Tableau d'honneur de la Faculté des études supérieures et postdorales, 2014-2015 / La maladie d’Alzheimer (MA) est la plus fréquente cause de démence mondialement et son incidence augmente en parallèle au vieillissement de la population. La pathogenèse de cette maladie neurodégénérative, actuellement sans traitement curatif, est associée à l’accumulation de bêta-amyloïde (Aβ) dans le parenchyme et la vasculature du cerveau en réponse à l’élimination déficiente de ce peptide neurotoxique. Des évidences épidémiologiques et expérimentales soulignent le rôle crucial du système immunitaire inné dans la MA. Plus précisément, plusieurs études suggèrent que les phagocytes mononucléaires (microglies, monocytes et macrophages cérébraux) constituent des cellules pivots pour combattre l’accumulation d’Aβ. Les études incluses dans cette thèse de doctorat avaient comme objectif d’accroitre les connaissances concernant le rôle des phagocytes mononucléaires dans la pathologie du modèle murin APP/PS1 de la MA. Un nombre croissant d’études indique que les Toll-like receptors (TLRs) seraient critiques pour la clairance d’Aβ par les phagocytes mononucléaires. Nous avons donc étudié le rôle de la protéine adaptatrice myeloid differentiation factor 88 (MyD88), qui permet la transduction du signal intracellulaire de la plupart des TLRs. Nous avons observé une augmentation des niveaux d’Aβ solubles et une aggravation des déficits cognitifs dans les souris APP/PS1 avec une signalisation MyD88 défectueuse, vraisemblablement suite à une réduction de la phagocytose des phagocytes mononucléaires et une coordination défaillante de la réponse immunitaire innée. La stimulation des TLRs pourrait ainsi avoir un grand potentiel thérapeutique pour la MA. Nous avons démontré que des injections systémiques répétées du ligand TLR4 monophosphoryl lipid A (MPL) dans les souris APP/PS1 ont mené à une réduction significative des niveaux d’Aβ et une amélioration des fonctions cognitives. Le MPL induit une forte réponse phagocytique des phagocytes mononucléaires tout en générant une réaction inflammatoire modérée. Finalement, en utilisant la microscopie intravitale biphotonique, nous avons dévoilé que les monocytes patrouilleurs en état basal sont attirés et rampent sur la face luminale de veines contenant de l’Aβ, capturent l’Aβ et ont la capacité de regagner à nouveau la circulation sanguine. L’ablation sélective des monocytes patrouilleurs dans les souris APP/PS1 a induit une augmentation significative des niveaux d’Aβ dans le cortex et l’hippocampe. Ensemble, ces données démontrent que les phagocytes mononucléaires sains et fonctionnels peuvent promouvoir l’élimination d’Aβ et constituent une cible thérapeutique prometteuse pour la MA. / Alzheimer’s disease (AD) is the most common cause of dementia worldwide and its incidence is rising in parallel with the aging population. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid-β (Aβ) in the brain parenchyma and cerebral vasculature in response to the impaired clearance of this neurotoxic peptide. Epidemiological and experimental evidence underline the crucial role of the innate immune system in AD. More precisely, several studies suggest that mononuclear phagocytes (microglia, monocytes and cerebral macrophages) constitute pivotal cells to fight the accumulation of Aβ. The studies enclosed in this doctoral thesis intended to better understand the role of mononuclear phagocytes on the pathology of the APP/PS1 mouse model of AD. Accumulating evidence indicates a critical role of Toll-like receptors (TLRs) in the clearance of Aβ by mononuclear phagocytes. We thus investigated the role of the myeloid differentiation factor 88 (MyD88), which is the adaptor protein for most of these innate immune receptors, transducing the intracellular signal from TLRs to the nucleus. We observed increased soluble levels of A oligomers and enhanced cognitive deficits in APP/PS1 mice with impaired MyD88 signalling, likely through reduced phagocytosis of mononuclear phagocytes and an impaired coordination of the innate immune response. Compounds that stimulate TLRs to clear Aβ may therefore have great therapeutic potential in AD patients. We demonstrated that repeated systemic injections of the TLR4 ligand monophosphoryl lipid A (MPL) in APP/PS1 mice led to a significant reduction in Aβ load and improved cognitive function. MPL induced a potent phagocytic response by mononuclear phagocytes while triggering a moderate inflammatory reaction. Finally, using live intravital two-photon microscopy, we discovered that patrolling monocytes in steady state are attracted and crawl onto the luminal walls of Aβ-positive veins, uptake Aβ and are able to circulate back into the bloodstream. Selective removal of patrolling monocytes in APP/PS1 mice induced a significant increase of Aβ load in the cortex and hippocampus. Overall, these studies demonstrate that healthy and functional mononuclear phagocytes can promote the elimination of Aβ and constitute a promising therapeutic target for AD.

Le virus de l'immunodéficience humaine de type 1 (VIH-1), agent causal du syndrome d'immunodéficience humaine (SIDA), est rapidement devenu pandémique suite à son identification en 1981. Les données statistiques publiées par l'Organisation des Nations Unies pour le Syndrome d'Immunodéficience Acquise (ONUSIDA) démontrent que, parmi les 34,3 à 41,4 millions de cas d'infection recensés en 2014, près de 70 % d'entre eux ont été répertoriés en Afrique subsaharienne et environ 6,5 % en Europe et en Amérique du Nord. L'Agence de la santé publique du Canada estimait, quant à elle, à près de 75 500 le nombre de personnes vivant avec le VIH-1 sur son territoire en 2014, dont environ 18 000 résidant au Québec. Malgré l'avènement de trithérapies contrôlant l'infection, à ce jour, aucune médicamentation n'en permet la guérison. La complexité de la pathogenèse virale menant à l'établissement d'une infection latente et à des comorbidités associées à l'état d'inflammation chronique est si considérable qu'aucun traitement ne peut encore enrayer la maladie. La recherche fondamentale et translationnelle, permettant de mieux comprendre le fonctionnement du système immunitaire en réponse à l'infection et d'associer plus efficacement ces avancées faites en laboratoire à la pratique médicale, sont donc primordiales. Les résultats présentés dans cette thèse abondent dans ce sens, puisqu'ils mènent à une meilleure compréhension du rôle que peut avoir le microbiote sur la susceptibilité à l'infection au VIH-1 des lymphocytes T CD4+ primaires, ce qui pourrait inspirer l'élaboration de composés médicamenteux limitant le dialogue entre les bactéries et les cellules permissives au virus. En effet, l'infection au VIH-1 peut être associée à un désordre du tractus gastro-intestinal menant à une translocation de bactéries de la lumière intestinale vers les muqueuses avoisinantes. Cette migration bactérienne semble influencer la susceptibilité à l'infection des lymphocytes T CD4+, mais encore aujourd'hui, tous les mécanismes ne sont pas parfaitement connus. Les deux études présentées dans ce manuscrit contribuent donc à l'avancement des connaissances dans le domaine puisqu'elles démontrent que 1) l'engagement du TLR2 semble entraîner la production de virus par les lymphocytes T CD4+ activés en favorisant l'activité de NF-B et la réplication virale chez les cellules CCR6+ et que 2) la sécrétion d'acétate (petit acide gras produit par la fermentation de composés non digestibles par les bactéries du tractus gastro-intestinal) semble promouvoir l'activation cellulaire et la diminution de l'activité des HDAC de classe 1 et 2 de sorte à accentuer l'intégration de l'ADN proviral dans le génome des lymphocytes T CD4+.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2007-2008. / Chaque année, 130 000 personnes subissent une lésion de la moelle épinière, entraînant une paralysie totale ou partielle de leurs membres. Le seul traitement approuvé, un glucocorticoïde, demeure controversé puisque le rôle de la réponse inflammatoire qui se développe à la suite d'une lésion est encore mal compris. Par l'étude d'un nerf périphérique pouvant régénérer de façon autonome, il est possible d'évaluer l'importance de l'inflammation dans le processus de régénérescence axonale. Notre étude porte sur le rôle des récepteurs Toll-like (TLRs) dans l'initiation de la réponse immunitaire, la dégénérescence Wallérienne et le recouvrement locomoteur suite à une lésion nerveuse périphérique. Les expériences furent effectuées en utilisant des animaux déficients en TLR2, TLR4 et MyD88, ou en employant des agonistes ou inhibiteurs des TLRs. Nos résultats suggèrent que les TLRs seraient activés par des molécules endogènes présentes ou relâchées lors d'une blessure physique et, par l'initiation d'une réponse immune, favoriseraient l'élimination des débris de myéline et la régénérescence nerveuse. / Each year, 130 000 people suffer a spinal cord injury, inducing a total or partial paralysis of their members. The only approved treatment, a glucocorticoid, is still controversial since the role of the inflammatory response that develops after injury is still poorly understood. With the study of a peripheral nerve that can regenerate in an autonomous manner, it is possible to evaluate the importance of inflammation in the process of axonal regeneration. Our study involves the role of the toll-like receptors (TLRs) in the initiation of the immune response, Wallerian degeneration and behavioural recovery after a peripheral nerve lesions. The experiments were performed using mice deficient for TLR2, TLR4 and MyD88, or by employing agonists or inhibitors of TLR signaling. Our results indicate that TLRs could be activated by endogenous molecules present or expressed following traumatic injury and, through initiation of immune responses, could permit the elimination of myelin debris and nerve regeneration.

Increasing evidence implicates the presence of bacteria in intrauterine tissues as an important risk factor for spontaneous preterm labor. Epigenetic alterations of innate immunity genes may increase the mother’s sensitivity to subclinical levels of bacteria. This study examined the presence of TET2, TLR-2, and TLR-9 in intrauterine tissue, and evaluated whether epigenetic alterations of these genes, as well as IL-8, changed their expression in human decidual tissue and a macrophage cell culture. Immunohistochemicalstaining was used to detect the presence of these proteins in intrauterine tissue. Gene expression changes were evaluated in stimulated monocytes and macrophages. Fluorescence immunohistochemistry was used to track translocation of TET2 in stimulated monocytes and macrophages. Secreted IL-8 concentration was detected with ELISA. Decidual expression of TET2, TLR-2, and TLR-9 increased in the order TNL < TL < sPTL < iPTL. This study found that TET2, TLR-2, TLR-9, and IL-8 are regulated by epigenetic mechanisms. This study was the first to report activation of TET2 involves its translocation from the cytosol to the nucleus in macrophages.

Lipopolissacarídeos (LPS), pode tanto proteger quanto exacerbar o desenvolvimento da asma. LPS inicia a ativação da resposta imune via ligação da molécula Toll-like receptor 4 (TLR4) que sinaliza por duas vias distintas, as moléculas adaptadoras MyD88 e TRIF. LPS é um adjuvante que induz resposta do tipo Th1, enquanto que o hidróxido de alumínio (Alum) desperta respostas Th2, porém, a mistura de ambos adjuvantes na indução da resposta alérgica pulmonar ainda não foi investigada. No presente estudo, nós determinamos o efeito de dois agonistas de TLR4, um natural (LPS) e outro sintético (ER-803022) adsorvidos ao Alum sobre o desenvolvimento de doença alérgica pulmonar. Os animais foram sensibilizados pela via subcutânea com os antígenos, Ovoalbumina (OVA) ou Toxóide Tetânico (TT) na presença ou ausência de agonistas de TLR4 co-adsorvidos ao Alum e desafiados com os respectivos antígenos pela via intranasal. Nossos resultados mostraram que a sensibilização com OVA ou TT e LPS coadsorvidos ao Alum, impede o estabelecimento da resposta alérgica mediada por linfócitos Th2, tais como, influxo de eosinófilos, produção de citocinas do tipo 2, hiperreatividade brônquica, secreção de muco, e produção de IgE ou IgG1 anafilática. Apesar dos níveis de IgG2a, isotipo associado com as respostas Th1 estarem aumentados, análise da histopatologia pulmonar não revelou um desvio para o padrão Th1 de inflamação. Verificamos que a presença das moléculas TLR4, MyD88, IL-12/IFN-g mas não TRIF foram necessários para LPS exercer seu efeito inibitório. O agonista sintético de TLR4, menos tóxico que LPS, também protegeu contra o desenvolvimento de inflamação alérgica pulmonar. Em conclusão, nosso trabalho esclarece o efeito da sinalização do TLR4 na sensibilização alérgica e indica que agonista sintético de TLR4 com baixa toxicidade, pode ser utilizado para modular a capacidade adjuvante do Alum e conseqüentemente diminuir a indução de alergias. / Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. LPS triggers immune responses through Toll-like receptor (TLR) 4 that in turn activates two major signaling pathways via either MyD88 or TRIF adaptor proteins. LPS is a pro-Th1 adjuvant while aluminum hydroxide (Alum) is a strong Th2 adjuvant, but the effect of mixing both adjuvants on development of lung allergy has not been investigated. We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways we used TLR4, MyD88, TRIF, or IL-12/IFN-g deficient mice. Mice were sensitized subcutaneously to allergens such as ovalbumin (OVA) or tetanus toxoid (TT) with or without TLR4 agonists coadsorbed onto Alum and challenged twice via intranasal route with the same allergens. The development of type 2 immunity was evaluated 24 h after last allergen challenge. We found that sensitization with OVA or TT plus LPS co-adsorbed onto Alum impaired allergeninduced Th2-mediated responses such as airway eosinophilia, type 2 cytokines secretion, airway hyperreactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, a Th1 affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. LPS impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules via the IL-12/IFN-g axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. TLR4 agonists co-adsorbed with allergen onto Alum down modulate Th2 immunity and prevent the development of polarized T cell-mediated airway inflammation. Thus, our work clarifies the effect of TLR4 signaling in allergic sensitization and indicates that TLR4 agonists with low toxicity might be useful for down regulating the pro-Th2 adjuvant activity of alum and consequently decrease the induction of allergy.

Recentemente, a expressão gênica dos receptores TLR foi encontrada em diversos tipos de células tumorais. A sua participação na biologia do câncer é controversa já que foram descritas ações pró e anti-tumorais após a ativação de sua sinalização. Na Leucemia Mielóide Crônica (LMC) nada se tem demonstrado. BCR-ABL é uma oncoproteína quimérica cujo sítio tirosina quinasa constitutivamente ativado promove inúmeras vias de sinalizações que desencadeia a transformação celular. Este trabalho se inicia com a hipótese de existir uma relação entre o oncogene BCR-ABL e a expressão dos receptores TLRs. Nós verificamos em células murinas TonB210.1 com expressão de BCR-ABL induzível por doxiciclina que Tlr1 e Tlr2 tem sua expressão gênica relativa aumentada na presença da oncoproteína. A regulação positiva de Tlr1 é dependente da ação tirosina quinasa de BCR-ABL. Também mostramos que as vias p38 e JNK estão reprimindo a expressão de Tlr1 induzida por BCR-ABL enquanto que a via ERK é utilizada pelo BCR-ABL para promovê-la. Por outro lado, observamos que a ligação de TLR1/TLR2 com seu agonista sintético Pam3CSK4 em células TonB210.1 BCR-ABL positivas induz um aumento da produção de IL-6 e leva ao aumento da resistência a morte quando induzida pelas drogas Ara-C e VP16. Em conclusão, estes resultados indicam que BCR-ABL esta regulando a expressão gênica de alguns TLRs. Por tanto esses dados contribuem para a compreensão sobre o comportamento de células tumorais BCR-ABL positivas em um contexto de infecção e por conseqüência, dão margem ao estudo de novos alvos de fator de risco para a LMC. / Recently, the gene expression of TLR receptors have been described in several kinds of tumour cells. Its participation in cancer biology is controversial because roles were already been described in pro and anti-tumoral activities after their signaling activation. In Chronic Myeloid Leukemia (CML) there are no published data. BCR-ABL is a quimeric protein and its tyrosine-kinase site is activated constitutively. Thus, many signaling pathways are activated and several cell processes are altered thereby resulting in cellular transformation. This work has started with the hypothesis that a putative relationship between the oncogene BCR-ABL and the expression of TLR receptors could exists. We verified in murine cells TonB210.1 BCR-ABL expression inducible by doxycicline that Tlr1 and Tlr2 have their relative gene expression up-regulated in the presence of the oncoprotein. Therefore the Tlr1 regulation is dependent of BCR-ABL tyrosine kinase action. Using MAPK inhibitors we showed that p38 and JNK pathways are suppressing the TLR1 induction by BCR-ABL while ERK pathway is used by the oncoprotein for promote it. On the other hand, we observed in TonB210.1 BCR-ABL positive cells that the binding of TLR1/TLR2 heterodimer to their synthetic agonist Pam3CSK4 induced an increased production of IL-6 and when these cells were induced by Ara-C and VP-16 drugs the apoptosis resistance increased. In conclusion, these results indicate that the oncoprotein regulates the gene expression of some TLRs. Therefore, this fact gives us data about the behavior of BCR-ABL positive tumor cells in the context of infection and in consequence the study of new risk factor targets for CML.

Les thérapies anticancéreuses ont apporté un gain largement reconnu en matière de réduction de la charge tumorale, de survie des patients et d’amélioration de leur qualité de vie, dans un certain nombre de cancers. Hélas, ces thérapies exercent un effet immunosuppresseur en détruisant les effecteurs ou en bloquant l’activité de certains facteurs biologiques impliqués dans le recrutement des acteurs du système immunitaire. D’autre part, plusieurs travaux ont permis de démontrer que ces traitements pouvaient avoir un effet contraire en générant ou en favorisant l’induction d’une réponse immunitaire anti-tumorale, soit par effet direct sur le recrutement et l’activation des effecteurs de l’immunité, soit en potentialisant les interactions cellulaires par des mécanismes biologiques. Ces derniers faisant intervenir les cytokines, la stimulation des TLR, l’augmentation des interactions entre cellules du SI; ce qui permet de passer d’une anergie immunologique vers un véritable système d’éradication des cellules cancéreuses.Dans notre laboratoire, nous avons essayé d’évaluer l’implication du système immunitaire dans la réponse thérapeutique induite par des agents cytotoxiques conventionnels. Ici, nous décrivons les effets d’un inhibiteur de cyclines kinases multi-cibles « CDKi PHA-793 887 » testé dans un essai de phase I mené sur deux sites en Europe. C’est le constat inattendu que 6 des 15 patients, traités par ce médicament (PHA-793887) ont développé de graves infections bactériennes et virales et que 6 d’entre eux ont présenté la réactivation du virus de l’herpès qui nous a conduit à étudier ces effets sur le système immunitaire et en particulier sur le dialogue entre cellules dendritiques (CD) et cellules natural killer (NK). Ce travail met en évidence que ce médicament inhibe le signalling des récepteurs toll-like (TLR) réduisant par conséquent l’interaction CD/NK in vitro. Enfin la stimulation des cellules des patients sous traitement démontre une réduction importante de ce signalling ex-vivo. Ainsi, cet effet immunosuppresseur inattendu a permis une réactivation virale chez 40% des patients. La deuxième partie de ce travail, concerne les effets du cyclophosphamide (CTX) utilisé à faible dose. L’injection d'une faible dose chez la souris ou d’un dosage métronomique chez l'homme, promeut la différenciation des cellules lymphocytaires vers Th17 (sécrétant de l’interleukine-17 (IL-17)) et Th1 (sécrétant de l’interféron-γ (IFN)). Ceux-ci ont été retrouvés dans le sang et dans des ascites carcinomateuses de patients. Ainsi, le CTX pourrait participer à la génération de réponses anti-tumorale via la différenciation Th 17 comme cela fut suggéré par de récentes études précliniques montrant l’existence d’une corrélation étroite entre le taux des lymphocytes Th17 infiltrant la tumeur et la destruction tumorale. / Cancer therapies have made a gain widespread recognition in the reduction of tumor burden, patient survival and improved quality of life in a number of cancers. Unfortunately, these therapies exert an immunosuppressive effect by killing effectors or blocking the activity of certain biological factors involved in recruiting of the immune system. On the other hand, several studies have shown that these treatments could have the opposite effect by generating or promoting the induction of antitumor immune response, either by direct effect on the recruitment and activation of effectors immunity, either by potentiating cellular interactions by biological mechanisms. The latter involving cytokines, TLR stimulation, increased interactions between cells of the IS; which toggles between immunological anergy to a real system to eradicate cancer cells. In our laboratory, we tried to evaluate the involvement of the immune system in the therapeutic response induced by conventional cytotoxic agents. Here, we describe the effects of an inhibitor of cyclin kinases multi-target "CDKIs PHA-793887" tested in a phase I trial conducted at two sites in Europe. This unexpected finding is that 6 of 15 patients treated with this drug (PHA-793887) developed severe bacterial and viral infections and six of them showed reactivation of the herpes virus that has led us to study these effects on the immune system and in particular on the dialogue between dendritic (DCs) and natural killer (NK) cells. This work shows that this drug inhibits the signaling of toll-like receptor (TLR) thereby reducing the interaction DC / NK in vitro. Finally, stimulation of the cells of treated patients demonstrated a significant reduction of this signaling ex vivo. Thus, this immunosuppressive effect has an unexpected viral reactivation in 40% of patients. The second part of this work concerns the effects of metronomic dose of cyclophosphamide (CTX). The injection of a low dose in mice or metronomic dosing in humans, markedly promotes the differentiation of CD4+ T helper 17 (Th17) cells that can be recovered in both blood and tumor beds. However, CTX does not convert regulatory T cells into Th17 cells and promotes cell differentiation into Th17 lymphocytes (secreting interleukin-17 (IL-17)) and Th1 (secreting interferon-γ (IFN)). These were found in blood and in ascites carcinoma patients. Thus, CTX may participate in the generation of antitumor responses through Th 17 differentiation as was suggested by recent preclinical studies showing the existence of a correlation between the rate of Th17 lymphocytes infiltrating the tumor and tumor destruction.

Hepatitis B virus (HBV) infection is one of the major causes of chronic and cancerous liver disease. Elimination of HBV from chronically infected patients by recombinant interferon α (IFNα) monotherapy shows that the mechanisms of the innate immunity play an important role in suppressing viral infection. However, the mechanisms of recognition of the HBV genome and its escape from the mechanisms of natural immunity are still little known. One of the principal factors enabling the virus to escape from cellular restriction mechanisms is the HBx viral protein. HBx is a 154 amino acid pleiotropic multifunctional protein affecting transcription, signal transduction, cell cycle, protein degradation, apoptosis, and chromosomal stability in the host cell. Previous results from our laboratory have shown that activation of the MEK1/2-ERK signaling pathway in plasmacytoid dendritic cells leads to inhibition of IFNα production. The aim of my work was to determine whether HBx activates the MEK1/2-ERK pathway and thus inhibits IFN type I production also in hepatocytes. For this purpose, I monitored HBx production in the Huh7 hepatoma cell line by transfecting the bicistronic plasmid pHBx- IRES-EGFP and Western blotting. Using the same method, I monitored activation of the MEK1/2-ERK signaling pathway by ERK...

Lipopolissacarídeos (LPS), pode tanto proteger quanto exacerbar o desenvolvimento da asma. LPS inicia a ativação da resposta imune via ligação da molécula Toll-like receptor 4 (TLR4) que sinaliza por duas vias distintas, as moléculas adaptadoras MyD88 e TRIF. LPS é um adjuvante que induz resposta do tipo Th1, enquanto que o hidróxido de alumínio (Alum) desperta respostas Th2, porém, a mistura de ambos adjuvantes na indução da resposta alérgica pulmonar ainda não foi investigada. No presente estudo, nós determinamos o efeito de dois agonistas de TLR4, um natural (LPS) e outro sintético (ER-803022) adsorvidos ao Alum sobre o desenvolvimento de doença alérgica pulmonar. Os animais foram sensibilizados pela via subcutânea com os antígenos, Ovoalbumina (OVA) ou Toxóide Tetânico (TT) na presença ou ausência de agonistas de TLR4 co-adsorvidos ao Alum e desafiados com os respectivos antígenos pela via intranasal. Nossos resultados mostraram que a sensibilização com OVA ou TT e LPS coadsorvidos ao Alum, impede o estabelecimento da resposta alérgica mediada por linfócitos Th2, tais como, influxo de eosinófilos, produção de citocinas do tipo 2, hiperreatividade brônquica, secreção de muco, e produção de IgE ou IgG1 anafilática. Apesar dos níveis de IgG2a, isotipo associado com as respostas Th1 estarem aumentados, análise da histopatologia pulmonar não revelou um desvio para o padrão Th1 de inflamação. Verificamos que a presença das moléculas TLR4, MyD88, IL-12/IFN-g mas não TRIF foram necessários para LPS exercer seu efeito inibitório. O agonista sintético de TLR4, menos tóxico que LPS, também protegeu contra o desenvolvimento de inflamação alérgica pulmonar. Em conclusão, nosso trabalho esclarece o efeito da sinalização do TLR4 na sensibilização alérgica e indica que agonista sintético de TLR4 com baixa toxicidade, pode ser utilizado para modular a capacidade adjuvante do Alum e conseqüentemente diminuir a indução de alergias. / Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. LPS triggers immune responses through Toll-like receptor (TLR) 4 that in turn activates two major signaling pathways via either MyD88 or TRIF adaptor proteins. LPS is a pro-Th1 adjuvant while aluminum hydroxide (Alum) is a strong Th2 adjuvant, but the effect of mixing both adjuvants on development of lung allergy has not been investigated. We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways we used TLR4, MyD88, TRIF, or IL-12/IFN-g deficient mice. Mice were sensitized subcutaneously to allergens such as ovalbumin (OVA) or tetanus toxoid (TT) with or without TLR4 agonists coadsorbed onto Alum and challenged twice via intranasal route with the same allergens. The development of type 2 immunity was evaluated 24 h after last allergen challenge. We found that sensitization with OVA or TT plus LPS co-adsorbed onto Alum impaired allergeninduced Th2-mediated responses such as airway eosinophilia, type 2 cytokines secretion, airway hyperreactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, a Th1 affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. LPS impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules via the IL-12/IFN-g axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. TLR4 agonists co-adsorbed with allergen onto Alum down modulate Th2 immunity and prevent the development of polarized T cell-mediated airway inflammation. Thus, our work clarifies the effect of TLR4 signaling in allergic sensitization and indicates that TLR4 agonists with low toxicity might be useful for down regulating the pro-Th2 adjuvant activity of alum and consequently decrease the induction of allergy.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T12:30:34Z No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:05:05Z (GMT) No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Made available in DSpace on 2016-10-22T13:05:05Z (GMT). No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) Previous issue date: 2008-02-15 / Nos países tropicais, as perdas causadas pela infestação de carrapatos em bovinos acarretam um grande impacto no sistema de produção animal. Recentes estudos têm mostrado a importância de fatores genéticos ligados a resistência a carrapato em Bos taurus indicus e Bos taurus taurus e que as citocinas têm um papel crítico na prevenção ou progressão de doenças. O objetivo desse trabalho foi avaliar os níveis de expressão dos genes IL-10 e IL-4 relacionados ao perfil imunológico Th2 associado à susceptibilidade ao carrapato e os genes IL-2 e IFN- relacionados ao perfil imunológico Th1 associado à resistência ao parasito. Além destes genes, analisou-se o perfil de expressão do gene TLR-2, importante no processo de reconhecimento de patógenos e os genes IL-8 e TNF-α importantes no processo inflamatório inicial. Seis animais mais resistentes e seis animais mais susceptíveis de uma população F2 de 332 animais, originária do cruzamento de animais F1(½ Holandês: ½ Gir), foram selecionados baseado na contagem de carrapatos e valor genético. Amostras de tecido foram coletadas de pele no 5° e 12° dias após a infestação para extração de RNA total. As PCRs em tempo real foram realizadas usando o gene GAPDH como controle endógeno. Os animais resistentes e susceptíveis apresentaram aumento de expressão do gene IL-10 no 5° (p<0,01) e 12 ° dias após a infestação (p<0,05). O gene IL-2, nos animais resistentes e susceptíveis, no 5° dia após a infestação não apresentou alteração da expressão sendo que 12° dia, em ambos os grupos de animais, este gene passou a ser mais expresso em relação ao animal controle sugerindo um perfil de resposta imunológica do tipo de Th2 nos animais resistentes e susceptíveis nos primeiros dias após a infestação. O gene IL-4 apresentou uma tendência ao aumento de expressão nos animais resistentes e susceptíveis em relação ao controle, sendo o perfil Th2 sugerido atribuído a IL-10 produzida por linfócitos T regulatórios (p>0,05). O gene TNF- apresentou aumento de expressão nos animais susceptíveis no 5° dia após a infestação com posterior diminuição no 12° dia após a infestação (p<0,05). Nos animais resistentes não foi observada alteração da expressão deste gene, isto sugere que ele possa estar mais atuante no início do processo inflamatório, logo após a fixação do carrapato. A mesma observação estende-se para o gene IL-8, em que não foi verificada alteração de expressão nos animais resistentes, embora nos animais susceptíveis este gene apresentou diminuição da expressão no 12° dia após a infestação (p<0,05). Quanto ao gene IFN-, não houve diferença de expressão entre os animais resistentes e susceptíveis, sendo que este gene parece não estar relacionado ao mecanismo de resistência. O gene TLR-2 apresentou diminuição da expressão em ambos os grupos de animais. Estes resultados sugerem que a resposta imune adquirida avaliada neste trabalho não apresenta papel preponderante no mecanismo de resistência e que resposta imune inata poderia está envolvida no mecanismo de resistência ao carrapato. Portanto, avaliação da resposta imunológica horas após a fixação do carrapato poderia nos fornecer resultados mais conclusivos. / In tropical countries losses caused by tick infestation in cattle lead to a major impact on animal production systems. Recent studies have shown the importance of genetic factors linked to tick resistance in Bos indicus and Bos taurus as well as the critical role in the prevention or progression of diseases mediated by cytokines. The aim of this work was to evaluate gene expression of IL-10 and IL-4 in relation to tick susceptibility associated with the Th2 profile and gene expression of IL-2 and IFN- in relation to tick resistance associated with the Th1 profile. In addition, the expression of TLR-2, important in the process the recognition of pathogens, and TNF-α and IL-8 genes, important in the initial inflammatory process, were evaluated. Six tick-resistant and six tick-susceptible animals from a F2 population of 332 animals, originated from the cross of F1 animals (½ Holstein: ½ Gir), were selected based on tick count and breeding value for tick resistance. Skin biopsies were collected in the 5th and 12th days after tick infestation. The GAPDH was used as endogenous control to normalize the amount of starting cDNA target in the real-time PCR assay. Both resistant and susceptible animals showed increased gene expression of IL-10 in the 5th and 12th days after infestation in relation to control animal (p<0.05). The IL-2 gene showed no change of expression in the 5th day after infestation for the resistant and susceptible animals. In the 12th post infestation, both resistant and susceptible animals showed increased gene expression in relation to control animal. These results suggest an enhancement of Th2 profile through the increase of IL-10 mRNA levels and a possible inhibition of the Th1 pattern in both groups (resistant and susceptible) starting 5 days after infestation and return to normal by day 12. Despite our results suggest the occurrence of the Th2 profile, the susceptible and resistant animals did not show variation on gene expression for IL-4 in relation to control animal. The susceptible animals showed increased expression of TNF-α in the 5th day after infestation. However, in the 12th day post infestation it was noted a decrease in the gene expression level. The resistant animals showed no change in the expression of this gene in relation to control animals suggesting that TNF-α could be more actively expressed in the early steps of the inflammatory process. Similarly, the resistant animals showed no variation in the expression of IL-8 while the susceptible animals showed increased expression in the 12th day post infestation. There were no differences of expression between resistant and susceptible animals in relation to IFN-γ what suggests that this gene might not be involved in the resistance mechanism. The TLR-2 gene showed decreased expression in both resistant and susceptible animals (p<0.05). Finally, there was no difference in expression between susceptible and resistant animals in relation to all selected genes in the 5th and 12th days after infestation. These results suggest that the acquired immunity evaluated in this work might not have preponderant role in the resistance mechanism. The innate immunity might be playing a major role in the bovine tick resistance/susceptibility mechanism in early hours after infestation.