The pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid β (Aβ) plaques, present in the majority of clinically diagnosed cases. Treatment options, such as the FDA-approved Lecanemab, target early-stage AD by promoting Aβ clearance via microglial (MG) phagocytosis, but no effective therapy exists for late-stage AD. Hyperhomocysteinemia (HHcy), prevalent in the elderly, is an established risk factor for AD, with previous studies linking it to various pathologies but not to Aβ dynamics or MG function. This study examined the hypothesis that HHcy-induced epigenetic changes impair MG clearance of Aβ, potentially exacerbating AD. It also investigated the role of IGF-1 (Insulin-like growth factor 1) in MG phagocytosis, considering the established influence of hypomethylation on both IGF-1 expression and AD progression. In this study, we probed the interplay among HHcy, Aβ accumulation, and MG phagocytosis in AD using Cbs-/- mice as a model. Our approach delineated an HHcy-induced hypomethylation status in both plasma and brain cortex. HHcy increased Aβ deposition without a corresponding rise in Aβ production in the 5xFAD x Cbs-/- mouse brains. This was characterized by elevated levels of soluble Aβ40 and insoluble Aβ42, alongside unaltered the β-secretase and s-APPβ expression. Single-nucleus multiomic profiling unveiled five distinct MG subclusters (MG_0 to MG_4) in the hippocampi of both Cbs-/- and control mice. MG_0, specific to control mice, involved activated MG phagocytosis. Subclusters MG_2 and MG_3, predominantly identified in Cbs-/- mice, exhibited decreased migration and phagocytosis. Subcluster MG_4, unique to Cbs-/- mice, displayed impaired cytoskeleton organization and dendritic development. HHcy suppressed MG Aβ phagocytosis activity in the mouse MG cell line SIM-A9 (ex vivo phagocytosis), freshly isolated MG from HHcy mice with Cbs-/- or a high-methionine (HM) diet (ex vivo phagocytosis), and in 3xTg-AD mice with an HM diet (in vivo phagocytosis). Through meta-analysis over transcriptomes of Cbs-/- mouse MG, human and mouse AD MG, Aβ phagocytosis model, and human AD methylome, we identified 5 differentially expressed genes (DEGs) (Flt1, Calponin 3, Igf1, Cacna2d4, and Celsr) in HHcy-suppressed phagocytic AD MG. All of them have been reported to regulate the migration and Aβ phagocytosis of MG and macrophages (MΦ). IGF-1 expression changes are the most significant between liver-specific Ahcy-/- (model of hypomethylation) and HHcy-altered phagocytic AD MG. In both AD and non-AD mice, the HHcy group exhibited significantly lower IGF-1 levels in the brain, plasma, and MG than the non-HHcy group. Additionally, a negative correlation was observed between Igf1 levels and MG Aβ phagocytosis. Our study underscored the critical role of HHcy in AD progression, particularly through its detrimental impact on MG Aβ phagocytosis and altered MG subclusters, leading to reduced Aβ clearance. We identified key genes, such as IGF-1, essential for MG function. These findings indicated that HHcy may exacerbate AD and potentially diminish the efficacy of treatments like Lecanemab, highlighting the importance of HHcy-targeted strategies in AD therapy. / Biomedical Sciences

Traumatic brain injury (TBI) elicits an immediate neuroinflammatory response that involves resident glia and infiltrating peripheral immune cells that coordinate tissue damage and functional deficits. The activation of resident microglial has been associated with a change in their morphology from a branched-like ramified cell to an ameboid state. This activation is thought to initiate a pro-inflammatory response leading to the release of neurotoxic, immune chemoattractant, and antigen-presenting signals. Subsequently, peripheral-derived immune cells (PICs), such as neutrophils and monocytes, travel to the site of injury and help coordinate this response. However, little is known regarding whether PICs influence the progressive activation state of microglia in the acute and chronic phases of injury. Overactivation of microglia can lead to neuroinflammation-mediated tissue damage and death or dysfunction of healthy neurons. Therefore, understanding how microenvironmental cues may regulate the microglial response may aid in strategies to retool their activation state in the brain. EphA4 receptor tyrosine kinase has been identified as a potential cell-to-cell contact protein on PICs that could be involved in the inflammatory changes following TBI. While microglial activation changes have been described in TBI models, the mechanistic role of infiltrating peripheral-derived immune cell (PIC) recruitment on microglial fate and function is not well understood. The purpose of my project is to gain a better understating of the temporospatial influence that EphA4-expressing PICs, specifically monocyte/macrophages, have on microglial proliferation, survival, activation phenotype, and debris clean-up using bone marrow GFP chimeric mice and the cortical contusion injury TBI model. / Doctor of Philosophy / Traumatic brain injury (TBI) triggers an immediate response from the brain's immune system, involving both local glial cells and immune cells from outside the brain. These cells work together to mediate the initial injury but, in some cases, cause development of a secondary injury. Microglia, the brain's resident immune cell, change their shape and behavior when activated by a TBI, becoming more aggressive and releasing inflammatory proteins. At the same time, immune cells from the bloodstream, like neutrophils and monocytes, rush to the injury site to assist. Yet, it's unclear how these immune cells affect microglia over time during the injury's acute and chronic phases. If microglia become too active, they can cause further damage to brain tissue and harm healthy neurons. Therefore, understanding the signals that control microglial activity could help us develop therapies to manage brain inflammation. One protein of interest in this process is the EphA4 receptor found on immune cells, which might play a crucial role in inflammation following TBI. While we know that microglia change post-TBI, we don't fully understand how the recruitment of immune cells from outside the brain affects them. My research aims to clarify how EphA4-expressing immune cells, especially monocytes/macrophages, influence microglia in terms of growth, behavior, and their ability to mediate a TBI.

Streptococcus suis de type 2 est un microorganisme pathogène d’importance chez le porc. Il est la cause de différentes pathologies ayant comme caractéristique commune la méningite. C’est également un agent émergeant de zoonose : des cas cliniques humains ont récemment été rapportés en Asie. Cependant, la pathogénèse de S. suis n’est pas encore complètement élucidée. Jusqu’à présent, la réponse pro-inflammatoire initiée par S. suis n’a été étudiée qu’in vitro. L’étude du choc septique et de la méningite requiert toujours des modèles expérimentaux appropriés. Au cours de cette étude, nous avons développé un modèle in vivo d’infection chez la souris qui utilise la voie d’inoculation intra-péritonéale. Ce modèle a servi à l’étude de la réponse pro-inflammatoire associée à ce pathogène, tant au niveau systémique qu’au niveau du système nerveux central (SNC). Il nous a également permis de déterminer si la sensibilité aux infections à S. suis pouvait être influencée par des prédispositions génétiques de l’hôte. Le modèle d’infection par S. suis a été mis au point sur des souris de lignée CD1. Les résultats ont démontré une bactériémie élevée pendant les trois jours suivant l’infection. Celle-ci était accompagnée d’une libération rapide et importante de différentes cytokines pro-inflammatoires (TNF-α, IL-6, IL-12p40/p70, IFN-ɣ) et de chémokines (KC, MCP-1 and RANTES), qui ont entraîné un choc septique et la mort de 20 % des animaux. Ensuite, pour confirmer le rôle de l’inflammation sur la mortalité et pour déterminer si les caractéristiques génétiques de l’hôte pouvaient influencer la réponse inflammatoire et l’issue de la maladie, le modèle d’infection a été étendu à deux lignées murines consanguines différentes considérées comme résistante : la lignée C57BL/6 (B6), et sensible : la lignée A/J. Les résultats ont démontré une importante différence de sensibilité entre les souris A/J et les souris B6, avec un taux de mortalité atteignant 100 % à 20 h post-infection (p.i.) pour la première lignée et de seulement 16 % à 36 h p.i. pour la seconde. La quantité de bactéries dans le sang et dans les organes internes était similaire pour les deux lignées. Donc, tout comme dans la lignée CD1, la bactériémie ne semblait pas être liée à la mort des souris. La différence entre les taux de mortalité a été attribuée à un choc septique non contrôlé chez les souris A/J infectées par S. suis. Les souris A/J présentaient des taux exceptionnellement élevés de TNF-α, IL-12p40/p70, IL-1β and IFN- γ, significativement supérieurs à ceux retrouvés dans la lignée B6. Par contre, les niveaux de chémokines étaient similaires entre les lignées, ce qui suggère que leur influence est limitée dans le développement du choc septique dû à S. suis. Les souris B6 avaient une production plus élevée d’IL-10, une cytokine anti-inflammatoire, ce qui suppose que la cascade cytokinaire pro-inflammatoire était mieux contrôlée, entraînant un meilleur taux de survie. Le rôle bénéfique potentiel de l’IL-10 chez les souris infectées par S. suis a été confirmé par deux approches : d’une part en bloquant chez les souris B6 le récepteur cellulaire à l’IL-10 (IL-10R) par un anticorps monoclonal anti-IL-10R de souris et d’autre part en complémentant les souris A/J avec de l’IL-10 de souris recombinante. Les souris B6 ayant reçu le anticorps monoclonal anti-IL-10R avant d’être infectées par S. suis ont développé des signes cliniques aigus similaires à ceux observés chez les souris A/J, avec une mortalité rapide et élevée et des taux de TNF-α plus élevés que les souris infectées non traitées. Chez les souris A/J infectées par S. suis, le traitement avec l’IL-10 de souris recombinante a significativement retardé l’apparition du choc septique. Ces résultats montrent que la survie au choc septique dû à S. suis implique un contrôle très précis des mécanismes pro- et anti-inflammatoires et que la réponse anti-inflammatoire doit être activée simultanément ou très rapidement après le début de la réponse pro-inflammatoire. Grâce à ces expériences, nous avons donc fait un premier pas dans l’identification de gènes associés à la résistance envers S. suis chez l’hôte. Une des réussites les plus importantes du modèle d’infection de la souris décrit dans ce projet est le fait que les souris CD1 ayant survécu à la septicémie présentaient dès 4 jours p.i. des signes cliniques neurologiques clairs et un syndrome vestibulaire relativement similaires à ceux observés lors de méningite à S. suis chez le porc et chez l’homme. L’analyse par hybridation in situ combinée à de l’immunohistochimie des cerveaux des souris CD1 infectées a montré que la réponse inflammatoire du SNC débutait avec une augmentation significative de la transcription du Toll-like receptor (TLR)2 et du CD14 dans les microvaisseaux cérébraux et dans les plexus choroïdes, ce qui suggère que S. suis pourrait se servir de ces structures comme portes d’entrée vers le cerveau. Aussi, le NF-κB (suivi par le système rapporteur de l’activation transcriptionnelle de IκBα), le TNF-α, l’IL-1β et le MCP-1 ont été activés, principalement dans des cellules identifiées comme de la microglie et dans une moindre mesure comme des astrocytes. Cette activation a également été observée dans différentes structures du cerveau, principalement le cortex cérébral, le corps calleux, l’hippocampe, les plexus choroïdes, le thalamus, l’hypothalamus et les méninges. Partout, cette réaction pro-inflammatoire était accompagnée de zones extensives d’inflammation et de nécrose, de démyélinisation sévère et de la présence d’antigènes de S. suis dans la microglie. Nous avons mené ensuite des études in vitro pour mieux comprendre l’interaction entre S. suis et la microglie. Pour cela, nous avons infecté des cellules microgliales de souris avec la souche sauvage virulente (WT) de S. suis, ainsi qu’avec deux mutants isogéniques, un pour la capsule (CPS) et un autre pour la production d’hémolysine (suilysine). Nos résultats ont montré que la capsule était un important mécanisme de résistance à la phagocytose pour S. suis et qu’elle modulait la réponse inflammatoire, en dissimulant les composants pro-inflammatoires de la paroi bactérienne. Par contre, l’absence d’hémolysine, qui est un facteur cytotoxique potentiel, n’a pas eu d’impact majeur sur l’interaction de S. suis avec la microglie. Ces études sur les cellules microgliales ont permis de confirmer les résultats obtenus précédemment in vivo. La souche WT a induit une régulation à la hausse du TLR2 ainsi que la production de plusieurs médiateurs pro-inflammatoires, dont le TNF-α et le MCP-1. S. suis a induit la translocation du NF-kB. Cet effet était plus rapide dans les cellules stimulées par le mutant déficient en CPS, ce qui suggère que les composants de la paroi cellulaire représentent de puissants inducteurs du NF-kB. De plus, la souche S. suis WT a stimulé l’expression de la phosphotyrosine, de la PKC et de différentes cascades liées à l’enzyme mitogen-activated protein kinase (MAPK). Cependant, les cellules microgliales infectées par le mutant déficient en CPS ont montré des profils de phosphorylation plus forts et plus soutenus que celles infectées par le WT. Finalement, la capsule a aussi modulé l’expression de l’oxyde nitrique synthétase inductible (iNOS) induite par S. suis et par la production subséquente d’oxyde nitrique par la microglie. Ceci pourrait être lié in vivo à la neurotoxicité et à la vasodilatation. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes sous-tendant l’induction de l’inflammation par S. suis, ce qui devrait permettre, d’établir éventuellement des stratégies plus efficaces de lutte contre la septicémie et la méningite. Enfin, nous pensons que ce modèle expérimental d’infection chez la souris pourra être utilisé dans l’étude de la pathogénèse d’autres bactéries ayant le SNC pour cible. / Streptococcus suis serotype 2 is an important swine pathogen responsible for diverse infections, meningitis being its most striking feature. In addition, it is an emerging agent of zoonosis, which has gained worldwide attention due to important outbreaks in Asia. Understanding the pathogenesis of S. suis infections still represents a challenge. Up to present, the pro-inflammatory response due to S. suis has only been studied in vitro, and there is still a great need of appropriate experimental models for both septic shock and meningitis. In the present study, we successfully developed an in vivo model of S. suis infection in adult mice infected by the intraperitoneal route. This model served to investigate the pro-inflammatory events that take place at both the systemic and Central Nervous System (CNS) levels associated with this important pathogen. In addition, this model was useful to determine if susceptibility to S. suis infection may be influenced by the genetic background of the host. The mouse model of S. suis infection was standardized in CD1 mice. Results showed sustained bacteremia during the 3 days post-infection (p.i.), accompanied by a quick and substantial release of different pro-inflammatory cytokines (TNF-α, IL-6, IL-12p40/p70, IFN-ɣ) and chemokines (KC, MCP-1 and RANTES) that lead to septic shock and 20% mortality in mice. Once the hallmark of the septic phase of S. suis infection was established in CD1 mice, research continued with the objective to confirm the role of inflammation in mortality and to determine if the genetic background of the host may influence the inflammatory response toward this pathogen and the further outcome of the disease. For this, the mouse model of S. suis infection was used with two genetically different inbred mouse strains, this is, C57BL/6 (B6) and A/J mice, which are considered as the prototype of Th1-type and Th2-type mice, respectively. Results demonstrated a striking susceptibility to S. suis infection in A/J mice in comparison to B6 mice, with 100% mortality in the former mice strain at 20 h p.i., and 16 % mortality at 36 h p.i. for the latter. Very interestingly, and similarly to CD1 mice, bacteremia did not seem to be responsible for the death of mice, as both mice strains presented similar amounts of bacteria in blood and organs. Thus, it was postulated that the higher mortality in S. suis-infected A/J mice was due to uncontrolled septic shock. In fact, A/J mice presented very high levels of TNF-α, IL-12p40/p70, IL-1β and IFN-ɣ, that significantly exceeded those found in B6 mice. Remarkably, chemokine levels were similar between strains, suggesting their limited participation in the development of septic shock by S. suis. A greater survival of B6 mice was partially related to a better regulation of the pro-inflammatory cytokine cascade, as they showed a higher production of the anti-inflammatory cytokine IL-10 than A/J mice. The potential beneficial role of the IL-10 in mice infected with S. suis was confirmed using two approaches: the first, by blockage of the cell receptor of IL-10 (IL-10R) with an anti-mouse IL-10R monoclonal antibody (Mab) in B6 mice and the second by administrating recombinant mouse (rm)IL-10 (rmIL-10) to A/J mice. B6 mice that received the IL-10R MAb treatment before challenge with S. suis developed a clinical acute disease similar to that observed with A/J mice, with a striking and rapid increase in mortality and higher levels of TNF-α in comparison to those of infected mice that did not receive the treatment. Controversially, treatment with rmIL-10 significantly delayed the onset of septic shock in A/J mice infected with S. suis. These results show that survival from S. suis septic shock requires a tight regulation of pro- and anti-inflammatory mechanisms, and that the latter should be activated at the same time or soon after the onset of the pro-inflammatory response. This part of the study may represent a first step in the identification of host genes associated with resistance against S. suis. One of the most important achievements of the mouse model of infection described in this project is the development of distinct clinical signs of neurological disease in CD1 mice from 4 days p.i. Indeed, in CD1 mice that survived sepsis due to S. suis infection, clinical signs of neurological disease and vestibular syndrome, which are quite similar to those observed in clinical cases of S. suis meningitis in both pigs and humans, were observed. Studies of the brains of infected CD1 mice using in situ hybridization combined with immunocytochemistry, demonstrated that the CNS inflammatory response began with a significant increase in the transcription of Toll-like receptor (TLR)2 and CD14 initially in the brain microvasculature and choroid plexuses, suggesting that S. suis may use these structures as portals of entry to the brain. There also was activation of NF-κB (as indicated by transcriptional activation of IκBα as a reporter system) and TNF-α, IL-1β and MCP-1, mainly in cells identified as microglia and to a lesser extent in astrocytes. These signals reached different brain structures, mainly the brain cortex, corpus callosum, hippocampus, choroid plexuses, thalamus, hypothalamus and meninges. All of these pro-inflammatory events were associated with extensive areas of inflammation and necrosis, severe demyelination and presence of antigens of S. suis inside microglia. In vitro studies were conducted in order to better understand the interactions of S. suis and microglia. For this, mouse microglia were infected with a virulent wild type (WT) strain of S. suis. Two isogenic mutants deficient in capsule (CPS) or hemolysin production (suilysin, SLY) respectively, were also included for comparative purposes. The CPS was important for S. suis resistance to phagocytosis, and it also modulated the inflammatory response by hiding pro-inflammatory components from the bacterial cell wall. On the other hand, the absence of SLY, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Studies with microglia helped to confirm previous findings in vivo in mice, as the WT S. suis strain induced the up-regulation of TLR2 and the production of several pro-inflammatory mediators, including TNF-α and MCP-1. As observed in mice, S. suis induced NF-kB translocation, which was more rapid for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-kB. Moreover, WT S. suis promoted phosphotyrosine, PKC and different mitogen-activated protein kinase (MAPK) events. However, microglia infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. Finally, the CPS also modulated S. suis-induced inducible nitrogen oxide synthase (iNOS) expression and further nitric oxide production in microglia, which could be related to neurotoxicity and vasodilatation in vivo. We are confident that our results may help to more fully understand the mechanisms underlying S. suis induction of inflammation, leading to the design of more efficient anti-inflammatory strategies for sepsis and meningitis. Finally, we believe this experimental model of infection in mice could also be useful for studying the pathogenesis of infections of the CNS, due to other bacteria.

A tuberculose (TB) é um grave problema de saúde pública. Somente no ano de 2004, cerca de 9 milhões de pessoas desenvolveram TB ativa e mais de 2 milhões de pessoas morreram da doença. O desenvolvimento de novos modelos experimentais de TB seriam de grande utilidade para para elucidar mecanismos fisiopatológicos da doença e testar esquemas terapêuticos para a prevenção e contenção da doença. Além disso, o desenvolvimento de novas vacinas torna-se indispensável como ferramenta de prevenção e controle da TB. A TB no sistema nervoso central (SNC), assim como em outros tecidos do organismo, promove a ativação de células inflamatórias. No SNC a micróglia desempenha este papel, sendo capaz de produzir ou ser influenciada por mediadores solúveis. Vários mediadores estão envolvidos nos mecanismos moleculares decorrentes da infecção e inflamação causados pela TB, entre eles: NFB, iNOS e VEGF. A ativação do NFB, um fator de transcrição citoplasmático que sob estímulo migra para o núcleo celular, tem íntima relação com a indução da iNOS e de VEGF. A resistência intracelular a patógenos, inclusive ao Mycobacterium tuberculosis, parece estar associada a expressão de iNOS em macrófagos. O óxido nítrico (NO) tem papel importante na comunicação intercelular, estimulando a síntese de mediadores inflamatórios, como as citocinas, e regulando sua própria produção endógena. Estas citocinas por sua vez também podem induzir a atividade do NFB e a expressão da iNOS e VEGF. O VEGF é um potente ativador de permeabilidade vascular e de angiogênese, envolvido na ruptura da barreira hemato-encefálica. Neste estudo, mostramos a caracterização morfológica e imuno-histoquímica de um modelo murino de TB no SNC, com a indução da doença pela inoculação de BCG. Com este modelo experimental obtivemos importantes resultados que podem esclarecer mecanismos envolvidos na fisiopatologia da neuro-TB humana. A indução de meningite e tuberculomas foi possível através da inoculação de 104 cfu de BCG no cerebelo de camundongos, por estereotaxia, e esta indução foi dependente do tempo. A confirmação do diagnóstico foi feita pela detecção de bacilos álcool-ácido resistentes (BAAR), nas lesões tuberculosas. Observamos, ao longo do tempo (1 a 6 dias; 1, 2, 4 e 8 semanas) o recrutamento de diferentes populações gliais (micróglia e astrócitos) no sítio de injeção. Houve aumento de produção e ativação NFB nas lesões tuberculosas, caracterizada pela translocação da molécula do citoplasma para o núcleo celular. Houve expressão de iNOS restrita às lesões tuberculosas, além do aumento de expressão de VEGF nestas lesões. Além disso, camundongos imunizados com a vacina gênica hsp65, contra a TB, não expressam VEGF em suas lesões. Esta vacina parece conferir um efeito protetor em nosso modelo experimental, reduzindo a expressão de VEGF, e consequentemente reduzindo seu efeito angiogênico decorrente do processo inflamatório. O recrutamento glial, e a produção de mediadores solúveis (NFB, iNOS e VEGF) pelo hospedeiro, em resposta à invasão do patógeno no SNC, parecem estar envolvidos na fisiopatologia da neurotuberculose, como demonstrado neste modelo experimental. Nosso modelo permitirá investigar fatores possivelmente responsáveis pelo desenvolvimento e manutenção de lesões tuberculosas no SNC. O objetivo final seria elucidar a fisiopatologia desta grave doença e compreender eventos moleculares envolvidos na produção de lesões. O conhecimento gerado poderá permitir o delineamento de terapias específicas e efetivas. / Tuberculosis (TB) is a serious public health problem; in 2004, 9 million people developed active TB and the disease killed 2 million patients. Development of experimental models and new vaccines are essential both to elucidate physiopathological mechanisms and to control the disease. This infection in the central nervous system (CNS), as in other tissues of the organism, activates inflammatory cells. In CNS, this role is performed by the microglia, which is capable of producing or be influenced by soluble mediators. Several mediators are involved in the molecular mechanisms of the infection and inflammation by mycobacteria , such as NFB, iNOS and VEGF. NFB activation, a cytoplasmic transcriptional factor that migrates to the cellular nucleus under stimuli, is involved with the iNOS and VEGF induction of expression. The intracellular resistance to Mycobacterium tuberculosis has been associated with iNOS expression in macrophage cells. Nitric oxide (NO) is crucial in intercellular communication, modulating the synthesis of mediators of inflammation, such as cytokines, and modulation itself. These cytokines induces NFB activity, and induces iNOS and VEGF expression. VEGF is a potent activator of vascular permeability and of angiogenesis and it is a factor involved in the breakdown of the blood brain-barrier in tuberculous meningitis. In this study, we showed the morphologic and immunohistochemistry characterization of an experimental model of TB in the CNS, with inoculation of BCG in mice. In this model we elicited important outcome that can elucidate mechanisms involved in the physiopathology of human neuron-TB. Induction of meningitis and tuberculomas were possible with stereotaxic inoculation of 104 cfu of BCG in mice cerebellum, in a time-dependent way. Diagnostic was confirmed by detection of alcohol-acid resistant bacilli (BAAR), in tuberculous lesions. We observed, the time-course (1 to 6 days; 1, 2, 4 e 8 weeks) of the recruitment of different glial populations (microglia and astrocytes) in the injection site. There was increased production and activation of NFB in the tuberculous lesions, it was characterized by its nuclear translocation from cytoplasm. There was iNOS expression only in the tuberculous lesions, and expression increased of VEGF in these lesions. Furthermore, mice immunizated with vaccine DNA-hsp65 there was no expression of VEGF in its lesions. This vaccine seems confer a protector effect in our experimental model, reducing the expression of VEGF, and then reducing its angiogenic effect derived from inflammatory process. Glial recruitment, and the soluble mediators production (NFB, iNOS e VEGF) by the host, producing in response to invasion of the pathogen in the CNS, has been involved in the pathophysiology of the neuro-TB, such as demonstrated in this experimental model. Our model will allow investigate possible factors responsible for the development and maintenance of tuberculous lesions in the CNS. The final aim is to elucidate the physiopathology of this serious illness and understand the molecular events involved in the production of the lesions. The knowledge created may permit to pave the way to delineate specific and effective therapies.

A Esclerose Lateral Amiotrófica (ELA) é a doença neurodegenerativa do neurônio motor que acomete indivíduos adultos e promove a perda progressiva das funções motoras. A evolução é rápida (2 a 5 anos) e culmina na morte por complicações e falência dos músculos respiratórios. Descrições recentes sugerem a contribuição de tipos celulares não neuronais, particularmente o astrócito e a microglia, para a morte do neurônio motor. O camundongo transgênico SOD1G93A, que carrega a SOD1 humana mutada, foi utilizado neste trabalho. Estudos comportamentais apontaram alterações motoras importantes no animal transgênico a partir de 90 dias de vida e permitiram selecionar, então, as idades pré-sintomáticas de 40 dias e 80 dias para os estudos moleculares. A análise da expressão gênica nos animais transgênicos e selvagens destas duas idades foi realizada por microarray utilizando-se a plataforma que contém o genoma completo do camundongo e detectou 492 e 1105 transcritos diferencialmente expressos nos animais de 40 e 80 dias, respectivamente. Estes resultados foram validados por PCR quantitativa (qPCR). As análises bioinformáticas dos resultados identificaram 17 e 11 vias moleculares super-representadas nas idades de 40 dias e 80 dias, respectivamente. Destas, as vias endocitose, sinapse glutamatérgica, proteólise mediada por ubiquitina, via de sinalização de quimiocina, fosforilação oxidativa, processamento e apresentação de antígeno e junção oclusiva foram comuns a ambas as idades. Ainda, as vias sinapse glutamatérgica e fagossomo foram sugeridas como potencialmente mais importantes em animais transgênicos de 40 dias e 80 dias, respectivamente. Transcritos específicos foram analisados em amostras enriquecidas de células (astrócito, microglia e neurônio motor) microdissecadas a laser do corno anterior da medula espinal dos animais. Os transcritos Cxcr4, Slc1a2 e Ube2i foram avaliados por qPCR nas amostras enriquecidas de astrócitos dos animais de 40 dias, enquanto que Cxcr4 e Slc17a6 foram avaliados nas amostras de neurônios motores dos animais desta idade. Cxcr4 apresentou expressão diminuída nos astrócitos transgênicos e aumentada nos neurônios destes animais. Slc1a2, Ube2i e Slc17a6 estavam aumentados nos tipos celulares estudados nos animais transgênicos. Tap2 e Tuba1a foram avaliados nas amostras enriquecidas de microglias dos animais de 80 dias e mostraram-se aumentados nas amostras dos transgênicos. Finalmente, Akt1 apresentou expressão diminuída nos neurônios motores microdissecados dos animais transgênicos em comparação aos selvagens. Os resultados sugerem que alterações na sinalização glutamatérgica podem exercer papel essencial em fases pré-sintomáticas mais precoces da doença (40 dias), enquanto que em fases pré-clínicas mais próximas ao aparecimento dos sintomas (80 dias), as respostas mais importantes parecem estar relacionadas à neuroimmunomodulação. Dessa forma, este trabalho aponta para novas perspectivas para o estudo da ELA / Amyotrophic Lateral Sclerosis (ALS) is an adult onset motor neuron neurodegenerative disease that leads to the progressive loss of muscular functions. It is a fast progression disorder (2 to 5 years) culminating in death by respiratory failure. Recent findings suggest that non neuronal cell types, especially astrocytes and microglia, might contribute to the neuronal death. The transgenic mouse SOD1G93A, carring human mutant SOD1, was used in this study. Behavioral studies pointed to the onset of the clinical symptoms occurring at 90 days in the animal model, thus, allowing the selection of the pre-symptomatic ages of 40 and 80 days to the molecular studies. Gene expression analysis of transgenic mice and their non-transgenic littermates at those ages was performed by using a microarray platform containing the whole mouse genome and has detected 492 and 1105 differentially expressed genes at 40 days and 80 days old mice, respectively. These results were validated by quantitative PCR (qPCR). Bioinformatic analysis of the results identified 17 and 11 over-represented molecular pathways at 40 days and 80 days, respectively. Of these, endocytosis, glutamatergic synapse, ubiquitin-mediated proteolysis, chemokine signaling pathway, oxidative phosphorylation, antigen processing and presentation and also tight junction were common to both ages. Furthermore, glutamatergic synapse and fagosome were suggested as potentially more important at 40 and 80 days, respectively. Specific transcripts were analyzed on enriched samples of cells (astrocytes, microglia and motor neuron) obtained by laser microdissection from the ventral horn of mouse spinal cord. The transcripts Cxcr4, Slc1a2 and Ube2i were evaluated by qPCR in enriched samples of astrocytes of the 40 days old mice, and Cxcr4 and Slc17a6 were analyzed in motor neuron samples at this age. Cxcr4 has been found decreased in astrocytes from transgenic mice and increased in the motor neurons of these animals. Slc1a2, Ube2i and Slc17a6 have increased in the cell type in which they were evaluated in the transgenic mice. Tap2 and Tuba1a were evaluated at microglia enriched samples of 80 days old mice and were found to be increased. Finally, Akt1 has decreased in enriched samples of motor neurons from 80 days old mice. The results suggest that glutamatergic signaling might play essential role in early stages of the disease (40 days), while in phases closer to the appearance of the symptoms (80 days), the neuroimmunomodulation takes place. Thus, this study points to new perspectives for ALS study

Disfunção autonômica, inflamação e estresse oxidativo são características da hipertensão. Investigamos a cronologia das adaptações fisiológicas e celulares induzidas pelo treinamento aeróbio em ratos espontaneamente hipertensos (SHR). SHR exibiam disfunção autonômica e, no núcleo Paraventricular no hipotálamo (PVN), estresse oxidativo e inflamação. Duas semanas de treinamento aeróbio normalizaram a função autonômica, estresse oxidativo, inflamação, ativação de microglia e conteúdo de HMGB no PVN. Após 8 semanas, SHR treinados apresentaram menor pressão arterial e resistência vascular periférica. Redução do conteúdo de HMGB1 consiste num mecanismo para explicar os benefícios do treinamento, já que infusão aguda intracerebroventricular de HMGB1 produziu disfunção autonômica e ativação de microglia pela sinalização do CxCr4. Assim, redução do estresse oxidativo e da inflamação induzida pelo treinamento contribui para a reversão da disfunção autonômica na hipertensão e a redução da liberação de HMGB1 explica estes benefícios. / Autonomic dysfunction, inflammation and oxidative stress are hallmarks in hypertension. We evaluated time-course of physiologic and cellular adaptations induced by aerobic training in spontaneous hypertensive rat (SHR). SHR showed autonomic dysfunction and, in the hypothalamic paraventricular nucleus (PVN), oxidative stress and inflammation. 2-weeks of aerobic training normalized autonomic function, oxidative stress, inflammation, microglia activation and HMGB1 content into the PVN. After 8-weeks, trained SHR exhibited lower arterial pressure and peripheral vascular resistance. Decrease of HMGB1 content is a mechanism to explain these training benefits, since HMGB1 intracerebroventricular acute infusion induced autonomic dysfunction, microglia activation through CxCr4 signaling. So, decrease of oxidative stress and inflammation induced by aerobic training contributes to reverse autonomic dysfunction in hypertension and decrease of HMGB1 content explains these benefits.

Streptococcus suis de type 2 est un microorganisme pathogène d’importance chez le porc. Il est la cause de différentes pathologies ayant comme caractéristique commune la méningite. C’est également un agent émergeant de zoonose : des cas cliniques humains ont récemment été rapportés en Asie. Cependant, la pathogénèse de S. suis n’est pas encore complètement élucidée. Jusqu’à présent, la réponse pro-inflammatoire initiée par S. suis n’a été étudiée qu’in vitro. L’étude du choc septique et de la méningite requiert toujours des modèles expérimentaux appropriés. Au cours de cette étude, nous avons développé un modèle in vivo d’infection chez la souris qui utilise la voie d’inoculation intra-péritonéale. Ce modèle a servi à l’étude de la réponse pro-inflammatoire associée à ce pathogène, tant au niveau systémique qu’au niveau du système nerveux central (SNC). Il nous a également permis de déterminer si la sensibilité aux infections à S. suis pouvait être influencée par des prédispositions génétiques de l’hôte. Le modèle d’infection par S. suis a été mis au point sur des souris de lignée CD1. Les résultats ont démontré une bactériémie élevée pendant les trois jours suivant l’infection. Celle-ci était accompagnée d’une libération rapide et importante de différentes cytokines pro-inflammatoires (TNF-α, IL-6, IL-12p40/p70, IFN-ɣ) et de chémokines (KC, MCP-1 and RANTES), qui ont entraîné un choc septique et la mort de 20 % des animaux. Ensuite, pour confirmer le rôle de l’inflammation sur la mortalité et pour déterminer si les caractéristiques génétiques de l’hôte pouvaient influencer la réponse inflammatoire et l’issue de la maladie, le modèle d’infection a été étendu à deux lignées murines consanguines différentes considérées comme résistante : la lignée C57BL/6 (B6), et sensible : la lignée A/J. Les résultats ont démontré une importante différence de sensibilité entre les souris A/J et les souris B6, avec un taux de mortalité atteignant 100 % à 20 h post-infection (p.i.) pour la première lignée et de seulement 16 % à 36 h p.i. pour la seconde. La quantité de bactéries dans le sang et dans les organes internes était similaire pour les deux lignées. Donc, tout comme dans la lignée CD1, la bactériémie ne semblait pas être liée à la mort des souris. La différence entre les taux de mortalité a été attribuée à un choc septique non contrôlé chez les souris A/J infectées par S. suis. Les souris A/J présentaient des taux exceptionnellement élevés de TNF-α, IL-12p40/p70, IL-1β and IFN- γ, significativement supérieurs à ceux retrouvés dans la lignée B6. Par contre, les niveaux de chémokines étaient similaires entre les lignées, ce qui suggère que leur influence est limitée dans le développement du choc septique dû à S. suis. Les souris B6 avaient une production plus élevée d’IL-10, une cytokine anti-inflammatoire, ce qui suppose que la cascade cytokinaire pro-inflammatoire était mieux contrôlée, entraînant un meilleur taux de survie. Le rôle bénéfique potentiel de l’IL-10 chez les souris infectées par S. suis a été confirmé par deux approches : d’une part en bloquant chez les souris B6 le récepteur cellulaire à l’IL-10 (IL-10R) par un anticorps monoclonal anti-IL-10R de souris et d’autre part en complémentant les souris A/J avec de l’IL-10 de souris recombinante. Les souris B6 ayant reçu le anticorps monoclonal anti-IL-10R avant d’être infectées par S. suis ont développé des signes cliniques aigus similaires à ceux observés chez les souris A/J, avec une mortalité rapide et élevée et des taux de TNF-α plus élevés que les souris infectées non traitées. Chez les souris A/J infectées par S. suis, le traitement avec l’IL-10 de souris recombinante a significativement retardé l’apparition du choc septique. Ces résultats montrent que la survie au choc septique dû à S. suis implique un contrôle très précis des mécanismes pro- et anti-inflammatoires et que la réponse anti-inflammatoire doit être activée simultanément ou très rapidement après le début de la réponse pro-inflammatoire. Grâce à ces expériences, nous avons donc fait un premier pas dans l’identification de gènes associés à la résistance envers S. suis chez l’hôte. Une des réussites les plus importantes du modèle d’infection de la souris décrit dans ce projet est le fait que les souris CD1 ayant survécu à la septicémie présentaient dès 4 jours p.i. des signes cliniques neurologiques clairs et un syndrome vestibulaire relativement similaires à ceux observés lors de méningite à S. suis chez le porc et chez l’homme. L’analyse par hybridation in situ combinée à de l’immunohistochimie des cerveaux des souris CD1 infectées a montré que la réponse inflammatoire du SNC débutait avec une augmentation significative de la transcription du Toll-like receptor (TLR)2 et du CD14 dans les microvaisseaux cérébraux et dans les plexus choroïdes, ce qui suggère que S. suis pourrait se servir de ces structures comme portes d’entrée vers le cerveau. Aussi, le NF-κB (suivi par le système rapporteur de l’activation transcriptionnelle de IκBα), le TNF-α, l’IL-1β et le MCP-1 ont été activés, principalement dans des cellules identifiées comme de la microglie et dans une moindre mesure comme des astrocytes. Cette activation a également été observée dans différentes structures du cerveau, principalement le cortex cérébral, le corps calleux, l’hippocampe, les plexus choroïdes, le thalamus, l’hypothalamus et les méninges. Partout, cette réaction pro-inflammatoire était accompagnée de zones extensives d’inflammation et de nécrose, de démyélinisation sévère et de la présence d’antigènes de S. suis dans la microglie. Nous avons mené ensuite des études in vitro pour mieux comprendre l’interaction entre S. suis et la microglie. Pour cela, nous avons infecté des cellules microgliales de souris avec la souche sauvage virulente (WT) de S. suis, ainsi qu’avec deux mutants isogéniques, un pour la capsule (CPS) et un autre pour la production d’hémolysine (suilysine). Nos résultats ont montré que la capsule était un important mécanisme de résistance à la phagocytose pour S. suis et qu’elle modulait la réponse inflammatoire, en dissimulant les composants pro-inflammatoires de la paroi bactérienne. Par contre, l’absence d’hémolysine, qui est un facteur cytotoxique potentiel, n’a pas eu d’impact majeur sur l’interaction de S. suis avec la microglie. Ces études sur les cellules microgliales ont permis de confirmer les résultats obtenus précédemment in vivo. La souche WT a induit une régulation à la hausse du TLR2 ainsi que la production de plusieurs médiateurs pro-inflammatoires, dont le TNF-α et le MCP-1. S. suis a induit la translocation du NF-kB. Cet effet était plus rapide dans les cellules stimulées par le mutant déficient en CPS, ce qui suggère que les composants de la paroi cellulaire représentent de puissants inducteurs du NF-kB. De plus, la souche S. suis WT a stimulé l’expression de la phosphotyrosine, de la PKC et de différentes cascades liées à l’enzyme mitogen-activated protein kinase (MAPK). Cependant, les cellules microgliales infectées par le mutant déficient en CPS ont montré des profils de phosphorylation plus forts et plus soutenus que celles infectées par le WT. Finalement, la capsule a aussi modulé l’expression de l’oxyde nitrique synthétase inductible (iNOS) induite par S. suis et par la production subséquente d’oxyde nitrique par la microglie. Ceci pourrait être lié in vivo à la neurotoxicité et à la vasodilatation. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes sous-tendant l’induction de l’inflammation par S. suis, ce qui devrait permettre, d’établir éventuellement des stratégies plus efficaces de lutte contre la septicémie et la méningite. Enfin, nous pensons que ce modèle expérimental d’infection chez la souris pourra être utilisé dans l’étude de la pathogénèse d’autres bactéries ayant le SNC pour cible. / Streptococcus suis serotype 2 is an important swine pathogen responsible for diverse infections, meningitis being its most striking feature. In addition, it is an emerging agent of zoonosis, which has gained worldwide attention due to important outbreaks in Asia. Understanding the pathogenesis of S. suis infections still represents a challenge. Up to present, the pro-inflammatory response due to S. suis has only been studied in vitro, and there is still a great need of appropriate experimental models for both septic shock and meningitis. In the present study, we successfully developed an in vivo model of S. suis infection in adult mice infected by the intraperitoneal route. This model served to investigate the pro-inflammatory events that take place at both the systemic and Central Nervous System (CNS) levels associated with this important pathogen. In addition, this model was useful to determine if susceptibility to S. suis infection may be influenced by the genetic background of the host. The mouse model of S. suis infection was standardized in CD1 mice. Results showed sustained bacteremia during the 3 days post-infection (p.i.), accompanied by a quick and substantial release of different pro-inflammatory cytokines (TNF-α, IL-6, IL-12p40/p70, IFN-ɣ) and chemokines (KC, MCP-1 and RANTES) that lead to septic shock and 20% mortality in mice. Once the hallmark of the septic phase of S. suis infection was established in CD1 mice, research continued with the objective to confirm the role of inflammation in mortality and to determine if the genetic background of the host may influence the inflammatory response toward this pathogen and the further outcome of the disease. For this, the mouse model of S. suis infection was used with two genetically different inbred mouse strains, this is, C57BL/6 (B6) and A/J mice, which are considered as the prototype of Th1-type and Th2-type mice, respectively. Results demonstrated a striking susceptibility to S. suis infection in A/J mice in comparison to B6 mice, with 100% mortality in the former mice strain at 20 h p.i., and 16 % mortality at 36 h p.i. for the latter. Very interestingly, and similarly to CD1 mice, bacteremia did not seem to be responsible for the death of mice, as both mice strains presented similar amounts of bacteria in blood and organs. Thus, it was postulated that the higher mortality in S. suis-infected A/J mice was due to uncontrolled septic shock. In fact, A/J mice presented very high levels of TNF-α, IL-12p40/p70, IL-1β and IFN-ɣ, that significantly exceeded those found in B6 mice. Remarkably, chemokine levels were similar between strains, suggesting their limited participation in the development of septic shock by S. suis. A greater survival of B6 mice was partially related to a better regulation of the pro-inflammatory cytokine cascade, as they showed a higher production of the anti-inflammatory cytokine IL-10 than A/J mice. The potential beneficial role of the IL-10 in mice infected with S. suis was confirmed using two approaches: the first, by blockage of the cell receptor of IL-10 (IL-10R) with an anti-mouse IL-10R monoclonal antibody (Mab) in B6 mice and the second by administrating recombinant mouse (rm)IL-10 (rmIL-10) to A/J mice. B6 mice that received the IL-10R MAb treatment before challenge with S. suis developed a clinical acute disease similar to that observed with A/J mice, with a striking and rapid increase in mortality and higher levels of TNF-α in comparison to those of infected mice that did not receive the treatment. Controversially, treatment with rmIL-10 significantly delayed the onset of septic shock in A/J mice infected with S. suis. These results show that survival from S. suis septic shock requires a tight regulation of pro- and anti-inflammatory mechanisms, and that the latter should be activated at the same time or soon after the onset of the pro-inflammatory response. This part of the study may represent a first step in the identification of host genes associated with resistance against S. suis. One of the most important achievements of the mouse model of infection described in this project is the development of distinct clinical signs of neurological disease in CD1 mice from 4 days p.i. Indeed, in CD1 mice that survived sepsis due to S. suis infection, clinical signs of neurological disease and vestibular syndrome, which are quite similar to those observed in clinical cases of S. suis meningitis in both pigs and humans, were observed. Studies of the brains of infected CD1 mice using in situ hybridization combined with immunocytochemistry, demonstrated that the CNS inflammatory response began with a significant increase in the transcription of Toll-like receptor (TLR)2 and CD14 initially in the brain microvasculature and choroid plexuses, suggesting that S. suis may use these structures as portals of entry to the brain. There also was activation of NF-κB (as indicated by transcriptional activation of IκBα as a reporter system) and TNF-α, IL-1β and MCP-1, mainly in cells identified as microglia and to a lesser extent in astrocytes. These signals reached different brain structures, mainly the brain cortex, corpus callosum, hippocampus, choroid plexuses, thalamus, hypothalamus and meninges. All of these pro-inflammatory events were associated with extensive areas of inflammation and necrosis, severe demyelination and presence of antigens of S. suis inside microglia. In vitro studies were conducted in order to better understand the interactions of S. suis and microglia. For this, mouse microglia were infected with a virulent wild type (WT) strain of S. suis. Two isogenic mutants deficient in capsule (CPS) or hemolysin production (suilysin, SLY) respectively, were also included for comparative purposes. The CPS was important for S. suis resistance to phagocytosis, and it also modulated the inflammatory response by hiding pro-inflammatory components from the bacterial cell wall. On the other hand, the absence of SLY, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Studies with microglia helped to confirm previous findings in vivo in mice, as the WT S. suis strain induced the up-regulation of TLR2 and the production of several pro-inflammatory mediators, including TNF-α and MCP-1. As observed in mice, S. suis induced NF-kB translocation, which was more rapid for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-kB. Moreover, WT S. suis promoted phosphotyrosine, PKC and different mitogen-activated protein kinase (MAPK) events. However, microglia infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. Finally, the CPS also modulated S. suis-induced inducible nitrogen oxide synthase (iNOS) expression and further nitric oxide production in microglia, which could be related to neurotoxicity and vasodilatation in vivo. We are confident that our results may help to more fully understand the mechanisms underlying S. suis induction of inflammation, leading to the design of more efficient anti-inflammatory strategies for sepsis and meningitis. Finally, we believe this experimental model of infection in mice could also be useful for studying the pathogenesis of infections of the CNS, due to other bacteria.

Disfunção autonômica, inflamação e estresse oxidativo são características da hipertensão. Investigamos a cronologia das adaptações fisiológicas e celulares induzidas pelo treinamento aeróbio em ratos espontaneamente hipertensos (SHR). SHR exibiam disfunção autonômica e, no núcleo Paraventricular no hipotálamo (PVN), estresse oxidativo e inflamação. Duas semanas de treinamento aeróbio normalizaram a função autonômica, estresse oxidativo, inflamação, ativação de microglia e conteúdo de HMGB no PVN. Após 8 semanas, SHR treinados apresentaram menor pressão arterial e resistência vascular periférica. Redução do conteúdo de HMGB1 consiste num mecanismo para explicar os benefícios do treinamento, já que infusão aguda intracerebroventricular de HMGB1 produziu disfunção autonômica e ativação de microglia pela sinalização do CxCr4. Assim, redução do estresse oxidativo e da inflamação induzida pelo treinamento contribui para a reversão da disfunção autonômica na hipertensão e a redução da liberação de HMGB1 explica estes benefícios. / Autonomic dysfunction, inflammation and oxidative stress are hallmarks in hypertension. We evaluated time-course of physiologic and cellular adaptations induced by aerobic training in spontaneous hypertensive rat (SHR). SHR showed autonomic dysfunction and, in the hypothalamic paraventricular nucleus (PVN), oxidative stress and inflammation. 2-weeks of aerobic training normalized autonomic function, oxidative stress, inflammation, microglia activation and HMGB1 content into the PVN. After 8-weeks, trained SHR exhibited lower arterial pressure and peripheral vascular resistance. Decrease of HMGB1 content is a mechanism to explain these training benefits, since HMGB1 intracerebroventricular acute infusion induced autonomic dysfunction, microglia activation through CxCr4 signaling. So, decrease of oxidative stress and inflammation induced by aerobic training contributes to reverse autonomic dysfunction in hypertension and decrease of HMGB1 content explains these benefits.

A Esclerose Lateral Amiotrófica (ELA) é a doença neurodegenerativa do neurônio motor que acomete indivíduos adultos e promove a perda progressiva das funções motoras. A evolução é rápida (2 a 5 anos) e culmina na morte por complicações e falência dos músculos respiratórios. Descrições recentes sugerem a contribuição de tipos celulares não neuronais, particularmente o astrócito e a microglia, para a morte do neurônio motor. O camundongo transgênico SOD1G93A, que carrega a SOD1 humana mutada, foi utilizado neste trabalho. Estudos comportamentais apontaram alterações motoras importantes no animal transgênico a partir de 90 dias de vida e permitiram selecionar, então, as idades pré-sintomáticas de 40 dias e 80 dias para os estudos moleculares. A análise da expressão gênica nos animais transgênicos e selvagens destas duas idades foi realizada por microarray utilizando-se a plataforma que contém o genoma completo do camundongo e detectou 492 e 1105 transcritos diferencialmente expressos nos animais de 40 e 80 dias, respectivamente. Estes resultados foram validados por PCR quantitativa (qPCR). As análises bioinformáticas dos resultados identificaram 17 e 11 vias moleculares super-representadas nas idades de 40 dias e 80 dias, respectivamente. Destas, as vias endocitose, sinapse glutamatérgica, proteólise mediada por ubiquitina, via de sinalização de quimiocina, fosforilação oxidativa, processamento e apresentação de antígeno e junção oclusiva foram comuns a ambas as idades. Ainda, as vias sinapse glutamatérgica e fagossomo foram sugeridas como potencialmente mais importantes em animais transgênicos de 40 dias e 80 dias, respectivamente. Transcritos específicos foram analisados em amostras enriquecidas de células (astrócito, microglia e neurônio motor) microdissecadas a laser do corno anterior da medula espinal dos animais. Os transcritos Cxcr4, Slc1a2 e Ube2i foram avaliados por qPCR nas amostras enriquecidas de astrócitos dos animais de 40 dias, enquanto que Cxcr4 e Slc17a6 foram avaliados nas amostras de neurônios motores dos animais desta idade. Cxcr4 apresentou expressão diminuída nos astrócitos transgênicos e aumentada nos neurônios destes animais. Slc1a2, Ube2i e Slc17a6 estavam aumentados nos tipos celulares estudados nos animais transgênicos. Tap2 e Tuba1a foram avaliados nas amostras enriquecidas de microglias dos animais de 80 dias e mostraram-se aumentados nas amostras dos transgênicos. Finalmente, Akt1 apresentou expressão diminuída nos neurônios motores microdissecados dos animais transgênicos em comparação aos selvagens. Os resultados sugerem que alterações na sinalização glutamatérgica podem exercer papel essencial em fases pré-sintomáticas mais precoces da doença (40 dias), enquanto que em fases pré-clínicas mais próximas ao aparecimento dos sintomas (80 dias), as respostas mais importantes parecem estar relacionadas à neuroimmunomodulação. Dessa forma, este trabalho aponta para novas perspectivas para o estudo da ELA / Amyotrophic Lateral Sclerosis (ALS) is an adult onset motor neuron neurodegenerative disease that leads to the progressive loss of muscular functions. It is a fast progression disorder (2 to 5 years) culminating in death by respiratory failure. Recent findings suggest that non neuronal cell types, especially astrocytes and microglia, might contribute to the neuronal death. The transgenic mouse SOD1G93A, carring human mutant SOD1, was used in this study. Behavioral studies pointed to the onset of the clinical symptoms occurring at 90 days in the animal model, thus, allowing the selection of the pre-symptomatic ages of 40 and 80 days to the molecular studies. Gene expression analysis of transgenic mice and their non-transgenic littermates at those ages was performed by using a microarray platform containing the whole mouse genome and has detected 492 and 1105 differentially expressed genes at 40 days and 80 days old mice, respectively. These results were validated by quantitative PCR (qPCR). Bioinformatic analysis of the results identified 17 and 11 over-represented molecular pathways at 40 days and 80 days, respectively. Of these, endocytosis, glutamatergic synapse, ubiquitin-mediated proteolysis, chemokine signaling pathway, oxidative phosphorylation, antigen processing and presentation and also tight junction were common to both ages. Furthermore, glutamatergic synapse and fagosome were suggested as potentially more important at 40 and 80 days, respectively. Specific transcripts were analyzed on enriched samples of cells (astrocytes, microglia and motor neuron) obtained by laser microdissection from the ventral horn of mouse spinal cord. The transcripts Cxcr4, Slc1a2 and Ube2i were evaluated by qPCR in enriched samples of astrocytes of the 40 days old mice, and Cxcr4 and Slc17a6 were analyzed in motor neuron samples at this age. Cxcr4 has been found decreased in astrocytes from transgenic mice and increased in the motor neurons of these animals. Slc1a2, Ube2i and Slc17a6 have increased in the cell type in which they were evaluated in the transgenic mice. Tap2 and Tuba1a were evaluated at microglia enriched samples of 80 days old mice and were found to be increased. Finally, Akt1 has decreased in enriched samples of motor neurons from 80 days old mice. The results suggest that glutamatergic signaling might play essential role in early stages of the disease (40 days), while in phases closer to the appearance of the symptoms (80 days), the neuroimmunomodulation takes place. Thus, this study points to new perspectives for ALS study

A tuberculose (TB) é um grave problema de saúde pública. Somente no ano de 2004, cerca de 9 milhões de pessoas desenvolveram TB ativa e mais de 2 milhões de pessoas morreram da doença. O desenvolvimento de novos modelos experimentais de TB seriam de grande utilidade para para elucidar mecanismos fisiopatológicos da doença e testar esquemas terapêuticos para a prevenção e contenção da doença. Além disso, o desenvolvimento de novas vacinas torna-se indispensável como ferramenta de prevenção e controle da TB. A TB no sistema nervoso central (SNC), assim como em outros tecidos do organismo, promove a ativação de células inflamatórias. No SNC a micróglia desempenha este papel, sendo capaz de produzir ou ser influenciada por mediadores solúveis. Vários mediadores estão envolvidos nos mecanismos moleculares decorrentes da infecção e inflamação causados pela TB, entre eles: NFB, iNOS e VEGF. A ativação do NFB, um fator de transcrição citoplasmático que sob estímulo migra para o núcleo celular, tem íntima relação com a indução da iNOS e de VEGF. A resistência intracelular a patógenos, inclusive ao Mycobacterium tuberculosis, parece estar associada a expressão de iNOS em macrófagos. O óxido nítrico (NO) tem papel importante na comunicação intercelular, estimulando a síntese de mediadores inflamatórios, como as citocinas, e regulando sua própria produção endógena. Estas citocinas por sua vez também podem induzir a atividade do NFB e a expressão da iNOS e VEGF. O VEGF é um potente ativador de permeabilidade vascular e de angiogênese, envolvido na ruptura da barreira hemato-encefálica. Neste estudo, mostramos a caracterização morfológica e imuno-histoquímica de um modelo murino de TB no SNC, com a indução da doença pela inoculação de BCG. Com este modelo experimental obtivemos importantes resultados que podem esclarecer mecanismos envolvidos na fisiopatologia da neuro-TB humana. A indução de meningite e tuberculomas foi possível através da inoculação de 104 cfu de BCG no cerebelo de camundongos, por estereotaxia, e esta indução foi dependente do tempo. A confirmação do diagnóstico foi feita pela detecção de bacilos álcool-ácido resistentes (BAAR), nas lesões tuberculosas. Observamos, ao longo do tempo (1 a 6 dias; 1, 2, 4 e 8 semanas) o recrutamento de diferentes populações gliais (micróglia e astrócitos) no sítio de injeção. Houve aumento de produção e ativação NFB nas lesões tuberculosas, caracterizada pela translocação da molécula do citoplasma para o núcleo celular. Houve expressão de iNOS restrita às lesões tuberculosas, além do aumento de expressão de VEGF nestas lesões. Além disso, camundongos imunizados com a vacina gênica hsp65, contra a TB, não expressam VEGF em suas lesões. Esta vacina parece conferir um efeito protetor em nosso modelo experimental, reduzindo a expressão de VEGF, e consequentemente reduzindo seu efeito angiogênico decorrente do processo inflamatório. O recrutamento glial, e a produção de mediadores solúveis (NFB, iNOS e VEGF) pelo hospedeiro, em resposta à invasão do patógeno no SNC, parecem estar envolvidos na fisiopatologia da neurotuberculose, como demonstrado neste modelo experimental. Nosso modelo permitirá investigar fatores possivelmente responsáveis pelo desenvolvimento e manutenção de lesões tuberculosas no SNC. O objetivo final seria elucidar a fisiopatologia desta grave doença e compreender eventos moleculares envolvidos na produção de lesões. O conhecimento gerado poderá permitir o delineamento de terapias específicas e efetivas. / Tuberculosis (TB) is a serious public health problem; in 2004, 9 million people developed active TB and the disease killed 2 million patients. Development of experimental models and new vaccines are essential both to elucidate physiopathological mechanisms and to control the disease. This infection in the central nervous system (CNS), as in other tissues of the organism, activates inflammatory cells. In CNS, this role is performed by the microglia, which is capable of producing or be influenced by soluble mediators. Several mediators are involved in the molecular mechanisms of the infection and inflammation by mycobacteria , such as NFB, iNOS and VEGF. NFB activation, a cytoplasmic transcriptional factor that migrates to the cellular nucleus under stimuli, is involved with the iNOS and VEGF induction of expression. The intracellular resistance to Mycobacterium tuberculosis has been associated with iNOS expression in macrophage cells. Nitric oxide (NO) is crucial in intercellular communication, modulating the synthesis of mediators of inflammation, such as cytokines, and modulation itself. These cytokines induces NFB activity, and induces iNOS and VEGF expression. VEGF is a potent activator of vascular permeability and of angiogenesis and it is a factor involved in the breakdown of the blood brain-barrier in tuberculous meningitis. In this study, we showed the morphologic and immunohistochemistry characterization of an experimental model of TB in the CNS, with inoculation of BCG in mice. In this model we elicited important outcome that can elucidate mechanisms involved in the physiopathology of human neuron-TB. Induction of meningitis and tuberculomas were possible with stereotaxic inoculation of 104 cfu of BCG in mice cerebellum, in a time-dependent way. Diagnostic was confirmed by detection of alcohol-acid resistant bacilli (BAAR), in tuberculous lesions. We observed, the time-course (1 to 6 days; 1, 2, 4 e 8 weeks) of the recruitment of different glial populations (microglia and astrocytes) in the injection site. There was increased production and activation of NFB in the tuberculous lesions, it was characterized by its nuclear translocation from cytoplasm. There was iNOS expression only in the tuberculous lesions, and expression increased of VEGF in these lesions. Furthermore, mice immunizated with vaccine DNA-hsp65 there was no expression of VEGF in its lesions. This vaccine seems confer a protector effect in our experimental model, reducing the expression of VEGF, and then reducing its angiogenic effect derived from inflammatory process. Glial recruitment, and the soluble mediators production (NFB, iNOS e VEGF) by the host, producing in response to invasion of the pathogen in the CNS, has been involved in the pathophysiology of the neuro-TB, such as demonstrated in this experimental model. Our model will allow investigate possible factors responsible for the development and maintenance of tuberculous lesions in the CNS. The final aim is to elucidate the physiopathology of this serious illness and understand the molecular events involved in the production of the lesions. The knowledge created may permit to pave the way to delineate specific and effective therapies.