Résumé des travaux de thèse Contrairement au dogme original décrit par van Furth sur le système des phagocytes mononucléés (MP), il est maintenant suggéré que les monocytes ne représentent plus uniquement des précurseurs de macrophages mais qu'ils exercent des fonctions immunologiques en tant que tel. Non seulement les monocytes sont capables de circuler dans les tissus sans se différencier en macrophages mais la plupart des macrophages tissulaires ont en réalité une origine embryonnaire. Je me suis intéressée à l'activité migratoire et aux fonctions effectrices des monocytes et des macrophages grâce à l'utilisation de l'imagerie intravitale. Cette approche nous a permis d'appréhender la dynamique des MP entre le réseau vasculaire et le parenchyme tissulaire. De cette façon nous avons pu mettre en évidence le rôle de CX3CR1 dans le contrôle du déploiement monocytaire dans un modèle d'inflammation aiguë. En parallèle, dans un modèle de métastases pulmonaires, nous avons démontré que les monocytes ne contribuent qu'à une partie des macrophages associés aux tumeurs (TAM) et que l'autre partie dérive de la prolifération des macrophages interstitiels du poumon. Ces différentes sous-populations de TAM semblent avoir des contributions différentes dans le développement tumoral et la réponse à la chimiothérapie. Mon travail a permis de mieux appréhender la biologie des monocytes au-delà de leur fonction comme simples précurseurs de macrophages et apportent des concepts nouveaux sur l'origine des TAM. Ces travaux ouvrent de nouvelles perspectives dans la compréhension des mécanismes biologiques gouvernés par les MP et dans la régulation la réponse inflammatoire. / Contrariwise to the original dogma established by van Furth on the Mononuclear Phagocyte System (MPs), it is now suggested that monocyte fate and functions should not be restricted to macrophage precursors. Experimental evidences show that monocytes can circulate in tissues without differentiating and most of the tissue associated macrophages have embryonic origin. My thesis work focused on migratory activity and effective functions of monocytes and macrophages based on intravital imaging technology. This approach represent a unique tool to study the dynamic of MPs between the vasculature and tissue parenchyma. Our work provided new insights in the role of CX3CR1 in the monocyte deployment during acute inflammatory response.In a model of pulmonary metastases, we also demonstrated that monocytes represent only one part of tumor associated macrophages (TAMs) and the other part is constituted by lung interstitial macrophages. These different subsets of TAMs seem to contribute differently in tumor development and response to chemotherapy.Overall my work provides new insights about monocyte biology beyond their macrophage precursor fate and presents new concepts on the origin of TAMs. It also adds new perspectives in the understanding of biological mechanism regulated by MPs and in the control of the inflammatory response.

Monócitos são células hematopoiéticas com função na imunidade inata e adquirida. De acordo com o estímulo que recebem, podem se diferenciar em macrófagos e potencializar suas funções efetoras, modulando a resposta imune e participando de vários processos fisiológicos e patológicos. Os macrófagos são muito heterogêneos e capazes de assumir diferentes fenótipos em resposta aos estímulos que recebem do microambiente. Em um ambiente gorvernado por interferon gama (IFN-γ), se diferenciam em células com aumentada capacidade de apresentação de antígenos e síntese de citocinas pró-inflamatórias (macrófagos M1). Por outro lado, quando são estimulados por interleucina-4 (IL-4), eles se diferenciam em um fenótipo antagonista com atividade de reparo (macrófagos M2). Dada a importância destas células no sistema imune, é necessário desenvolver e otimizar técnicas que sirvam de ferramentas para estudar a diferenciação de macrófagos em seus perfis fenotípicos bem como seu papel em doenças humanas. Assim, o objetivo deste estudo foi padronizar e comparar dois diferentes protocolos de isolamento de monócitos descritos na literatura e analisar seu impacto sobre a diferenciação de macrófagos. Isolamos monócitos do sangue periférico de cinco indivíduos saudáveis pelas técnicas de aderência e seleção positiva. Os monócitos foram diferenciados a macrófagos com suplementação de M-CSF. Depois da indução aos perfis M1 e M2, avaliamos marcadores de superfície celular, expressão de mRNA de citocinas, secreção de quimiocinas e fagocitose. Observamos que os métodos utilizados para isolar monócitos possuem diferentes purezas, mas que monócitos isolados por ambos métodos foram capazes de ser diferenciados a macrófagos em seus perfis M1 e M2. Análises de citometria de fluxo mostraram que há uma diminuição da expressão de CD14, principalmente em macrófagos M2, e manutenção (M2) ou aumento (m1) da expressão de HLA-DR. Monócitos (CD80-CD86high) induzidos aos fenótipo M1 são caracterizados pela regulação positiva de CD80 e regulação negativa de CD86 (CD80++CD86+). O perfil M2 foi caracterizado pela expressão de CD206high e ausência de CD163- (CD206highCD163-). A expressão do mRNA revelou que IL-1β e TNF-α foram marcadores de M1 e TGF-β e CCL18 foram marcadores de M2. Além disso, quimiocinas inflamatórias como CXCL9, CXCL10 e CCL5 foram significativamente aumentadas em macrophagos M1. Macrófagos M1 e M2 são ativos e funcionais como demonstrado no ensaio de fagocitose. Embora ambos métodos utilizados para isolar monócitos tiveram purezas diferentes, ambas técnicas forneceram monócitos capazes de serem diferenciados a macrófagos. / Monocytes are hematopoietic cells with a major role in innate and adaptative immunity. According to the stimulus they receive, they can differentiate into macrophages and enhance effector functions by modulating the immune response and participating in various physiological and pathophysiological processes. Macrophages are very heterogeneous and are able to assume different phenotypes in response to the different stimuli they receive from the microenvironment. In a proinflammatory milieu ruled by interferon gamma (IFN-γ), they differentiate into cells with increased capacity to present antigens and synthesis of proinflammatory cytokines (M1 macrophages). On the other hand, when they are stimulated with interleukin 4 (IL-4), they differentiate into an antagonist phenotype with repair activity (M2 macrophages). Given the importance of these cells in the immune system, it is necessary to develop and optimize techniques that serve as useful tools for studying the differentiation of macrophages in their different phenotypic profiles as well as their roles in human diseases. In this regard, the aim of this study was to standardize and compare two different human monocyte isolation protocols described in the literature and analyze their impact on macrophage differentiation. We isolated peripheral blood monocytes from five healthy subjects by the adherence technique and positive selection. Monocytes were differentiated into macrophages with M-CSF supplementation. After M1 or M2 induction, we evaluated cell surface markers, mRNA cytokine expression, chemokine secretion and phagocytosis. We found that the methods used to isolate monocytes have different purities, but monocytes isolated from both methods were able to differentiate into the M1 and M2 profile. The monocyte and macrophage flow cytometry analysis demonstrated that CD14 decreased expression, mainly in M2 macrophages, and maintained (M2) or increased (M1) the HLA-DR expression. Monocytes (CD80-CD86high) induced to an M1 phenotype were characterized by upregulation of CD80 and down regulation of CD86. (CD80++CD86+) The M2 profile was characterized by the expression of CD206high and absence of CD163- (CD206highCD163-). The mRNA expression revealed that IL-1β and TNF-α were M1 markers and TGF-β and CCL18 were M2 markers. Further more, inflammatory chemokines as CXCL9, CXCL10 and CCL5 were significantly increased in M1 macrophages. M1 and M2 macrophages were active and functional as shown in the phagocytic assay. Although methods used for the isolation of monocytes yielded different purities, both techniques provided monocytes able to differentiate to macrophages.

Monócitos são células hematopoiéticas com função na imunidade inata e adquirida. De acordo com o estímulo que recebem, podem se diferenciar em macrófagos e potencializar suas funções efetoras, modulando a resposta imune e participando de vários processos fisiológicos e patológicos. Os macrófagos são muito heterogêneos e capazes de assumir diferentes fenótipos em resposta aos estímulos que recebem do microambiente. Em um ambiente gorvernado por interferon gama (IFN-γ), se diferenciam em células com aumentada capacidade de apresentação de antígenos e síntese de citocinas pró-inflamatórias (macrófagos M1). Por outro lado, quando são estimulados por interleucina-4 (IL-4), eles se diferenciam em um fenótipo antagonista com atividade de reparo (macrófagos M2). Dada a importância destas células no sistema imune, é necessário desenvolver e otimizar técnicas que sirvam de ferramentas para estudar a diferenciação de macrófagos em seus perfis fenotípicos bem como seu papel em doenças humanas. Assim, o objetivo deste estudo foi padronizar e comparar dois diferentes protocolos de isolamento de monócitos descritos na literatura e analisar seu impacto sobre a diferenciação de macrófagos. Isolamos monócitos do sangue periférico de cinco indivíduos saudáveis pelas técnicas de aderência e seleção positiva. Os monócitos foram diferenciados a macrófagos com suplementação de M-CSF. Depois da indução aos perfis M1 e M2, avaliamos marcadores de superfície celular, expressão de mRNA de citocinas, secreção de quimiocinas e fagocitose. Observamos que os métodos utilizados para isolar monócitos possuem diferentes purezas, mas que monócitos isolados por ambos métodos foram capazes de ser diferenciados a macrófagos em seus perfis M1 e M2. Análises de citometria de fluxo mostraram que há uma diminuição da expressão de CD14, principalmente em macrófagos M2, e manutenção (M2) ou aumento (m1) da expressão de HLA-DR. Monócitos (CD80-CD86high) induzidos aos fenótipo M1 são caracterizados pela regulação positiva de CD80 e regulação negativa de CD86 (CD80++CD86+). O perfil M2 foi caracterizado pela expressão de CD206high e ausência de CD163- (CD206highCD163-). A expressão do mRNA revelou que IL-1β e TNF-α foram marcadores de M1 e TGF-β e CCL18 foram marcadores de M2. Além disso, quimiocinas inflamatórias como CXCL9, CXCL10 e CCL5 foram significativamente aumentadas em macrophagos M1. Macrófagos M1 e M2 são ativos e funcionais como demonstrado no ensaio de fagocitose. Embora ambos métodos utilizados para isolar monócitos tiveram purezas diferentes, ambas técnicas forneceram monócitos capazes de serem diferenciados a macrófagos. / Monocytes are hematopoietic cells with a major role in innate and adaptative immunity. According to the stimulus they receive, they can differentiate into macrophages and enhance effector functions by modulating the immune response and participating in various physiological and pathophysiological processes. Macrophages are very heterogeneous and are able to assume different phenotypes in response to the different stimuli they receive from the microenvironment. In a proinflammatory milieu ruled by interferon gamma (IFN-γ), they differentiate into cells with increased capacity to present antigens and synthesis of proinflammatory cytokines (M1 macrophages). On the other hand, when they are stimulated with interleukin 4 (IL-4), they differentiate into an antagonist phenotype with repair activity (M2 macrophages). Given the importance of these cells in the immune system, it is necessary to develop and optimize techniques that serve as useful tools for studying the differentiation of macrophages in their different phenotypic profiles as well as their roles in human diseases. In this regard, the aim of this study was to standardize and compare two different human monocyte isolation protocols described in the literature and analyze their impact on macrophage differentiation. We isolated peripheral blood monocytes from five healthy subjects by the adherence technique and positive selection. Monocytes were differentiated into macrophages with M-CSF supplementation. After M1 or M2 induction, we evaluated cell surface markers, mRNA cytokine expression, chemokine secretion and phagocytosis. We found that the methods used to isolate monocytes have different purities, but monocytes isolated from both methods were able to differentiate into the M1 and M2 profile. The monocyte and macrophage flow cytometry analysis demonstrated that CD14 decreased expression, mainly in M2 macrophages, and maintained (M2) or increased (M1) the HLA-DR expression. Monocytes (CD80-CD86high) induced to an M1 phenotype were characterized by upregulation of CD80 and down regulation of CD86. (CD80++CD86+) The M2 profile was characterized by the expression of CD206high and absence of CD163- (CD206highCD163-). The mRNA expression revealed that IL-1β and TNF-α were M1 markers and TGF-β and CCL18 were M2 markers. Further more, inflammatory chemokines as CXCL9, CXCL10 and CCL5 were significantly increased in M1 macrophages. M1 and M2 macrophages were active and functional as shown in the phagocytic assay. Although methods used for the isolation of monocytes yielded different purities, both techniques provided monocytes able to differentiate to macrophages.

Apesar de apenas 10% dos indivíduos infectados por Mycobacterium tuberculosis desenvolverem tuberculose, essa doença causa a morte de milhares de pessoas anualmente. Sabendo que o background genético do hospedeiro está relacionado com suscetibilidade/resistência à infecção por M. tuberculosis, nosso grupo vem avaliando diferenças na resposta imunológica entre camundongos C57BL/6 resistentes e BALB/c suscetíveis. No presente estudo, nós nos propusemos a avaliar populações de macrófagos M1 e M2 em animais com capacidade diferente de elaborar resposta imune celular nessa infecção. Animais C57BL/6, que elaboram resposta imune celular de maior magnitude, e BALB/c foram infectados com M. tuberculosis, e avaliados aos 30 e 70 dias de infecção. Observamos maior porcentagem de células CD11b+Ly-6C+, que caracterizam monócitos inflamatórios, no pulmão de animais BALB/c com 70 dias de infecção comparada à porcentagem detectada em animais C57BL/6 com mesmo período de infecção. Houve correlação positiva significativa entre número de bacilos e expressão gênica para iNOS aos 30 dias de infecção comparando ambas as linhagens. Ao contrário da nossa hipótese inicial, observamos maior porcentagem de células CD11b+F4/80+CD206+, que caracteriza o fenótipo de macrófagos M2, no pulmão de animais C57BL/6 com 70 dias de infecção comparada à porcentagem detectada em animais BALB/c com mesmo período de infecção, havendo correlação inversa significativa de células CD11b+F4/80+CD206+ e número de bacilos aos 70 dias de infecção. Não houve diferença na análise funcional de macrófagos M1 e M2 obtidos a partir de precursores da medula óssea, comparando as duas linhagens de camundongos. Dessa forma, experimentos com o propósito de estudar a função dessas células in vivo serão conduzidos para avaliar se os macrófagos M2 participam da resposta protetora que auxilia na eliminação dos bacilos ou na resposta protetora que auxilia no reparo tecidual do hospedeiro. / Despite only 10% of individuals infected with Mycobacterium tuberculosis develop tuberculosis, this disease causes millions of deaths annually. We know that the host genetic background is associated with susceptibility/resistance to M. tuberculosis-infection. Therefore, our group has studied differences in the immunologic response between resistant C57BL/6 mice and susceptible BALB/c mice. In the present study, our aim was to evaluate populations of M1 and M2 macrophages in mice that generate different magnitude of cellular immune response in this infection. C57BL/6 mice, which elaborate a higher immune cellular response, and BALB/c mice were infected with M. tuberculosis, and evaluated 30 and 70 days post infection. We observed higher percentage of CD11b+Ly-6C+ cells, which characterize inflammatory monocytes, in the lung of BALB/c mice with 70 days of infection compared to the percentage detected in C57BL/6 animals in the same period of infection. There was a significant positive correlation between CFU number and iNOS genic expression comparing both mouse strains 30 days post infection. On the contrary to our initial hypothesis, we observed higher percentage of CD11b+F4/80+CD206+ cells, which characterize M2 macrophage phenotype, in the lung of Day 70-infected C57BL/6 animals compared with the percentage detected in BALB/c mice at the same time of infection. There was a significant inverse correlation between CD11b+F4/80+CD206+ cells and bacillus number at 70 days post infection. There was no difference in the functional analysis of M1 and M2 macrophages obtained from precursors of bone marrow, comparing both mouse strains. Therefore, experimental assays with the aim to study whether M2 macrophages contribute for the protective immune response that induce bacillus clearance or contribute to the protective immune response that promote host tissue repair will be performed in an attempt to understand better the role of these cells in the M. tuberculosis-infection.

Monócitos são células hematopoiéticas com função na imunidade inata e adquirida. De acordo com o estímulo que recebem, podem se diferenciar em macrófagos e potencializar suas funções efetoras, modulando a resposta imune e participando de vários processos fisiológicos e patológicos. Os macrófagos são muito heterogêneos e capazes de assumir diferentes fenótipos em resposta aos estímulos que recebem do microambiente. Em um ambiente gorvernado por interferon gama (IFN-γ), se diferenciam em células com aumentada capacidade de apresentação de antígenos e síntese de citocinas pró-inflamatórias (macrófagos M1). Por outro lado, quando são estimulados por interleucina-4 (IL-4), eles se diferenciam em um fenótipo antagonista com atividade de reparo (macrófagos M2). Dada a importância destas células no sistema imune, é necessário desenvolver e otimizar técnicas que sirvam de ferramentas para estudar a diferenciação de macrófagos em seus perfis fenotípicos bem como seu papel em doenças humanas. Assim, o objetivo deste estudo foi padronizar e comparar dois diferentes protocolos de isolamento de monócitos descritos na literatura e analisar seu impacto sobre a diferenciação de macrófagos. Isolamos monócitos do sangue periférico de cinco indivíduos saudáveis pelas técnicas de aderência e seleção positiva. Os monócitos foram diferenciados a macrófagos com suplementação de M-CSF. Depois da indução aos perfis M1 e M2, avaliamos marcadores de superfície celular, expressão de mRNA de citocinas, secreção de quimiocinas e fagocitose. Observamos que os métodos utilizados para isolar monócitos possuem diferentes purezas, mas que monócitos isolados por ambos métodos foram capazes de ser diferenciados a macrófagos em seus perfis M1 e M2. Análises de citometria de fluxo mostraram que há uma diminuição da expressão de CD14, principalmente em macrófagos M2, e manutenção (M2) ou aumento (m1) da expressão de HLA-DR. Monócitos (CD80-CD86high) induzidos aos fenótipo M1 são caracterizados pela regulação positiva de CD80 e regulação negativa de CD86 (CD80++CD86+). O perfil M2 foi caracterizado pela expressão de CD206high e ausência de CD163- (CD206highCD163-). A expressão do mRNA revelou que IL-1β e TNF-α foram marcadores de M1 e TGF-β e CCL18 foram marcadores de M2. Além disso, quimiocinas inflamatórias como CXCL9, CXCL10 e CCL5 foram significativamente aumentadas em macrophagos M1. Macrófagos M1 e M2 são ativos e funcionais como demonstrado no ensaio de fagocitose. Embora ambos métodos utilizados para isolar monócitos tiveram purezas diferentes, ambas técnicas forneceram monócitos capazes de serem diferenciados a macrófagos. / Monocytes are hematopoietic cells with a major role in innate and adaptative immunity. According to the stimulus they receive, they can differentiate into macrophages and enhance effector functions by modulating the immune response and participating in various physiological and pathophysiological processes. Macrophages are very heterogeneous and are able to assume different phenotypes in response to the different stimuli they receive from the microenvironment. In a proinflammatory milieu ruled by interferon gamma (IFN-γ), they differentiate into cells with increased capacity to present antigens and synthesis of proinflammatory cytokines (M1 macrophages). On the other hand, when they are stimulated with interleukin 4 (IL-4), they differentiate into an antagonist phenotype with repair activity (M2 macrophages). Given the importance of these cells in the immune system, it is necessary to develop and optimize techniques that serve as useful tools for studying the differentiation of macrophages in their different phenotypic profiles as well as their roles in human diseases. In this regard, the aim of this study was to standardize and compare two different human monocyte isolation protocols described in the literature and analyze their impact on macrophage differentiation. We isolated peripheral blood monocytes from five healthy subjects by the adherence technique and positive selection. Monocytes were differentiated into macrophages with M-CSF supplementation. After M1 or M2 induction, we evaluated cell surface markers, mRNA cytokine expression, chemokine secretion and phagocytosis. We found that the methods used to isolate monocytes have different purities, but monocytes isolated from both methods were able to differentiate into the M1 and M2 profile. The monocyte and macrophage flow cytometry analysis demonstrated that CD14 decreased expression, mainly in M2 macrophages, and maintained (M2) or increased (M1) the HLA-DR expression. Monocytes (CD80-CD86high) induced to an M1 phenotype were characterized by upregulation of CD80 and down regulation of CD86. (CD80++CD86+) The M2 profile was characterized by the expression of CD206high and absence of CD163- (CD206highCD163-). The mRNA expression revealed that IL-1β and TNF-α were M1 markers and TGF-β and CCL18 were M2 markers. Further more, inflammatory chemokines as CXCL9, CXCL10 and CCL5 were significantly increased in M1 macrophages. M1 and M2 macrophages were active and functional as shown in the phagocytic assay. Although methods used for the isolation of monocytes yielded different purities, both techniques provided monocytes able to differentiate to macrophages.

A flagelina é reconhecida pelo receptor TLR5, e pelos receptores NLRs, NLRC4/Naip5. Estes últimos induzem ativação de caspase-1 e liberação de IL-1b e IL-18, além da morte do macrófago por piroptose. Para investigar os mecanismos moleculares envolvidos nesses processos, MOs peritoneais foram estimulados com flagelina de B. subtilis e S. typhimurium em sua forma livre (capaz de ativar TLR5), ou inserida em vesículas lipídicas (capaz de ativar os NLRs). Demonstramos que ambas as flagelinas citosólicas induzem produção de IL-1b e morte celular, embora a flagelina de S. typhimurium tenha mostrado maior potencial em induzir produção de IL-1b e IL-6 pelos MOs. Verificamos que a produção de IL-1b e lise celular de MOs se mostraram eventos subseqüentes à formação de poros na membrana celular. Embora a liberação de IL-1b após reconhecimento de ambas as flagelinas ser um fenômeno dependente do eixo NLRC4/caspase-1, a morte celular induzida pelas flagelinas citosólicas ocorre na ausência destas moléculas, ao contrário do que prevê a literatura atual. / Flagellin is recognized by TLR5, and NLRs NLRC4/Naip5. The latter induce activation of caspase-1 and release of IL-1b and IL-18, besides the death of macrophages by pyroptosis. To investigate the molecular mechanisms involved in these processes, peritoneal MOs were stimulated with flagellin from B. subtilis and S. typhimurium in its free form (activating TLR5), or inserted into lipid vesicles (able to activate NLRs). We demonstrated that both cytosolic flagellins induce the production of IL-1b and cell death, while the flagellin of S. typhimurium has shown greater potential to induce production of IL-1b and IL-6 by MOs. We found that the production of IL-1b and cell lysis by MOs are subsequent events proven by the formation of pores in cell membranes. Although the release of IL-1b after recognition of both flagellins is a phenomenon dependent on the axis NLRC4/caspase-1, cell death induced by cytosolic flagellin occurs in the absence of these molecules, unlike that provided by the current literature.

RANKL et RANK sont membres de la superfamille des TNF et de la superfamille des TNF-récepteurs, respectivement. Ils sont connus pour jouer un rôle important dans la régulation de la masse osseuse et dans le développement et la fonction du système immunitaire. Cependant des questions restent. Nous avons utilisé des souris génétiquement modifiées pour répondre à certaines de ces questions, en particulier en utilisant une souris dont les cellules stromales réticulaires marginales manquent RANKL dans les ganglions lymphatiques. Les résultats obtenus lors de cette thèse fournissent de nouvelles informations importantes sur l'impact positif de RANKL stromal sur les macrophages des ganglions lymphatiques concomitantes avec une fonction des cellules B amélioré et une pathogénicité virale réduit. Nous avons constaté que RANKL stromal régule l'expression de lymphotoxine et CXCL13, deux molécules clés de l'homéostasie des cellules B et de l'intégrité cellulaire des organes lymphoïdes secondaires. L’activité du RANKL semble suivre une hiérarchie temporelle sur lymphotoxine/TNFα, vu que le phénotype causé par le déficit en RANKL a une pénétrance augmenté avec l'âge. De plus, nous démontrons que RANKL active les cellules endotheliales lymphatiques des ganglions lymphatiques et on a trouvé que l'intégrine ITGA2b est un nouvel indicateur pour les cellules endotheliales lymphatiques activés. Ainsi, avec MAdCAM-1, ITGA2b sert comme un nouveau marqueur pour les cellules endothéliales lymphatiques qui sont constitutivement activés par le RANKL stromal. Au total, les données confirment l'importance de RANKL pour l'homéostasie des ganglions lymphatiques et dévoile les mécanismes ci-inconnus des fonctions de RANKL. À la lumière de cela et le fait que RANKL est sensible aux hormones féminines, nous avons étudié le rôle de RANKL dans le syndrome de Sjögren, une maladie inflammatoire chronique des glandes salivaires et lacrymales avec une forte polarisation de sexe féminin. Nous apportons la preuve que la neutralisation du RANKL réduit la taille des organes lymphoïdes tertiaire. En perspective, une éventuelle diaphonie entre les cellules endothéliales lymphatiques et les macrophages ou les cellules réticulaires marginales reste à clarifier. En outre, d'autres travaux sont nécessaires pour élucider le mécanisme par lequel RANKL stimule les maladies inflammatoires chroniques présentant des structures lymphoïdes tertiaires, afin de faire RANKL une nouvelle cible pour la thérapie. / RANKL and RANK are members of the TNF-superfamily and TNF-receptor superfamily, respectively. They are known to play an important role in the regulation of bone mass and in the development and the function of the immune system. However questions still remain. We have used genetically modified mice to address some of these questions, in particular by using a mouse whose lymph node marginal reticular stromal cells lack RANKL. The results obtained during this PhD provide important new insights into the positive impact of stromal RANKL on lymph node macrophages concomitant with enhanced B cell function and reduced viral pathogenicity. We found that stromal RANKL regulates lymphotoxin and CXCL13 expression, two key molecules for B cell homeostasis and secondarylymphoid organ cellular integrity. RANKL activity seems to follow a temporal hierarchy over lymphotoxin/TNFα, as the phenotype caused by stromal RANKL-deficiency has increased penetrance with age. Furthermore, we demonstrate that RANKL activates lymph node lymphatic endothelial cells and found that the integrin ITGA2b is a new indicator for activated lymphatic endothelial cells. Thus, together with MAdCAM-1, ITGA2b serves as a novel marker for those lymphatic endothelial cells that are constitutively activated by stromal RANKL. Altogether, the data reinforce the importance of RANKL for the lymph node homeostasis and uncover here to unknown mechanisms of RANKL functions.In light of this and the fact that RANKL is responsive to female hormones, we studied the role of RANKL in the Sjögrens syndrome, a chronic inflammatory disease of salivary and lacrimal glands with a strong female sex bias. We provide evidence that RANKL neutralization reduces tertiary lymphoid organ size. On the perspective side, a possible cross talk between lymph node lymphatic endothelial cells and macrophages or marginal reticular cells remains to be clarified.Furthermore, further work is required to elucidate the mechanism by which RANKL stimulates chronic inflammatory diseases presenting tertiary lymphoid structures, in order to make RANKL a new target for therapy.

Le tabagisme actif par la mère expose le fœtus en développement à des agents qui peuvent traverser la barrière placentaire et interférer avec les fonctions placentaires. Un large éventail de fonctions immunologiques, pourrait être compromises. Dans cette étude, nous avons évalué l'effet de l'extrait de la fumée de cigarette (CSE) sur les macrophages isolés à partir de placentas humains (pMφs), qui sont les principaux partenaires de l'immunité de fœto-maternelle innée. J’ai pu montrer que le CSE inhibe la formation des cellules géantes multinucléées (MGC). Cette propriété du CSE est spécifique aux macrophages car la fusion des macrophages dérivés des monocytes est inhibée lors de la formation de granulomes in vitro. J’ai également étudié l'absorption de particules et la production de cytokines par pMφs exposés au CSE. Le CSE a inhibé l'absorption des particules de zymosan, mais pas celle du zymosan opsonisé, ce qui suggère qu’il interfère avec les récepteurs phagocytaires et non phagocytaires. Le CSE augmente la libération de TNF et d'IL-33, et une diminue celle de l'IL-10, ce qui montre que l'équilibre entre les cytokines est affecté par le CSE. En outre, l’expression des métalloprotéinases telles que les MMP-1, MMP-10 et MMP-12, connues pour être impliquées dans le remodelage des tissus et la fusion des macrophages est dérégulée. Enfin, j’ai montré que la nicotine, l'un des principaux composés de tabac, n'a pas affecté les propriétés fonctionnelles des pMφs. / Active smoking by the mother exposes the developing fetus to agents that can cross the placental barrier and interfere with placental functions. A wide range of immunological functions, including innate and adaptive immune responses, might be impaired. In this study, we assessed the effect of cigarette smoke extract (CSE) on macrophages isolated from human placentas (pMφs), which are major partners of innate feto-maternal immunity. I showed that CSE significantly inhibited the formation of multinucleated giant cells (MGCs). This property of CSE is specific to macrophages because the fusion of monocyte-derived macrophages is inhibited during the in vitro formation of granulomas. I also investigated particle uptake and cytokine production by pMφs exposed to CSE. CSE inhibited the uptake of zymosan, but not that of opsonized zymosan, suggesting that it interferes with phagocytic receptors, not with the phagocytic machinery of pMφs. CSE increased the release of Tumor Necrosis Factor and interleukin-33, and decreased that of interleukin-10, demonstrating that the balance between inflammatory and anti-inflammatory cytokines is affected by CSE. Furthermore, CSE enhanced the expression of metalloproteinase (MMPs) genes such as MMP-1, MMP-10 and MMP-12, known to be involved in tissue remodeling including macrophage fusion. Finally, I showed that nicotine, one of the major compounds of tobacco, did not affect the functional properties of pMφs.

En élevage intensif, les porcs sont exposés à de nombreux facteurs de stress qui pourraient favoriser la survenue d’infections et contribuer à l’utilisation massive d’antibiotiques. Les facteurs de stress activent principalement l’axe hypothalamo-hypophyso-surrénalien et l’axe sympatho-adréno-médullaire qui contribuent au retour à l’état d’équilibre. Les médiateurs de ces deux axes, les glucocorticoïdes et les catécholamines, peuvent moduler les réponses immunitaires d’un individu. Dans ce contexte, les objectifs de ma thèse étaient de décrire (i) les effets d’un stress social aigu chez des porcelets forts et faibles répondeurs à une stimulation à l’ACTH sur des paramètres immunitaires et (ii) les effets des catécholamines sur les macrophages porcins. Un mélange de loge durant 1h avec des congénères non familiers induit une mobilisation leucocytaire mais altère la plupart des fonctions immunitaires analysées. Si la majorité des paramètres immunitaires testés ne diffère pas entre les porcelets des 2 groupes, les porcelets avec un axe corticotrope fort semblent plus résilients au stress ce qui encourage à poursuivre cette stratégie de sélection génétique pour produire des porcs robustes. Les effets des catécholamines sur les macrophages ont ensuite été étudiés plus spécifiquement in vitro. L’activation du récepteur β2-adrénergique affecte la sécrétion de cytokines pro-inflammatoires induite par le LPS et diminue l’expression des marqueurs pro-inflammatoires par les macrophages. L’ensemble des résultats obtenus avec des approches in vivo et in vitro devrait permettre une meilleure compréhension des relations entre stress et immunité chez le porc. / In intensive husbandry, pigs are exposed to multiple stressful events, which are thought to impair immune defences and may contribute to the prophylactic use of antibiotics. Stressors mainly activate hypothalamo-pituitary-adrenocortical (HPA) and sympatho-adreno-medullar axes, which both contribute to restore homeostasis. Glucocorticoids and catecholamines are mediators of these two pathways and may modulate immune responses. In this context, my PhD project aimed (i) to describe acute social stress effects on immune traits in piglets high and low responders to ACTH stimulation and (ii) to analyze catecholamine effects on porcine macrophages in vitro. One-hour mixing with unfamiliar conspecifics increases leucocyte mobilization and affects most of tested immune functions. Although most immune parameters do not differ between piglets from both groups, stress effects were less pronounced in piglets with a strong HPA axis. Thus, selecting piglets with a strong HPA axis seems a valuable tool to produce robust animals. Catecholamine effects were then more specifically studied on macrophages in vitro. β2-adrenergic receptor activation affects LPS-induced pro-inflammatory cytokine secretion and decreases pro-inflammatory marker expression in porcine macrophages. Altogether these data obtained using in vivo and in vitro experiments should allow a better understanding of the relationships between stress and immunity in pigs.

La fièvre Q se traduit par de graves conséquences obstétricales chez la femme enceinte. La voie principale de la contamination humaine par Coxiella burnetii, l’agent de la fièvre Q, est constituée des aérosols provenant des placentas d’animaux infectés. La nature des cellules placentaires cibles de C. burnetii reste totalement inconnue. J’ai montré que C. burnetii infecte les trophoblastes des lignées BeWo et JEG et que les bactéries se répliquent fortement dans les cellules BeWo et survivent dans les cellules JEG. Une analyse par microarray montre que C. burnetii induit une réponse inflammatoire dans les cellules BeWo qui lui est spécifique. Ces résultats suggèrent que les trophoblastes pourraient constituer une niche pour C. burnetii. Les macrophages placentaires pourraient également servir de réservoir pour C. burnetii. J’ai montré que les macrophages placentaires CD14+ sont des macrophages qui présentent des caractéristiques phénotypiques, transcriptionnelles et fonctionnelles différentes de celles des monocytes circulants et des macrophages dérivés de monocytes. Ils forment en outre des cellules géantes multinucléées qui pourraient réguler l’activité cytolytique des macrophages dans le contexte placentaire puisque les macrophages placentaires CD14+ ne sont ni de type M1 ni de type M2. / In pregnant women Q fever presents obstetrical complications. The principal way of human contamination by Coxiella burnetii, the agent of Q fever, is due to aerosols from placentas of infected animals. The nature of placenta cells that are targeted by C. burnetii remains unknown. I showed that C. burnetii infects BeWo and JEG trophoblastic cells and that organisms intensively replicated in BeWo cells and survived in JEG cells. A microarray analysis showed that C. burnetii induced a specific inflammatory response in BeWo cells. These results suggest that trophoblasts may serve as a reservoir for C. burnetii. Placenta macrophages placentaires may also targeted by C. burnetii. I showed that placenta CD14+ macrophages were characterized by phenotypic, transcriptional and functional properties different from those of circulating monocytes and monocyte-derived macrophages. In addition, placenta CD14+ macrophages differentiate into multinucleated giant cells that may regulate the cytolytic activity of macrophages in the placenta context since placenta CD14+ macrophages were not polarized in M1 or M2 macrophages. While M1/M2 polarization of macrophages is well established, that of monocytes remains an important question. We activated monocytes with canonical agonists of M1 and M2 profiles in macrophages using microarrays. The early response, 6 hours, of monocytes corresponded to a type M1/M2 response but the delayed response, 18 hours, did not correspond to the M1/M2 dichotomy, demonstrating a new level of heterogeneity of myeloid cells.