Chaque année, au moins 300 millions de personnes sont touchées par le paludisme et plus d’1 million d’entre elles en décèdent. Le neuropaludisme reste une complication mortelle, notamment chez les jeunes enfants d’Afrique. En dehors de la résistance aux antipaludiques, un défi de la lutte contre le paludisme est de définir la place des traitements protecteurs de l’hôte associés aux antipaludiques. En effet, le taux de mortalité résiduelle au cours du neuropaludisme est d’environ 20%, malgré un traitement adapté. Ce travail décrit les modèles expérimentaux de neuropaludisme, la place des neuroprotecteurs dans cette pathologie, puis, l’efficacité de deux neuroprotecteurs, l’érythropoïétine recombinante et les statines, au cours d’un neuropaludisme expérimental. Enfin, la pertinence de la neuroprotection sera documentée par une étude de preuve de concept chez l’Homme en zone d’endémie. / There are at least 300 million cases of malaria each year, resulting in more than a million deaths. Cerebral malaria is the most severe complication of malaria especially in young African children. Besides drug resistance, another challenge in the fight against malaria is the protective treatment of the host combined to the conventional antimalarial treatment. This opportunity came with the consideration of cerebral malaria residual case fatality rate of about 20%, despite a timely adequate antimalarial treatment. This work describes the available experimental model of cerebral malaria, the place of neuroprotective therapies in this disease and the efficacy of two neuroprotective drugs, recombinant erythropoietin and statins, during experimental cerebral malaria. Last, a proof-of-concept study documented the relevance of neuroprotection during human cerebral malaria in endemic area

Clinical findings have linked multiple risk factors and associated pathologies to Alzheimer\''s disease (AD). Amongst them are vascular risk factors such as hypertension and pathologies such as stroke. Coexistence of AD and these associated pathologies worsenes dementia, the clinical hallmark of the disease, as compared to pure AD. One general common denominator of these associated pathologies is the presence of hypoxic tissue conditions. It was asked the question, whether there exists a mutual, causal interaction between hypoxia and AD pathology, that could explain the clinical observations. Alternatively, the worsened clinical state of multiple brain pathologies could \"simply\" be the consequence of multimorbidity, i.e. accumulated disease load, without any causal interaction between the constituents. To approach this question whether hypoxia influences AD progression, use was made of a murine animal model of AD (transgenic mice: APPswe, PSEN1dE). Animals of two ages (8 and 14 months, \"young\" and \"old\" respectively) and two genotypes (transgenic and wild- type) were either treated under hypoxia or normoxia, corresponding to 8% and 21% oxygen, for 20 consecutive days. The resulting changes in the brain were assessed with a variety of techniques, namely by histology, ELISA, dot and Western blotting. Additional experiments in primary cell cultures were performed. Animals exposed to hypoxia showed an increased hematocrit (HCT), weight loss, reactive angiogenesis, but no infarctions. This illustrates that our hypoxic treatment put significant stress on the animals, without causing major pathologies. A large number of variables exists that could potentially be measured to assess the effect of hypoxia on AD. The focus was put on three of them: First, there is the Abeta1-42- protein, known to be the Abeta- isoform associated with the most detrimental disease progression. In AD, the self-combinatory Amyloid- beta peptide (Abeta) accumulates in the brain in so- called plaques, which is a main histologic finding of the disease. Its quantity was determined through histology and ELISA. Secondly, it was attempted to estimate the structural quality of the Abeta- protein by assessing the amount of A!- oligomers present. Abeta- protein does self- accumulate in various grades of complexity, i.e. as monomer, oligomer or fibril. Since oligomers are known to be the most neurotoxic \"species\" of the Abeta- protein, it was hypothesized that under hypoxic treatment their quantity could increase. And third, the organism\''s response to the Abeta- protein stimulus was investigated. Microglial cells have been described as the first cells to encounter the Abeta- protein \"threat\" in the shape of plaques, i.e. Abeta- protein aggregates. They then try to encapsulate and subsequently degrade them. Therefore, the attention was put on this cellular population. It was asked whether hypoxia could change the Abeta- protein quantity in the brain. This was assessed in two ways: First histologically, by staining for Abeta- protein depositions and quantifying them. Second, an ELISA was performed. Our findings state that hypoxic treatment does not alter the Abeta1-42 protein load in the brain, neither in young nor old animals, as assessed by histology and by total ELISA quantification of Abeta1-42 protein. Since hypoxia did not alter the quantity of the Abeta- protein, it was asked whether it influenced it qualitatively? If hypoxia increased oligomer formation, this change in the spectrum of the Abeta- species could, without any change in total Abeta- protein load, lead to increased neurotoxicity in animals under hypoxia. Initial experiments showed that oligomer formation in the brain seems to increase. However, this was not statistically significant and future experiments are necessary to evaluate this hypothesis further. It was then asked, whether hypoxia alters the cellular response to the protein. The total number of microglia in the hippocampal dentate gyrus, our structure of interest for practical purposes, and, it can be argued, by extension the brain, changes dynamically with various factors. First, transgenic animals present an increase in microglia. Second, microglia increase with age. Third, microglia decrease under hypoxia, but only do so significantly in old animals. Next, a parameter called \"plaque occupancy\" was coined to assess the microglia function to confront Abeta- plaques. Plaque occupancy is defined as the number of microglia in spatial proximity to one square millimeter of Abeta- plaque. This means, that microglia restricting one plaque are counted, and then normalized to this plaque\''s area. It was hypothesized that hypoxia would decrease plaque occupancy. Indeed, plaque occupancy roughly halved under hypoxia. Summarizing, our results demonstrate that long- term exposure to hypoxia significantly reduces the number of microglia. The reduced number results in significantly reduced plaque occupancy and compromizes the function of microglia to confront Abeta- plaques. The Abeta1-42 load, however, is not affected. On the other hand, Abeta shows an increased trend towards oligomer formation. A variety of possible explanations to these phenomena have been presented, that in our opinion deserve further investigation.

Sepsis is a clinical syndrome characterized by the systemic activation of inflammatory and coagulation pathways in response to microbial infection of normally sterile parts of the body. Despite considerable advances in our understanding of sepsis pathophysiology, sepsis remains the leading cause of death in non-coronary intensive care units (ICU) with a global disease burden between 15 and 19 million cases per year (Dellinger et al., 2008). Severe sepsis, defined as sepsis associated with organ dysfunction is associated with mortality rates of 33% to 45%. The incidence of severe sepsis continues to increase by 1.5% per annum due to the aging population, a rise in the prevalence of comorbidities, and the wider use of immunosuppressive agents and invasive procedures (Angus et al., 2001). Over the past several decades, many potential treatments for sepsis have shown early promise, yet have failed to improve survival in over 100 Phase II and Phase III clinical trials (Marshall, 2014) suggesting that some fundamental knowledge is lacking in our understanding of sepsis pathophysiology. Emerging studies on cell-free DNA (cfDNA), DNA released extracellularly into the circulation, demonstrate that cfDNA is a crucial link between inflammation and coagulation . In various conditions characterized by excessive inflammatory responses or aberrant prothrombotic responses, cfDNA has been implicated in exacerbating disease pathology (Atamaniuk, Kopecky, Skoupy, Säemann, & Weichhart, 2012; Fuchs, Brill, & Wagner, 2012; Swystun, Mukherjee, & Liaw, 2011). In clinical sepsis, levels of cfDNA upon admission into the ICU have strong prognostic value in predicting mortality (Dwivedi et al., 2012; Saukkonen et al., 2008). However, it is unclear whether these increases in cfDNA are an epiphenomenon during sepsis progression, or whether cfDNA actively plays a role in sepsis pathophysiology. In this work, in vivo studies were conducted to characterize the role of cfDNA in sepsis, the effects of DNase administration, and the potential mechanism by which cfDNA is released during experimental sepsis. In addition, mortality studies were conducted to identify surrogate markers of death to promote the design of humane and ethical animal studies in conducting sepsis research. Polymicrobial sepsis was induced via a surgical procedure whereby the cecum is exteriorized, ligated and punctured twice to introduce a continuous source of microorganisms, a model termed cecal ligation and puncture (CLP). In our CLP sepsis model, levels of cfDNA increased in a time-dependent manner. These increases accompanied an early pro-inflammatory response marked by increased pro-inflammatory IL-6, a transient increase in anti-inflammatory IL-10, and elevated lung myeloperoxidase (MPO) activity. Septic mice with elevated cfDNA levels also had high bacterial loads in the lungs, blood, and peritoneal cavity fluid. Organ damage was also observed in mice following CLP surgery versus mice subjected to the non-septic sham control surgery marked by increased levels of creatinine and alanine aminotransferase (ALT) indicative of kidney and liver injury, respectively. Histological analyses further confirmed lung and kidney damage following CLP surgery. Changes in coagulation were also observed in septic mice as mice subjected to CLP had sustained increases in thrombin-antithrombin (TAT) complexes. In addition, plasma from CLP-operated mice had increased thrombin generation (i.e. increased endogenous thromin potential, increased peak thrombin, decreased time to peak, and decreased lag time) mediated by FXIIa and enhanced by platelets. Following CLP-induced sepsis, elevations in cfDNA levels accompanied pro-inflammatory and pro-coagulant responses. The effects of in vivo DNase treatment in septic mice were time-dependent. Early DNase treatment when cfDNA levels were low resulted in an exaggerated pro-inflammatory response marked by increased plasma IL-6 levels and increased lung damage. In contrast, delayed DNase treatment at time-points when cfDNA levels were elevated suppressed inflammation characterized by an increase in anti-inflammatory IL-10 and reductions in cfDNA, IL-6, lung MPO, and ALT activity. Furthermore, delayed DNase administration resulted in decreased bacterial load in the lungs, blood, and peritoneal cavity fluid. Delayed DNase treatment also resulted in blunted pro-coagulant responses as levels of TAT complexes were suppressed and thrombin generation from septic mouse plasma was normalized. Moreover, DNase treatment when cfDNA levels were elevated increased survival in CLP-operated mice by 80% and reduced lung and liver damage. These findings suggest that administration of DNase when cfDNA levels are elevated may reduce pro-inflammatory and pro-coagulant responses and that delayed DNase treatment may infer protection in the CLP model of sepsis. One mechanism by which cfDNA is released is via the formation of neutrophil extracellular traps (NETs). Upon inflammatory stimulation, some neutrophils release chromatin material and antimicrobial proteins (i.e. neutrophil elastase, MPO, and histones) in an active process termed NETosis. Although NETs ensnare bacteria and exert antimicrobial properties, NETs may also exert harmful effects on the host by activating inflammation and coagulation. While some in vitro evidence suggest that neutrophils are the main source of cfDNA released following inflammatory stimulation, others have reported that neutrophils are not the main source of circulating cfDNA following septic challenge. To determine whether NETs contribute to cfDNA released during CLP sepsis, genetically modified mice that are incapable of forming NETs, PAD4-/- mice, were used. Levels of cfDNA in PAD-/- mice were significantly lower than cfDNA levels in C57Bl/6 mice following CLP surgery, suggesting that NETs were a source of cfDNA in our model. Levels of IL-6, MPO, and bacterial load in the lungs, blood, and peritoneal cavity were significantly reduced, indicating that NETs exert pro-inflammatory effects in CLP sepsis. Thrombin generation was also suppressed in PAD4-/- mice which suggests that NETs contribute to thrombin generation following CLP sepsis. NETs contribute to increases in circulating cfDNA and may exacerbate pathology by driving pro-inflammatory and pro-coagulant responses in CLP-induced sepsis. Appreciating the implications of conducting research using animals, it is pertinent that researchers ensure the highest ethical standards and design animal studies in the most humane, yet scientifically rigorous manner. Using mortality studies, we validated the utility of physiological and phenotypic markers to assess disease severity and predict death in murine sepsis. Temperature via a rectal probe monitor and sepsis scoring systems which assess components such as orbital tightening, level of consciousness, and activity were effective surrogate markers of death. These tools offer a non-invasive assessment of disease progression which do not artificially exacerbate sepsis pathology and immediate information regarding any changes in the health status. Surrogate markers of death also provide reliable monitoring to meet increasing standards of ethical, humane animal research and a feasible and cost-efficient means to obtain vital signs in small rodents. We have proposed a scoring system which can be used for assessing disease severity, endpoint monitoring, and predicting death to obviate inhumane methods of using death as an endpoint in sepsis studies. In summary, cfDNA levels are elevated in CLP-induced sepsis and these elevations accompany pro-inflammatory and pro-coagulant responses. NETosis may be a mechanism by which cfDNA is released and NETs may drive inflammation and coagulation in CLP sepsis. Delayed DNase administration may suppress inflammation and coagulation and may be protective in polymicrobial sepsis. In future animal sepsis studies, surrogate markers of death and a sepsis scoring system can be used in place of death as an endpoint to raise the standards in conducting ethical, humane sepsis research. / Thesis / Doctor of Philosophy (PhD)

No description available.

La sclérodermie (SSc) est une maladie rare affectant les personnes génétiquement prédisposées d’une réponse immunitaire défectueuse. Malgré les derniers avancements et développements dans le domaine, l’étiologie et la pathogénèse de la maladie demeurent peu comprises. Par ailleurs, il y a un ralentissement dans la compréhension de cette maladie à cause du manque de modèle animal représentatif de la SSc humaine. Malgré plusieurs lacunes, les souris traitées avec la bléomycine ou portant des modifications génétiques (TSK-1) sont très utilisées dans les études précliniques de la SSc mais elles ne présentent pas toutes les caractéristiques de cette maladie. Pour contribuer à la recherche sur la SSc, la stagiaire postdoctorale Dre Heena Mehta a développé dans le laboratoire du Dre Sarfati en collaboration avec le Dr Senécal, un modèle de souris expérimental induit par l’immunisation de cellules dendritiques (DCs) chargées de peptides de la protéine topoisomérase I (TOPOIA et TOPOIB). Dans le but de caractériser ce modèle murin et d’établir un profil immunitaire, j’ai concentré mes analyses principalement sur les caractéristiques de la SSc telles que la fibrose, l’inflammation, l’hyper-γ-globulinémie polyclonale, la vasculopathie ainsi que de l’expression de cytokines. Brièvement, l’immunisation de souris avec les DCs chargées avec la topoisomérase I (TOPOI) a induit l’inflammation pulmonaire et cutanée, en plus de la fibrose sous forme diffuse (dcSSc). Les souris présentaient également des symptômes de la vasculopathie ainsi que des taux élevés d’anticorps polyclonaux. Les résultats démontraient que les peptides TOPOIA étaient efficaces dans l’induction de la fibrose et de la réponse inflammatoire alors que les peptides TOPOIB étaient surtout impliqués dans la fibrose cutanée. En plus de nos résultats, les observations préliminaires sur le profil de cytokines tissulaires suggéraient que ce modèle pourrait remplacer ou complémenter les autres modèles animaux de SSc. / Systemic sclerosis (SSc) is a rare disease of unknown etiology that affects people that have a genetic predisposition to autoimmunity. Despite the latest advancement and development in the field, the mechanisms underlying disease development remain poorly understood. The lack of animal model that encompasses the cardinal features of human systemic sclerosis is a major cause of the slowdown in the understanding of this disease. In fact, some mouse models such as the bleomycin induced-SSc and TSK-1 mouse are widely used in preclinical studies of scleroderma. However, these models have several shortcomings since these mice do not display all the cardinal features of the disease found in humans. To contribute to the research of SSc, postdoctoral fellow Dre Heena Mehta has developed in Dre Sarfati’s laboratory in collaboration with Dr Senécal, an experimental murine model of SSc induced by dendritic cells loaded with topoisomerase I peptide. In order to characterise the model and establish an immune profile of our experimental mice, my analysis focused mainly on the cardinal features of scleroderma such as fibrosis, inflammation and polyclonal hyper-γ-globulinemia, vasculopathy and cytokines gene expression. Hence, immunization with dendritic cells loaded topoisomerase I peptides (TOPOIA and TOPOIB) induced pulmonary and dermal inflammation together with diffuse form of fibrosis. The mice also showed symptoms of vasculopathy and high levels of polyclonal antibodies. These results showed that TOPOIA peptides are effective in inducing fibrosis and inflammatory response while TOPOIB peptides are involved in skin fibrosis. Together with the results, the preliminary data on cytokine profile in tissue suggested that our mouse model could possibly replace/complement other current animal models of scleroderma.

A bovinocultura brasileira possui grande ênfase no mercado nacional. Doenças que acometem rebanhos comprometem o mercado além de gerarem grandes prejuízos econômicos. O Clostridium novyi tipo B provoca necrose hepática em bovinos através da produção da alfa toxina, uma potente exotoxina que reduz a produtividade através de alterações como hemoglobinúria, redução do apetite, febre, letargia, diminuição da produção de leite e sangue nas fezes. Conter o microrganismo causador torna-se uma busca necessária tanto do ponto de vista econômico quanto social. Entretanto, o controle da doença ainda é realizado por vacinas formuladas com múltiplos antígenos. A emulsão pode ser uma alternativa promissora para a melhoria da adsorção de antígenos nas formulações vacinais. Camundongos da linhagem Swiss foram utilizados a fim de se avaliar aspectos clínicos e validar resultados referentes a composição de uma nova formulação vacinal contendo adjuvante Montanide ISA 61 VG e antígeno inativado de C. novyi. Os testes de caracterização e antigenicidade indicaram a presença da proteína alfa toxina na composição avaliada. A imunogenicidade do antígeno inativado em emulsão W/O (água/óleo) foi verificada e a proporção empregada (40/60) mostrou ser ideal no uso de múltiplos antígenos, apresentando inocuidade, estabilidade do produto, liberação controlada e estímulo da resposta imune. A determinação da concentração de antígeno foi averiguada pela relação antígeno ativo e inativado com soros de animais doentes, visto a eficácia vacinal de 40%. A adequação da concentração de alfa toxina inativada na emulsão mostrou ser necessária para atingir melhores valores de proteção animal. Análises de hemograma, bioquímicas e morfologia de fígado, baço e coxa contribuíram para elucidar os efeitos da emulsão e comprovar necrose hepática nos grupos não imunizados, além de sugerir avanços na adsorção de vacinas. Os resultados possibilitaram o estabelecimento de um modelo murino de infecção de C. novyi com futuras aplicações relacionadas à produção vacinal com múltiplos antígenos emulsificados para controle das clostridioses. / Brazilian cattle breeding has great emphasis on the national market. Diseases that affect herds compromise the market as well as generate great economic losses. Clostridium novyi type B causes hepatic necrosis in cattle through the production of alpha toxin, a potent exotoxin that reduces productivity through changes such as hemoglobinuria, reduced appetite, fever, lethargy, decreased milk and stool production. Containing the causative micro-organism becomes a necessary quest both economically and socially. However, control of the disease is still performed by vaccines formulated with multiple antigens. The emulsion may be a promising alternative for the improvement of antigen adsorption in vaccine formulations. Swiss strain mice were used to evaluate clinical aspects and validate results regarding the composition of a new vaccine formulation containing Montanide ISA 61 VG adjuvant and C. novyi inactivated antigen. Characterization and antigenicity tests indicated the presence of the alpha toxin protein in the evaluated composition. The immunogenicity of antigen inactivated in W / O emulsion (water / oil) was verified and the ratio employed (40/60) showed to be ideal in the use of multiple antigens, presenting innocuousness, product stability, controlled release and stimulation of the immune response. The determination of the antigen concentration was investigated by the active antigen ratio and inactivated with sera from sick animals, since the vaccine efficacy was 40%. The suitability of the inactivated alpha toxin concentration in the emulsion was shown to be necessary to achieve better animal protection values. Hemogram, biochemical and liver, spleen and thigh morphology contributed to elucidate the effects of the emulsion and to verify hepatic necrosis in the nonimmunized groups, in addition to suggesting advances in the adsorption of vaccines. The results allowed the establishment of a murine model of C. novyi infection with future applications related to the vaccine production with multiple emulsified antigens to control clostridia.

Physical exercise has been implicated in several immunophysiological improvements especially during the aging process when an immunocompromised status could be established. Toxoplasma gondii is a protozoan parasite that causes a widespread opportunistic infection, which may present severe consequences, particularly to the fetus and immunocompromised patients. It is estimated that one-third of the human population worldwide has been infected by this parasite, being the reactivation during immunesenescence an unexplored public health issue. The present study was designed to assess the immunophysiological differences between exercised versus sedentary C57BL/6 male mice that have been infected by T. gondii. Before the two key set of experiments, several other sets were carried out to the prospection of the main found data of this study. There were two main sets of experiments: in the first set, the animals were infected after exercising and three groups were formed: experimental groups - infected sedentary (IS, n=6); infected exercised (IEx, n=6) and control group - non-infected sedentary (NIS, n=6). When splenocytes were stimulated by T. gondii-soluble tachyzoite antigens, it was found an significant increasing (P<0.0001) in interferon-gamma (IFN-&#61543;) production concomitant with any changes in IL-10, allowing a greater IFN-&#61543;/IL-10 ratios, produced by the cells from exercised group compared to sedentary one. However, there was no difference concerning quantification of T. gondii genomic DNA by qPCR and immunohistochemistry analysis in brain cysts (P>0.05). In order to further investigate the consequences of these data for the host, a second set of experiments was performed, when the animals were infected before exercising and four groups of animals were established for comparison purpose, as follows: experimental groups - infected sedentary (IS, n=7); infected exercised (IEx, n=6) and control groups - non-infected sedentary (NIS, n=6) and non-infected exercised (NIEx, n=6). It was found that exercised group survived longer (P<0.05) than sedentary one. In both sets of experiments, mice have been submitted to moderate exercises: running (14m/min; 3x/week) and strength (60-80% of one-repetition maximum; 2x/week). In summary, moderated aerobic and strength exercises are able to modulate immune responses against T. gondii infection, being these immunological features beneficial to the host. / A prática regular de exercício físico tem sido relacionado com melhoras imunopatológicas, especialmente durante o processo de envelhecimento quando pode haver um estado de imunocomprometimento. O Toxoplasma gondii é um parasito protozoário que causa infecção oportunista de forma global, podendo apresentar consequências severas, particularmente ao feto e pacientes imunocomprometimentos. É estimado que um terço da população mundial esteja atualmente infectado por este parasito, sendo a reativação durante a imunossenescência um tópico em saúde pública não explorado. O presente estudo foi projetado para avaliar parâmetros de diferenças imunológicas entre camundongos machos C57BL/6 exercitados e sedentários que tenham sido infectados por T. gondii. Antes dos experimentos chaves vários outros conjuntos de experimentos foram realizados para prospecção dos principais achados desta tese. Houve dois conjuntos de experimentos principais: no primeiro conjunto de experimentos, os animais foram infectados após o início dos exercícios formando-se três grupos: um grupo controle - sedentário não infectado (NIS, IS, n=4); dois grupos infectados - um sedentário (IS, n=4) e um exercitado (IEx, n=6). Quando esplenócitos foram estimuladas com antígeno de taquizoíto solúvel de T gondii (STAg), encontrou-se uma maior produção de IFN-&#61543;, concomitante à uma manutenção das taxas de IL-10, que permitiu uma maior razão de IFN-&#61543;/IL10 (P<0,0001) para o grupo exercitado comparado com o sedentário. No entanto, não houve diferenças no que diz repeito a quantificação de DNA genômico de T. gondii pelas análises de qPCR e imunohistoquímica de cistos cerebrais (P>0,05). Com objetivo de se pesquisar posteriormente as consequências destes dados para o hospedeiro, um segundo conjunto de experimentos foram realizados, quando os animais foram infectados antes do início dos exercícios, e quatro grupos foram estabelecidos para comparação deste propósito, como se segue: dois grupos controles não infectados - sedentário (NIS, n=6) e exercitado (NIEx, n=6), e dois grupos infectados - sedentário (IS, n=7) e exercitado (IEx, n=6). Encontrou-se que o grupo infectado exercitado teve sua longevidade aumentada (P<0,05) comparado com o infectado sedentário. Em ambos os conjuntos de experimentos, os camundongos foram submetidos à exercícios moderados: de corrida (14 m/min; 3 x/semana) e resistido (60-80% de uma repetição máxima; 2x/semana). Em síntese, exercícios físicos aeróbicos e resistidos moderados são capazes de modular a resposta do sistema imune no combate da infecção por T. gondii sendo estas características imunológicas benéficas para o hospedeiro. / Doutor em Imunologia e Parasitologia Aplicadas

Un enjeu majeur en cancérologie est de réduire le risque de développement métastatique et de rechute. La transition épithélio-mésenchymateuse (EMT), processus physiologique au cours de l'embryogenèse, est un mécanisme central de la carcinogenèse, contribuant de façon précoce à la transformation et la dissémination des cellules tumorales via l'inhibition de la surveillance cellulaire (apoptose et senescence) et l'acquisition de capacités migratoires et invasives. Une autre caractéristique des cancers est la capacité d'échapper à la réponse immunitaire, puissante barrière anti-tumorale. Mais les cellules tumorales entretiennent des relations complexes avec le système immunitaire. Alors que la propension de l'inflammation et des cellules immunitaires innées à favoriser le développement tumoral et l'échappement immunitaire, via l'induction de l'EMT et le maintien d'un microenvironnement immuno-suppresseur, a été bien étudiée, le rôle éventuel de la réponse immunitaire adaptative dans la promotion de l'EMT est quant à lui peu connu. Grâce au développement d'un modèle murin de lignée tumorale mammaire plastique surexprimant l'oncogène Her2/Neu, ce travail démontre in vivo la capacité des cellules tumorales à subir l'EMT, induite par la réponse immunitaire médiée par les lymphocytes T. La déplétion spécifique des lymphocytes T (LT) CD4 restaure le phénotype épithélial de la tumeur, indiquant que les LT CD4 médient une réponse immunitaire induisant l'EMT. En retour, l'EMT confère aux cellules tumorales la capacité de modeler l'immunité comme le recrutement de neutrophiles. Ce travail apporte un nouvel éclairage sur les interactions entre cellules tumorales et système immunitaire / Current clinical challenge in many carcinomas is to reduce the risk of metastasis development and cancer recurrence. Epithelial-mesenchymal transition (EMT), a physiological process during embryogenesis, is a central mechanism in oncogenesis. EMT induction contributes to early transformation and dissemination through inhibition of cellular surveillance (apoptosis and senescence) and increased migrative and invasive behavior. Another necessary hallmark of cancer is the ability of tumor cells to evade immune surveillance, a powerful barrier against tumor progression. But cancer cells enjoy intricate relations with the immune system. Whereas inclination of inflammation and innate immune cells to favor tumor development and immune escape, via EMT induction and immunosuppressive microenvironment maintenance, has been well investigated, the role of adaptive immune response in EMT promotion is understudied. Based on the development of a plastic murine mammary tumor cell line model overexpressing Her2/Neu oncogene, this study demonstrate in vivo that tumor cells keep an epithelial phenotype in adaptive immunodeficient mice but undergo EMT under the pressure of T-cell mediated immune response, characterized by loss of epithelial EpCAM marker and acquisition of mesenchymal features and EMT transcriptomic signature. CD4 T cell depletion but not CD8 restores the epithelial phenotype of tumors, suggesting that CD4 T cells mediate an immune response that could lead ton EMT induction. In return, EMT confers the ability of tumor cells to shape immunity like intra-tumor neutrophil infiltration. This work shed a new light on interactions between tumor cells and immune system

La mucoviscidose est une maladie génétique présentant des altérations ioniques causées par une protéine membranaire défectueuse ou inexistante la CFTR entraînant des perturbations métaboliques. Nous sommes intéressés à l’altération du métabolisme des acides gras polyinsaturés (AGPI) n-3 et n-6, caractérisé par une déficience en acides linoléique (LA) et docosahexaénoïque (DHA) et une élévation de l’acide arachidonique (AA). Ce déséquilibre peut être préjudiciable à l’état des patients du fait du rôle clé de certains AGPI à différents niveaux de la vie. Nos travaux ont portés sur la recherche des bénéfices d’un vecteur riche en un AGPI n-3 d’intérêt, le DHA, dans le contexte de la mucoviscidose à travers deux approches : une approche par supplémentation réalisée chez un modèle murin présentant la mutation la plus répandue chez les patients, et une approche sur un modèle d’entérocyte humain afin d’étudier de façon plus approfondie l’intérêt du supplément sur la fonctionnalité cellulaire (absorption des nutriments lipidiques). Nos résultats nous ont conduits à différentes conclusions : les souris delF508 nourries avec du Peptamen Junior présentent une perturbation du métabolisme des n-6 (LA, AA) dès l’âge de 3 mois ; cette altération apparaît mais plus tard sous régime standard. Par contre, quelque soit le régime alimentaire notre modèle ne développe pas de déficience en DHA jusqu’à l’âge d’1 an. L’évolution du statut en AGPI n-6 est donc dépendante du régime et de l’âge. L’apport de glycérophospholipides enrichis en DHA administré à faible dose permet de corriger les perturbations en AGPI n-6 et d’augmenter le rapport en DHA/AA dans la plupart des organes. Ce vecteur s’avère potentiellement intéressant en nutrithérapie chez le patient. Un enrichissement physiologique en DHA de l’entérocyte ne modifie pas ses capacités absorptives qui sont plutôt régulées par le type de particules véhiculant les produits de lipolyses, micelles de sels biliaires versus liposomes. / Cystic fibrosis is a genetic disease showing ionic alterations, caused by an absent or defective membrane protein CFTR leading to metabolic disturbances. We are interested in n-6 and n-3 polyunsaturated fatty acid (PUFA) metabolism alteration characterized by a linoleic (LA) and docosahexaenoic (DHA) acids deficiency and high level of arachidonic acid (AA). This imbalance can affect patient health due to the key role of some PUFAs in different levels of life. Our studies focused on finding the benefits of a vector enriched in one interesting n-3 PUFA, DHA, in context of cystic fibrosis, through two approaches: a nutritional supplementation conducted in a mouse model with the most common mutation of cystic fibrosis patients, and an approach on a model of human enterocyte to investigate more thoroughly the effect of the vector on cell functionality (absorption of lipid nutrients). Our results led us to different conclusions: del F508 mice fed with Peptamen Junior have a metabolic disorder of the n-6 PUFA (LA, AA) at the age of 3 months, but this alteration appears later under standard regime . But, whatever the diet, our model does not develop a DHA deficiency to the age of 1 year. The changing status of n-6 is dependent on diet and age. Glycerophospholipids enriched in DHA given at low doses can correct disturbances in PUFA n-6 and increase the DHA/AA ratio in most organs. This vector is potentially interesting for a patient nutritherapy. A physiological DHA enrichment of enterocyte does not alter its absorption capacity, which is rather regulated by the type of particles carrying the lipolysis products, bile salt micelles versus liposomes.

La sclérose systémique (SSc) est une maladie auto-immune chronique incurable caractérisée par une présentation clinique complexe et hétérogène. Notre laboratoire a développé un modèle murin de fibrose pulmonaire et cutanée qui est induit par l’immunisation répétitive avec des cellules dendritiques chargées avec des peptides de la topoisomérase I, et qui partage de nombreuses caractéristiques avec la SSc humaine. Premièrement, nous avons caractérisé la maladie expérimentale quant à sa persistance à long terme (objectif 1) et son caractère progressif (objectif 2). Une cascade de coagulation dérégulée est impliquée dans le développement de la fibrose dans la SSc. La thrombine, un médiateur clé de la coagulation, semble contribuer à ce processus. Deuxièmement, nous avons étudié l’efficacité d’un inhibiteur de la thrombine, i.e. dabigatran, dans ce modèle (objectif 3). Le microbiote intestinal semble jouer un rôle déterminant dans plusieurs pathologies, y compris les maladies auto-immunes. Troisièmement, nous avons évalué l’effet de la manipulation du microbiote des souris par l’administration de streptomycine (objectif 4). Les souris immunisées développent une maladie persistante et la fibrose observée est précédée d’une phase inflammatoire. Le dabigatran aggrave la fibrose pulmonaire et cutanée lorsqu’administré durant la période inflammatoire et n’a aucun effet protecteur durant la phase fibrotique. La manipulation du microbiote par la streptomycine aggrave l’atteinte pulmonaire lorsque l’antibiothérapie est donnée en début de vie et exacerbe l’atteinte cutanée lorsqu’administrée à l’âge adulte. Notre modèle expérimental représente donc un outil important pour évaluer différentes approches thérapeutiques pour la SSc de par sa persistance et son caractère progressif. En se basant sur nos résultats, le dabigatran ne semble pas constituer un choix thérapeutique adéquat pour traiter la fibrose chez les patients atteints de SSc. L’exposition à la streptomycine à certaines périodes de la vie affecte différentiellement le développement et les manifestations cliniques de la maladie expérimentale. / Systemic sclerosis (SSc) is an incurable and chronic autoimmune disease characterized by a complex and heterogeneous clinical presentation. Our laboratory has developed a mouse model of lung and skin fibrosis that shares many features with human SSc, and is induced by repeated immunization with dendritic cells loaded with peptides of topoisomerase I. First, the long term persistence (objective 1) and progressive nature (objective 2) of this experimental disease model was characterized. A dysregulated coagulation cascade is implicated in the development of fibrosis in SSc. Thrombin, a key mediator of coagulation, appears to contribute to this process. Next, the efficacy of dabigatran, a thrombin inhibitor, to ameliorate lung and skin fibrosis was studied in this model (objective 3). Intestinal microbiota appears to play a key role in several diseases including autoimmune diseases. Finally, the effect of manipulating gut microbiota by administration of streptomycin on disease pathogenesis was evaluated in this model (objective 4). Immunized mice developed persistent fibrosis that was preceded by an inflammatory phase. Dabigatran aggravated pulmonary and skin fibrosis when administered during the inflammatory period and was not protective when given during the fibrotic phase. Manipulation of intestinal microbiota by streptomycin aggravated lung fibrosis when it was given early in life and exacerbated skin disease when administered in adulthood. Our model of experimental SSc with progressive and persistent disease represents an important tool to evaluate different therapeutic approaches for SSc. Furthermore, our results caution against the use of dabigatran as a therapeutic option to treat fibrosis in patients with SSc. Exposure to streptomycin for certain periods of life differentially affects the development and clinical manifestations of experimental SSc.