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During the period of bone marrow engraftment and immune reconstitution after hematopoietic cell transplantation (HCT), patients are at high riskto opportunistic infections, including invasive fungal infections of the lung, such as invasive aspergillosis caused by Aspergillus fumigatus. These infections pose potentially life-threatening complications necessitating the development of new strategies to mitigate the risks. Alveolar macrophages (AM), along with alveolar epithelial cells (AEC), constitute the initial defense against fungal pathogens in the lung upon inhalation and penetration of fungal conidia into the lower respiratory tract to reach alveolar spaces. In this thesis, our objective was to investigate the interaction between the host and A. fumigatus, focusing on the role of AM in the initial immune response. Furthermore, we examined how specific cytokines modulate AM responses to A. fumigatus in vivo. To mimic the invasive A. fumigatus infection in immunocompromised patients, we utilized mouse models of allo-HCT (8 Gy TBI, C57Bl/6BALB/c) combined with intratracheal A. fumigatus infection. We monitored the survival, clinical performance and investigated local host-pathogen interaction using flow cytometry and 3D light sheet fluorescence microscopy. We observed that allo-HCT recipients exhibited better survival rates when infected with A. fumigatus 6 days after allo-HCT rather than 4 days after allo-HCT. Notably, the lungs of late-infected mice demonstrated a higher frequency, proliferation potential and phagocytic activity of tissue-resident AM compared to neutrophils and monocytes. These observations suggest that tissue-resident AM play a crucial role in protecting mice from A. fumigatus infection. To confirm this, we selectively depleted AM, which resulted in increased susceptibility to uncontrolled infection. In our study, we observed a similar pattern for AM frequency and activity in the lungs of early-infected mice. However, it appeared that AM alone were not sufficiently effective in protecting the mice from the infection. To address this, we investigated the potential of cytokines to enhance AM function and provide earlier protection. Among the cytokines tested, including M-CSF, IL-34, GM-CSF and G-CSF, we focused on M-CSF and IL-34, both of which act on the CSF-1 receptor. In vitro experiments revealed that M-CSF, but not IL-34, increased AM migration speed (0.52 vs. 0.3 µm/min, respectively) and versatility (diffusion co-efficient of 0.78 vs. 0.6 µm2/min, respectively) and improved their ability to kill fungal pathogens. Concequently, we treated allo-HCT recipients with M-CSF and infected them with A. fumigatus intratracheally 4 days after allo-HCT. M-CSF administration led to a twofold increase in myelopoiesis and a 1.5-fold expansion of tissue-resident AM. Moreover, M-CSF enhanced AM killing capacity, resulting in the protection of 90% of allo-HCT recipients from lethal A. fumigatus infection on day 4 after allo-HCT. Additionally, M-CSF improved lung tissue integrity and reduced serum levels of pro-inflammatory cytokines. It is worth noting that M-CSF treatment did not protect against lethal aspergillosis when AM were locally depleted in allo-HCT recipients, highlighting the crucial role of a functional pool of tissue-resident AM in protecting against pulmonary infections and maintaining lung function. In conclusion, our findings strongly support the potential clinical application of M-CSF to enhance tissue-resident AM responsiveness, preserve lung function, and provide early protection against A. fumigatus infections in allo-HCT recipients / Im Zeitfenster der Knochenmarkstransplantation und der Immunrekonstitution sind Patienten, die sich einer hämatopoetischen Zelltransplantation (HCT) unterziehen, anfällig für opportunistische Infektionen, wie z. B. invasive Pilzinfektionen der Lunge. Die invasive Aspergillose, die durch Aspergillus fumigatus verursacht wird, stellt ein lebensbedrohliches Risiko dar und erfordert dringend neue Ansätze zur Bekämpfung dieser Infektionsgefahr. Insbesondere gewebeständige Alveolarmakrophagen (AM) gelten als erste Immunzellen, gelten als erste Immunzellen, die nach Inhalation und Eindringen durch die Atemwege in die Alveolarräume auf Pilzerreger treffen. Gemeinsam mit den Alveolarepithelzellen (AEC) bilden die AM eine erste Verteidigungslinie gegen diese Erreger. Das Ziel dieser Arbeit war die Untersuchung der Interaktion zwischen Wirt und A. fumigatus sowie der ersten Immunantwort der Lunge. Ein besonderer Fokus lag dabei auf den AM und ihrer Rolle in dieser Reaktion. Zudem untersuchten wir, wie definierte Zytokine die AM-Antworten auf A. fumigatus in vivo beeinflussen. Wir stellten fest, dass Transplantationsempfänger eine bessere Überlebensrate aufwiesen, wenn sie 6 Tage nach der allo-HCT mit A. fumigatus infiziert wurden, im Vergleich zu 4 Tagen nach der allo-HCT. Auffällig war, dass die Lungen der Tag+6-infizierten Mäuse eine höhere Häufigkeit, Proliferationspotenzial und phagozytische Aktivität der gewebeständigen AM im Vergleich zu neutrophilen Granulozyten und Monozyten aufwiesen. Diese Beobachtungen legten nahe, dass gewebeeigene AM eine entscheidende Rolle beim Schutz der Mäuse vor einer A. fumigatus-Infektion spielen. Zur Bestätigung dieser Beobachtung eliminierten wir gezielt die AM in der Lunge, was signifikant die Anfälligkeit für eine letale invasive Aspergillose erhöhte. Ein ähnliches Muster beobachteten wir für die AMHäufigkeit und -Aktivität in den Lungen von am Tag+4 nach allo-HCT infizierten Mäusen. Es schien jedoch, dass AM allein nicht ausreichend wirksam waren, um die Mäuse vor der Infektion zu schützen. Daher untersuchten wir das Potenzial von Zytokinen, die AM-Funktion zu verbessern und einen früheren Schutz zu bieten. Unter den getesteten Zytokinen, einschließlich M-CSF, IL-34, GM-CSF und G-CSF, konzentrierten wir uns auf M-CSF und IL34, die beide spezifisch an den CSF-1-Rezeptor binden. In-vitro-Experimente zeigten, dass M-CSF, im Gegensatz zu IL-34, die Migrationsgeschwindigkeit der AM (0,52 bzw. 0,3 µm/min) sowie ihre Beweglickeit (Diffusionskoeffizient von 0,78 bzw. 0,6 µm2/min) erhöhte und ihre Fähigkeit zur Abtötung von Pilzerregern verbesserte. Dementsprechend behandelten wir allo-HCT-Empfänger mit M-CSF und infizierten sie anschließend intratracheal mit A. fumigatus 4 Tage nach der allo-HCT Die Verabreichung von M-CSF steigerte die Myelopoese zweifach und den Pool der gewebeständigen AM in der Lunge 1,5-fach. M-CSF erbesserte die Abtötungskapazität der AM, was dazu führte, dass 90% der allo-HCT-Empfänger am Tag 4 nach der allo-HCT vor einer tödlichen A. fumigatus-Infektion geschützt waren. Darüber hinaus verbesserte M-CSF die Integrität des Lungengewebes und reduzierte die Serumspiegel proinflammatorischer Zytokine. Es ist anzumerken, dass die Behandlung mit M-CSF keinen Schutz vor einer tödlichen Aspergillose bot, wenn die AM lokal eliminiert wurden, was die entscheidende Rolle eines funktionalen Pools gewebeständiger AM beim Schutz vor Lungeninfektionen und der Aufrechterhaltung der Lungenfunktion betont. Zusammenfassend unterstützen unsere Ergebnisse stark die potenzielle klinische Anwendung von M-CSF zur Verbesserung der Reaktionsfähigkeit gewebeeigener AM, Erhaltung der Lungenfunktion und Bereitstellung eines frühzeitigen Schutzes vor A. fumigatus-Infektionen bei allo-HCT-Empfängern.

Los macrófagos son células fagocíticas pertenecientes al sistema inmunitario que participan tanto en la respuesta innata como en la adaptativa. En presencia de M-CSF, su estímulo mitogénico específico, proliferan, mientras que el tratamiento con estímulos activadores como el IFN-γ o el LPS hace que detengan su proliferación, se activen y pasen así a ejercer sus funciones específicas. En este contexto, existen dos tipos de citocinas con capacidad de activar al macrófago, las llamadas citocinas de tipo Th1 y las de tipo Th2. Las citocinas de tipo Th1 como el IFN-γ inducen en los macrófagos un patrón de activación llamado "clásico" cuya finalidad es provocar una respuesta de tipo proinflamatoria que acabará por eliminar la presencia de agentes patógenos. Por el contrario, las citocinas de tipo Th2 como la IL-4 inducen una activación "alternativa" en los macrófagos, cuya finalidad es la de llevar a cabo la reparación y remodelación tisular tras la respuesta proinflamatoria. En este estudio hemos observado, por primera vez, que la activación alternativa del macrófago cumple también la dicotomía proliferación/activación observada bajo estímulos clásicos e inhibe la proliferación del macrófago en el umbral entre las fases G1 y S del ciclo celular. En este sentido, hemos visto que tanto el IFN-γ como la IL-4 inducen un alargamiento en el tiempo de la cinética de activación de la MAPK ERK-1/2, encargada de regular la proliferación del macrófago en respuesta al M-CSF. Esta MAPK es regulada por la fosfatasa MKP-,1 quien se encarga de defosforilarla. Así, en presencia del M-CSF, MKP-1 se induce para regular a ERK-1/2 mientras que el IFN-γ y la IL-4 bloquean su expresión de forma dependiente de STAT. La inhibición directa de MKP-1 mediante tecnología de RNA de interferencia induce también un alargamiento de la actividad de ERK-1/2 y, en consecuencia, una parada de la proliferación, corroborando así los resultados obtenidos previamente. También hemos analizado el estado de activación de los complejos cdk2/cicE, encargados de la progresión del ciclo celular entre las fases G1 y S. Hemos visto que, mientras que el M-CSF induce la expresión de la ciclina E y, en consecuencia, la activación del complejo cdk2/cicE, tanto el IFN-γ como la IL-4 inhiben a ambos, explicando así el mecanismo utilizado por estas citocinas para mantener la parada en G1. Este bloqueo de la actividad cdk2/cicE es llevado a cabo gracias a la acción del inhibidor de cdk p21Waf1. Sin embargo, aunque ambas citocinas inducen la expresión de esta proteína, utilizando células knockout para p21Waf1 hemos demostrado que sólo la parada inducida por la IL-4 es dependiente de esta proteína, lo cual sugiere que el IFN-γ y la IL-4 utilizan vías de señalización distintas para ejercer funciones celulares similares. Por otro lado, hemos analizado también la implicación de la vía de las MAPK en la activación alternativa del macrófago. En este aspecto hemos demostrado que, aunque la expresión de los principales genes responsables de dicha activación es dependiente de STAT6, la acción de JNK1 ejerce un efecto sinérgico sobre su inducción, ya que la inhibición de esta quinasa resulta en una disminución de la expresión de dichos genes. El mecanismo mediante el cual JNK1 ejerce esta función no está aún del todo claro. Sin embargo, los resultados obtenidos mediante ensayos de inmunoprecipitacion de cromatina sugieren que JNK1 podría estar fosforilando a determinados cofactores en las zonas promotoras de algunos genes, colaborando así con la actividad del factor de transcripción STAT6. / Macrophages play a crucial role as regulators of immune response. They are originated from bone marrow stem cells and, in response of growth factors and particularly to macrophage colony-stimulating factor (M-CSF), they proliferate and differentiate. We have previously observed that activation of extracellularly regulated kinase (ERK) 1/2 is required for macrophage proliferation in response to growth factors. A short and early pattern of ERK activity correlated with the proliferative response. In contrast, slightly prolonged patterns of activity of these kinases were induced by signals that lead to macrophage activation and growth arrest. IFN-γ is the main endogenous Th1 type macrophage activator and IL-4 is the main Th2 type one. While M-CSF withdrawal arrested the cell cycle at early G1, treatment of macrophages with IFN-γ or IL-4 stopped the cell cycle later, at the G1/S boundary. IFN-γ and IL-4 induced the expression of the Cdk inhibitor p21Waf1. Using knockout mice and siRNA experiments, we found that p21Waf1 is required for IL-4- but not for IFN-γ-dependent inhibition of proliferation. Moreover, both IFN-γ and IL-4 elongate the activity pattern of the MAPK ERK-1/2 through the inhibition of the phosphatase MKP-1, which in fact results in a decrease of proliferation. Moreover, inhibition of MKP-1 expression using siRNA technology or synthetic inhibitors also led to elongated ERK activity and significant blockage of M-CSF-dependent proliferation. On the other hand, we have also analyzed the relationship between IL-4 and the MAPK pathway on the context of the alternative activation of macrophages. The IL-4 signaling pathway involves activation of STAT-6. However, IL-4 has also the capability to activate members of the MAPK family. In primary bone marrow-derived macrophages, we have observed strong activation of JNK-1 at early time points of IL-4 stimulation, whereas weak activation of ERK-1/2 and p38 were detected at a more delayed stage. By using selective inhibitors and knockout models, we have explored the contributions of MAPK activation to the macrophage response to IL-4. Our findings indicate that JNK-1 regulate IL-4-mediated expression of genes typically involved in alternative activation such as arginase 1.

Preeclampsia is an important disease during pregnancy and causes significant maternal and fetal mortality and morbidity. Despite intense research efforts, the etiology and pathogenesis of the disease remain largely unknown. Since placentas from preeclamptic patients are smaller than normal, and cytokine growth factors are suggested to be important in placental growth, the effects of macrophage-colony stimulating factor (M-CSF) on human trophoblast cells were examined. While term trophoblast cells did not respond to M-CSF, those from early trimester and choriocarcinoma cells showed enhanced growth after treatment. In addition, the serum level of M-CSF in hypertensive pregnant women at the second trimester were significantly lower than those of normal pregnant women. These data suggest possible roles of M-CSF in preeclampsia. When M-CSF was administered to pregnant rats on days 8-11, rats had smaller placentas at day 12 and increased fetal resorption rate at day 20. The effects of interleukin-12 (IL-12) was also examined on days 8-11. While placental development was normal at both days 12 and 20, fetuses were significantly smaller at day 20. To remedy the difficulties and dangers associated with obtaining human placentas, I characterized endovascular trophoblast cell behavior in pregnant rats. In normal pregnancy, rat trophoblast cells simulated all features of human endovascular trophoblast behavior including selective invasion into the spiral arteries, retrograde migration, embedding, and secretion of PAS-positive materials as well as IIphysiological changes," In pregnancy terminated with a certain type of spontaneous fetal resorption, defective endovascular trophoblast cell behavior was observed, which was similar to that reported in preeclamptic pregnancy. Finally, the roles of cytoskeleton on trophoblast cell locomotion were investigated in vivo with a cytoskeleton-disrupting agent, cytochalasin B. This treatment impaired trophoblast cell invasion at day 12 and induced smaller fetuses at day 20, suggesting the importance of cytoskeleton in trophoblast movement. In conclusion, the results suggest the importance of the use of appropriate specimens and endpoints in the study of pregnancy, and rats may serve as a suitable animal model for the study of endovascular trophoblast cell behavior with clinical relevance to preeclampsia. / Ph. D.

Les macrophages associés aux tumeurs (TAM) orchestrent l'inflammation nécessaire à la croissance tumorale (propriétés de type M1) et favorisent les métastases et l'angiogenèse (caractéristiques de type M2). Cependant, la nature des facteurs capables de conférer des propriétés M1 et M2 aux macrophages humains demeure inconnue. L'acide lactique (AL) est un métabolite produit par les cellules tumorales connu pour moduler les fonctions des cellules présentes dans le microenvironnement tumoral. Dans cette étude, nous avons analysé son impact sur la différenciation des monocytes humains. Les résultats montrent que l’AL induit la différentiation des monocytes (cultivées en présence de GM-CSF) en macrophages présentant un phénotype atypique (CD14high CD163high IL-10low IL-12low) et, de manière intéressante, des caractéristiques phénotypiques M1 (production de cytokines inflammatoires) et M2 (production de facteurs de croissance, expression de gènes prototypiques M2). Un profil similaire est obtenu lorsque les monocytes sont cultivés avec des cellules cancéreuses primaires glycolytiques. Ces effets de l'AL sur la polarisation des macrophages nécessitent l'entrée du lactate dans les cellules (via le transporteur MCT-1) et son oxydation en pyruvate et sont médiés par une stabilisation de HIF-1α et une consommation autocrine de M-CSF.Ces résultats (i) identifient l'AL comme un médiateur induisant la génération de macrophages humains présentant des caractéristiques M2 et des propriétés inflammatoires et (ii) renforcent l'intérêt de l’utilisation des inhibiteurs de la glycolyse aérobie pour moduler les fonctions des TAM. / In established tumors, tumor-associated macrophages (TAM) orchestrate unresolving cancer-related inflammation (M1-related properties) and favor tumor development, metastasis and angiogenesis (M2-like properties). However, to date, the nature of the polarization factor(s) able to confer M1 and M2 functional properties to human macrophages remains unknown.Lactic acid (LA), a metabolite produced at high levels in most established tumors, can impact the phenotype and functions of cells present in the tumor microenvironment. In this study, we analyzed the impact of LA on the human monocyte differentiation. Results showed that LA skews monocytes (differentiated in the presence of GM-CSF) into macrophages (GM+LA-Mφ) exhibiting an atypical CD14high CD163high IL-10low IL-12low phenotype. Interestingly they harbor M1 and M2 phenotypic features, as assessed the production of a wide variety of inflammatory and growth factors and the expression of prototypic M2-like genes. A similar profile is induced by culturing monocytes with glycolytic human primary cancer cells. These effects of LA on macrophage polarization require the entry of lactate into the cells (via the monocarboxylate transporter 1) and its oxidation into pyruvate and are mediated via HIF-1α stabilization and autocrine M-CSF consumption by differentiating cells. These results identify tumor-derived LA as a missing link reconciling the M2-like features of TAM with their inflammatory properties. They also reinforce the interest of aerobic glycolysis inhibitors to modulate the functions of TAM.

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Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

Astronauts are at risk of losing 1.0% to 1.5% of their bone mass for every month they spend in space despite their adherence to diets and exercise regimens designed to protect their musculoskeletal systems. This loss is the result of microgravity-related impairment of osteocyte and osteoblast function and the consequent upregulation of osteoclast-mediated bone resorption. This review describes the ontogeny of osteoclast hematopoietic stem cells and the contributions macrophage colony stimulating factor, receptor activator of the nuclear factor-kappa B ligand, and the calcineurin pathways make in osteoclast differentiation and provides details of bone formation, the osteoclast cytoskeleton, the immune regulation of osteoclasts, and osteoclast mechanotransduction on Earth, in space, and under conditions of simulated microgravity. The article discusses the need to better understand how osteoclasts are able to function in zero gravity and reviews current and prospective therapies that may be used to treat osteoclast-mediated bone disease.

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