Resultados anteriores do nosso grupo mostraram que a melatonina (MLT) age em monócitos aumentando sua sensibilidade à IL-4 e, consequentemente, levando estas células a se diferenciarem em células dendríticas - quando in vitro e estimulados com IL-4 e GM-CSF (mo-DCs) com um perfil fenotípico e funcional mais ativado. Outros estudos do nosso grupo mostraram que mo-DCs de pacientes portadoras de câncer apresentam desvios funcionais capazes de comprometer a ativação de linfócitos por este tipo celular. Um outro apontamento de nosso grupo foi para o fato de que, muito provavelmente relacionado a estes desvios, está um comprometimento da via IL-4/STAT-6/SOCS-5, que se mostrou alterada em pacientes com leucemia linfóide crônica. Baseado nestes resultados, o presente trabalho objetivou investigar os efeitos da MLT na diferenciação in vitro de mo-DCs de doadoras saudáveis e de pacientes portadoras de câncer de mama, sob a hipótese de que este hormônio poderia agir na via IL-4/STAT-6/SOCS-5 de modo a gerar mo-DCs com fenótipo e função relacionados à maior ativação. Para tanto, monócitos provenientes do sangue periférico de indivíduos saudáveis e de pacientes foram tratados com MLT (2,5 nM 2 horas) e induzidos à diferenciação em mo-DCs; no quinto dia as células foram ativadas com LPS (100 ng/ml 24 horas). As análises, por citometria de fluxo, do fenótipo e das citocinas ao longo da diferenciação de mo-DCs não apontou efeitos consistentes acerca do tratamento com MLT, mas indicou maior expressão de CD83+ nos monócitos das pacientes (p=0,014) e maior concentração da citocina IL-12p70 no sobrenadante das culturas de mo-DCs destes indivíduos, ao final da diferenciação (p=0,02). Para a análise da capacidade linfoestimuladora das mo-DCs foi realizada co-cultura destas células com linfócitos T (LT) alogeneicos (1 DC: 30 LT) por 5 dias. Não houve diferença na indução de proliferação de LT estimulados por mo-DCs de saudáveis nem de pacientes. A MLT mostrou efeito apenas nas co-culturas com células saudáveis aumentando as concentrações de TNF, IFN-γ e IL-2 nos sobrenadantes. Nas co-culturas com células de pacientes sem tratamento, houve maior nível de IL-2 e IL-10, se comparadas com saudáveis. Os monócitos foram também tratados com MLT (2.5 nM) ou IL-4 (50 ng/mL) por 15 minutos, para avaliação da expressão de STAT-6, pSTAT-6 e SOCS-5. Não foi constatado efeito da MLT nesse caso, mas os monócitos de pacientes apresentaram maior expressão de STAT-6, porém menor de pSTAT-6, comparados com saudáveis. Tomados em conjunto, os resultados globais indicam algumas diferenças fenotípicas e funcionais entre monócitos de pacientes e controles, mas não entre suas mo-DCs. Quanto à MLT, os resultados apontam para o fato de que o hormônio gera alterações apenas em células provenientes de indivíduos saudáveis e somente com relação às citocinas encontradas nos sobrenadantes das co-culturas. / Previous studies at our lab have shown that melatonin (MLT) acts on monocytes increasing their sensitivity to Interleukin-4 (IL-4) and, consequently, generating dendritic cells (DCs) when in vitro and treated with IL-4 and GM-CSF (mo-DCs) phenotically and functionally more activated. Other studies from our group have shown that mo-DCs obtained from cancer patients have functional biases capable of compromising the activation of T lymphocytes. Another result from our group pointed to the fact that part of the mo-DCs\' functional bias in cancer patients could be attributed to the decrease in STAT-6 signaling, a pathway that is activated by IL-4 and down regulated by SOCS-5, whose levels, in turn, were found elevated in cancer patients\' monocytes. Based on these results, the present work aimed to investigate the effects of MLT on the in vitro differentiation of healthy donors and breast cancer patients mo-DCs, under the hypothesis that this hormone could act on the IL-4/STAT-6/SOCS-5 axis generating better activated mo-DCs. Peripheral blood monocytes from healthy donors and breast cancer patients were treated with MLT (2,5 nM 2 hours) and induced to differentiate into mo-DCs; on the fifth day cells were activated with LPS (100 ng/ml 24 hours). Flow cytometry analysis of phenotype and cytokines in supernatants during mo-DCs differentiation didnt show MLT effects, but indicated higher frequency of CD83+ (p=0,014) monocytes among mononuclear cells of the patients, when compared with healthy donors. In addition, higher concentration of IL-12p70 was found in mo-DCs cultures (sixth day - p=0,02). The capacity to stimulate allogeneic T lymphocytes was assessed by co-cultures (1DC : 30LT), maintained for 5 days. Results indicate an effect of MLT only in order to increase the TNF, IFN-γ and IL-2 concentrations in supernatants of co-cultures with healthy donors mo-DCs. Patients cells showed differences only when there was no hormone treatment, showing higher levels of IL-10 in the co-culture supernatants, when compared to healthy controls. Monocytes were also treated with MLT (2,5 nM) or IL-4 (50 ng/mL) for 15 minutes to evaluate STAT-6, pSTAT-6 and SOCS-5 expression. Results showed an increase of STAT-6 in patients monocytes and a lower capacity of these cells to phosphorylate this molecule, even when in presence of IL-4. Together, the results point to some phenotypic and functional differences between patients and healthy donors monocytes, but it was not shown in their mo-DCs. Results also point to the fact that MLT generates changes only in healthy donors cells and those effects were only seen with cytokines found in cultures supernatants.

Les macrophages associés aux tumeurs (TAMs) proviennent des monocytes circulants attirés par l'inflammation chronique due à la tumeur. Ces monocytes vont se différencier en une variété de macrophages en fonction des médiateurs présents dans le microenvironnement tumoral. Ainsi, il est admis que les macrophages peuvent stimuler la croissance tumorale (macrophages classiquement appelés M2), mais peuvent aussi reconnaitre et éliminer spécifiquement les cellules transformées (macrophages M1). Cette dichotomie fonctionnelle est dépendante du stade de développement cancéreux et plus spécifiquement de l'évolution du microenvironnement tumoral. De manière générale, l'infiltration et la densité élevée des TAMs au niveau de la tumeur sont corrélées à un mauvais pronostic et de ce fait les TAMs sont considérés comme une cible thérapeutique importante. Dans ce travail de thèse, nous nous sommes attachés dans un premier temps à caractériser le phénotype et les fonctions des macrophages au cours de l'évolution de la tumeur. La mise en place d'un modèle murin préclinique de lymphome T (cellules EL4) nous a permis de montrer que les macrophages présents à un stade tardif du développement tumoral, sont capables de stimuler la prolifération des cellules tumorales. Ces macrophages produisent des facteurs pro-angiogéniques, comme le TGF-ß, et immunosuppresseurs, comme l'IL-6, l'IL-10 et l'Indoléamine dioxygénase (IDO). De manière intéressante, nous avons démontré la capacité de l'IL-6, sécrétée en grande quantité dans l'ascite tumorale, à induire la différenciation des macrophages résidents en macrophages M2 pro-tumoraux. Inversement, nous avons montré que des macrophages murins résidents activés par l'IFN-? ou l'IL-13, cytokines non présentes dans l'ascite tumorale, acquièrent un phénotype anti-tumoral. Alors que l'IFN-? est connu pour induire un macrophage M1 présentant un puissant potentiel tumoricide, nous montrons que l'IL-13, qui polarise les macrophages vers un phénotype alternatif (M2), peut aussi différencier les macrophages vers un phénotype anti-tumoral. Dans ce contexte, nous avons étudié les mécanismes anti-tumoraux des macrophages polarisés par l'IL-13 vis-à-vis des cellules lymphoblastoïdes T. De façon originale, nous avons montré que les macrophages activés par l'IL-13 induisent la nécrose des cellules EL4, en produisant des radicaux libres oxygénés (RLO) et en déplétant l'arginine par l'augmentation de l'activité arginase. Nous avons également montré que les récepteurs Mannose et Dectine-1(Récepteurs Lectine de type-C), fortement exprimés par les macrophages activés par l'IL-13, reconnaissent les cellules tumorales et déclenchent la production des RLO en activant la voie Syk-P47phox. Ils induisent aussi la surexpression de l'arginase en activant la libération d'acide arachidonique et la production des HETEs (acide hydroxy-eicosatétraénoïque), agonistes naturels du récepteur nucléaire PPAR?. De plus, nous avons pu valider, in vivo, la capacité de l'IL-13 à reprogrammer les macrophages pro-tumoraux associés au lymphome T vers un phénotype anti-tumoral et à induire une régression de la tumeur. Ces résultats ont été confirmés par la diminution de la charge tumorale des souris porteuses de lymphome T, après un transfert adoptif des macrophages activés par l'IL-13. Enfin, nous avons établi que l'IL-13 active aussi les fonctions anti-tumorales des macrophages dérivés de monocytes humains contre diverses cellules tumorales humaines. Ce travail illustre la complexité du paradigme M1/M2 dans la description des fonctions pro- et anti-tumorales des macrophages. Ainsi, nos travaux démontrent la capacité de l'IL-13 à stimuler les défenses anti-tumorales des macrophages dans le cadre d'un lymphome T. Ce travail montre également que la modulation efficace des récepteurs Mannose et Dectine-1 par l'IL-13 pourrait permettre d'orienter l'interaction " cellules tumorales-macrophages " pour le bénéfice de l'hôte. / Tumor-associated macrophages (TAMs) are derived from circulating monocytes attracted by the chronic inflammation in the tumor. These monocytes differentiate into a variety of macrophages according to the mediators present in tumor microenvironment. Thus, it is known that macrophages can stimulate tumor growth (usually called M2 macrophages) but may also, in other circumstances, specifically recognize and eliminate transformed cells (M1 macrophages). This functional dichotomy is dependent on the stage of cancer development and specifically on tumor microenvironment. In general, the infiltration and the high density of TAMs in the tumor are associated with poor prognosis and consequently, TAMs might be an important therapeutic target. In this work, we first focused on the phenotype and the functions of the macrophages associated to tumor progression. The use of a preclinical mouse model of T-cell lymphoma (EL4 cells) has shown that macrophages associated to the late stage of tumor development stimulate EL4 tumor cell proliferation. These macrophages produce pro-angiogenic factors like TGF-ß and immunosuppressive mediators, such as IL-6, IL-10 and Indoleamine dioxygenase (IDO). Interestingly, we have shown that IL-6, highly secreted in tumor ascites, can improve protumoral functions of resident macrophages. Conversely, murine resident macrophages activated by IFN-? or IL-13, cytokines not detected in the tumor ascites, acquire an antitumor phenotype. While IFN-? is known to induce M1 macrophages with a powerful tumoricidal potential, we report that IL-13, which stimulate alternative phenotype (M2) of macrophages, can induce antitumor functions of macrophages. In this context, we studied the antitumor mechanisms of IL-13-activated macrophages against T-lymphoma cells. We report that IL-13-activated macrophages exert antitumor activity by promoting T-lymphoma cell necrosis through ROS release and arginase induced-L-arginine depletion. In fact, L-arginine degrading enzymes have been suggested as antitumor agents against multiple tumor lineages auxotrophic for L-arginine. We have also shown that the activation of IL-13-activated macrophages antitumor functions is dependent on the tumor cell recognition by mannose and dectin-1 receptors (C-type Lectin Receptors). Indeed, after recognition, these receptors that are overexpressed by IL-13-activated macrophages, activate Syk-P47phox pathway for ROS production and arachidonic acid-HETEs (hydroxy-eicosatetraenoic acid)-PPAR? axis for arginase activity. Moreover, IL-13 improves T-cell lymphoma regression in tumor-bearing mice through its ability to reprogram TAMs toward cytotoxic effectors. This was supported by a decrease of tumor burden in tumor-bearing mice after adoptive transfer of IL-13-activated macrophages. Finally, we established that IL-13 activates human monocyte-derived macrophages to become tumoricidal against various human tumor cells. This work shows the complexity of the M1/M2 paradigm in the description of pro- and antitumor functions of macrophages. Thus, our findings demonstrate the ability of IL-13 to stimulate macrophage antitumor activities, in the context of T-cell lymphoma. This work also suggests that effective modulation of mannose and dectin-1 receptors by IL-13 might shift the "tumor cells-macrophages" interaction for the host benefit.

No description available.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-01-02T16:42:12Z No. of bitstreams: 1 Tese Lidiane.pdf: 1449534 bytes, checksum: 483272938ee77a60ad54caa8f898d243 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-01-24T12:15:04Z (GMT) No. of bitstreams: 1 Tese Lidiane.pdf: 1449534 bytes, checksum: 483272938ee77a60ad54caa8f898d243 (MD5) / Made available in DSpace on 2018-01-24T12:17:42Z (GMT). No. of bitstreams: 1 Tese Lidiane.pdf: 1449534 bytes, checksum: 483272938ee77a60ad54caa8f898d243 (MD5) Previous issue date: 2017-08-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introduction: acute bronchiolitis (AB) is an inflammatory disease of the airways considered the most common pathology of the lower respiratory tract in childhood. Responsible for a large number of hospitalizations in infants is one of the leading respiratory diseases worldwide, raising the costs of health care in infants. According to epidemiological data, between 75,000 and 125,000 children are hospitalized in the United States each year with infections caused by respiratory syncytial virus (RSV), accounting for approximately 25% of pediatric pneumonia and up to 70% of hospitalizations for acute bronchiolitis. Children with deficiencies in cell-mediated immunity can develop more severe and prolonged infections. Activation of the mTOR, STAT-3 and STAT-6 signaling pathways have been identified as key regulators in different functions of the immune system. The aim of this study was to investigate the relationship between the gene expression of mTOR, STAT-3, STAT-6 and AB severity. Methods: it is a cohort study that included a group of infants less than 12 months old with AB admitted to a tertiary hospital in Porto Alegre, Brazil. Nasopharyngeal lavage was collected from all patients and stored in Trizol solution at -80?C at the Biomedical Research Institute (IPB) of PUCRS for subsequent extraction of RNA and cDNA synthesis. Specific primers were used to verify the relative expression of mTOR, STAT-3 and STAT-6 by means of real-time PCR. The results obtained were correlated with AB severity markers such as hospitalization time and wheezing time. Results: for the analysis of expression of the mTOR signaling protein and transcription factors STAT-3 and STAT-6, 23 patients hospitalized with AB were included. A general correlation was made between clinical markers (days of hospitalization and days of wheezing) and expression of signaling pathways. Data were stratified according to severity markers and showed a trend towards decreased mTOR expression in patients with a wheezing time equal to or greater than 5 days (r = -0.702 and p = 0.024). However, the STAT-3 and STAT-6 signaling pathways were not correlated with AB severity factors when applied in this group of patients. Conclusion: transcription factors are essential for generating effective immune responses. mTOR, STAT-3 and STAT-6 participate in the expression of a variety of genes in response to cellular stimuli and may play a key role in the manifestation of the disease. Our data demonstrate the decrease in mTOR expression, with improvement in clinical markers of severity, but other studies are needed to reinforce this finding. / Introdu??o: a bronquiolite aguda (BA) ? uma doen?a inflamat?ria das vias a?reas considerada a patologia mais comum do trato respirat?rio inferior na inf?ncia. Respons?vel por um grande n?mero de hospitaliza??es em lactentes ? uma das principais doen?as respirat?rias em todo o mundo, elevando os custos de cuidados de sa?de em lactentes. Segundo dados epidemiol?gicos, entre 75.000 e 125.000 crian?as s?o hospitalizadas nos Estados Unidos anualmente com infec??es causadas pelo v?rus sincicial respirat?rio (VSR), respondendo por aproximadamente 25% das pneumonias pedi?tricas e at? 70% das interna??es por bronquiolite aguda (BA). As crian?as com defici?ncia na imunidade mediada por c?lulas podem desenvolver infec??es mais graves e prolongadas. A ativa??o das vias de sinaliza??o mTOR, STAT-3 e STAT-6 t?m sido identificadas como reguladores-chave em diferentes fun??es do sistema imune. O objetivo deste estudo foi investigar a rela??o entre a express?o g?nica de mTOR, STAT-3, STAT-6 e a gravidade da BA. M?todos: trata-se de um estudo de coorte onde foi inclu?do um grupo de lactentes de idade inferior a 12 meses, com BA, internados em um hospital terci?rio de Porto Alegre, Brasil. Foi coletado lavado nasofar?ngeo de todos os pacientes e armazenados em solu??o de Trizol ? temperatura de -80?C no Instituto de Pesquisas Biom?dicas (IPB) da PUCRS para posterior extra??o de RNA e s?ntese de cDNA. Foram utilizados os iniciadores espec?ficos para verificar a express?o relativa de mTOR, STAT-3 e STAT-6 por meio de PCR em tempo real. Os resultados obtidos foram correlacionados com marcadores de gravidade da BA como tempo de interna??o e tempo de sibil?ncia. Resultados: para a an?lise da express?o da prote?na de sinaliza??o mTOR e fatores de transcri??o STAT-3 e STAT-6, foram inclu?dos 23 pacientes hospitalizados com BA. Foi realizada uma correla??o geral entre os marcadores cl?nicos (dias de interna??o e dias de sibil?ncia) e a express?o das vias de sinaliza??o. Os dados foram estratificados de acordo com os marcadores de severidade e mostraram uma tend?ncia para a diminui??o da express?o de mTOR em pacientes com tempo de sibil?ncia igual ou superior a 5 dias (r = -0,702 e p = 0,024). No entanto, as vias de sinaliza??o STAT-3 e STAT-6 n?o foram correlacionadas com fatores de gravidade da BA quando aplicadas neste grupo de pacientes. Conclus?o: os fatores de transcri??o s?o essenciais para gerar respostas imunes eficazes. O mTOR, STAT-3 e STAT-6 participam na express?o de uma variedade de genes em resposta a est?mulos celulares e podem desempenhar um papel-chave na manifesta??o da doen?a. Nossos dados demonstram a diminui??o da express?o de mTOR, com melhora dos marcadores cl?nicos de gravidade, por?m outros estudos s?o necess?rios para refor?ar este achado.