La réparation osseuse est assurée par le recrutement constant de cellules souches/progéniteurs ostéo-compétents de nature hématopoïétique (CSH/PH), et mésenchymateuse (CSM). Une approche prometteuse pour le traitement des défauts osseux graves consisterait à favoriser le recrutement et la mobilisation des CSH/PH et des CSM à partir de la moelle osseuse vers le site de lésion. Plusieurs facteurs environnementaux sont connus pour moduler la prolifération, la mobilisation et la différenciation des progéniteurs ostéocompétents, dont l’hypoxie et l’hypodynamie (absence de contraintes mécaniques). Le but de ce travail de thèse a été d’investiguer in vivo l’impact de l’hypoxie respiratoire et de l’absence de contraintes mécaniques, appliquées séparément ou ensemble sur i) la mobilisation des progéniteurs ostéocompétents et sur ii) la réparation d’un défaut osseux cavitaire fémoral chez la souris. Sur un modèle murin dépourvu de défaut osseux, nos données montrent que l’hypoxie respiratoire est un agent mobilisateur des progéniteurs ostéocompétents, et qu’elle pourrait donc potentiellement exercer des effets bénéfiques sur la réparation osseuse. Toutefois, les effets de l’hypoxie sont modulés selon le statut hypodynamique ou non de l’animal. L’absence de contraintes mécaniques limite la mobilisation des progéniteurs érythrocytaires et mésenchymateux initiée par l’hypoxie, suggérant un effet potentiellement délétère de l’hypodynamie en condition hypoxique dans le contexte de la réparation osseuse. Chez les souris opérées, nous confirmons que l’hypoxie respiratoire déclenchée lors des phases de remodelage améliore la réparation du défaut osseux cavitaire. Une mobilisation des progéniteurs mésenchymateux et hématopoïétiques du fémur contra-latéral vers le fémur opéré est noté, mais le mode d’action de l’hypoxie passerait plutôt par une accélération du mécanisme de réparation dans la zone lésée. De façon intéressante, nous montrons que l’hypodynamie ne diminue pas le bénéfice apporté par l’hypoxie respiratoire à la réparation osseuse. En conclusion, ce travail de thèse identifie l’hypoxie respiratoire comme un candidat thérapeutique pertinent pour l’amélioration de la réparation osseuse. Bien que la perte des contraintes mécaniques module la biologie des cellules ostéoprogénitrices en absence de lésion, l’hypodynamie ne semble pas influencer la consolidation osseuse dans le cadre d’une amélioration de la réparation par un épisode hypoxique tardif. / Many environmental factors are known to influence bone cell fate, including proliferation, mobilization and differentiation of osteoprogenitor cells deriving from both hematopoietic and mesenchymal lineages. Among these factors, hypoxia and unloading (lack of mechanical loading / hypodynamia) are of particular interest. This study aims at investigating the impact of short-term hypobaric hypoxia and hindlimb unloading applied alone or in combination i) on the mobilization of osteocompetent progenitor cells on mice and ii) on the healing in a mouse model of surgical metaphyseal bone defect.In mice free of bone defect, our data indicate that respiratory hypobaric hypoxia acts as a mobilizing stimulus for osteoprogenitor cells. However, the effects of hypoxia in the bone marrow depend on whether mice are subjected to hindlimb unloading or not: hypodynamia tends to restrain the mobilization of both mesenchymal and erythroid progenitors under hypoxia. This suggests a potential detrimental influence of hypodynamia in the course of bone healing in hypoxic condition.In mice with surgery, we showed that hypobaric hypoxia during the remodelling process strongly enhances bone healing. A mobilization of both mesenchymal and hematopoietic progenitors is detected from the contralateral femur to the operated femur. In the lesion area, an acceleration of the repair process is evidenced. Interestingly, hindlimb unloading does not exert any negative influence on bone repair in our animal model. In conclusion, this study identifies delayed hypobaric hypoxia as a potent candidate to enhance bone healing. Even if unloading exerts significant effects on the biology of osteoprogenitor cells on mice free of bone defect, its influence is not detrimental for bone repair.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Acredita-se que os primeiros progenitores da hematopoese definitiva surjam da diferenciação do endotélio da aorta dorsal, na altura da região da Aorta-Gônada-Mesonefros (AGM). Com o intuito de estudar esta região e o fenótipo das células do endotélio da aorta dorsal nesta posição topográfica, ovos galados de Gallus gallus domesticus L. foram incubados em chocadeira, classificados em estádios de E16 a E25 e processados histotecnologicamente para obtenção de secções seriadas na altura da região AGM. Estas passaram por coloração por Hematoxilina-Eosina, histoquímica para PAS, PAS-diastase e Alcian Blue pH 1.0 e pH 2.5, histoquímica por lectinas fluoresceinadas e imunofluorescência para moléculas de superfície, citoesqueleto e matriz extracelular. Foi observada hipertrofia endotelial no assoalho da aorta nos estádios observados, o qual se apresentava positivo ao PAS, com ocorrência frequente de vacuolizações basais PAS negativas, e o surgimento ocasional de grupamentos celulares intravasculares. Nestes, as células que se destacavam da membrana basal do endotélio expressavam progressivamente mais material PAS positivo, o qual, no entanto, em nenhum momento pareceu se tratar de glicogênio. Em relação às glicosaminoglicanas, notamos a presença predominante de ácido hialurônico por todo o mesênquima da região e em outras estruturas como periferia da notocorda, tubo neural e mesoderma lateral. Ocorreu co-expressão de fibronectina e α-actina de músculo liso em células circunjacentes à aorta, na face ventral do vaso. GFAP e BMP-4 são expressas entre as células do tubo neural e em sua periferia, assim como na notocorda do embrião. As lectinas Abrus precatorius, Lens culinarise Ricinus communis mostraram-se positivas principalmente na região subedotelial do assoalho da aorta nos estádios observados neste trabalho. Bandeiraea simplicifolia exibiu pouca marcação na aorta dorsal e a Arachis hypogeae foi negativa. Outras estruturas da região AGM também expressaram resíduos de açúcares revelados por estas lectinas, tais como: notocorda, tubo neural, mesênquima, intestino primitivo e saco vitelínico. Estes resultados acrescentam elementos morfológicos e bioquímicos ao conhecimento sobre a região AGM de embriões de galinha e sobre o endotélio, possivelmente hemogênico, da aorta dorsal. / Nowadays it is known that firsts definitive hematopoietic progenitors arise from endothelium differentiation of dorsal aorta, at Aorta-Gonad-Mesonephros (AGM) site. In order to study those events and cells phenotype of dorsal aorta endothelium in this topographical site, fertilized eggs of Gallus gallus domesticus L. were incubated, from E16 to E25 and to be processed by histotechonology to obtain serial sections of the AGM site. After this, they were stained with Hematoxilin-Eosin, histochemistry to PAS, diastase PAS and Alcian Blue pH 1.0 and pH 2.5, to obtain a better overview and characterization from cells, also, histochemistry to fluorescein lectins and immunofluorescense to surface molecules, cytoeskeleton andextracellular matrix were performed. Our results showed endothelial hypertrophy of the aorta floor in the stages analyzed, which shown positive for PAS, with frequent appearance of PAS negatives basal vacuolizations and an occasional intravascular cell clusters arise of. It was also observed that cells which were separated from endothelium basal membrane shown progressively were more PAS positive which however in any time seems to be glycogen. Regard of glycosaminoglycans we noted the main presence of hialuronic acid for all the mesenchymes site and in others structures like notochord periphery, neural tube and lateral mesoderm. It was observed fibronectin and smooth muscle α-actin co-expression on aorta surrounding walls, at vessels ventral face. GFAP and BMP-4 are express between the cells of neural tube and surrounding it, as well as at embryo notochord. The lectins Abrus precatorius, Lens culinaris and Ricinus communis showed mostly positive expression on the sub endothelium site of the dorsal aorta floor at the stages analyzed in this work. Bandeiraea simplicifolia showed low expression on dorsal aorta and in Arachis hypogeae it was negative. Other structures of AGMs site also expressed sugar residues revealed by these lectins like: notochord, neural tube, mesenchyme, primitive gut and yolk sac.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Acredita-se que os primeiros progenitores da hematopoese definitiva surjam da diferenciação do endotélio da aorta dorsal, na altura da região da Aorta-Gônada-Mesonefros (AGM). Com o intuito de estudar esta região e o fenótipo das células do endotélio da aorta dorsal nesta posição topográfica, ovos galados de Gallus gallus domesticus L. foram incubados em chocadeira, classificados em estádios de E16 a E25 e processados histotecnologicamente para obtenção de secções seriadas na altura da região AGM. Estas passaram por coloração por Hematoxilina-Eosina, histoquímica para PAS, PAS-diastase e Alcian Blue pH 1.0 e pH 2.5, histoquímica por lectinas fluoresceinadas e imunofluorescência para moléculas de superfície, citoesqueleto e matriz extracelular. Foi observada hipertrofia endotelial no assoalho da aorta nos estádios observados, o qual se apresentava positivo ao PAS, com ocorrência frequente de vacuolizações basais PAS negativas, e o surgimento ocasional de grupamentos celulares intravasculares. Nestes, as células que se destacavam da membrana basal do endotélio expressavam progressivamente mais material PAS positivo, o qual, no entanto, em nenhum momento pareceu se tratar de glicogênio. Em relação às glicosaminoglicanas, notamos a presença predominante de ácido hialurônico por todo o mesênquima da região e em outras estruturas como periferia da notocorda, tubo neural e mesoderma lateral. Ocorreu co-expressão de fibronectina e α-actina de músculo liso em células circunjacentes à aorta, na face ventral do vaso. GFAP e BMP-4 são expressas entre as células do tubo neural e em sua periferia, assim como na notocorda do embrião. As lectinas Abrus precatorius, Lens culinarise Ricinus communis mostraram-se positivas principalmente na região subedotelial do assoalho da aorta nos estádios observados neste trabalho. Bandeiraea simplicifolia exibiu pouca marcação na aorta dorsal e a Arachis hypogeae foi negativa. Outras estruturas da região AGM também expressaram resíduos de açúcares revelados por estas lectinas, tais como: notocorda, tubo neural, mesênquima, intestino primitivo e saco vitelínico. Estes resultados acrescentam elementos morfológicos e bioquímicos ao conhecimento sobre a região AGM de embriões de galinha e sobre o endotélio, possivelmente hemogênico, da aorta dorsal. / Nowadays it is known that firsts definitive hematopoietic progenitors arise from endothelium differentiation of dorsal aorta, at Aorta-Gonad-Mesonephros (AGM) site. In order to study those events and cells phenotype of dorsal aorta endothelium in this topographical site, fertilized eggs of Gallus gallus domesticus L. were incubated, from E16 to E25 and to be processed by histotechonology to obtain serial sections of the AGM site. After this, they were stained with Hematoxilin-Eosin, histochemistry to PAS, diastase PAS and Alcian Blue pH 1.0 and pH 2.5, to obtain a better overview and characterization from cells, also, histochemistry to fluorescein lectins and immunofluorescense to surface molecules, cytoeskeleton andextracellular matrix were performed. Our results showed endothelial hypertrophy of the aorta floor in the stages analyzed, which shown positive for PAS, with frequent appearance of PAS negatives basal vacuolizations and an occasional intravascular cell clusters arise of. It was also observed that cells which were separated from endothelium basal membrane shown progressively were more PAS positive which however in any time seems to be glycogen. Regard of glycosaminoglycans we noted the main presence of hialuronic acid for all the mesenchymes site and in others structures like notochord periphery, neural tube and lateral mesoderm. It was observed fibronectin and smooth muscle α-actin co-expression on aorta surrounding walls, at vessels ventral face. GFAP and BMP-4 are express between the cells of neural tube and surrounding it, as well as at embryo notochord. The lectins Abrus precatorius, Lens culinaris and Ricinus communis showed mostly positive expression on the sub endothelium site of the dorsal aorta floor at the stages analyzed in this work. Bandeiraea simplicifolia showed low expression on dorsal aorta and in Arachis hypogeae it was negative. Other structures of AGMs site also expressed sugar residues revealed by these lectins like: notochord, neural tube, mesenchyme, primitive gut and yolk sac.

Les infections et l’activation du système immunitaire stimulent l’hématopoïèse. L’activation des récepteurs Toll-like (TLRs) des cellules souches hématopoïétiques, par leur reconnaissance de motifs moléculaires portés par des agents infectieux, en oriente la différenciation vers les voies myéloïdes, renforçant la capacité de notre organisme à lutter contre les infections. Ici, nous avons étudié si les agonistes TLRs peuvent, au contraire, induire au sein de la moelle osseuse l’émergence de progéniteurs hématopoïétiques présentant des propriétés immunorégulatrices. Nous montrons que l’incubation de moelle osseuse de souris en présence de l’agoniste TRL-9, CpG-B, entraîne l’émergence d’une population de progéniteurs au stade pro-B (appelée CpG-proBs). Le transfert adoptif de seulement 60,000 CpG-proBs par receveur, à l’apparition des premiers signes cliniques, confère une protection à long terme dans deux modèles expérimentaux de maladies auto-immunes, le Diabète de Type I (T1D) et l’Encéphalomyélite Auto-immune Expérimentale (EAE). La migration, la différenciation, et les mécanismes cellulaires et moléculaires de cette population protectrice sont décrits et comparés entre ces deux modèles. Dans les deux modèles, les CpG-proBs migrent vers le tissu cible de la réponse auto-immune et se différencient en cellules B matures régulatrices. Dans le T1D, l’interféron-γ (IFN-γ) produit par les cellules T s’avère essentiel pour induire la surexpression de FasL à la surface des CpG-proBs, entraînant l’apoptose des cellules T effectrices. De plus, l’IFN-γ produit par les CpG-proBs réduit la production par les cellules T de l’IL-21, une cytokine pathogène majeure dans le T1D. La descendance des CpG-proBs est composée de précurseurs transitionnels B, de cellules B de la zone marginale et de cellules B folliculaires, exprimant de forts niveaux de FasL et toujours capables d’induire l’apoptose des cellules T, prolongeant ainsi le contrôle des cellules effectrices T auto-immunes in vivo. Dans l’EAE, l’IFNγ est indirectement responsable de la rétention des cellules T, par l’internalisation de CCR7, au sein des ganglions lymphatiques, inhibant ainsi leur migration au système nerveux central (SNC). Dans la moelle épinière, tissu cible de l’EAE, les CpG-proBs se différencient en cellules B220+CD5+CD1dhiCD11b+, secrétant la cytokine anti-inflammatoire IL-10. Enfin, la mobilisation des progéniteurs hématopoïétiques par un cocktail de facteurs hématopoïétiques confère à une sous-population multipotente au stade MPP2 la propriété d’augmenter l’expansion des Foxp3+ Tregs et de prévenir la survenue du diabète de type 1. Nous montrons que les MPP2 mobilisés s’avèrent également capables d’exercer un effet protecteur envers l’EAE. Leur capacité à induire l’expansion de Treg Foxp3+ au sein du SNC et à la périphérie joue un rôle essentiel dans la protection des souris envers l’EAE, puisque la déplétion des Treg abolit la protection déjà établie. Pour conclure, nous avons mis en évidence que diverses stimulations de l’hématopoïèse induisent l’émergence de nouvelles populations de progéniteurs hématopoïétiques qui présentent des propriétés immunorégulatrices et constituent de nouveaux outils de thérapie cellulaire des maladies auto-immunes. / It is well known today that various infectious events or other stimuli of the immune system can trigger hematopoiesis. The hematopoeitic stem and/or progenitor cells express on their cell surface Toll-like receptors which can recognize molecular motifs of infectious agents. The stimulation of TLRs on hematopoietic stem cells favors their differentiation into myeloid lineages, reinforcing the capacity of our body to fight against the pathogens. Herein, we have investigated whether the stimulation of TLRs can induce, instead, the emergence within the bone marrow of selective progenitor cells with immunoregulatory properties. We show that incubation of bone marrow cells with the TLR-9 ligand CpG-B can induce a pro-B cell population (named CpG-proBs) whose adoptive transfer at low numbers of 60,000 cells provided long-lasting protection in two models of autoimmune diseases, Type I Diabetes (TID) and Experimental Autoimmune Encephalomyelitis (EAE) at the onset of clinical signs. The migration, differentiation and molecular mechanism of action of this protective population is described and compared between these two models. In both models, the CpG-proBs migrate to the target tissue of autoimmune responses and differentiate into more mature regulatory B cells. In TID, IFN-γ produced by both T and CpG-proB cells is essential for the upregulation of FasL at the surface of CpG-proBs, inducing the apoptosis of the effector T cells. In addition, IFN-γ reduced the T-cell production of IL-21, a major pathogenic cytokine in TID. The progeny of the adoptively transferred CpG-proBs, including transitional precursors B cells, marginal zone and follicular B cells, display high expression of FasL, promote apoptosis of effector T cells and prolong the control of autoimmune effector T cells in vivo. In EAE, IFN-γ was responsible for the restriction of T cells to the lymph nodes, inhibiting their homing to the CNS. IFN-γ indirectly induced the internalization of CCR7, a receptor required for the migration across the blood-brain barrier. In the spinal cord (target tissue in EAE), CpG-proBs differentiated into B220+CD5+CD1dhiCD11b+ cells secreting the anti-inflammatory cytokine IL-10. Finally, hematopoietic progenitor populations mobilized to the periphery by a cocktail of G-CSF and Flt3l, at the stage of MPP2, have already been shown to protect against TID by expanding the Foxp3+ Tregs. We evaluated them in the EAE model, showing that the ability of these mobilized progenitor cells to trigger the expansion of Foxp3+ Treg within the CNS and the periphery was necessary for providing protection to EAE mice since Treg depletion abrogated the protection once established. In conclusion, we provide evidence for the emergence of new populations of hematopoietic progenitor cells which can display immunoregulatory properties and might be used for cell therapy of autoimmune diseases.