La NAFLD (non alcoholic fatty liver disease) ou stéatopathie dysmétabolique est la manifestation hépatique du syndrome métabolique. Elle regroupe l’ensemble des lésions hépatiques liées à l’obésité en dehors de toute consommation d’alcool : la stéatose, la NASH (non alcoholic steatohepatitis), la fibrose, la cirrhose et le carcinome hépatocellulaire. Les systèmes immunitaires inné et adaptatif participent activement à la pathologie. J’ai étudié deux axes : l’axe du récepteur des glucocorticoïdes-GILZ dans les cellules de Kupffer et CXCR4-CXCL12 dans les lymphocytes T CD4+.Les cellules de Kupffer (CK) jouent un rôle clé dans la pathologie de la NASH. GILZ (glucocorticoid induced leucine zipper) est exprimé par les monocytes/macrophages et est sous le contrôle du récepteur aux glucocorticoïdes (GR). De plus, GILZ intervient dans l’inhibition des processus inflammatoires. J’ai montré que l’obésité entraîne une diminution de l’expression du GR et de GILZ dans les CK. En utilisant du RU486, un antagoniste spécifique du GR, j’ai prouvé que la diminution de l’expression du GR entraîne la diminution de l’expression de GILZ et sensibilise les CK au LPS. Ce mécanisme joue un rôle déterminant dans le développement de l’inflammation hépatique au cours de l’obésité, en modulant la réponse inflammatoire des CK. Le recrutement de cellules inflammatoires dans le foie est un élément clé de la progression de la NASH. Les lymphocytes T CD4+ issus de souris obèses ont des propriétés chimiotactiques accrues dépendantes de CXCR4. J’ai montré que la NASH augmente les propriétés migratoires dépendantes de CXCR4 des lymphocytes T CD4+ chez l’homme et la souris dans trois modèles murins de NASH. Le traitement de souris obèses par de l’AMD3100, un antagoniste de CXCR4, permet de diminuer le recrutement hépatique de lymphocytes. L’augmentation du chimiotactisme des lymphocytes T CD4+ n’était pas dû, ni à une augmentation de l’expression de CXCR4 et CXCR7, ni même de CXCL12 au niveau du foie. J’ai montré que ce mécanisme dépendait de l’augmentation de l’affinité de CXCR4 pour CXCL12.Ainsi, j’ai mis en évidence deux axes participant à l’inflammation hépatique au cours de l’obésité. Ces axes représentent de nouvelles cibles thérapeutiques potentielles. / NAFLD (non alcoholic fatty liver disease) is the hepatic manifestation of metabolic syndrome. It encompasses the entire spectrum of obesity-related liver lesions : steatosis, NASH (non alcoholic steatohepatitis), fibrosis, cirrhosis and hepatocellular carcinoma. Innate and adaptative immune systems participate actively to the pathophysiology.I studied two axis : the glucocorticoid receptor-GILZ axis in Kupffer cells and CXCR4-CXCL12 in CD4+ T lymphocytes.Kupffer cells (KC) play a key role in pathophysiology of NASH. GILZ (glucocorticoid induced leucine zipper) is expressed by monocytes/macrophages and is under the control of glucocorticoid receptor (GR). Moreover, GILZ takes part in inhibition of inflammatory processes. I showed that obesity induces a decreased expression of GR and GILZ in KC. Using RU486, a GR antagonist, I proved that decreased expression of GR induces the decreased expression of GILZ and sensitize KC to LPS. This mechanism plays a decisive role in initiation of liver inflammation in obesity, modulating inflammatory response of KC. In obese mice, recruitment of inflammatory cells into the liver is a key element in the progression of NASH. CD4+ T lymphocytes from obese mice have enhanced CXCR4-dependent chemotactic properties. I showed that NASH enhances CXCR4-dependent chemotactic properties of CD4+ T lymphocytes in patients and in three mouse models of NASH. Obese mice treatment with AMD3100, a CXCR4 antagonist, decreases lymphocytes recruitement into the liver. Enhanced chemotactic properties of CD4+ T lymphocytes were not due to increased expressions of nor CXCR4 and CXCR7, neither CXCL12 in the liver. I showed that this mechanism was dependent of an increased affinity of CXCR4 to CXCL12.Therefore, I highlighted two axis participating to obesity-related liver inflammation. These axis represent new potential therapeutic targets.

O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1α/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 µg/animal, i.p.) ou pela injeção de LPS (100 µg/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 µg/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1α e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 µm. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 µg/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1α/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1α estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos. / The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

Le sang provenant d’un cordon ombilical (SCO) représente une bonne source de cellules souches hématopoïétiques (CSH) pour des transplantations. Cependant, le nombre de cellules souches contenues dans ce sang est souvent insuffisant pour greffer un adulte. Le mécanisme intervenant dans la domiciliation de ces cellules au sein de la moelle osseuse (MO) est encore mal compris. On sait que l’interaction entre la chimiokine SDF-1 et le récepteur CXCR4, présent sur les cellules CD34+ de SCO, mène à la migration de ces cellules en direction de la MO. Nous pensons que l’augmentation de la proportion de cellules qui réussit à se greffer pourra pallier au problème du nombre. Les produits de dégradation, C3a et le C3desarg,, issus du système du complément, sont connus pour favoriser la réponse de cellules exprimant CXCR4 vers SDF-1. Nous avons analysé l’effet du C3adesarg, molécule non anaphylatoxique, sur la migration cellulaire vers SDF-1, de même que sur la prise de greffe des cellules CD34+ issues de SCO suite à une transplantation sur des souris NOD/SCIDyC-. Nos expériences ont démontré que le C3a ainsi que le C3adesarg augmentaient tous les deux la réponse des cellules CD34+ vers SDF-1. Toutefois, nous n’avons pas pu démontrer que ces molécules liaient directement le récepteur CXCR4. Par contre, le composé C3adesarg favorise la prise de greffe des cellules CD34+ de SCO. Il serait donc un bon candidat pour poursuivre une optimisation de ses propriétés. Nous avons également constaté que suite à une transplantation chez la souris, les cellules CD34+ de SCO subissent une hausse d’expression transitoire de leur CXCR4 environ quatre jours après la greffe. Cette hausse d’expression coïncide avec la multiplication des cellules CD34+ dans la MO. Nous avons également confirmé qu’une cellule CD34+ avec une forte expression de CXCR4 était dans un état prolifératif. Nos données suggèrent que l’interaction directe avec les cellules stromales soit responsable de cette hausse d’expression de CXCR4. / Since the first successful cord blood (CB) transplant was performed there has been a gradual increase in the use of CB for haematopoietic stem cell (HSC) transplantation, but the number of stem cells per CB is in general too low to ensure successful transplantation in adult patients. We would like to bypass the limitation of insufficient number of these cells in CB by enhancing the engraftment efficiency. The chemokine stromal-derived factor (SDF)-1, that binds to its receptor, CXCR4, plays an important and unique role in regulating the trafficking of HSC and their homing/retention in bone marrow (BM), but molecular regulatory mechanism of niches for HSC maintenance remains unclear. The complement C3 cleavage fragments, C3a and C3adesarg, modulate the responsiveness of CXCR4-expressing cell lines to SDF-1. We assessed the effect of the non anaphylatoxic complement fragment, C3adesarg, on SDF-1 responsiveness and engraftment of CB-HSC transplantation in a NOD/SCIDyC- mouse model. Complement breakdown products C3a and C3adesarg both increase the responsiveness of CD34+ cells to SDF-1. We find no evidence for direct interaction of complement fragments with CXCR4. Our data suggest that C3adesarg might contribute to optimize CB-HSC homing to bone marrow, and therefore efficacy of cord blood transplantation. We quantified the number of CXCR4 on the surface of CB-CD34+ after transplantation in mice. Our results showed that there is a transient overexpression of CXCR4 on the surface of HSC CD34+ found in the BM of NOD/SCIDyC- mice after 4-5 days post-injection. This transient overexpression correlated with multiplication of CD34+ cells in the BM. We confirm that the cells with an overexpression of CXCR4 are in a proliferation state. Our data suggested that this transient overexpression is caused by an interaction with the stomal cells.

Les récepteurs couplés aux protéines G (RCPG) sont une famille très diversifiée de protéines membranaires capables de répondre à un grand nombre de signaux chimiques tels que des photons, des molécules odorantes, ou des hormones. En plus de cette diversité, l’étude des RCPG montre que des associations protéiques spécifiques multiplient les possibilités de signalisation de chacun de ces récepteurs. En permettant d’atténuer, de potentialiser, ou de générer une nouvelle voie de signalisation, l’association des RCPG en oligomères s’avère une importante source de diversité. L’utilisation du transfert d’énergie de résonance de bioluminescence (BRET) qui permet de détecter les interactions protéiques a révélé de nombreuses associations de RCPG. Durant cette thèse, des outils ont été développés pour combiner efficacement le BRET à des essais de complémentation de protéines (PCA) dans le but de savoir si l’oligomérisation des RCPG pouvait impliquer plus de deux récepteurs. Les résultats présentés montrent que les récepteurs de chimiokines CXCR4 et CCR2 forment des homo et hétéro tétramères, et que l’activation d’un dimère CCR2 peut moduler la conformation d’un dimère CXCR4 par un changement conformationnel trans-récepteur. La coopérativité négative de liaison de ligand qui a été démontrée auparavant entre CXCR4 et CCR2 dans des lymphocytes T CD4+ exprimant les récepteurs de manière endogène confirme la validité biologique de cette interaction. Les données présentées suggèrent également que ces complexes peuvent engager les effecteurs Gαi et β-arrestine2, indiquant qu’ils représentent la forme fonctionnelle de ces récepteurs. Enfin, nous avons pu confirmer que chaque récepteur de l’hétérodimère CXCR4-CCR2 est impliqué dans l’engagement des effecteurs lors de l’activation de CCR2. Un autre niveau de complexité dans la signalisation des RCPG est atteint par leur capacité à coupler de multiples protéines G. La liaison du facteur dérivé des cellules stromales (SDF-1) au récepteur CXCR4 permet la migration des lymphocytes T par une voie de signalisation dépendante de la protéine Gαi. Nous avons pu démontrer en revanche que la migration des cellules de cancer du sein était initiée par un couplage de CXCR4 à la voie Gα13-Rho pour former des métastases dans des organes distants. Enfin, un dernier niveau de régulation des RCPG a été abordé par l’étude de la phosphorylation de CXCR4 suite à son activation, qui permet la désensibilisation du récepteur et l’engagement de voies de signalisation dépendantes de la β-arrestine. Il apparaît que la désensibilisation de la voie du calcium serait médiée par la phosphorylation de CXCR4 par les kinases des RCPG (GRK) GRK2 et GRK6 et le recrutement de β- arrestine2, alors GRK3, GRK6 et la β-arrestine1 potentialiseraient l’activation des kinases régulées par les signaux extracellulaires (ERK1/2). Nous suggérons également que c’est la phosphorylation de l’extrémité C-terminale de CXCR4 qui permettrait son association avec la β-arrestine. / G protein-coupled receptors (GPCRs) are a diverse family of membrane proteins capable of responding to a large number of extracellular stimuli including photons, odorant molecules and hormones. In addition to this diversity, it has been shown that GPCRs form specific protein:protein interactions, multiplying the signalling possibilities of each of these receptors. With the ability to diminish, to potentiate or even generate new signalling pathways, the oligomeric association of GPCRs plays an important role in generating this diversity. The use of bioluminescence resonance energy transfer (BRET), which allows the detection of interactions among proteins, has revealed numerous associations between GPCRs. During this thesis, tools have been developed that effectively combine BRET with protein complementation assays (PCA) with the goal of determining if interactions between GPCRs could involve more than two receptors. The results show that the chemokine receptors CXCR4 and CCR2 form both homo and hetero tetramers, and that the activation of a dimer of CCR2 can modulate the conformation of a CXCR4 dimer through a transreceptor conformational change. Negative cooperativity of ligand binding has previously been demonstrated between CXCR4 and CCR2 in CD4+ T lymphocytes endogenously expressing the receptors, confirming the biological validity of this interaction. The data presented also suggests that these complexes can engage the effector proteins Gαi and β- arrestin 2, indicating that they represent a functional form of the receptors. Furthermore, we have confirmed that each receptor of the CXCR4-CCR2 heterodimer is implicated in the engagement of effectors during the activation of CCR2. An additional level of complexity in GPCR-promoted signaling exists in their capacity to couple of multiple G proteins. Binding of stromal cell-derived factor-1 (SDF-1) to CXCR4 is known to promote T lymphocyte migration through a Gαi-dependent signalling pathway. In addition to this mechanism, we have demonstrated that breast cancer cell migration can initiated by a coupling of CXCR4 to the Gα13-Rho pathway, leading to the formation of metastases in distant organs. Finally, a novel level of GPCR regulation was revealed through the study of CXCR4 phosphorylation following its activation, which leads to the desensitization of the receptor and the engagement of β-arrestin-dependent signalling pathways. It appears that the desensitization of calcium signalling is mediated through the phosphorylation of CXCR4 by the GPCR kinases (GRKs) GRK2 and GRK6 and the recruitment of β-arrestin 2, whereas GRK3, GRK6 and β-arrestin 1 potentiate the activation of extracellular regulated kinase (ERK1/2). We also propose that the phosphorylation of the far C-terminal tail of CXCR4 is required for the interaction between the receptor and β-arrestin. / Thèse réalisée en cotutelle avec l'université Montpellier2 dans le laboratoire de pharmacologie moléculaire de Jean-Philippe Pin à l'institut de génomique fonctionnelle (IGF), Montpellier, France.

L’interaction d’un ligand avec un récepteur à sept domaines transmembranaires (7TMR) couplé aux protéines G, mène à l’adoption de différentes conformations par le récepteur. Ces diverses conformations pourraient expliquer l’activation différentielle des voies de signalisation. Or, le lien entre la conformation et l’activité du récepteur n’est pas tout à fait claire. Selon les modèles classiques pharmacologiques, comme le modèle du complexe ternaire, il n’existe qu’un nombre limité de conformations qu’un récepteur peut adopter. Afin d’établir un lien entre la structure et la fonction des récepteurs, nous avons choisi dans un premier temps, le récepteur de chimiokine CXCR4 comme récepteur modèle. Ce dernier, est une cible thérapeutique prometteuse, impliqué dans l’entrée du VIH-1 dans les cellules cibles et dans la dissémination de métastases cancéreuses. Grâce au transfert d’énergie par résonance de bioluminescence (BRET) nous pouvons détecter les changements conformationnels des homodimères constitutifs de CXCR4 dans les cellules vivantes. En conséquence, nous avons mesuré les conformations de mutants de CXCR4 dont les mutations affecteraient sa fonction. Nous montrons que la capacité des mutants à activer la protéine Galphai est altérée suite au traitement avec l’agoniste SDF-1. Notamment, ces mutations altèrent la conformation du récepteur à l’état basal ainsi que la réponse conformationnelle induite suite au traitement avec l’agoniste SDF-1, l’agoniste partiel AMD3100 ou l’agoniste inverse TC14012. Ainsi, différentes conformations de CXCR4 peuvent donner lieu à une activation similaire de la protéine G, ce qui implique une flexibilité des récepteurs actifs qui ne peut pas être expliquée par le modèle du complexe ternaire (Berchiche et al. 2007). Également, nous nous sommes intéressés au récepteur de chimiokine CCR2, exprimé à la surface des cellules immunitaires. Il joue un rôle important dans l’inflammation et dans des pathologies inflammatoires telles que l’asthme. CCR2 forme des homodimères constitutifs et possède différents ligands naturels dont la redondance fonctionnelle a été suggérée. Nous avons étudié le lien entre les conformations et les activations d’effecteurs (fonctions) de CCR2. Notre hypothèse est que les différents ligands naturels induisent différentes conformations du récepteur menant à différentes fonctions. Nous montrons que les réponses de CCR2 aux différents ligands ne sont pas redondantes au niveau pharmacologique et que les chimiokines CCL8, CCL7 et CCL13 (MCP-2 à MCP-4) sont des agonistes partiels de CCR2, du moins dans les systèmes que nous avons étudiés. Ainsi, l’absence de redondance fonctionnelle parmi les chimiokines liant le même récepteur, ne résulterait pas de mécanismes complexes de régulation in vivo, mais ferait partie de leurs propriétés pharmacologiques intrinsèques (Berchiche et al. 2011). Enfin, nous nous sommes intéressés au récepteur de chimiokine CXCR7. Récemment identifié, CXCR7 est le deuxième récepteur cible de la chimiokine SDF-1. Cette chimiokine a été considérée comme étant capable d’interagir uniquement avec le récepteur CXCR4. Notamment, CXCR4 et CXCR7 possèdent un patron d’expression semblable dans les tissus. Nous avons évalué l’effet de l’AMD3100, ligand synthétique de CXCR4, sur la conformation et la signalisation de CXCR7. Nos résultats montrent qu’AMD3100, tout comme SDF-1, lie CXCR7 et augmente la liaison de SDF-1 à CXCR7. Grâce au BRET, nous montrons aussi qu’AMD3100 seul est un agoniste de CXCR7 et qu’il est un modulateur allostérique positif de la liaison de SDF-1 à CXCR7. Aussi, nous montrons pour la première fois le recrutement de la beta-arrestine 2 à CXCR7 en réponse à un agoniste. L’AMD3100 est un ligand de CXCR4 et de CXCR7 avec des effets opposés, ce qui appelle à la prudence lors de l’utilisation de cette molécule pour l’étude des voies de signalisation impliquant SDF-1 (Kalatskaya et al. 2009). En conclusion, nos travaux amènent des évidences qu’il existe plusieurs conformations actives des récepteurs et appuient les modèles de structure-activité des récepteurs qui prennent en considération leur flexibilité conformationnelle. / Ligand binding to 7TMRs is thought to induce conformational changes within the receptor that translate into activation of downstream effectors. The link between receptor conformation and activity is still poorly understood, as current models of receptor activation fail to take an increasing amount of experimental data into account. Classical pharmacological models such as the ternary complex model are based on the concept that receptors can only adopt a limited number of conformations. To clarify structure-function relationships in 7TMRs, first we studied chemokine receptor CXCR4. This receptor is an important drug target, involved in HIV-1 entry and cancer metastasis. Bioluminescence Resonance Energy Transfer (BRET) allows us to directly probe conformational changes within pre-formed CXCR4 homodimers in live cells. Using BRET, we measured the conformation of CXCR4 mutants and we also monitored their function by measuring their ability to induce Galphai activation. The analyzed mutants had substitutions in locations which are pivotal molecular switches for receptor conformation and activation. We show that agonist induced Gi activation is altered for most mutants. These mutations also alter CXCR4’s conformation at basal conditions (in absence of ligand) and in the presence of the agonist, SDF-1, the partial agonist, AMD3100 and the inverse agonist, TC14012. Moreover, different conformations of active receptors were detected in the presence of SDF-1, suggesting that different receptor conformations are able to trigger Galphai activity. These data provide biophysical evidence for different active receptor conformations, that cannot be explained by classical models of receptor function (Berchiche et al. 2007). Furthermore, the second part of our work focused on chemokine receptor CCR2. Mainly expressed on immune cells, CCR2 is involved in many inflammatory and vascular diseases. This receptor binds seven natural ligands that have been referred to as redundant. We set out to explore whether the different chemokine ligands of CCR2 receptor induce different conformational changes leading to different functional consequences. Our results show that the different natural ligands of CCR2 are not pharmacologically redundant. Moreover, chemokines CCL8, CCL7 and CCL13 (MCP-2 to MCP-4) are partial agonists of CCR2, at least in the systems we used. Our results support the validity of models for receptor-ligand interactions in which different ligands stabilize different receptor conformations also for endogenous receptor ligands, demonstrating that these natural ligands are not pharmacologically and functionally redundant (Berchiche et al. 2011). As the third part of this work, we studied chemokine receptor CXCR7, the alternative receptor for SDF-1. Until recently, CXCR4 was the only receptor known to bind SDF-1. Moreover, the expression patterns are similar for receptors CXCR4 and CXCR7. Therefore, we investigated the conformational and functional consequences of the synthetic inhibitor of CXCR4, AMD3100, on CXCR7. We show that AMD3100 also binds the alternative SDF-1 receptor, CXCR7. SDF-1 or AMD3100 alone trigger beta-arrestin recruitment to CXCR7, which we identify as a previously unreported signalling pathway of CXCR7. In addition, AMD3100 has positive allosteric effects on SDF-1 binding to CXCR7, on SDF-1-induced conformational rearrangements in the receptor dimer as measured by BRET, and on SDF-1-induced beta-arrestin recruitment to CXCR7. The finding that AMD3100 not only binds CXCR4, but also to CXCR7, with opposite effects on the two receptors, call for caution in the use of this compound as a tool to dissect SDF-1 effects on the respective receptors in vitro and in vivo. Finally, these data provide biophysical evidence for different active receptor conformations, and support models of 7TMR structure-activity relationships that take conformational heterogeneity into account.

Die vorliegende Arbeit beinhaltet die Generierung und Evaluation von Natürlichen Killerzellen, die EGFRvIII-positive und SDF-1alpha sekretierende primäre Glioblastomzellen aufspüren, erkennen und effizient abtöten können. Die Kombination der gelenkten Zytotoxizität mit einer optimierten Migration von Effektorzellen des Immunsystems wird auf Grundlage der in dieser Arbeit gewonnenen Daten als ein vielversprechender Ansatz für eine zukünftige Therapie des primären Glioblastoms vorgeschlagen.

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O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1α/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 µg/animal, i.p.) ou pela injeção de LPS (100 µg/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 µg/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1α e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 µm. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 µg/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1α/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1α estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos. / The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

L’épidémiologie moléculaire des virus VIH-1 en France est caractérisée par une augmentation constante de la diversité virologique et de la fréquence des virus de sous-types non-B chez les patients en primo-infection. Entre 1997 et 2007, 28.4% des 591 patients suivis étaient infectés par des virus de sous-types non-B. De plus, 49 patients (8.3%) étaient infectés par des souches de sous-types différents sur les gènes pol et env, témoignant d’évènements de recombinaisons entre ces gènes. Ces virus recombinants étaient isolés à la fois chez des patients originaires d’Afrique sub-saharienne (28.3%) et des sujets caucasiens (6.3%). Ces résultats témoignent des échanges de souches virales entre les populations d'origine africaine et caucasienne, contribuant encore à augmenter la diversité virologique dans ces deux populations. Parmi 131 virus de sous-types non-B, 12.2% étaient classés comme ayant un tropisme CXCR4 par méthode génotypique, mais seulement 0.8% par méthode phénotypique, indiquant d’une part la faible proportion de virus non-B de tropisme CXCR4 en primo-infection en France, et d’autre part le manque de spécificité des méthodes génotypiques de détermination du tropisme pour ces sous-types, rendant nécessaire la mise au point d’autres algorithmes spécifiques pour ces virus.L’analyse de 987 virus isolés dans la cohorte entre 1999 et 2010 a mis en évidence que 12.7% d’entre eux étaient regroupés en "clusters" de transmission. Les patients en primo-infection contribuent donc de façon significative à la propagation de l’épidémie de VIH en France, particulièrement les hommes homo/bi-sexuels, avec une fréquence augmentant au cours de la période récente (2006-2010).La comparaison des quasi-espèces virales circulant concomitamment chez 8 patients en primo-infection (« receveurs ») et leurs 8 partenaires sexuels respectifs (« donneurs ») a révélé dans tous les cas la transmission d’un virus unique, présent de façon minoritaire parmi les sous-populations virales du donneur. La transmission virale muqueuse implique donc une sélection génétique drastique. / High genetic diversity is a major characteristics of HIV-1. In France, although subtype B strains are still predominant, the proportion of non-B viruses isolated in patients at the time of primary HIV-1 (PHI) infection increases over time. Between 1997 and 2007, 28.4% of patients were infected with non-B subtypes strains. Forty-nine viruses showed different phylogenies between the pol and env genes, indicating that recombinations have occurred in 8.3% of cases. These recombinants were isolated both in patients from Sub-Saharan Africa (28.3%) and in white subjects (6.3%).The phenotypic analysis of viral tropism of 131 non-B strains showed a very low (0.8%) proportion of CXCR4-tropic strains (X4 strains) at the time of PHI. Compared to phenotypic tests, genotypic predictions can overestimate (12.2% versus 0.8%) the proportion of X4 strains in non-B subtypes.The phylogenetic analysis of 987 strains isolated in 1999-2010 showed that 12.7% of PHI cosegregated into 56 transmission chains. PHIs are a significant source of onward transmission, especially in men having sex with men, with increasing frequency during the recent years (10.2% in 1999-2006 versus 15.2% in 2006-2010, p=0.02).The comparison of the viral quasispecies isolated in plasma and PBMC samples from 8 patients at the time of PHI ("recipients") and their transmitting partners ("donors") suggested that a severe genetic bottleneck occurrs during HIV-1 heterosexual and homosexual transmission. Indeed, we observed in all cases the transmission of a single variant, which was derived from an infrequent variant population within the blood of the donor. The proportion of X4 quasispecies in donors were higher in case of X4 versus CCR5-tropic viral transmission, suggesting that X4 transmission may be associated with a threshold of X4 circulating quasispecies in donors.

L’objectif de ce travail de thèse était de développer des outils pour détecter et traiter le cancer àun stade précoce. Nous avons donc entrepris la synthèse de nouveaux radiopharmaceutiques ciblantspécifiquement le récepteur CXCR4, en utilisant le savoir-faire et l'expertise de notre groupe dans lasynthèse et la fonctionnalisation des polyazacycloalcanes. Nous avons travaillé simultanément surdeux aspects : l’agent chélatant et la molécule vectrice.Dans un premier temps, les travaux ont concerné la conception, la synthèse et la caractérisationde nouveaux macrocycles à fort potentiel pour la chélation du cuivre et du gallium. Nous avons toutd’abord développé une nouvelle voie de synthèse permettant d’accéder à des dérivés homocyclènesC-functionnalisés. Nous nous sommes ensuite intéressés aux dérivés du 1,4,7-triazacyclononane(TACN). En optimisant une voie de synthèse déjà développée au laboratoire, nous avons facilitél’accès à des dérivés TACN N- et C-fonctionnalisés. Nous avons ainsi préparé une série denouveaux agents chélatants bifonctionnels adaptés pour la complexation du cuivre ou du gallium, envariant la nature de la fonction de greffage et des bras coordinants. Nous avons également réalisé lasynthèse de nouveaux cryptands en série cyclène et nous avons étudié leur propriété decomplexation vis à vis du cuivre.Dans un second temps, nous avons développé une nouvelle famille d’agents imageants duCXCR4 en modifiant la structure des AMD3100 et AMD3465. Ce travail a tout d’abord nécessité lamise au point de nouvelles méthodes de fonctionnalisation de ces structures. Nous avons ainsi pupréparer de nouveaux synthons porteur d’une fonction de greffage dans les deux séries. Nous avonsensuite introduit différentes sondes imageantes, telles que des chélates adaptés pour la complexationdu cuivre, gallium et indium ainsi que des sondes fluorescentes de type bodipy / The objective of this thesis work was to develop CXCR4-targeted tools to localize and treat cancer at an early stage. In this line, we investigated the synthesis of new target-specific radiopharmaceuticals. The work focused on two main axes, i.e. the chelating agent and the carrier, by using the know-how and the expertise of our group in polyazacycloalkanes synthesis and functionalization. In the first part, we were interested in developing new macrocyclic scaffolds of high potential for copper and gallium chelation. We first focused on the development of a new powerful route towards selectively functionalized constrained homocyclens. The second part was based on C-functionalized 1,4,7-triazacyclononane (TACN) and its derivatives. From a synthetic route previously developed in our group, we were able to facilitate and optimize the synthesis of selectively N- and C-functionalized TACN. By varying the grafting functions and the pendant coordinating arms, we prepared several really promising bifunctional chelating agents for copper and gallium chelation. We also investigated the synthesis of new cryptands based on cyclen and we studied their properties towards copper complexation. In the second part of this thesis work, we were interested in generating a new family of imaging agents based on well-known CXCR4 antagonists, i.e. AMD3100 and AMD3465. The access towards these agents first required the preparation of original building blocks by modification of the AMD3100 and AMD3465 cores. The conjugation of such platforms onto the appropriate probe enabled the synthesis of various systems for optical and nuclear imaging. Thus, we were able to introduce a bodipy dye and several chelators adapted for gallium, copper and indium chelation