 e TLR4 na modulacao da BAG2 promovida a degradacao de p-Tau em celulas SH-SY5Y. Neste estudo utilizamos LPS e pIpC para ativar TLR4 e TLR3 respectivamente. Linha de celulas SH-SY5Y foi usadae estabelecendo que o Tau induza a formacao de microtubulos sem limites que esta de acordo com a sua funcao como uma proteina associada amicro tubulos em DA. E tambem, utilizaram-se as celulas SH-SY5Y indiferenciadas uma vez que a expressao de BAG2 e inferior e indiferenciada, em comparacao com celulas diferenciadas. Neste estudo, a ativacao de TLR3 com diferentes concentracoes dem pIpC (1, 10, 50 e 100¿Êg/ml) resultou na regulacao negativa significativa de p-Tau em BAG2 expressao excessiva. Por outro lado, a ativacao de TLR4 com diferentes concentracoes de LPS (10, 50 e 100¿Êg/ml) resultaram em diminuicao dependente da dose na BAG2 expressao excessiva. Alem disso, usou-se JSH-23 para a inibicao de NF-kB. JSH-23 a uma concentracao de 25¿ÊM mostraram regulacao negativa significativa em p-Tau independente do BAG2 expressao excessiva e/ou a estimulacao de TLR3 e TLR4. Inibicao de NF-kB com JSH-23 25¿ÊM resultou na supra regulacao de transcricao significativa BAG2 endogena resultando na degradacao de ptau. Em contraste, a inibicao do NF-kB resultou na supra regulacao de HMW p-tau. BAG2 exogeno sobre a estimulacao TLR4 resultou nodecrescimo da regulacao de HMW p-tau. No entanto JSH-23 endogena induzida BAG2 falhou para regular negativamente HMW p-tau. A ativacao de TLR3/TLR4 nao mostrou toxicidade celular, nem a fosforilacao tau nas celulas SHSY5Y. Alem disso, a estimulacao por LPS na presenca/ausencia de sobre expressao BAG2 nao participa na proliferacao celular. No geral, os resultados demonstram a modulacao BAG2 em TLR4 e TLR3 estimulacao e inibicao de NF-kB degradacao em p-Tau e ou HMW p-Tau em DA. / Alzheimer¿s disease (AD) is a neurodegenerative disorder caused by progressive loss of synapses and neurons, characterized by memory dysfunction and global cognitive impairment. The abnormally hyperphosphorylated, insoluble, filamentous tau protein was shown to be the main component of NFTs, a pathological hallmark of AD. Neuroinflammation is the pathological secondary inducer response to deposition of A and neuronal cell death associated with AD. Considerable research has documented the involvement of Toll-like receptors (TLRs) in neuroinflammation, contributing to the pathogenesis of AD. All TLRs activate conserved inflammatory pathways, further activating nuclear factor-kB (NF-kB) resulting in the release and production of pro-inflammatory cytokines. Among the TLRs family, several studies have indicated the involvement of TLR3 and TLR4 contributing to neuroinflammation. Furthermore, a body of data indicates the involvement of BAG2 in phosphorylated Tau degradation. In this context we investigated the role of TLR3 and TLR4 in modulation of BAG2 promoted p-Tau degradation in SH-SY5Y cells. In this study we used LPS and pIpC to activate TLR4 and TLR3 respectively. SH-SY5Y cell line was used since it is established that the Tau induces the formation of microtubule bundles which is in accord with its function as a microtubule-associated protein in AD. And also, we used undifferentiated SH-SY5Y cells since the expression of BAG2 is lower in undifferentiated as compared to differentiated cell. In this study, activation of TLR3 with different concentration of pIpC (1, 10, 50 and 100ìg/mL) resulted in significant downregulation of p-Tau on BAG2 overexpression. On the other hand, activation of TLR4 with different concentration of LPS (10, 50 and 100ìg/mL) resulted in dose dependent decrease on BAG2 overexpression. . Further we used JSH-23 for NF-kB inhibition. JSH-23 at a concentration of 25ìM showed significant downregulation in p-Tau independent of BAG2 overexpression and/or on TLR4 and TLR3 stimulation. NF-kB inhibition with JSH-23 25ìM resulted in significant upregulation of endogenous BAG2 transcript resulting in p-Tau degradation. In contrast, NF-kB inhibition resulted in upregulation of HMW p-Tau. Exogenous BAG2 on TLR4 stimulation resulted in downregulation of HMW p-Tau. However JSH-23 induced endogenous BAG2 failed to downregulate HMW p-Tau. Activation of TLR3/TLR4 neither showed cell toxicity nor Tau phosphorylation in SH-SY5Y cells. Further LPS stimulation in presence/absence of BAG2 overexpression does not participate in cell proliferation. Overall, our findings demonstrate BAG2 modulation on TLR4 and TLR3 stimulation, and NF-kB inhibition in p-Tau and/or HMW p-Tau degradation in AD.

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Toll-like receptor (TLR)-mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre-treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)-induced protection was lost in TLR3 knockout mice. In poly (I:C)-treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF-κB activation in the brain. Poly (I:C) also decreased I/R-induced apoptosis by attenuation of Fas/FasL-mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase-3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)-induced interaction between Fas and FADD as well as caspase-3 and -8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co-association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase-3 and -8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.

No description available.

Les virus influenza aviaires faiblement pathogènes (IAFP) ciblent principalement les épithéliums des voies respiratoires et intestinales chez les poulets (Gallus gallus) infectés. Cependant, les virus influenza aviaires hautement pathogènes (IAHP) mènent à une maladie systémique fatale avec une localisation particulière aux endothéliums. L’objectif de cette thèse a été d’explorer les relations entre la réplication des virus influenza aviaires (IA) et la réponse antivirale de l’hôte dans deux modèles cellulaires originaux obtenus chez le poulet : des cellules épithéliales pulmonaires (CLEC213) et des cellules endothéliales d’aortes (chAEC). Les résultats clés sont les suivants : (i) la réplication productive des virus IA dans les chAEC dépend du clivage de l’hémagglutinine et de l’échappement viral à la réponse immunitaire innée ; (ii) les CLEC213 sont très permissives aux virus IA et présentent une faible réponse antivirale médiée par la signalisation TLR3 et MDA5 ; (iii) les fonctions régulatrices de SOCS1 et SOCS3, sur le signal des interférons et des cytokines, sont conservées chez le poulet. Nous proposons que certains virus IA peuvent exploiter les fonctions pro-virales de SOCS1 et SOCS3 à leur avantage de manière spécifique au type cellulaire. / Low pathogenic avian influenza (LPAI) viruses essentially target the epithelia of the respiratory and intestinal tract in the infected chicken host (Gallus gallus). However, highly pathogenic avian influenza (HPAI) viruses induce a peracute fatal systemic disease and exhibit a striking endothelial cell tropism. The objective of the present thesis was to explore the interdependencies of AI virus replication and the antiviral host response in two novel avian cell culture models: chicken lung epithelial cells (CLEC213) and chicken aortic endothelial cells (chAEC). The salient findings from this study are that (i) productive AI virus replication in chAEC is dependent on hemagglutinin cleavability and appears to be related to innate immune escape; (ii) CLEC213 are highly permissive to AI virus infection, due to a cell type-specific diminished TLR3- and/or MDA5-mediated antiviral signaling response; (iii) the interferon and cytokine regulatory functions of SOCS1 and SOCS3 are conserved in the chicken. Based on our data, we propose a model that predicts that certain AI viruses may exploit the proviral functions of SOCS1 and SOCS3 in a cell type-specific manner.

EHV-1 is a double-stranded DNA virus whereas EAV is a positive sense, single-stranded RNA virus. Therefore, genetically, they are very different from one another. However, both these viruses are endotheliotropic and thus, infect and replicates in equine endothelial cells resulting in vasculitis. Vasculitis is central to the pathogenesis of these two viruses. Thus, the main objective of this thesis was to investigate the inflammatory and innate immune responses of EECs that contribute towards the development of vasculitis following infection with EHV-1 and EAV in-vitro. Since proinflammatory cytokines and chemokines produced by endothelial cells play a significant role in the development of vasculitis, we investigated their gene expression as well as secretion. Results from this study showed that the proinflammatory response of EECs induced by EAV is relatively less when compared with the corresponding results from EHV-1 infected EECs. Furthermore, EAV elicits a lower type I interferon response in EECs when compared with EHV-1. Further investigations revealed an active role played by TLR 3 in inducing the proinflammatory response in EHV-1 infected EECs during the first 6 hours of infection but not in EAV infected EECs. Analyzing the whole transcriptome of EHV-1 and EAV infected EECs revealed a complex pattern of gene regulation and cellular pathways related to cellular immune, inflammatory and apoptotic responses. Finally, we investigated host genetic factors associated with EHV-1 induced myeloencephalopathy but found no evidence for a recessive allele influencing the development of EHM following EHV-1 infection for any genetic locus was identified. However, more complex host-pathogen interactions are possible.

Diverses études ont souligné le rôle prépondérant du dialogue entre les cellules NK et les cellules dendritiques au cours des réponses immunes. Cependant, les récepteurs impliqués dans ce processus restent incertains. Au cours de ce travail, nous nous sommes attachés à identifier les récepteurs mis en jeu lors de la reconnaissance virale à l’aide de modèles humains et murins. Pour cela, nous avons mimé l’infection virale en utilisant deux ARN bicaténaires synthétiques – poly(AU) et poly(IC) – et montré qu’ils sont tous deux capables d’activer TLR3 mais que seul poly(IC) engage les hélicases RIG-I et MDA5. Les deux ARN induisent l’activation des cellules NK au sein des PBMC humaines, mais seul poly(IC) induit la production d’IFN-gamma. Les DC myéloïdes (mDC) sont requises pour cette activation sans nécessité d’un contact cellulaire entre les cellules NK et les mDC. En outre, les IFN de type I et l’IL-12 secrétés par les DC sont respectivement nécessaires à l’initiation du potentiel lytique et à la production d’IFN-gamma. Poly(IC), au contraire de poly(AU), a une action synergique avec l’IL-12 produite par les mDC pour induire la production d’IFN-gamma en agissant directement sur les cellules NK. Enfin, l’activation conjointe de TLR3 et des hélicases RLR sur les mDC et RIG-I sur les cellules NK, nécessaire à la production d’IFN-gama en réponse à l’ARN bicaténaire, a été confirmée à l’aide de souris déficientes pour TLR3 et Cardif et d’un ligand spécifique de RIG-I. En conclusion, nous rapportons pour la première fois la nécessité pour un composé microbien d’engager deux familles de récepteurs sur deux populations cellulaires distinctes pour induire une réponse innée éfficace. / Crosstalk between NK cells and DC is critical for the response to the microbial mimic poly(IC) but the dsRNA receptors involved in each cell types remained to be defined. We show herein that two dsRNA, poly(AU) and poly(IC), similarly engaged TLR3 while only poly(IC) triggered the RIG-I and MDA-5 helicases. Both dsRNA triggered NK cell activation within PBMC but only poly(IC) induced IFN-gamma. mDC were required for NK cell activation by the two dsRNA, suggesting that they triggered at least TLR3 on mDC. DsRNA induction of cytolytic potential and IFN-gamma production in NK cells did not require contact with mDC but was dependent on the secretion of type I IFN and IL-12, respectively. Poly(IC) but not poly(AU) synergized with mDC-derived IL-12 for high IFN-gamma production by acting directly on NK cells. Finally, the requirement of TLR3 and the RLR on mDC and the involvement of the RIG-I but not TLR3 on NK cells for the production of IFN-gamma induced by dsRNA was confirmed using TLR3 and Cardif deficient mice and RIG-I specific activator. This cooperation was further confirmed using inactivated FLU virus infected-target cells both in human and mouse system demonstrating that NK cells were able to sense viral material by a direct transfer from infected cells likely through lytic immunological synapse without prior infection of NK cells. Thus, we report for the first time the requirement of cotriggering