A tuberculose ainda é pontuada como uma doença de impacto mundial, sendo considerada desde 1993 um problema de saúde pública global. Uma das grandes preocupações é a contínua prevalência de cepas da Mycobacterium tuberculosis multidroga resistentes, especialmente o genótipo hipervirulento W-Beijing. Acredita-se que este genótipo apresenta alguma vantagem seletiva em relação a outros genótipos da M. tuberculosis, além de estar associado à falha terapêutica, tuberculose extrapulmonar, resistência à vacinação pela BCG e acentuada capacidade de disseminação. Estas cepas apresentam variável capacidade de sobrevivência dentro de macrófagos e do granuloma, modulando vias metabólicas específicas que culminam no escape do sistema imunológico e sucesso na infecção. Buscando entender esta vantagem seletiva e capacidade de persistência na infecção, este estudo teve como objetivo analisar e comparar o perfil transcriptômico de macrófagos infectados com as cepas da M. tuberculosis, W-Beijing 1471 e H37Rv. Os RNAs mensageiros dos macrófagos infectados foram sequenciados em plataforma HiScan Genome Analyzer Illumina. Foram gerados aproximadamente 30 milhões de sequências por amostra, em leituras single reads, com mais de 70% de sequências com valores de score Q de qualidade superior ou igual a 30. Foram mapeados e analisados 35.581 transcritos. Em média, 63% dos genes não apresentaram diferenças nos valores de expressões, 19% tiveram suas expressões reduzidas e 18% dos genes foram classificados como mais expressos, para todas as amostras de macrófagos sequenciadas. Após as análises terciárias e validação por PCR em tempo real, as amostras infectadas com a cepa W-Beijing 1471 apresentaram um aumento nas expressões de IFNs da classe I (p<0,001) e aumento exacerbado de TNF-alfa (p<0,001), comparativamente ao controle e as amostras infectadas com a cepa padrão H37Rv. Aditivamente foi observado um aumento nas expressões de duas quinases, RIPK1 e RIPK3 e de moléculas envolvidas na indução e controle de espécies reativas de oxigênio (ROS), que em infecções por bactérias intracelulares, estão correlacionadas com a morte de macrófagos por necroptose. A cepa hipervirulenta da M. tuberculosis, W-Beijing 1471, apresentou reduzida persistência intramacrofágica e induziu morte precoce dos macrófagos ao quinto dia de infecção. A morte observada nos macrófagos foi associada a ativação de IFNs da classe I/TNF-&#945;/RIPK1/RIPK3 e ROS, indicando necroptose. Ainda, foi observado um aumento na expressão do receptor TLR3 nas amostras infectadas com a cepa W-Beijing, comparativamente as amostras controles e infectadas com a cepa H37Rv. É provável que a ativação inicial dos IFNs da classe I tenha ocorrido via TLR3 através do reconhecimento de dsRNAs da M. tuberculosis. / Since 1993 the tuberculosis is considered as a disease of worldwide impact and a problem of public health. A major concern is the continuing prevalence of multidrug resistant Mycobacterium tuberculosis strains, especially the hypervirulent W - Beijing genotype. It is believed that this genotype has a selective advantage over other genotypes of M. tuberculosis and has being associated with treatment failure, extrapulmonary tuberculosis, resistance to BCG vaccination and marked ability to spread. These strains have varying ability to survive within macrophages and granuloma, modulating specific metabolic pathways that culminate in the escape of the immune system response. To understand this selective advantage and ability to persist in infection, this study aimed to analyze and compare the transcriptomic profile of macrophages infected with strains of M. tuberculosis W - Beijing 1471 and H37Rv. The mRNAs of infected macrophages were sequenced in HiScan Illumina Genome Analyzer platform. Were generated approximately 30 million sequences per sample, in single-reads readings. More than 70 % of sequences had values of Q score superior or equal to 30. Were mapped and analyzed 35,581 transcripts. On average, 63% of the genes showed no differences in the expressions, 19% were downregulated and 18% were upregulated, for all samples sequenced macrophages. After tertiary analysis and validation by real-time PCR, samples infected with the strain W -Beijing in 1471 showed an increase in expression of IFN class I (p <0.001) and exacerbated increase of TNF- alpha (p < 0.001) when compared to the control samples and those infected with standard strain H37Rv. Additively was observed an increase in expressions of the two kinases RIPK1 and RIPK3 and molecules involved in the induction and control of reactive oxygen species (ROS). In infections by intracellular bacteria, activation of RIPK1, RIPK3, ROS and TNF-&#945;, are correlated with death of macrophages by necroptosis. The hypervirulent M. tuberculosis W - Beijing 1471 strain showed reduced persistence inside macrophage and induced early death of macrophages in the fifth day of infection. The death observed in macrophages was associated with activation of IFNs class I/TNF-&#945;/RIPK1/RIPK3 and ROS, indicating necroptosis. Also was observed an increase in the expression of TLR3 receptor in infected samples with W-Beijing 1471 strain compared to controls and those infected with H37Rv strain. Probably the initial activation of IFNs class I occurred by TLR3 through the recognition of M. tuberculosis dsRNAs.

Com o avanço da idade observamos a queda na eficiência do Sistema Imunológico, estando relacionada ao aumento da morbidade e mortalidade em idosos. Dentre as células do sistema imunológico encontramos os macrófagos que garantem ao organismo a capacidade de defesa contra infecções, proliferação de células tumorais e reparo de tecidos. Uma das formas de reverter ou até mesmo restaurar algumas das funções imunológicas comprometidas com o processo de envelhecimento é a utilização da prática de exercício aeróbio moderado. Por este motivo, estudamos o efeito do treinamento moderado em natação sobre a função e o metabolismo de macrófagos de ratos envelhecidos. Em macrófagos obtidos da cavidade peritoneal, observamos uma melhora da capacidade funcional, através do aumento das funções de aderência, quimiotaxia e produção de peróxido de hidrogênio (H2O2) e óxido nítrico (NO-), que foram acompanhadas pelo aumento no metabolismo de glicose (aumento de consumo e da enzima hexoquinase), contribuindo para a melhora da função imune no envelhecimento. / Disorders of the immune function contribute to the high incidence of infections and cancer among elderly people. Macrophages play a crucial role in immune response, destroying bacteria, parasites, viruses and tumour cells through various mechanisms of action. Exercise is able to induce changes and modulate the immune response. The aim of the present work was to evaluate the function and metabolism of macrophages obtained from old rats submitted to moderate exercise training. Sedentary adult (2 – 4 months), old (15 – 18 months) and trained old rats were studied. The results show an increase in the function of macrophages obtained from the peritoneal cavity of trained old rats compared with old rats, regarding chemotaxis, hydrogen peroxide and nitric oxide production, as well a enhanced glucose consumption and increased maximal activity of the enzymes hexoquinase and glutaminase. In summary, our results indicate that exercise (moderate training) stimulates some functional aspects of macrophages of old rats, with a concomitant increase in glucose metabolism.

Avaliamos o papel dos leucotrienos (LTs), as vias de sinalização e o efeito da insulina na fagocitose via FcgR por macrófagos alveolares (MAs) de ratos sadios (RS) e diabéticos (RD). Vimos que a) MAs de RD fagocitam menos que os de RS; b) a fagocitose é dependente de LTs endógenos em RS mas não em RD; c) a adição de LTB4 ou LTD4 aos MAs em cultura aumenta a fagocitose em RS e RD; d) MAs de RS e RD produzem quantidades equivalentes de LTB4 e LTC4; e) a adição de insulina aos MAs aumenta a capacidade fagocitica em ambos os grupos; f) em RS, a fagocitose via FcgR induz fosforilação de Akt e PKC-d, que é amplificada por LTs endógenos, enquanto que em RD ocorreu fosforilação somente da PKC-d. A foforilação de Akt e PKC-d amplificada por LTs produzidos sob estímulo do FcgR em MAs de RS parece ser, de alguma forma, dependente da ação da insulina, pois MAs provenientes de RD fagocitam menos, a fagocitose não é dependente de LTs endógenos e o estímulo via FcgR não é capaz de ativar a Akt. / We evaluated the role of leukotrienes (LTs), the signaling pathways and the effect of insulin in phagocytosis via FcgR by alveolar macrophages (AMs) from healthy (HR) and diabetic (DR) rats. The results showed that: a) AMs from DR showed lower phagocytic capacity than AMs from healthy rats; b) the phagocytosis was dependent of endogenous LTs in AMs from HR but not DR; c) addition of LTB4 and LTD4 to cultures enhanced the phagocytosis by AMs from HR and DR; d) AMs from HR and DR rats produced similar levels of LTB4 and LTC4; e) addition of insulin to AMs enhanced the phagocytic capacity in HR and DR; f) in HR, the phagocytosis via FcgR induced Akt and PKC-d phosphorylation, which is amplified by endogenous LTs, whereas in DR, only PKC-d was phosphorylated. The phosphorylation of Akt and PKC-d, amplified by LTs produced under FcgR engagement in AMs from HR, seems to be, in a way, dependent of insulin action, because AMs from DR have lower phagocytic capacity, the phagocytosis is not dependent of endogenous LTs and the FcgR engagement is not capable to activate Akt.

A artrite induzida por pristane (PIA) em camundongos AIRmax homozigotos para o alelo R e S do gene Slc11a1 foi usada para avaliar a influência do polimorfismo deste gene na resposta imune, mais especificamente na ativação de macrófagos peritoneais durante a PIA. Estudos anteriores mostraram que a presença do alelo S do gene Slc11a1 aumentou a incidência e a severidade da PIA em AIRmaxSS, sugerindo que este gene ou outro próximo esteja interagindo com o loci da inflamação para modular a PIA. O tratamento com pristane nos animais AIRmaxSS induziu infiltrado intenso composto por linfócitos, monócitos/macrófagos e neutrófilos. Macrófagos AIRmaxSS apresentaram perfis de expressão gênica e celular exacerbados durante a PIA, com expressão/produção elevada de H2O2, NO, IL-1b, IL-6, TNF-a e várias quimiocinas. Entretanto, o alelo R do gene Slc11a1 foi capaz de regular a intensidade de ativação do macrófago de forma mais eficiente que o alelo S e controlar desenvolvimento da artrite. Houve acometimento do rim, pulmão e timo durante a PIA. Nossos dados sugerem que o gene Slc11a1 modula a ativação dos macrófagos envolvidos na suscetibilidade a PIA e estas linhagens representam um modelo murino alternativo para o estudo da artrite reumatoide. / Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S allele was used in this study to characterize the role of Slc11a1 polymorphisms on immune response, more specifically in the activation of peritoneal macrophages during PIA. Previous reports showed the presence of S allele of Slc11a1 increased the incidence and severity PIA in AIRmaxSS, suggesting that this gene or another closed-linked gene interacts with inflammatory loci to modulate PIA. Pristane treatment induced intense infiltration of lymphocytes, monocytes/macrophages and neutrophils in AIRmaxSS animals. AIRmaxSS macrophages demonstrated exacerbated cellular and gene expression profiles during PIA, with higher expression/production of H2O2, NO, IL-1b, IL-6, TNF-a and chemokines. However, Slc11a1 R allele could be regulating macrophage activation intensity more efficiently than the S allele and control the development of arthritis. There was involvement of kidney, lung and thymus during PIA. Our data suggest that the Slc11a1 gene modulates macrophage activation involved in PIA susceptibility and these lines represent an alternative murine model of rheumatoid arthritis.

Camundongos AIRmax e AIRmin exibem diferenças significativas no número médio de leucócitos migrantes e no conteúdo protéico do exsudato inflamatório produzido por partículas de poliacrilamida. Um dos fatores preponderantes para a maior capacidade inflamatória da linhagem AIRmax é a maior produção de neutrófilos maduros pela medula óssea. Assim, considerando a diferente capacidade de produção leucocitária, entre as linhagens AIRmax e AIRmin, nos propomos a estudar comparativamente nestas linhagens, o processo de mielopoese in vitro em resposta ao GM-CSF e ATRA. Verificamos que as células da medula óssea dos animais AIRmax apresentaram maior potencial proliferativo e maiores níveis de expressão de genes envolvidos nos estágios iniciais da mielopoese, do que os animais AIRmin. Além disso, o conteúdo protéico das células em cultura revelou diferenças quantitativas e qualitativas de proteínas provavelmente envolvidas no processo de mielopoese. / Mice AIRmax and AIRmin exhibit significant differences in the average number of migrating leukocytes and in the protein content of inflammatory exudate produced by polyacrylamide particles. This higher inflammatory capacity of the AIRmax mice is due to three convergent factors: higher local production of chemotactic factors, increased resistance of locally infiltrated neutrophils to spontaneous apoptosis and larger production of mature neutrophils by the bone marrow. Thus, considering the differential capacity of leukocyte production between AIRmax and AIRmin mice, we are studying comparatively the myelocytic differentiation process in vitro. We verified that the BM cells of AIRmax mice showed higher proliferation levels. In addition by qPCR technique it was verified a larger expression of genes involved in the initial stages of myelopoiesis in the AIRmax BM cultures. The analysis of BM cellular protein content by 2D gel electrophoresis revealed quantitative and qualitative differences between two mouse lines.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2016-08-16T15:20:59Z No. of bitstreams: 1 barbarabrunamunizfiqueiredo.pdf: 1234783 bytes, checksum: b2e6590135c597d4befcbab1e5b68fe1 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-08-17T13:05:50Z (GMT) No. of bitstreams: 1 barbarabrunamunizfiqueiredo.pdf: 1234783 bytes, checksum: b2e6590135c597d4befcbab1e5b68fe1 (MD5) / Made available in DSpace on 2016-08-17T13:05:50Z (GMT). No. of bitstreams: 1 barbarabrunamunizfiqueiredo.pdf: 1234783 bytes, checksum: b2e6590135c597d4befcbab1e5b68fe1 (MD5) Previous issue date: 2016-02-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A tuberculose (TB) é a segunda maior causa de morte por doença infecciosa no mundo. O Mycobacterium bovis é um dos causadores de TB que se dispersa quando o indivíduo infectado tosse ou espirra liberando gotículas contendo as micobactérias. As micobactérias têm preferência por tecidos bem oxigenados pelo pulmão onde irão desenvolver a infecção e ativação do sistema imunológico. Um dos primeiros tipos celulares envolvidos na tentativa de controle da infecção são os macrófagos residentes que irão fagocitar as micobactérias e produzir espécies reativas nitrogênio e oxigênio na tentativa de destruir o patógeno além de produzir citocinas que irão auxiliar na ativação da resposta imune adquirida. O tratamento da TB é feito através do uso de antibióticos, mas no período pré-antibióticos, a vitamina D (1,25(OH)2D3) era utilizada como forma de tratamento. A vitamina D é um hormônio esteroide que já se mostrou capaz de afetar a resposta imunológica a doenças infecciosas como a TB por meio da produção de catalecidina. Dessa forma, o objetivo do presente trabalho foi avaliar o efeito da vitamina D na atividade microbicida de macrófagos infectados com as linhagens atenuada (BCG) e selvagem (ATCC19274) de M. bovis. Foram utilizadas células da linhagem RAW 264.7 infectadas com as linhagens BCG e ATCC19274, tratadas com 1nM, 10nM e 100nM da forma ativa de vitamina D (1,25(OH)2D3). O teste de viabilidade celular demonstrou que as concentrações de 1,25(OH)2D3 utilizadas não são tóxicas para as células. Através da técnica de ELISA observamos que a 1,25(OH)2D3 tende a diminuir a produção das citocinas IL-6 e IL10. A contagem dos bacilos fagocitados realizada após coloração por Ziehl-Neelsen demonstrou que há diminuição na carga bacilar de forma proporcional ao aumento da concentração de 1,25(OH)2D3 corroborando com a diminuição da viabilidade das bactérias determinada pela contagem de CFU em lisado celular. Em relação à dosagem de óxido nítrico (NO), foi observado aumento proporcional à concentração de 1,25(OH)2D3. Em conclusão, esses dados sugerem que a diminuição na carga bacilar e menor número de CFU no lisado celular possa ser consequência do tratamento com 1,25(OH)2D3 que afeta algum mecanismo microbicida dos macrófagos, e que este mecanismo afetado pela 1,25(OH)2D3 possa ser a produção de óxido nítrico. / Tuberculosis (TB) is the second leading cause of death from infectious disease worldwide. The Mycobacterium bovis is one of the TB causing agent that disperses when the infected person coughs or sneezes releasing droplets containing mycobacteria. This mycobacteria have a preference for tissues well oxygenated such as lungs where they will develop the infection, initiating the immune response. One of the first cell types involved in controlling the infection are the resident macrophages which will phagocytose mycobacteria and within the phagosome, producing reactive species nitrogen and oxygen in an attempt to destroy the pathogen, in addition to cytokines production, which construct the acquired immune response. TB treatment is done by the use of antibiotics, but in the pre-antibiotic period, vitamin D was used as a treatment. Vitamin D is a steroid hormone that has been shown to affect the immune response to infectious diseases such as TB by catalecidina production. Thus, the objective of this study was to evaluate the effect of vitamin D in the microbicidal activity of macrophages infected with the attenuated (BCG) and virulent (ATCC19274) strains of M. bovis. For this study were used cell line RAW 264.7 infected with the BCG strains and ATCC19274, treated with 1nM, 10nM and 100nM of the active form of vitamin D. The cell viability test showed that the concentrations used are non-toxic. Through ELISA we observed that vitamin D tends to decrease the production of IL-6 and IL-10 cytokines. The bacilli count after Ziehl-Neelsen staining demonstrated that there is a reduction in bacterial load in proportion to the concentration of vitamin D, confirming the reduction of viability of bacteria determined by counting CFU. The dosage of nitric oxide (NO) showed increased proportional to the concentration of vitamin D. We can conclude that a reduction in bacterial load and CFU count may be a result of treatment with vitamin D that affects some microbicidal mechanism of macrophage, and that this mechanism affected by vitamin D may be nitric oxide production.

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-&#947;) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no citoplasma, vesículas e núcleo. Curiosamente, verificamos MPO em células isoladas e também em agrupamentos celulares de duas ou mais células. Por citometria de fluxo identificamos macrófagos, linfócitos B1 e agrupamentos celulares como células que contém MPO. A mobilização de MPO durante a ativação celular, a presença de MPO em linfócitos e a presença de MPO e IDO em núcleos são informações novas que sugerem novas atividades para estas enzimas. / Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-&#947; mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by flow cytometry. The MPO mobilization during cell activation, the presence of MPO in lymphocytes and the presence of MPO and IDO in nuclei are new informations to suggest new activities for these enzymes.

Current smoking cessation guidelines recommend nicotine replacement therapy (NRT) to assist pregnant smokers to quit, but this is without strong evidence for effectiveness and safety. Nicotine, the main addictive component of tobacco, is known to exert physiological effects by binding to its receptor, the nicotinic acetylcholine receptor (nAChR). Recent studies have identified the presence of nAChRs in non-neuronal cells, and in macrophages, functional alteration upon stimulation with nicotine has been documented. To understand the impact of in utero nicotine exposure on various immune cell disposition and function, we designed preliminary studies using an in vivo model of P. aeruginosa infection. In this model, pregnant mice were exposed to nicotine and after weaning, offspring were infected intra-tracheally and humanely killed 5 days later. Nicotine-exposed mice had a greater weight reduction post-infection. This was accompanied by a decreased number of neutrophil, resident macrophages, and B lymphocytes in the lungs, while the number of B lymphocytes in the lymph nodes were greater than that of the control group. In the lung lavage fluids, IL-6, MCP-1, and TNFα concentrations were elevated in nicotine-exposed mice. In an in vitro system using bone marrow-derived macrophages, a significantly reduced production of IFNγ was observed in nicotine-exposed mice when cells were stimulated with LPS. To characterize and compare gene expression in macrophages isolated from neonates developmentally exposed to nicotine, we designed a clinical study to recruit pregnant mothers who 1) did not smoke during pregnancy, 2) smoked throughout pregnancy, or 3) used NRT during pregnancy. We found that successful RNA isolation can be achieved from neonatal tracheal aspirate samples and cell number and reagent volumes were important determinants of acceptable RNA quality and quantity. Together, these preliminary findings demonstrate a possible alteration in immune response as a result of in utero nicotine exposure and sets a groundwork for future studies in identifying mechanisms underlying the impact of developmental nicotine exposure.

La leucémie lymphoïde chronique (LLC) est l'hémopathie maligne la plus fréquente des pays occidentaux. Elle se caractérise par une accumulation de cellules leucémiques dans le sang, la moelle osseuse et les organes lymphoïdes secondaires. Malgré les nouvelles thérapies, la LLC reste incurable. Dans la LLC, comme dans tout cancer, le microenvironnement tumoral, un milieu complexe contenant des cellules immunitaires pouvant être pro-tumorales, prend une part croissante aussi bien dans la physiopathologie que le pronostic. La recherche de nouvelles thérapies ciblant les interactions des cellules leucémiques avec ce microenvironnement représente aujourd'hui une piste prometteuse pour traiter cette pathologie. Les " nurse-like " cells (NLC) semblaient être les macrophages associés aux tumeurs (TAM) de la LLC. Ces cellules, retrouvées dans les ganglions lymphatiques de patients, favorisent in vitro la survie des cellules leucémiques. Elles partagent avec les TAM de nombreuses caractéristiques telles que des capacités immunosuppressives, un profil transcriptomique proche et une implication dans la résistance aux traitements. Nous l'avons confirmé par une analyse fonctionnelle et phénotypique. Nos travaux indiquent clairement que les NLC sont les TAM de la LLC avec une forte expression de CD68 et de CD163 et qu'elles favorisent la survie des cellules leucémiques. Suite à ce travail, nous avons déterminé l'impact pronostique des NLC dans la LLC, en émettant l'hypothèse que, comme dans d'autres cancers, une forte infiltration par les TAM pouvait être de mauvais pronostic. Nous avons alors montré que le taux de NLC présentes au sein du ganglion lymphatique chez le patient est corrélé à la progression de la LLC. Les NLC relarguent également un facteur soluble le CD163 soluble (sCD163) dans le sang des patients, dont le taux est corrélé à des marqueurs de mauvais pronostic dans la LLC tels que le statut IgHV non muté, le caryotype complexe et les mutations de TP53. Enfin, le taux de sCD163 est un marqueur pronostique indépendant dans la LLC : un fort taux étant associé à un raccourcissement du temps avant retraitement, de la survie sans progression et de la survie globale des patients. Ensuite nous avons étudié l'impact d'une thérapie innovante, l'ibrutinib, un inhibiteur de tyrosine kinase ciblant spécifiquement la Bruton Tyrosine Kinase, sur ces NLC. In vitro, les monocytes se différencient toujours en NLC et leur phénotype n'est pas modifié. De plus, ces cellules sont toujours capables de protéger les cellules leucémiques de l'apoptose. Finalement, nous avons mis en évidence qu'elles pouvaient participer à la chimiorésistance, en protégeant in vitro les cellules leucémiques de l'ibrutinib mais pas du dasatinib, de l'idélalisib, du vénétoclax, de la bendamustine et du rituximab. L'ensemble de nos résultats indique que cibler les NLC est une piste prometteuse dans le traitement de la LLC. Les interactions cellulaires entre NLC et cellules leucémiques pourraient représenter une cible thérapeutique intéressante. Nous avons montré qu'in vitro le contact entre NLC et cellules leucémiques est nécessaire à la survie de ces dernières. Suite à une étude transcriptomique, nous avons mis en évidence plusieurs couples moléculaires potentiellement impliqués dans ces interactions. Seul le couple LFA3/CD2 s'est révélé être nécessaire dans ce contact via un mécanisme dépendant d'Akt et son blocage inhibe totalement les effets pro-survie des NLC sur les cellules leucémiques. En conclusion, les NLC via des interactions dépendantes du couple LFA-3/CD2 protègent les cellules leucémiques de l'apoptose in vitro. De plus, elles participent à la chimiorésistance en protégeant aussi les cellules leucémiques de l'ibrutinib. Elles relarguent également du sCD163, dosable dans le sérum, un facteur pronostique indépendant de la LLC. La recherche de nouvelles thérapies plus spécifiques, ciblant les NLC constitue donc une piste thérapeutique intéressante dans la LLC. / Chronic lymphocytic leukemia (CLL) is the most common hemopathy in western countries. This pathology is characterized by an accumulation of leukemic cells in blood, bone marrow and secondary lymphoid organs. Despite new therapies, CLL is still incurable. In CLL, the microenvironment, a complex media containing immune cells witch can favor the tumor, takes now a large place in the physiopathology and the prognosis. The research of new therapies targeting interactions between CLL cells and microenvironment is a promising runway to find new drugs in CLL. "Nurse-like" cells (NLC) should be the tumor associated macrophages (TAM) of CLL. This cells, found in the lymph node of CLL patients, protect CLL cells against in vitro apoptosis. They share with TAM many characteristics including immunosubversive proprieties chemoresistance induction and a close gene expression profiling. We confirmed that by functional and phenotypical analysis. Our results indicate that NLC are the TAM of CLL with a high expression of CD68 and CD163 and that NLC can protect CLL cells against in vitro apoptosis. Thus, we focused on the clinical impact of NLC in CLL. Because in several cancers a high infiltration of TAM is correlated with a poor clinical outcome, we hypothesized that NLC infiltration should be associated with CLL outcome. We showed that infiltration of NLC in the lymph node can be correlated with the progressivity of CLL. Moreover, NLC release a soluble factor, the sCD163 (soluble sCD163), in the blood of CLL patients. High levels of this factor, can be correlated with previously established prognostic makers in CLL such as TP53 mutations, unmutatted IgHV status and complex karyotype. Finally, sCD163 was an independent prognostic marker in CLL and high levels are associated with shorter time to next treatment, progression free survival and overall survival. Next, we studied the impact of ibrutinib, a new therapy in CLL, on NLC. Ibrutinib is a specific tyrosine kinase inhibitor witch targets specifically the Bruton Tyrosine Kinase. In vitro, NLC differentiated from ibrutinib treated patients have the same phenotype as NLC from untreated patients and are still able to protect CLL cells against apoptosis. To end, we tested if in vitro NLC could protect CLL cells against chemotherapy and showed that NLC protect CLL cells against ibrutinib but not against idelalisib, dasatinib, venetoclax, bendamustin and rituximab. All our previous studies reveal that NLC are a good target to find new therapies in CLL. We focused our work on the interaction between NLC and CLL cells. First, we demonstrated that the NLC/CLL cells contact is necessary to prevent CLL cells death in vitro. Next, by gene expression profiling, we screened several couple of molecules potentially implicated in these interactions. Finally, only LFA-3/CD2 couple was necessary for this contact through an Akt pathway dependent and the inhibition of this couple totally inhibited the pro-survival effect of NLC on CLL cells. To conclude, NLC protect CLL cells from in vitro apoptosis through LFA-3/CD2. Moreover, they protect CLL cells against ibrutinib and so facilitate the chemoresistance. They release sCD163, an independent marker in CLL, which can be measured in the serum. The research of new targets, which can be considerate as more specific, targeting NLC is still an interesting way to find new therapies in CLL.

L’ADN méthyltransférase 3a (DNMT3A) relie environnement et phénotype par la méthylation des dinucléotides CpG, qu’on les trouve en particulier dans les régions promotrices des gènes. Hypométhylation de ces CpG est associée à l’activation de la transcription, qui permet le contrôle de l'expression génique dans des états physiologiques et pathologiques. La plupart de nos connaissances sur l’implication de Dnmt3a en pathologie concernent le cancer, quelques données montrent sa contribution à d’autres pathologies. L’athérosclérose est la maladie cardiovasculaire la plus fréquente. Plusieurs facteurs de risque contribuant à son apparition, sont liés à L’environnement. En particulier, les dyslipidémies, largement influencées par le régime alimentaire. Par ailleurs, d’abondantes données décrivent la contribution des cellules inflammatoires à la physiopathologie de cette maladie. Jusqu'à présent, un nombre croissant de données suggère un rôle de la méthylation de l’ADN dans l'athérosclérose, mais à ce jour, le rôle de Dnmt3a dans la régulation du cholestérol et le développement initial des plaques n'a pas été étudié.Nos résultats suggèrent que l’inactivation de Dnmt3a dans les monocytes/macrophages ne modifie pas le développement initial des plaques d’athérome et n’a pas d’influence sur la polarisation des macrophages in vitro. En parallèle, nous avons démontré que l’inactivation de Dnmt3a dans les hépatocytes conduit à une différence significative de cholestérolémie plasmatique qui n’est pas liée à une dérégulation des gènes majeurs impliqués dans le métabolisme du cholestérol. En revanche, nous avons mis en évidence une activation des réponses inflammatoires. / DNA methyltransferase 3a (DNMT3A) links environment to phenotypes via catalysis of CpG dinucleotides, notably found in genes promoter regions, methylation and whose hypomethylation is associated with gene transcriptional activation thus enabling the control of gene expression in physiologic and pathologic states. Most of our knowledge about its’ role in disease occurrence are based on articles demonstrating its’ implication in human cancers. Limited data from mouse studies illustrates its’ contribution to certain pathologies. Atherosclerosis constitutes the single most important contributor to the growing burden of cardiovascular disease. Risk factors contribute to disease occurrence, where most are related to environmental influences, notably Dyslipidaemia, a key initiator of atherosclerosis. Abundant data link hypercholesterolemia to atherogenesis, on the other hand, contribution of inflammatory mechanisms that couple dyslipidaemia to atheroma formation has been also appreciated. So far, a growing number of data suggests a role of Dnmt3a in atherosclerosis but to date, its role in cholesterol regulation and early plaque formation has not been clearly elucidated. Our results suggested that deletion of Dnmt3a in monocyte/macrophages does not affect the formation of early atherosclerostic plaque nor does it impact the polarization of macrophages in vitro. In parallel, we have also demonstrated that the deletion of Dnmt3a in hepatocytes leads to significant elevation in TC levels. We were not able to relate this elevation to dysregulation of major genes involved in Cholesterol regulation. On the other hand, we noticed activation of hepatic inflammatory responses.