5 Anglický abstrakt Experimental autoimmune encephalomyelitis (EAE) is widely accepted as a murine model of human multiple sclerosis autoimmune disease. Murine EAE is usually actively induced by immunization with a suitable myelin antigen. Following immunization, CD4+ T helper lymphocytes Th1 and Th17 accumulate in the nervous tissue and via the production of cytokines, they mediate an inflammatory reaction and the subsequent destruction of myelin. The main goal of this study was the induction of EAE with clinically observable symptoms and the observation of changes in the counts and phenotypes of cells, mainly NK and T cells. NK cells express a wide range of inhibitory and activation receptors from the C-lectin-like receptor superfamily. The specific ligand of the activating NKR-P1C isoform is still unknown and thus this receptor's involvement in EAE was also observed. Another goal was the use of medication with regard to the disease progress improvement. For the purposes of this study, two inbred murine strains with distinct NKR-P1 surface expression were used - the SJL/J strain (expressing inhibitory NKR-P1B) and C57BL/6 (expression activating NKR-P1C). SJL mice elicited a relapse-remitting of EAE, while C57BL/6 had chronic EAE. Both mouse strains exerted changes in the counts of NK...

O Sistema Nervoso Central (SNC) humano é formado por cerca de 86,1 bilhões de neurônios entre o encéfalo e a medula espinhal. O desenvolvimento pré-natal humano (tempo da concepção ao nascimento) possui cerca de 38 semanas, e é dividido na fase embrionária que corresponde ao período das 8 semanas iniciais da gestação, seguido pela fase fetal. A fase embrionária é o período mais vulnerável à ocorrência de anormalidades congênitas. Por ser um órgão com grande período de desenvolvimento, o SNC está sujeito às alterações genéticas, epigenéticas e ambientais. Durante a fase de implantação do embrião, o DNA é mais vulnerável às influências externas, como à fumaça do cigarro, aumentando o risco de retardo do desenvolvimento fetal, o risco de morte súbita pós-natal e de anormalidades do sistema imune. Neste contexto, o objetivo deste trabalho é avaliar os efeitos da exposição à fumaça do cigarro sobre o processo de neuroinflamação da prole de camundongos C57BL/6 expostos à fumaça do cigarro durante a gestação e desafiados ou não com LPS. Para tanto, camundongos C57BL/6 fêmeas prenhes foram expostas à fumaça do cigarro desde o plug vaginal até o nascimento da prole. No 3º dia de vida, os filhotes foram separados para três linhas de trabalho: 1) in vivo: os animais foram desafiados com LPS pelo período de 4h, seguidos de eutanasia e análises de PCR Array do SNC. 2) in vitro: os encéfalos dissecados foram utilizados para a preparação de cultura mista de glia e da cultura enriquecida com neurônio. Após a maturação celular, as células foram estimuladas com LPS 100 ng/mL e, após 24h, foram realizados ensaios de CBA, citometria de fluxo, PCR, dosagem de NO, avaliação de morte celular e metilação global. 3) Encefalomielite Autoimune Experimental (EAE): após o desmame, os animais foram mantidos em suas caixas moradia por 8 semanas sem nenhum estímulo externo, e então foram imunizados com MOG35-55 para o desenvolvimento da EAE. Nos experimentos in vivo observamos o aumento da transcrição de genes relacionados ao processo inflamatório, como interleucinas e quimiocinas. Em relação aos experimentos in vitro observamos maior crescimento de células astrocitárias (astrogliose), e células da microglia com aumento de moléculas co-estimuladoras (CD80 e CD86) bem como da transcrição e concentração de citocinas pró-inflamatórias e produção de NO. Em cultura enriquecida de neurônio, foi observado aumento na porcentagem de células em apoptose no grupo exposto à fumaça do cigarro desafiados ou não com LPS. O bloqueio da atividade da microglia pela minociclina reverteu a apoptose e diminuiu a produção de NO minimizando a morte celular. Em relação aos experimentos de EAE, os animais expostos à fumaça do cigarro no período gestacional, quando imunizados na vida adulta apresentam aumento no grau da doença bem como maior persistência da mesma quando observado escore clínico, além de acompanhados de um grau maior de infiltrado celular e desmielinização. Desta forma podemos concluir que a exposição à fumaça do cigarro durante o período gestacional leva a uma programação fetal com aumento da resposta neuroinflamatória frente a um estimulo sistêmico, trazendo consequências na vida adulta. / The human central nervous system (CNS) is made up of about 86.1 billion neurons between the brain and the spinal cord. The human prenatal development (time from conception to birth) is about 38 weeks, and is divided into the embryonic phase that corresponds to the period of the initial 8 weeks of gestation, followed by the fetal phase. The embryonic stage is the period most vulnerable to the occurrence of congenital abnormalities. Because it is an organ with a long period of development, the CNS is subject to genetic, epigenetic and environmental changes. During the embryo implantation phase, DNA is more vulnerable to external influences such as cigarette smoke, increasing the risk of delay on fetal development, risk of sudden postnatal death, and abnormalities of the immune system. In this context, the aim of this work is to evaluate the effects of exposure to cigarette smoke on the neuroinflammation process of offspring of C57BL/6 mice exposed to cigarette smoke during gestation and challenged or not with LPS. For this, pregnant female C57BL/6 mice were exposed to cigarette smoke from vaginal plug to offspring birth. On the 3rd day of life the offspring were separated into three lines of work: 1) in vivo: the animals were challenged with 1mg/Kg LPS and after 4h they followed to euthanasia; PCR analysis of the CNS was made in this period. 2) in vitro: dissected encephalons were used for the preparation of mixed culture of glia and the culture enriched with neuron. After cell maturation, the cells were stimulated with 100 ng/mL LPS and, after 24 hours, CBA, flow cytometry, PCR, NO assay, cell death and global methylation assays were performed. 3) Experimental Autoimmune Encephalomyelitis (EAE): After weaning, the animals were kept in their housing for 8 weeks without any external stimulus, and then were immunized with MOG35-55 for the development of EAE. In the in vivo experiments we observed increased transcription of genes related to the inflammatory process, such as interleukins and chemokines. In vitro experiments showed higher growth of astrocytes (astrogliosis) and microglia cells with increased stimulatory molecules (CD80 and CD86) as well as the transcription and concentration of proinflammatory cytokines and NO production. In the enriched neuron culture, an increase in the percentage of cells in apoptosis was observed in the group exposed to cigarette smoke challenged or not with LPS. Blocking microglial activity by minocycline reversed apoptosis and decreased NO production by minimizing cell death. The EAE experiments shows that the animals exposed to cigarette smoke in the gestational period, when immunized in adulthood, present an increase in the degree of the disease as well as a greater persistence of the disease; The higher as the clinical score higher is the degree of cellular infiltration and demyelination. In this way we can conclude that the exposure to cigarette smoke during the gestational period leads to a fetal programming with increased neuroinflammatory response to a systemic stimulus and that this is able to last until the adult stage.

Encefalomielite Autoimune Experimental (EAE) é um modelo animal para o estudo da Esclerose Múltipla, doença autoimune na qual células do sistema imune atacam a bainha de mielina e os oligodendrócitos, levando a desmielinização, perda axonal e morte neuronal. Astrócitos são importantes na doença e na modulação da neuroinflamação. O estrógeno apresenta ação protetora, porém a ação via receptor acoplado a proteína G (GPER) é pouco conhecido. Como GPER está presente nos astrócitos, o objetivo deste projeto foi verificar se o tratamento com G1 (agonista seletivo de GPER) seria capaz de modular o processo inflamatório presente no SNC. O tratamento com o G1 (3mg/Kg, via subcutânea, durante três dias, iniciando no 5º dia após a indução da EAE) atenuou o escore clínico no pico da doença e alterou a morfologia dos astrócitos da medula espinhal, tanto na substância branca como na cinzenta, sugerindo um efeito anti-inflamatório do G1. Estudos subsequentes in vitro foram feitos para tentar elucidar possíveis vias de ativação relacionadas ao GPER. / Experimental autoimmune encephalomyelitis (EAE) is an animal model for the study of Multiple Sclerosis, an autoimmune disease in which cells of the immune system attack the myelin sheath and oligodendrocytes, leading to demyelination, axonal loss, and neuronal death. Astrocytes are important in disease and modulation of neuroinflammation. Estrogen has protective action, but the action by G protein-coupled estrogen receptor (GPER) is little known. GPER is present in astrocytes, the objective of this project was to verify if treatment with G1 (selective agonist of GPER) would be able to modulate the inflammatory process present in the CNS. Treatment with G1 (3 mg / kg, subcutaneously for three days, beginning on the 5th day after EAE induction) attenuated the clinical score at the peak of the disease and altered the morphology of the spinal cord astrocytes both in white matter and Suggesting an anti-inflammatory effect of G1. Subsequent in vitro studies have been done to try to elucidate possible pathways of activation related to GPER.

INTRODUÇÃO: Atualmente as células da microglia têm recebido grande atenção dentro da resposta imune, isto devido ao fato de que sua ativação por citocinas inflamatórias é capaz de promover a infiltração e destruição do sistema nervoso central (SNC) durante algumas doenças, principalmente no caso da esclerose múltipla (EM). Além de seu papel pró-inflamatório, já demonstrou-se que estas também são capazes de expressar moléculas supressoras como a indoleamina-2,3-dioxigenase (IDO), capaz de suprimir a proliferação de células T. Contudo, ainda pouco se sabe sobre seu verdadeiro papel na patogenia da EM. Recentemente tem sido descrita uma população de células T chamadas Th17, capaz de secretar grandes quantidades de IL-17, IL-21 e GM-CSF possuindo uma importância fundamental na patogenia da EM e de seu modelo murino, a EAE. Nesse contexto, a relação entre as Th17 e as células da microglia pode nos fornecer dados importantes acerca dos mecanismos envolvidos nas lesões observadas no SNC. OBJETIVO: Este trabalho teve como objetivo melhor elucidar a relação existente entre a expressão das moléculas imunes por células da microglia e a ação que estas promovem sobre as células T. MÉTODOS: Utilizamos culturas de células da microglia de linhagem, chamadas C8-B4, assim como cultura primária de células da microglia obtidas a partir sistema nervoso de camundongos C57BL/6 adultos. Caracterizamos o perfil imune da microglia, avaliando a transcrição de genes para citocinas através de PCR em tempo real assim como a expressão de suas moléculas ativadoras por citometria de fluxo. A avaliação da IDO se deu através da expressão da mesma por células da microglia ativadas ou não por LPS ou IFN-?. Ja sua capacidade funcional foi medida através da atividade proliferativa de linfócitos T CD4 específicos para MOG 35-55. RESULTADOS: Nossos resultados demonstraram que as células de ambas as culturas possuem a capacidade de expressar diversas moléculas imunes, tanto pró quanto anti-inflamatórios. Dentre estas observamos TLR-4, TLR-2, IL-6, IL-10 e TGF-?. Além disso, confirmamos a expressão da enzima IDO por estas células. O bloqueio de tal enzima impede o controle que a microglia tem sobre a proliferação dos linfócitos T CD4, tanto in vitro quanto in vivo. No modelo in vivo tal efeito repercute em uma encefalomilite mais severa, onde o quadro clínico do animal não regride. CONCLUSÃO: Os resultados aqui obtidos nos dão a certeza da influência das microglias dentro do contexto inflamatório, afirmando sua capacidade de modular a resposta imune. Além disto, fica clara a importância da enzima IDO, cuja ação dentro do controle de uma autoimunidade demonstra ser altamente necessária / INTRODUCTION: Microglia cells has gained great attention recently because its activation by inflammatory cytokines can promote infiltration and destruction of Central Nervous System (CNS) during some disease, mainly in the case of Multiple Sclerosis (MS). On the other hand, these cells may also express suppressor molecules such as the indoleamine-2,3-dioxygenase (IDO), able to suppress T cell proliferation. However, still little is known about its role in MS pathogenesis. Recently it has been described a new population of T cells called Th17, able to secrete high amounts of IL-17, IL-21 and GM-CSF, with a fundamental importance on MS and its murine model, EAE. In this context, the relationship between Th17 and microglia cells can provide us important data about the mechanisms involved in the establishment of CNS lesions. OBJECTIVES: This work had the objective to better elucidate the relationship between the expression of some molecules by microglia and its role T cell activation. METHODS: Through a cellular lineage knowing as C8-B4 and primary cultures of microglia obtained from CNS of adult mice C57BL6 we investigated the transcription of several genes for cytokines and membrane expression of several pattern recognition receptors. The IDO evaluation was performed after activation with LPS or rIFN-?. Its functional capacity was measured trough its action over T cell proliferation. RESULTS: Our results demonstrated that both cells have the capacity of express several immune molecules, both pro and anti-inflammatory. Among this, we observed TLR-4, TLR-2, IL-6, IL-10 and TGF-?. We also confirmed IDO expression by these cells. The blockade of such enzyme prevents the control of microglia above T CD4 lymphocytes proliferation, both in vitro and in vivo. Using the in vivo model, IDO blocker rendered a encephalomyelitis more severe. Conclusion: The results here obtained give us the certainty of microglia influence in inflammatory context, stating its capacity of modulating the immune response

Das Proteasom ist das zentrale proteolytische System in eukaryontischen Zellen, welches die Mehrzahl der intrazellulären Proteine abbaut. Da viele essentielle Prozesse in der Zelle proteolytisch reguliert werden, besitzt das Proteasom eine außerordentliche biologische Bedeutung. Die Erforschung des Proteasoms im ZNS steht erst am Anfang, dennoch zeigen zahlreiche Untersuchungen, dass Inhibition bzw. Störung des Ubiquitin-Proteasom-Systems mit vielen neurologischen oder neurodegenerativen Erkrankungen einhergeht. Deshalb wurde in der vorliegenden Arbeit nach Veränderungen des Proteasoms in Entzündungsprozessen im ZNS am Beispiel der experimentellen autoimmunen Encephalomyelitis (EAE) in der Maus gesucht. Schwerpunkt der Untersuchungen war das Proteasom in Astrozyten. Astrozyten stellen die größte Gruppe unter den Gliazellen dar und besitzen vielfältige Funktionen, zu denen neben klassischen housekeeping Funktionen auch Aufgaben bei der Immunantwort zählen. Der enge und für Neurone essentielle Kontakt prädestiniert Astrozyten, neuronale Erkrankungen mit auszulösen und zu modulieren. In dieser Arbeit wurden in primär isolierten Astrozyten Immunproteasomen (IP) detektiert. Durch Experimente mit der Astrozytenzelllinie TSA-3 konnte gezeigt werden, dass Astrozyten im unstimulierten Zustand nur Standardproteasom besitzen, auf Stimulation jedoch mit der Bildung von IP reagieren. Das Fehlen von IP in Astrozyten unter in vivo Bedingungen deckte sich mit den Strukturanalysen von Proteasomen aus dem Großhirn von Mäusen verschiedener Altersstufen, den mRNA-Expressionsanalysen sowie immunhistologischen Untersuchungen von Hirngewebe aus EAE Mäusen. Die aus dem Großhirn isolierten Proteasomen nach Induktion einer EAE durch Myelin-Oligodendrocyten-Glycoprotein (MOG) enthielten keine IP. Dennoch erfolgt eine Aktivitätsveränderung im Proteasom vor dem Auftreten der ersten EAE Symptome, die in vitro zu einer effizienteren Epitopgenerierung aus einem MOG-Peptid führt. / The proteasome is the central proteolytic system in all eukaryotic cells catalysing the degradation of the majority of intracellular proteins. Since many essential processes are proteolytically controlled, the proteasome is of crucial biological importance. Yet numerous investigations show that many neurological or neurodegenerative diseases go along with inhibition and/or changes of the ubiquitin-proteasome-system. Therefore the present thesis investigates the proteasome system during inflammatory processes in the CNS, namely during experimental autoimmune encephalomyelitis (EAE), a widely used animal model for human multiple sclerosis. Main focus of the investigations was the proteasome in astrocytes. Astrocytes embody the largest group of glial cells in the CNS and possess various functions. Apart from classical housekeeping functions astrocytes take part in the immune reaction in the CNS. Their close and essential contact to neurons predestines astrocytes to cause and modulate neural diseases. In the present work immune proteasome subunits were detected in primary astrocytes isolated from newborn mice. On the other hand, when grown under resting conditions the murine astrocyte cell line, TSA-3, contains standard proteasome only, however, when treated with interferon gamma, these cells produce immune proteasomes, too. Subunit analyses of proteasomes isolated from the cerebrum of mice of different age, measurement of the mRNA expression level of proteasome subunits as well as immune-histological investigations of brain tissue from mice confirmed the absence of immune proteasome in astrocytes under in vivo conditions. Proteasomes isolated from mouse brain after induction of EAE by active immunization with myelin oligodendrocyte glycoprotein (MOG) did not contain immune subunits. Nevertheless an activity change in the proteasomes isolated from brains before onset of EAE was observed, which lead to a more efficient epitope generation from MOG peptide.

Die intrazelluläre Weiterleitung von Interferonsignalen von der Zytoplasmamembran zum Zellkern wird vermittelt über den Signaltransduktor und Aktivator der Transkription 1 (STAT1), welcher in seiner tetrameren Form als Transkriptionsfaktor an Immunantworten beteiligt ist. In diesem Projekt wurde der Protomerenaustausch zwischen STAT1-Dimeren unter kinetischen Gesichtspunkten untersucht und dabei dieser Prozess als ein potentiell geschwindigkeitsbestimmender Schritt des Aktivierungs-/Inaktivierungs-Zyklus von STAT1 identifiziert. Die Daten unterstützen einen alternativen Mechanismus für den Wechsel zwischen der parallelen und antiparallelen Konformation von STAT1-Dimeren basierend auf der Dissoziation und nachfolgenden Reassoziation von Protomeren, bei dem reziproke Interaktionen innerhalb des N-terminalen Domänendimers zur Stabilisierung eines intermediären Konformationsübergangs nicht benötigt werden. Durch Bindung an spezifische DNA-Zielbereiche, als Gamma-aktivierte Sequenzen (GAS) bezeichnet, wird die Dynamik des Protomerenaustauschs wesentlich beeinträchtigt. In der Sequenz des für das zytoskelettale Strukturprotein Ezrin kodierenden humanen EZR-Gens wurde mittels in silico Analyse ein doppeltes GAS-Motiv als mögliche STAT1-Zielsequenz identifiziert und die Bindung von STAT1-Dimeren an jedes der beiden Elemente sowie eine moderate Geninduktion bestätigt. Allerdings zeigen Mäuse mit einer N-terminalen Substitutionsmutation von STAT1, welche die kooperative DNA-Bindung beeinträchtigt, sowie auch ein kompletter funktioneller Knockout des Stat1-Gens keine veränderte Expression von Ezrin und Moesin in Knochenmarkszellen verglichen mit Mäusen, die das Wildtyp-Molekül exprimieren. In einem Myokardinfarktmodell durch Ligatur des Ramus interventricularis anterior zeigen männliche Mäuse mit Expression der Interferon-γ-irresponsiven STAT1-Mutante höhere Überlebensraten, während weibliche Tiere vor den nachteiligen Effekten des kardialen Remodellings in der frühen Phase geschützt sind. In entzündlichen myokardialen Infiltraten dieser Tiere wurde ein geringfügig höheres Expressionsniveau an tyrosinphosphoryliertem STAT1 nachgewiesen, während die Gesamtproteinmenge an STAT1 gegenüber dem Wildtyp reduziert war. Zellen aus lymphatischen Organen STAT1-defizienter Tiere mit experimenteller autoimmuner Enzephalomyelitis, die als Modell einer T-Helfer-Zell-vermittelten Autoimmunerkrankung verwendet wurde, zeigten einen hyperproliferativen Phänotyp und sezernierten größere Mengen an IFNγ und IL-17A. Injektion dieser Mäuse mit Lipopolysaccharid während der Induktionsphase der experimentellen autoimmunen Enzephalomyelitis hob den hyperproliferativen Phänotyp vollständig auf. Zusammenfassend demonstrieren die Ergebnisse aus dieser Arbeit die Bedeutung einer kooperativen DNA-Bindung und Tetramerstabilisierung von STAT1 im Zusammenspiel komplexer immunologischer Prozesse auch in Abwesenheit infektiöser Pathogene und unterstreichen zudem die Schlüsselrolle von tyrosinphosphoryliertem STAT1 bei der Verknüpfung zwischen angeborenem und erworbenem Immunsystem.

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Endogenous retroviruses (of the HERV-W family) represent about 8% (1%) of our genome. Their endogenous and exogenous forms (MSRV, Multiple Sclerosis-associated RetroVirus) can alter the regulation of the immune system and be involved in inam- matory and autoimmune pathologies (Multiple Sclerosis). The MSRV envelope protein (ENV) stimulates T lymphocytes by acting as a superantigen. It also interacts with mono- cytes and dendritic cells via membrane receptors, thereby provoking inammatory cytokine production. Our studies are based on the characterisation of the immunological cascade leading from the interaction of the viral envelope with its receptor to the pathological inammatory reaction. The work presented in this thesis combines an in vitro cellular and molecular approach with an in vivo validation using an animal model (mouse). The chosen animal model is Experimental Autoimmune Encephalomyelitis (EAE) in which the complete Freund's adjuvant can be replaced by ENV. Its effects on the murine organism are evaluated on several levels: analysis of behaviour (clinical score) and brain (IRM), cellular and molecular analysis of the immune system. Furthermore, we want to generate a transgenic mouse model expressing different ENVs (MSRV/HERV) under the control of different promoters since MSRV/HERV are found only in great apes. This model, of which the rst steps of elaboration are part of this work, will allow us to study the behaviour of the ENV over-expressing animals as well as their brain and the effects on the immune system.

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