Tanto a invasão local como o comprometimento de linfonodos cervicais tem grande impacto na sobrevida de pacientes portadores de carcinomas epidermóides de cabeça e pescoço. Em nosso trabalho nós primeiramente determinamos a expressão dos receptores de quimiocinas de CXCR1 a CXCR5, além de CCR7 e CX3CR1 pelo método do ensaio de proteção à ribonuclease (RPA) em 98 fragmentos de tumores primários, 91 fragmentos de mucosas adjacentes e 26 linfonodos comprometidos e correlacionamos estes dados com parâmetros anátomo-patológicos e sobrevida. CXCL12 ligante do receptor CXCR4 e CCL19 e CCL21 ambos ligantes de CCR7 foram determinados em 38 fragmentos de tumores, 33 mucosas adjacentes e 25 linfonodos comprometidos pela técnica de real-time PCR. Os tumores primários apresentam expressão aumentada do mRNA de CXCR1 (P=0.013), CXCR3 (P=0.008) e CXCR4 (P=0.025). Não observamos correlações entre status linfonodal ou tamanho de tumor. Os linfonodos comprometidos expressam mais mRNA dos receptores de quimiocinas CXCR4, CXCR5, CCR7 e CX3CR1 (todos com P<0.0001) em comparação aos tumores comprometidos. Observamos um aumento de sobrevida (P=0.048) e uma tendência a aumento de sobrevida livre de doença (P=0.074) nos pacientes negativos para a expressão de CX3CR1 (n=17) em comparação aos pacientes positivos (n=21) somente no subgrupo de pacientes portadores de carcinomas da cavidade oral. O mesmo foi observado com os pacientes CCR7 negativos também no subgrupo de pacientes portadores de carcinomas da cavidade oral, tanto em sobrevida global (P=0.024) como para sobrevida livre de doença (P=0.049). Em relação aos ligantes de quimiocinas observamos um aumento do mRNA de CCL21 em linfonodos comprometidos em relação aos tumores primários (P=0.059). Concluímos que a interação quimiotática entre CCR7 e de seu ligante CCL21, poderia ser um mecanismo de atração de células tumorais para os linfonodos em tumores de cavidade oral, além disso a negatividade da expressão do mRNA de CCR7 e CX3CR1 são candidatos marcadores de uma melhor sobrevida em carcinomas epidermóides de cavidade oral. / Local invasion and lymph nodal spread impact in the outcome of Head and Neck squamous cell carcinoma (HNSCC) patients (pts). We determined CXCR1-5, CCR7 and CX3CR1 mRNA expression by means of RNAse protection assay in 98 HNSCC primary tumors and 91 adjacent mucosa and 26 metastatic lymph nodes, correlating this data with outcome. CXCL12 and CCL19/CCL21, ligands for CXCR4 and CCR7, were determined in 38 tumor fragments, 33 adjacent mucosas and 25 de metastatic lymph nodes, by means of Quantitative Real-Time PCR. Tumors presented higher CXCR1 (P=0.013), CXCR3 (P=0.008) and CXCR4 mRNA (P=0.025) expression as compared to mucosa. No correlations are observed neither lymph nodal status nor tumor size impacted on chemokine receptor expression. Metastatic lymph nodes expressed more CXCR4, CXCR5, CCR7 and CX3CR1 (P<0.0001) as compared to matched tumors. We found a longer overall survival (OS) (P=0.048) and a trend toward longer disease free survival (DFS) (P=0.074) in CX3CR1 negative (n=17) as compared to positive pts (n=21) only in oral subgroup. The same occurred for CCR7 negative oral SCC, in terms of OS (P=0.024) and DFS (P=0.049). We conclude that, of the chemokine receptors here studied, CCR7 and CX3CR1 mRNA expression seems to better reflect outcome in oral subsite only. In addition, CCL21, a CCR7 ligand mRNAs is more expressed in metastatic lymph nodes than tumors (P=0.059). Further studies are warranted to confirm these results.

L’objectif de ma thèse a été d’étudier l’expression et l’activité de deux récepteurs de chimiokine dans deux désordres immunitaires, CCR1 dans le modèle murin NZB/W de néphrite lupique et CXCR4 dans la Lymphopénie T CD4+ Idiopathique (LCI), un déficit immunitaire rare chez l’Homme. Le Lupus Erythémateux Disséminé est une maladie autoimmune, chronique et inflammatoire dont le développement est caractérisé par une perte progressive de la fonction rénale associé notamment à une infiltration leucocytaire. Je me suis intéressé à la contribution de CCR1 et de ses ligands CCL3/CCL5 au recrutement leucocytaire intra-rénal chez la souris NZB/W néphritique. Nos résultats révèlent une augmentation de l’expression et de la fonction de CCR1 à la surface des cellules T (LT), des phagocytes et des neutrophiles issues de souris néphritiques. Un traitement aigu par un antagoniste non-peptidique de CCR1 administré par voie orale a réduit l’infiltration rénale des LT et des macrophages. L’inhibition de CCR1 à long terme a permis de diminuer l’accumulation rénale des LT CD4+ effecteurs/mémoires, des monocytes inflammatoires Ly6C+ et des macrophages polarisés M1 ou M2, a amélioré les atteintes tubulo-interstitielles et glomérulaires, a retardé l’apparition d’une protéinurie fatale et in fine a prolongé la survie des souris NZB/W. Ainsi, la combinaison d’approches pharmacologiques et fonctionnelles nous a permis de dévoiler un rôle pathogénique de CCR1, dans la progression de la néphrite lupique chez la souris NZB/W. La LCI est un déficit immuno-hématologique hétérogène et d’étiologie inconnue associant un nombre faible et persistant de LT CD4+ circulants et des infections opportunistes sévères en particulier d’origine fongique. Nos analyses multi-paramétriques par cytométrie en flux ont permis de révéler une perte d’expression et de fonction de CXCR4 à la membrane des LT de 17 des 20 patients étudiés. Ces données suggèrent que l’anomalie de CXCR4 constitue un trait biologique commun de la LCI. Notre approche transcriptomique a également permis d’identifier des signatures spécifiques de l’expression de gènes associés au seuil d’activation du TCR et à l’immuno-sénescence dans la LCI. Nos analyses phénotypiques et fonctionnelles ont confirmé ces observations et rapportent pour la première fois que les LT circulants résiduels de patients présentent un profil sénescent (exemple : perte d’expression des molécules de co-stimulation CD27 et CD28), des anomalies de réponse du TCR in vitro et une érosion des télomères. Sur un plan mécanistique, nous avons montré que les anomalies de signalisation intrinsèque aux LT des patients sont causées par l’expression accrue de la DUal-Specific Phosphatase 4 (DUSP4). Par conséquent, nos travaux révèlent une sénescence précoce des LT de patients souffrant de LCI, qui pourrait résulter d’une hyperstimulation chronique. Ce phénomène semble dépendre de la surexpression de DUSP4, dont la modulation pourrait constituer une piste thérapeutique dans la LCI afin d’améliorer les stratégies vaccinales. / My PhD works focused on the expression and function of chemokine receptors in two immune disorders, namely CCR1 in the lupus-prone NZB/W mouse model and CXCR4 in the Idiopathic CD4+ T-cell lymphopenia, a rare human immune defect.Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with renal leukocyte infiltration. During my PhD, I investigated the role of the CCR1 chemokine receptor in this process during the progression of nephritis in NZB/W mice. We found that peripheral T-cells, mononuclear phagocytes and neutrophils from nephritic NZB/W mice were more responsive to CCR1 ligands than the leukocytes from younger prenephritic mice. Short-term treatment of nephritic NZB/W mice with a orally available CCR1 antagonist decreased renal infiltration by T-cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4+ T-cells, Ly6C+ inflammatory monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. Altogether, these findings highlight a pivotal role for CCR1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.ICL is heterogeneous immunological syndrome of unclear molecular mechanisms, characterized by a profound and persistent CD4+ T-cell defect and by opportunistic infections in particular of fungal origin. We detected using multiparametric flow-cytometry analyses, reduced levels of CXCR4 expression and chemotactic function on T-cells from 17 of 20 ICL patients. These results suggest that the impaired membrane expression and function of CXCR4 is a common biologic trait of ICL. Using a transcriptomic approach, we also identified an ICL-specific T-cell gene expression signature characteristic of low TCR sensitivity and accelerated T-cell aging. Phenotypic and functional analyses of circulating T cells confirmed these observations and extended them to include an expansion of terminally-differentiated T cells with hallmarks of aging including loss of the co-stimulatory molecules CD27 and CD28, defective in vitro TCR responses, and telomere erosion. Mechanistically, we further showed that intrinsic T-cell signaling defects were caused by higher expression of DUal-Specific Phosphatase 4 (DUSP4). These findings suggest that premature T-cell senescence occurs in ICL as a result of chronic T-cell activation. This is in part due to abnormally high DUSP4 expression in CD4+ T cells, the modulation of which may constitute a novel therapeutic avenue in ICL to improve vaccination strategies.

No description available.

CXCR4, récepteur de la chimiokine SDF-1 (stromal cell-derived factor 1) joue un rôle capital dans l’hématopoïèse normale mais aussi dans la biologie de la cellule leucémique. Ce récepteur est exprimé à la surface des blastes et participe à « l’ancrage » de la cellule souche leucémique (CSL) au sein de la niche médullaire. Les interactions de la CSL avec le micro-environnement sont source de signaux de survie et de résistance à l’apoptose. La première partie de ce travail correspond à deux analyses en cytométrie en flux de l’expression de CXCR4 et de molécules d’adhérence sur des échantillons diagnostiques de LAM (leucémie aiguë myéloïde). Ce travail confirme la valeur pronostique péjorative de l’expression de CXCR4 et propose un modèle de stratification pronostique des patients, en fonction de leur phénotype d’adhérence. La deuxième partie s’intéresse à l’identification de potentielles cibles thérapeutiques dans un modèle de LAL à chromosome Philadelphie, pathologie au pronostic sombre malgré les progrès thérapeutiques liés aux ITK (inhibiteurs de tyrosine kinase). L’inhibition de CXCR4 par l’AMD3100 permet de potentialiser l’efficacité de l’aracytine et du dasatinib dans un modèle de co-culture stromale avec la lignée SUPB15. Une deuxième piste de ciblage thérapeutique de la LAL Phi+ est l’inhibition de la protéine chaperone HSP90. Une expression forte de HSP90 (dans les LAL Phi+ par rapport aux LAL Phi-) s’associe à une plus grande cytotoxicité du 17-AAG. En conclusion, CXCR4 est un récepteur clé de la cellule leucémique. L’étude de son niveau d’expression permet des stratifications pronostiques des patients et son blocage en fait une cible thérapeutique prometteuse / CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. The first part of this work consists of two flow cytometry analyses of CXCR4 and adhesion molecules expression in patients with AML (acute myeloid leukaemia) at diagnosis. The results confirm that CXCR4 expression is associated with poor prognosis and this work proposes to stratify patients, according to their adhesive phenotype, in order to establish risk-adapted strategies. The second part deals with the identification of potential therapeutic targets in a model of ALL with chromosome Philadelphia. Despite therapeutic improvements with the ITK (tyrosine kinase inhibitors) era, long term survival remains poor. The inhibition of CXCR4 by the AMD3100 enhances the sensitivity of SUPB15 cell line to cytarabine and dasatinib therapy in a model of stromal co-culture. A second way of therapeutic targeting of the ALL Phi + is the inhibition of the heat-shock protein HSP90. High percentage of HSP90-positive cells (in Ph+ ALL samples) is associated with high sensitivity to 17-AAG. In conclusion, CXCR4 appears as a key receptor of the leukaemic cell. The analysis of its level of expression allows prognostic stratifications and its blockade represents a promising therapeutic target

Mieloma Múltiplo é a segunda doença com maior prevalência nas doenças malignidades hematológica, incurável com média de sobrevivência de 3-5 anos. MM é uma malignidade das células do plasma caracterizada pela destruição e reabsorção óssea e supressão da formação do osso. A quimiocina SDF-1 (CXCL12) e seu receptor CXCR4 têm um importante papel direcional na migração, homing das células do plasma em mieloma múltiplo e mobilização das células de MM para fora da medula óssea. A talidomida tem sido usada com êxito no tratamento de pacientes com mieloma múltiplo. Neste estudo verificamos o efeito da talidomida na expressão da quimiocina SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo e em linhagem de células de mieloma múltiplo humano (RPMI-8226) tratados e sem tratamento de talidomida. Nossos resultamos mostraram uma expressão heterogênea na expressão da quimiocina SDF-1 e seu receptor CXCR4 nos pacientes com mieloma múltiplo estudado (n= 79). Entretanto, pacientes com mieloma múltiplo tratados com talidomida mostraram uma baixa expressão da quimiocina SDF-1 e seu receptor CXC4 quando comparados com pacientes recém diagnosticados para mieloma múltiplo e pacientes com mieloma múltiplo tratados com outros medicamentos. Nossos resultados sugerem que o tratamento com talidomida induz uma baixa regulação na expressão no ligante SDF-1 e seu receptor CXCR4 em pacientes com mieloma múltiplo / Multiple Myeloma (MM) is a second most prevalent hematological malignancy and remains incurable with a median survival of 3-5 years. MM is a plasma cell malignancy characterized by devastating bone destruction due to the enhanced bone resorption and suppressed bone formation. The chemokine stromal-derived factor-1 (SDF-1) and its receptor CXCR4 play an important role in directional migration, homing of plasma cells in multiple myeloma (MM) and mobilization of MM cells out of the bone marrow. The drug thalidomide has been successfully used in the treatment of patients with MM. In this study, we assessed the effect of thalidomide on SDF-1 and CXCR4 expression in MM patients and human myeloma-derived cell line, RPMI 8226 treated with or without thalidomide. A heterogeneous expression pattern of chemokines SDF-1 and CXCR4 receptor were observed for all MM patients studied. However, patients treated with thalidomide showed a significantly decrease in expression of SDF-1 and CXCR4 as compared to newly diagnosed MM patients and MM patients treated with other drugs. RPMI 8226 cell line treated with 10, 20 and 100µM thalidomide also demonstrated decrease in SDF-1 and CXCR4 expression as compared with cell control (RPMI-8226 without thalidomide). Ours results indicate that thalidomide therapy induces down-regulation of CXCR4 and its ligand SDF-1 in multiple myeloma

Tanto a invasão local como o comprometimento de linfonodos cervicais tem grande impacto na sobrevida de pacientes portadores de carcinomas epidermóides de cabeça e pescoço. Em nosso trabalho nós primeiramente determinamos a expressão dos receptores de quimiocinas de CXCR1 a CXCR5, além de CCR7 e CX3CR1 pelo método do ensaio de proteção à ribonuclease (RPA) em 98 fragmentos de tumores primários, 91 fragmentos de mucosas adjacentes e 26 linfonodos comprometidos e correlacionamos estes dados com parâmetros anátomo-patológicos e sobrevida. CXCL12 ligante do receptor CXCR4 e CCL19 e CCL21 ambos ligantes de CCR7 foram determinados em 38 fragmentos de tumores, 33 mucosas adjacentes e 25 linfonodos comprometidos pela técnica de real-time PCR. Os tumores primários apresentam expressão aumentada do mRNA de CXCR1 (P=0.013), CXCR3 (P=0.008) e CXCR4 (P=0.025). Não observamos correlações entre status linfonodal ou tamanho de tumor. Os linfonodos comprometidos expressam mais mRNA dos receptores de quimiocinas CXCR4, CXCR5, CCR7 e CX3CR1 (todos com P<0.0001) em comparação aos tumores comprometidos. Observamos um aumento de sobrevida (P=0.048) e uma tendência a aumento de sobrevida livre de doença (P=0.074) nos pacientes negativos para a expressão de CX3CR1 (n=17) em comparação aos pacientes positivos (n=21) somente no subgrupo de pacientes portadores de carcinomas da cavidade oral. O mesmo foi observado com os pacientes CCR7 negativos também no subgrupo de pacientes portadores de carcinomas da cavidade oral, tanto em sobrevida global (P=0.024) como para sobrevida livre de doença (P=0.049). Em relação aos ligantes de quimiocinas observamos um aumento do mRNA de CCL21 em linfonodos comprometidos em relação aos tumores primários (P=0.059). Concluímos que a interação quimiotática entre CCR7 e de seu ligante CCL21, poderia ser um mecanismo de atração de células tumorais para os linfonodos em tumores de cavidade oral, além disso a negatividade da expressão do mRNA de CCR7 e CX3CR1 são candidatos marcadores de uma melhor sobrevida em carcinomas epidermóides de cavidade oral. / Local invasion and lymph nodal spread impact in the outcome of Head and Neck squamous cell carcinoma (HNSCC) patients (pts). We determined CXCR1-5, CCR7 and CX3CR1 mRNA expression by means of RNAse protection assay in 98 HNSCC primary tumors and 91 adjacent mucosa and 26 metastatic lymph nodes, correlating this data with outcome. CXCL12 and CCL19/CCL21, ligands for CXCR4 and CCR7, were determined in 38 tumor fragments, 33 adjacent mucosas and 25 de metastatic lymph nodes, by means of Quantitative Real-Time PCR. Tumors presented higher CXCR1 (P=0.013), CXCR3 (P=0.008) and CXCR4 mRNA (P=0.025) expression as compared to mucosa. No correlations are observed neither lymph nodal status nor tumor size impacted on chemokine receptor expression. Metastatic lymph nodes expressed more CXCR4, CXCR5, CCR7 and CX3CR1 (P<0.0001) as compared to matched tumors. We found a longer overall survival (OS) (P=0.048) and a trend toward longer disease free survival (DFS) (P=0.074) in CX3CR1 negative (n=17) as compared to positive pts (n=21) only in oral subgroup. The same occurred for CCR7 negative oral SCC, in terms of OS (P=0.024) and DFS (P=0.049). We conclude that, of the chemokine receptors here studied, CCR7 and CX3CR1 mRNA expression seems to better reflect outcome in oral subsite only. In addition, CCL21, a CCR7 ligand mRNAs is more expressed in metastatic lymph nodes than tumors (P=0.059). Further studies are warranted to confirm these results.

Grâce à la production de nanoparticules lipidiques, les SELN (Synthetic Exosomes-Like Nanoparticles), mimant la composition des exosomes produits par les cellules tumorales pancréatiques humaines SOJ-6, nous avons démontré que les effets apoptotiques des exosomes naturels étaient dus aux lipides. En effet, nous avons montré que les SELN dont le rapport rafts/phospholipides est le plus élevé, interagissent avec les cellules SOJ-6 au niveau des rafts et perturbent la voie Notch. Cela conduit à la diminution de l'expression du facteur de survie Hes-1, qui est accentuée par la perte d'activité du complexe PTEN-GSK-3β. Ces dérégulations induisent l'apoptose dépendante de la mitochondrie des cellules SOJ-6, caractérisée par l'augmentation du ratio Bax/Bcl-2, l'activation de la caspase 9 et la dégradation de l'ADN. En revanche, les cellules MiaPaCa-2 résistent aux SELN, ce qui s'explique par leur caractère indifférencié. Ainsi la surexpression de marqueurs de cellules souches tels que l'ALDH et CXCR4 leur confèrent une grande résistance. Elles sont toutefois sensibles à la cyclopamine un inhibiteur de la voie Hedgehog, dont les effets sont atténués si les cellules MiaPaCa-2 sont préincubées avec les SELN, prouvant que ces cellules mettent en place des voies de survie leur permettant d'échapper à l'apoptose. Nos investigations ont montré que dans les cellules MiaPaCa-2, sous l'effet des SELN, l'activation de la voie canonique NF-кB permet d'induire la transcription du gène codant SDF-1α, seul ligand connu du récepteur CXCR4. Le facteur produit et sécrété active de manière auto et paracrine une voie de survie Akt-dépendante. / It has been previously reported that exosomes released by the human pancreatic tumoral cell line SOJ-6 could induce their own apoptosis. Thanks to the production of lipid nanoparticles, SELN (Synthetic Exosomes-Like Nanoparticles) mimicking the lipid composition of natural exosomes, we have shown that lipids were responsible for the observed effects. Indeed, we showed that SELN with the higher ratio rafts/phospholipids could interact with SOJ-6 cells at the level of the rafts to perturb the Notch pathway, preferentially localized in these lipid microdomains. This induces a decreased expression of the main target of this pathway, the survival factor Hes-1. This decrease is intensified by the activation of the complex PTEN-GSK-3β. These deregulations drive cells towards the mitochondria-dependent apoptosis as shown by the increase of the ratio Bax/Bcl-2, the caspase 9 activity and the DNA fragmentation. Whereas MiaPaCa-2 cells are resistant to SELN, which is explained by their stem-like cell phenotype, contrarily to the well-differentiated SOJ-6 cell line. Although the over-expression of some stem cell markers, such as ALDH and CXCR4 is responsible for their resistance, they remain sensitive to the cyclopamine, a Hedgehog inhibitor. We found out that MiaPaCa-2 cells pre-incubation with SELN could protect them from the inhibitory effect of the cyclopamine, meaning that upon SELN incubation, a survival pathway is triggered in MiaPaCa-2 cells. So we showed that, upon SELN incubation, the canonical NF-кB pathway is activated in MiaPaCa-2 cells to promote SDF-1α expression. Once released, SDF-1α interacts with its receptor CXCR4 to activate an Akt-dependent survival pathway.

Das Chemokine SDF-1α und sein Rezeptor CXCR4 sind in eine Vielzahl biologischer Prozesse, wie der Organogenese, der Hämatopoese und der Immunantwort involviert. Die Entdeckung des alternativen SDF-1α-Rezeptors CXCR7 bewirkte eine erneute Untersuchung der Funktion des SDF-1-Systems in diesen Vorgängen. CXCR7 ist in der Lage, je nach Gewebe- oder Zelltyp, als \'Scavenger\'-Rezeptor, Modulator des CXCR4 oder selbstständig aktiver Rezeptor zu agieren. In dieser Arbeit wurde untersucht, inwiefern beide Rezeptoren im Verlauf der Entwicklung der Muskulatur exprimiert werden, welche Aufgabe sie dabei übernehmen und ob sich Rückschlüsse auf die Muskelregeneration daraus ableiten lassen. Hierfür erfolgten in vitro-Untersuchungen an C2C12-Zellen und die anschließende Analyse der Expression von CXCR4, CXCR7 und SDF-1α in der sich entwickelnden Gliedmaßenmuskulatur von Wildtyp- und mdx-Mäusen. Die Untersuchung von C2C12-Zellen zeigte in allen Differenzierungsstadien eine detektierbare Menge von CXCR4 und eine zunehmende Expression des CXCR7. Die Behandlung der Zellen mit SDF-1α führte zu einer Phosphorylierung von Erk1/2 und PKCζ/λ und hemmte gleichzeitig deren Differenzierung. Nach einer Blockierung des CXCR4 mit seinem pharmakologischen Antagonist AMD3100 oder nach Hemmung der Expression durch spezifische siRNA blieb die Aktivierung des Signalweges aus und der hemmende Einfluss des SDF-1α auf die Differenzierung verschwand vollständig. Im Gegensatz dazu blieben nach der pharmakologischen Blockierung oder durch siRNA vermittelten Expressionshemmung des CXCR7 alle SDF-1α induzierten Effekte vollständig erhalten. Eine Untersuchung des Signalweges am dritten Tag der Differenzierung zeigte keine Aktivierung von Erk1/2. Ebenso blieb Erk1/2 nach einer Hemmung der Expression des CXCR4 unphosphoryliert. Im Gegensatz dazu fand nach einer Hemmung der Expression des CXCR7 mit spezifischer siRNA erneut eine Aktivierung des Signalweges statt. Weiterhin konnte in vivo festgestellt werden, dass die Expression des CXCR4 in der Muskulatur während der embryonalen Entwicklung am höchsten ist und bereits kurz nach der Geburt stark abnimmt, wenn die sekundäre Muskelentwicklung abgeschlossen ist. Die Expression des CXCR7 hingegen steigt perinatal an und bleibt bis zum Erwachsenenalter bestehen. Zusammengefasst zeigen diese Ergebnisse, dass CXCR4 aktiv das Signalgeschehen von SDF-1α in der Myogenese vermittelt und somit zur Differenzierungshemmung von Myoblasten beiträgt. CXCR7 hingegen wirkt als passiver SDF-1α-Scavenger und induziert somit durch eine Modulierung der extrazellulären SDF-1α-Konzentration die Differenzierung. In Übereinstimmung mit der Rolle des SDF-1α-Systems bei der Muskelentwicklung, konnte eine kontinuierliche SDF-1α- Expression im Bindegewebe um pränatale und im Endomysium von postnatalen und adulten Muskelfasern festgestellt werden. Diese SDF-1α-Expression stieg ebenso wie die CXCR4-Expression bei der Analyse der Muskulatur von dystrophin-defizienten Mäusen an und zeigte somit, dass dieses System auch für die Proliferation von Muskelvorläuferzellen in der regenerativen Muskulatur eine wichtige Rolle spielt. Bemerkenswerter Weise hatte diese Muskeldystrophie keinen Einfluss auf die Expression des CXCR7 und beeinflusst vermutlich dessen Funktion über einen anderen Mechanismus. Durch die offensichtlich enge Kontrolle von Muskelentwicklung und Regeneration durch CXCR4, CXCR7 und deren Liganden SDF-1α, bilden diese ein vielversprechendes therapeutisches Ziel für bestimmte Muskelerkrankungen. / The chemokine, SDF-1α, and its receptor, CXCR4, are assumed to control a multitude of biological processes such as organogenesis, haematopoesis, and immune responses. The previous demonstration that SDF-1α additionally binds to the chemokine receptor, CXCR7, currently urges a re-evaluation of the function of the SDF-1 system in these processes. In fact, depending on the tissue and cell type, CXCR7 either acts as a scavenger receptor, a modulator of CXCR4 or an independent active receptor. This thesis is dedicated to answer the following questions: Are both SDF-1α receptors expressed during muscle development? What is the actual function of these receptors during myogenesis? Is there a role of the SDF-1 system in muscle regeneration? To adress these issues both in vitro studies with the myoblast cell line, C2C12, as well as in vivo analyses on the expression of CXCR4, CXCR7 and SDF-1α in developing and regenerating limb muscles have been performed. At all stages of differentiation, C2C12 cells exhibited measurable amounts of CXCR4. In addition, in the course of differentiation C2C12 cells showed increasing expression levels of CXCR7. Treatment of the cells with SDF-1α resulted in the phosphorylation of Erk1/2 and PKCζ/λ and subsequently blocked their myogenic differentiation. Following inactivation of CXCR4 either by its antagonist, AMD3100, or by specific siRNA, SDF-1α failed to activate both pathways and in addition no longer inhibited the myogenic differentiation of C2C12 cells. By contrast, inactivation of CXCR7 remained without effects on SDF-1α-induced cell signalling and resulting inhibitory effects on myogenic differentiation. Interestingly, SDF-1α also failed to activate Erk1/2 signalling in differentiated C2C12 cells. Cell signalling in differentiated C2C12 cells was, however, restored following inhibition of CXCR7 expression, but not following inhibition of CXCR4 expression. The in vivo analysis further revealed that in limb muscles expression of the CXCR4 is highest during embryonic development with a decrease in expression levels shortly after birth when secondary muscle development is completed. Vice versa, expression levels of CXCR7 increased perinatally and remained high into adulthood. In summary, these findings unravel that CXCR4 actively mediates SDF-1α-signalling during myogenesis. The findings further provide evidence that CXCR7 acts as a scavenger receptor during myogenesis which controls myogenic differentiation by modulating extracellular SDF-1α concentration. In further agreement with a major role of SDF-1α in muscle development, SDF-1α is continously expressed by the endomysium of postnatal and adult muscle fibers. Moreover, expression of SDF-1α as well as CXCR4 is massively increased in muscles of dystrophin-deficient mice further implying that the SDF-1 system plays an equally important role during muscle development and regeneration. The pivotal role of SDF-1α in muscle development and regeneration points to the SDF-1 system as a promising therapeutical target for certain muscle diseases.

Background Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. Design and Methods In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. Results Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4. Conclusions Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.

Le cancer endométrioïde de l’ovaire (CEO) ainsi que l’implant endométriosique sont deux tumeurs caractérisées par la présence de cellules épithéliales et stromales ressemblant aux cellules endométriales. Des études antérieures ont mis en évidence des anomalies immunologiques communes à la cellule épithéliale endométriale (CEE), à la cellule endométriosique (CENDO) et à la cellule du CEO (CCEO) suggérant une origine commune endométriale de l’endométriose (ENDO) et du CEO. Selon la théorie de l'implantation, la migration des cellules endométriales vers d'autres sites ectopiques constitue un événement important dans l'étiopathogénie de l’endométriose (ENDO). Récemment, il a été démontré que CXCL12/CXCR4, un axe œstrogèno-dépendant, joue un rôle important dans l’homéostasie cellulaire et contrôle la migration des cellules dans les conditions normales et pathologiques. Ainsi, l'implication des œstrogènes dans la régulation de ce système nous amène à nous questionner sur l’effet du bisphénol A (BPA), un xéno-œstrogène utilisé dans une large gamme de produits, sur l’expression de CXCL12/CXCR4 et sur la migration de la CEE et son développement ectopique en tumeur. Dans ce projet, nous avons utilisé des CEE primaires provenant d’endomètres et d’implants endométriosiques, ainsi que deux lignées néoplasiques (endométriale et endométrioïde ovarienne). Nous avons étudié le rôle du BPA dans l'expression du couple CXCL12/CXCR4 par des méthodes classiques d’ELISA, RT-PCR en temps réel, Western blot et immunocytochimie. La prolifération a été évaluée par méthode colorimétrique et la migration par la technique des deux chambres de Boyden. Le dosage du BPA a été fait par chromatographie en phase gazeuse. Les principaux résultats montrent que l’expression basale du CXCR4 au niveau des différents types de cellules est augmentée par le traitement au BPA en comparaison aux contrôles. Ces résultats démontrent, une fois de plus, une possible origine commune de l’ENDO et CEO. D’autre part, nous avons démontré que le traitement au BPA entraine une augmentation de la migration et de la prolifération des CEE selon un axe CXCL12/CXCR4 dépendant. Ceci pourrait être un mécanisme impliqué dans la genèse de l’endométriose et suggère que le BPA pourrait être le facteur responsable par l’activation de l’axe CXCL12/CXCR4. Ce travail a aussi permis d’identifier une anomalie inhérente au tissu endométrial de patientes endométriosiques et qui consiste en une augmentation de l’expression du récepteur CXCR4, ce qui pourrait constituer un biomarqueur de diagnostic de l’ENDO. En dernier lieu, compte tenu de l'absence de lien entre les concentrations de BPA et celles du CXCL12, la réponse chimiotactique qui serait responsable de la migration de la CEE vers la cavité pelvienne pourrait être dépendante d'autres facteurs du microenvironnement péritonéal, ce qui appuie la nature multifactorielle de l’étiopathogénie de endométriose.