Orientador: Vânia Niéto Brito de Souza / Resumo: A infecção pelo Mycobacterium leprae (ML) estimula um processo de desdiferenciação e proliferação das células de Schwann (SCs) que pode contribuir para a disseminação do bacilo. Os macrófagos (MOs) são células efetoras da resposta imune que promovem a eliminação de patógenos, entretanto, na hanseníase são colonizados pelo ML. Sabe-se que os MOs podem apresentar uma polarização funcional na qual os MOs M1 apresentam características pró-inflamatórias e microbicidas enquanto os MOs M2 atuam na reparação tecidual e possuem perfil anti-inflamatório. SCs infectadas pelo ML produzem mediadores capazes de interferir com a função dos MOs aumentando sua sobrevida e promovendo sua migração. Embora diferentes programas funcionais tenham sido observados em MOs de pacientes com formas polares da hanseníase a influência de SCs nesse processo não é sabida. Neste estudo avaliamos se SCs infectadas pelo ML podem interferir na polarização de MOs murinos derivados de medula óssea. Para tanto, culturas primárias de SCs murinas foram infectadas experimentalmente com bacilos viáveis e cocultivadas com MOs. Nossos resultados indicam que a produção de óxido nítrico foi baixa nas culturas de MOs após a infecção com o bacilo, mas mostrou-se aumentada nas coculturas de MOs e SCs infectadas pelo ML. A infecção com ML não induziu produção significante das citocinas IL-6, IL-10 e TNF em culturas de MOs e SCs, entretanto, a interação entre MOs e SCs infectadas com o bacilo resultou em aumento na produção de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mycobacterium leprae (ML) infection stimulates dedifferentiation and proliferation of Schwann cells (SCs) that may contribute to the spread of the bacillus. Macrophages (MOs) are effector cells of the immune response that promote the elimination of pathogens, however, in leprosy they are colonized by ML. It is known that MOs can present a functional polarization in which M1 MOs show pro-inflammatory and microbicidal activities while M2 MOs act in tissue repair presenting an anti-inflammatory profile. SCs infected by ML produce mediators able to interfere with MOs function, increasing their survival and promoting their migration. Although different functional programs have been observed in MOs from patients with polar forms of leprosy, the influence of SCs in this process is not known. In this study we evaluated whether SCs infected with ML could interfere in the polarization of murine MOs derived from bone marrow. For this purpose, primary cultures of murine SCs were experimentally infected with viable bacilli and co-cultivated with MOs. Our results indicate that nitric oxide production was low in cultures of MOs after infection with the bacillus, but it was increased in the co-cultures of MOs and ML-infected SCs. The infection with ML did not induce significant production of IL-10, TNF and IL-6 in cultures of MOs and SCs, however, the interaction between MOs and ML infected-SCs resulted in increased production of cytokines, mainly IL-10, inducing a decrease in the TNF/IL-10 ... (Complete abstract click electronic access below) / Mestre

Background Ultrasmall superparamagnetic particles of iron oxide (USPIO)- enhanced magnetic resonance imaging (MRI) can detect tissue-resident inflammatory macrophages and identify cellular inflammation. Clinical studies using this non-invasive technique are now emerging. Objectives The aims of this thesis were (i) to determine whether USPIO-enhanced MRI can detect and serially monitor myocardial inflammation after myocardial infarction (MI) using single and repeated USPIO administration, (ii) to report a range of normal R2* (1/T2*) values at 1.5 tesla (T) and 3 T in healthy myocardium and other tissues before and after USPIO administration, (iii) to determine whether USPIO-enhanced MRI can detect myocardial inflammation in acute myocarditis, and (iv) to determine whether USPIO-enhanced MRI can detect myocardial inflammation in patients with a prior cardiac transplant. Methods Thirty-one patients were recruited following acute MI and followed up for 3 months with repeated T2 and USPIO-enhanced T2* mapping 3 T MRI. Twenty healthy volunteers were recruited: 10 imaged each at 1.5 T and 3 T. T2 and USPIO-enhanced T2* mapping MRI was conducted. Fourteen patients with suspected acute myocarditis underwent T2 and USPIO-enhanced T2* mapping 3 T MRI, with further imaging at 3 months. Eleven patients with prior cardiac transplant underwent T2 and USPIO-enhanced T2* mapping 1.5 T MRI with further imaging at 3 months. Regions of interest within the myocardium, along with other tissues, were selected for analysis. Pre-contrast T2 values, and the change in R2* due to USPIO from baseline to 24 hours after USPIO were compared for each region of interest. Results In patients with MI, USPIO uptake in the infarct zone peaked at days 2-3, and greater USPIO uptake was detected in the infarct zone compared to remote myocardium in the first 2 weeks after myocardial infarction. In contrast, T2-defined myocardial oedema peaked at days 3-9 and remained increased in the infarct zone throughout the 3-month follow up period. Histology confirmed colocalisation of iron and macrophages within the infarcted, but not the non-infarcted, myocardium. In healthy volunteers, we reported a range of normal myocardial and tissue R2* values at baseline, and following USPIO. Tissues showing greatest USPIO enhancement were organs of the reticuloendothelial system: the liver, spleen and bone marrow. Myocarditis was confirmed in 9 of the 14 suspected cases of myocarditis. There was greater myocardial oedema, but no demonstrable difference in USPIO enhancement, in inflamed myocardial regions in patients with myocarditis when compared to healthy myocardium. We recorded an improvement in cardiac function and reduced imaging measures of inflammation after 3 months. Ten patients with cardiac transplant were retained for analysis. Measures of myocardial oedema were greater in patients with cardiac transplant than healthy volunteers. There was no difference in the change in R2* due to USPIO between patients with transplantation and healthy volunteers. Imaging recordings did not change when repeated at 3 months. Conclusions Myocardial macrophage activity can be detected using USPIO-enhanced MRI in the first 2 weeks following acute MI. This observed pattern of cellular inflammation is distinct, and provides complementary information to, the more prolonged myocardial oedema detectable using T2 mapping. In patients with acute myocarditis, USPIO-enhanced MRI does not provide additional clinically relevant information to standard clinical MRI sequences. This suggests that tissue-resident macrophages do not provide a substantial contribution to the myocardial inflammation in this condition. Stable patients with cardiac transplantation have increased myocardial T2 values, consistent with resting myocardial oedema or fibrosis. In contrast, USPIO-enhanced MRI is normal and stable over time suggesting the absence of chronic macrophage-driven cellular inflammation. In conclusion, this imaging technique holds promise as a non-invasive method of assessing and monitoring macrophage-driven myocardial inflammation after myocardial infarction with potential application to diagnosis, risk stratification and assessment of novel anti-inflammatory therapeutic interventions. It remains to be determined whether USPIO-enhanced MRI may be able to identify myocardial inflammation in other myocardial inflammatory conditions including acute cardiac transplant rejection.

Orientador: Carlos Roberto Daleck / Banca: Mirela Tinucci Costa / Banca: Renée Laufer Amorim / Resumo: Tendo em vista a relação da cicloxigenase-2 com a progressão do câncer, objetivou-se neste trabalho avaliar a expressão da atividade desta enzima com a imunorreatividade de macrófagos no diagnóstico e prognóstico de mastocitoma canino. Para a realização deste estudo foram selecionadas 24 amostras de mastocitomas e 5 amostras de tecido cutâneo sem alterações patológicas (grupo controle). Os espécimes foram divididos em grupos: GO ¬ grupo controle (n=5), G1 -mastocitomas grau I (n=8) , G2 - mastocitomas grau 11 (n=8), G3 - mastocitomas grau 11I (n=8). A avaliação da expressão da COX-2 e macrófagos foi conduzida por imunoistoquímica, utilizando-se o complexo avidina-biotina (ABC). Os resultados mostraram que os dois anticorpos imunorreagiram com tecidos caninos normais ou neoplásicos. A expressão da COX-2 mostrou marcação crescente, conforme a agressividade do tumor e houve significância (P<O,05) entre os grupos GO e G1; GO e G2; GO e G3; G1 e G3. A imunomarcação de macrófagos foi decrescente em relação à gradação histológica e-houve significância (P<O,05) entre os grupos GO e G1; GO e G2; GO e G3; G1 e G3; G2 e G3. Assim como a expressão de macrófagos, a sobrevida dos animais foi decrescente em relação ao grau de malignidade do tumor e inversamente proporcional a marcação de COX-2. / Abstract: Considering the relationship between cyclooxygenase-2 (COX-2) with the cancer evolution, this study aimed to assessment the expression of this enzyme with the immunoreactive of macrophage in the diagnosis and prognostic of canine mast cell tumor. Twenty four mast cell tumors samples were selected for the accomplishment of this study and tive samples of normal skin tissue (control group - GO). The samples were divided in groups: GO ¬ control group (n=5), G1 - mastocytoma grade I (n=8), G2 - mastocytoma grade 11 (n=8), G3 - mastocytoma grade 11I (n=8). The evaluation of the COX-2 and macrophage expression was achieved by immunohistochemistry, by means of complex avidine-biotine (ABC). The COX-2 expression increased, as the tumor grade progression; moreover. it had relevancy (P<O,05) among the groups: GO and G1, GO and G2, GO and G3, G1 and G3. The macrophage immunoreactive was decreased in relation of histological grade and it had a relevancy (P<O,05) among the groups: GO and G1, GO and G2. GO and G3. G1 and G3, G2 and G3. As macrophage expression, animal survival was decreased in relation of the histological grade of tumor and proportional reversed COX-2 expression. / Mestre

No description available.

Made available in DSpace on 2016-08-15T20:31:35Z (GMT). No. of bitstreams: 1 CaioRMO_DISSERT.pdf: 4090638 bytes, checksum: b7507f10f52ebe87761d7eafda429ed0 (MD5) Previous issue date: 2010-08-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / EP24.15 (endopeptidase 24.15, EC.3.4.24.15) also know as thimet-oligopeptidase is a metal dependent peptidase involved in the metabolism of neuropeptides as well as MHC-I peptides and more recently important as regulator of protein/protein interactions. The aim of this work is to verify the tissue and cellular distribution of EP24.15 in the lymphoid tissues (thymus, spleen, lymph nods) by means of immunohistochemistry technique. The analysis revealed that EP24.15 is expressed throughout the studied organs, nevertheless the tissue distribuition showed some areas that contained several labeled cell and others devoid of labeling. The cytoplasmic labeling was more frequent if compared to nuclear one. Macrophage is the cell type more fequently labled. The results suggest that it can be involved in the MHC-I peptide metabolism as well as CTLs differentiation. / EP24.15 (endopeptidase 24.15, EC.3.4.24.15) também conhceida por thimet-oligopeptidase é uma metaloendopeptidase que está envolvida no metabolismo de neuropeptídeos, bem como na apresentação de antígenos via MHC-I e mais recentemente como um importante regulador das interações proteína/proteína. O objetivo do presente trabalho é verificar a distribuição tecidual e celular da EP24.15 nos tecidos linfóides (timo, baço e linfonodo) através da técnica de imunohistoquímica. A análise dos resultados revelou que a EP24.15 foi expressa em todos os órgãos estudados, no entanto a distribuição tecidual mostrou áreas com várias células marcadas e outras sem marcação. A distribuição citoplasmática foi mais frequente que a nucelar. O marcrófago foi o tipo celular mais frequentemente marcado. Os resultados sugerem que a enzima pode estar envolvida no metabolismo de peptídeos via MHC-I bem como na diferenciação de CTLs.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-30T21:17:29Z No. of bitstreams: 1 Juliana Perrone Bezerra De Menezes Avaliacao do papel... - 2005.pdf: 32348298 bytes, checksum: 109d71b2fb835421caaa135becd309de (MD5) / Made available in DSpace on 2012-11-30T21:17:29Z (GMT). No. of bitstreams: 1 Juliana Perrone Bezerra De Menezes Avaliacao do papel... - 2005.pdf: 32348298 bytes, checksum: 109d71b2fb835421caaa135becd309de (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Micobactérias ambientais podem ser encontradas em água, solo, poeira, alimentos e animais. A importância do estudo dessas micobactérias tem aumentado nos últimos anos, principalmente, devido a predisposição de pacientes com imunodeficiência à infecção por essas espécies de micobactéria. Além disso, a exposição a micobactérias ambientais pode constituir um dos fatores associados à baixa eficácia da imunização com a vacina BCG. As manifestações da doença, assim como a manutenção da infecção micobateriana, dependem da interação entre a micobactéria e o sistema imune do hospedeiro. O presente trabalho teve como objetivo avaliar a resposta de macrófagos peritoneais de camundongos susceptíveis BALB/c infectados com M intracellulare ou M fortuitum. Macrófagos peritoneais de camundongos BALB/c foram infectados por M intracellulare ou M fortuitum e as diferenças entre essas duas espécies quanto à capacidade de infectar e sobreviver no interior de macrófagos primários, tratados ou não com IFN-y, e produzir óxido nítrico foram avaliadas. Foi observado que os macrófagos infectados com M fortuitum apresentam um maior percentual de células infectadas que aqueles infectados com M. intracellulare, após 4, 24 e 48 horas de infecção. Entretanto, tanto M. fortuitum quanto M intracellulare são capazes de sobreviver no interior de macrófagos peritoneais, pois não há alteração da carga bacilar dessa duas espécies de micobactéria ao longo da infecção. Observamos ainda que M. intracellulare induziu uma maior produção de óxido nítrico por macrófagos primários infectados e tratados por IFN-y que M fortuitum. No entanto, o pré-tratamento com IFN-y não alterou o percentual de células infectadas nem a viabilidade de M intracellulare ou M. fortuitum. Os dados obtidos neste trabalho mostram que, in vitro, M. fortuitum e M. intracellulare interagem de formas distintas, levando á diferentes respostas do macrófago e a destinos intracelulares distintos. Além disso, mostramos que M intracellulare e M. fortuitum são resistentes ao óxido nítrico produzido por macrófagos após ativação por IFN-y. / Environmental mycobacteria are found in water, soil, dust, food and animals. Environmental Mycobacterium importance has increased in the last few years, mostly because of immunodeficient patient predisposition to infection. Moreover, exposure to environmental mycobacteria could be associated to low levels of protection induced by immunization with BCG. Disease manifestations as well as infection outcome depend on interaction between mycobacteria and host immune system. The goal of this work was to evaluate peritoneal macrophage response, from the susceptible BALB/c mice, to M. intracellulare or M. fortuitum infection. Peritoneal inflammatory macrophages, pre-activated or not with IFN-y, were infected by M. intracellulare or M fortuitum and diferences between these two species related to the capacity to infect macrophages, to survive intracellularly and to induce NO production were evaluated. It was observed that the percentage of M. fortuitum-xnÍQoXQá cells was higher related to M. intracellulare-míecieá ones, after 4, 24 and 48 hours of infection. In addition, both M. fortuitum and M. intracellulare presented the ability to survive in peritoneal macrophages. It was also observed that in response to IFN-y activation, M. intracellulare induced higher NO production thanM fortuitum. However, pre-activation with IFN-y did not modify, neither the percentage of M. intracellulare and M. fortuitum infected cells, nor intracellular bacillum survival. These data demonstrate that, in vitro., M. fortuitum and M. intracellulare differently interact with macrophages, inducing diferent macrophage reponses and that both M. intracellulare and M fortuitum are resistant to NO production upon IFN-y activation.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2013-03-21T18:15:02Z No. of bitstreams: 1 André_C_Ferreira.pdf: 895655 bytes, checksum: fffce754856c9d5194a6fac09f960736 (MD5) / Made available in DSpace on 2013-03-21T18:15:02Z (GMT). No. of bitstreams: 1 André_C_Ferreira.pdf: 895655 bytes, checksum: fffce754856c9d5194a6fac09f960736 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A malária é uma doença parasitária causada por protozoários do gênero Plasmodium e representa um grande problema de saúde pública. Sabe-se que metade da população mundial vive em áreas endêmicas, onde esta doença gera cerca de 500 milhões de casos clínicos e em torno de 1 milhão de mortes anualmente. O processo infeccioso da malária é desencadeado por uma resposta imunoinflamatória exacerbada do hospedeiro caracterizada por migração e acúmulo de leucócitos, produção de citocinas pró-inflamatórias e mediadores químicos. Neste processo, as leucointegrinas exercem um papel extremamente importante mediando a adesão, migração e sinalização celular. Neste grupo de integrinas, a CD11d/CD18, que foi descrita mais recentemente está envolvida em diversos eventos patológicos como aterosclerose, dano neuronal e outros. Resultados preliminares de nosso grupo mostraram que animais deficientes (CD11d-/-) para esta integrina apresentam uma maior sobrevida à infecção com Plasmodium berghei Anka (PbA). Entretanto, ainda não se conhece o papel desta integrina na fisiopatologia da malária. O presente trabalho mostrou que a integrina CD11d/CD18 é dinamicamente expressa em macrófagos diferenciados da medula óssea de animais infectados com PbA. Ensaios in vitro com essas células, demonstraram que esta integrina não afeta a capacidade proliferativa dessas células. Além disso, avaliamos parâmetros importantes para a ativação celular, como produção de espécies reativas de oxigênio e citocinas pró- e anti-inflamatórias. Observamos que macrófagos de animais CD11d-/- produzem quantidades significantemente mais baixas de malondialdeído e de TNF- , e níveis altos de IL-10 em relação aos macrófagos de animais CD11d+/+. Além disso, a produção de prostaglandina E2 foi significantemente diminuída em macrófagos provenientes de animais CD11d-/-. Esses dados indicam que a integrina possui um papel importante na modulação da resposta imune. Verificamos ainda que macrófagos de animais CD11d-/- apresentaram uma diminuição na sua capacidade fagocítica de hemácias parasitadas em relação aos macrófagos de animais CD11d+/+. Entretanto, a ausência desta integrina não afetou a capacidade destes macrófagos de fagocitar hemozoína sintética (sHz). Estes resultados juntos sugerem que a integrina CD11d está envolvida no processo de ativação celular dos macrófagos, modulando a resposta imune do hospedeiro na fisiopatologia da malária, o que torna esta molécula um potencial alvo para ações terapêuticas. / Malaria is a parasitic disease caused by protozoa of the genus Plasmodium and is a major public health problem. It is known that half the world population lives in endemic areas where this disease causes about 500 million clinical cases and around 1 million deaths annually. The process of malaria infection is initiated by a host immunoinflammatory response exacerbated characterized by the migration and accumulation of leukocytes, the production of proinflammatory cytokines and chemical mediators. In this process, leukointegrins play a very important role in mediating adhesion, migration and cell signaling. In this group of integrins, CD11d/CD18, which has been described more recently, is involved in gravel pathological events such as atherosclerosis, neuronal damage and others pathologies. Preliminary results from our group have shown that animals deficient (CD11d-/-) for this integrin have a higher survival to infection with Plasmodium berghei Anka (PbA). However, we still do not know the role of CD11d/CD18 integrin in the pathophysiology of malaria. The present study demonstrated that the integrin is dynamically expressed in differentiated bone marrow macrophages from animals infected with PbA. Experiments with these cells in vitro have demonstrated that the integrin does not affect the proliferative capacity of these cells. Furthermore, we evaluated parameters important to cellular activation, such as production of reactive oxygen species, proinflammatory and anti-inflammator cytokines. Observed that macrophages, from deficient mice for the integrin CD11d-/-, produce significantly lower amounts of malondialdehyde and TNF- , and high levels of IL-10 in relation to the macrophages from animals CD11d+/+. Furthermore, the production of prostaglandin E2 was significantly reduced by macrophages from animals CD11d-/-. These data indicate that integrin plays an important role in modulating the immune response. Also verified that macrophages from animals CD11d-/- showed a decrease in their phagocytic ability of infected erythrocytes to macrophages compared to animals CD11d+/+. However, absense of this integrin did not affect the ability of macrophages to phagocytize synthetic hemozoin – sHz. These results together suggest that CD11d integrin is involved in cellular activation of macrophages by modulating the host immune response in the pathophysiology of malaria, which makes this molecule a potential target for therapeutic actions.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-10-29T13:31:11Z No. of bitstreams: 1 Beatriz Rocha Simone Dias Vacúolos....pdf: 43303992 bytes, checksum: 310fce5b6b7557207067a51be3ca3c64 (MD5) / Made available in DSpace on 2014-10-29T13:31:11Z (GMT). No. of bitstreams: 1 Beatriz Rocha Simone Dias Vacúolos....pdf: 43303992 bytes, checksum: 310fce5b6b7557207067a51be3ca3c64 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A Leishmania é um parasito intracelular obrigatório que vive e se multiplic adentro dos vacúolos parasitóforos em macrófagos no hospedeiro vertebrado. Apesar dos vacúolos induzidos por diferentes espécies de Leishmania apresentarem semelhanças bioquímicas, esses compartimentos apresentam diferenças significativas nos seus tamanhos. Os vacúolos parasitóforos induzidos por Leishmania mexicana e Leishmania amazonensis apresentam grandes dimensões e contêm uma grande quantidade de amastigotas, enquanto que os induzidos por Leishmania major e Leishmania donovani são pequenos e com pouco espaço ao redor das amastigotas. Estudos recentes demonstraram que compartimentos induzidos por microrganismos intracelulares são capazes de interagir com a via autofágica e esta pode controlar ou promover o estabelecimento da infecção a depender da natureza do microrganismo. Até o momento, poucos estudos foram realizados para avaliar o papel da autofagia na biogênese e maturação dos vacúolos parasitóforos induzidos por Leishmania. Recentemente, foi demonstrado que em macrófagos de camundongos BALB/c, a indução de autofagia provoca um aumento na carga parasitária de L. amazonensis, no entanto, não é capaz de aumentar a carga parasitária de L. major. Além disso, estudos indicam que vacúolos parasitóforos de L. mexicana adquirem macromoléculas do citoplasma da célula hospedeira por meio de microautofagia. Uma vez que L. amazonensis integra o mesmo complexo que L. mexicana, nossa hipótese é que vacúolos parasitóforos induzidos por L. amazonensis interagem com a via autofágica.Assim, o presente estudo tem como bjetivo verificar e comparar a participação da autofagia na infecção por L. amazonensis ou L. major em macrófagos murinos. Para este fim, avaliamos quanto a características autofágicas, os vacúolos parasitóforos induzidos por L. amazonensis ou L. major em macrófagos de camundongo CBA e analisamos a influência da superexpressão de LC3 sobre a sobrevivência de L. amazonensis ou L. major em macrófagos infectados. Inicialmente, macrófagos de camundongos CBA foram infectados com L. amazonensis ou L. major e incubados com ysoTracker, marcador de compartimentos lisossomais, ou DQ-BSA, marcador de compartimentos degradativos. Além disso, foi avaliada a presença de LAMP, proteína lisossomal, e LC3, proteína específica de autofagossomo, na membrana destes vacúolos. Em seguida, a co- localização dos parasitos com os vacúolos parasitóforos contendo estes marcadores foi quantificada. Nossos resultados demostraram um maior percentual de co-localização tanto do LysoTracker como doDQ-BSAcom parasitos em vacúolos no interior de macrófagos infectados com L. major em comparação com aqueles infectados com L. amazonensis. No entanto, não houve diferença no percentual de co-localização de LAMP com L. major ou L. amazonensis e foi observado um maior percentual de co-localização do LC3 com parasitos em macrófagos infectados com L. amazonensis em comparação com aqueles infectados com L. major. Posteriormente, avaliamos o efeito da superexpressão da LC3 na infecção por L. amazonensis ou L. major. Células de linhagem macrofágica RAW foram transfectadas com o plasmídeo contendo a sequência codificante para a LC3 e infectadas com L. amazonensis ou L. major. Nós observamos uma reduçãono percentual de infecção por L. amazonensis e L. major nas células RAW- pmRFP-LC3 em comparação às controle. Essa diminuição na infecção se deu por inibição da fagocitose de L. amazonensis e L. major pois os parasitos continuam a interagir com a membrana das células RAW-pmRFP-LC3, mas não são internalizadas. Em conjunto, estes dados demonstram que os vacúolos parasitóforos de L. amazonensis e L. major interagem com compartimentos da via autofágica de forma distinta e que a superexpressão de LC3 reduz a fagocitose de L. amazonensis e L. major por células RAW, o que resulta na redução da infecção / Leishmania is an intracellular parasite that lives and multiplies within parasitophorous vacuoles in macrophages in the vertebrate host. Despite the fact that vacuoles induced by different species of Leishmania present biochemical similarities, these compartments have significant differences in their sizes and composition. The parasitophorous vacuoles induced by Leishmania mexicana and Leishmania amazonensis are large and contain a large number of amastigotes, while vacuoles induced by Leishmania major and Leishmania donovani are small and tight. Recent studies have demonstrated that depending on the type of intracellular microorganism, the induced compartments can interact with the autophagic pathway and control or promote the establishment of infection. To date, few studies have been conducted to evaluate the role autophagic process plays in the biogenesis and maturation of parasitophorous vacuoles induced by Leishmania. Recently, it has been demonstrated that in macrophages of BALB/c mice, the induction of autophagic causes an increase in parasitic load of L. amazonensis, but not L. major. Furthermore, other studies indicate that L. mexicana-induced parasitophorous vacuoles acquire macromolecules from the cytoplasm of the host cell through microautophagy. Once L. amazonensis belongs to the same complex that L. mexicana, our hypothesis is that L. amazonensis-induced parasitophorous vacuoles interact with the autophagic pathway. Thus, the present study aims to evaluate and compare the role autophagic process plays in Leishmania infection. We evaluated L. amazonensis- or L. major-induced parasitophorous vacuoles regarding their autophagic characteristics and we analyzed the influence of the overexpression of LC3 on the survival of parasites in infected macrophages. Initially, macrophages of CBA mice were infected with L. amazonensis or L. major and incubated with a marker of lysosomal compartments, LysoTracker, or a marker of degrading compartments, DQ-BSA. In addition, we evaluated the presence of the lysosomal membrane protein, LAMP-1, and a protein specific of autophagossomes, LC3 in the membrane of these vacuoles. Then, the colocalization of parasites with the marker labeled-compartments was quantified. Our results demonstrated a higher percentage of colocalization of both LysoTracker and DQ-BSA with parasites in vacuoles within macrophages infected with L. major in comparison with those infected with L. amazonensis. However, there was no difference in the percentage of colocalization of LAMP with L. major or L. amazonensis. We also observed a higher percentage of LC3-co-localizing with parasites in macrophages infected with L. amazonensis in comparison with those infected with L. major. Subsequently, we evaluated the effect of overexpression of LC3 in macrophages infected with L. amazonensis or L. major. RAW cells were transfected with the plasmid containing the coding sequence for the LC3 (RAW-pmRFP-LC3) and then were infected with L. amazonensis or L. major stationary phase promastigotas. A reduction was observed in the percentage of infected RAW-pmRFP-LC3 cells with L. amazonensis and L. major compared to control cells. This decrease in the percentage of infected cells is due to the inhibition of phagocytic ability of RAW-pmRFP-LC3 cells, since the parasites continue to interact with cell membrane, but is not internalized. Together, these findings show that L. amazonensis- and L. major-induced parasitophorous vacuoles interact differently with compartments of the autophagic pathway and that the overexpression of LC3 reduces phagocytosis of both L. amazonensis and L. major by RAW-pmRFP-LC3 cells resulting in the reduction of infection.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T14:23:59Z No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T14:24:19Z (GMT) No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) / Made available in DSpace on 2016-02-04T14:24:19Z (GMT). No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10 sobre a de TNF. Macrófagos derivados de medula óssea de camundongos deficientes no gene HO-1 têm menor carga parasitária, quando infectados por L. chagasi em comparação aos macrófagos de camundongos selvagens. Além disso, pacientes com LV apresentam maiores níveis de heme-oxigenase 1 e de heme no soro. Nossas observações indicam que heme é capaz de induzir necroptose em macrófagos humanos, e de que moléculas da via da necroptose estão associadas com a resistência na infecção por Leishmania. A molécula RIPK1 controla a replicação de Leishmania por um mecanismo independente da produção de IL-1β, enquanto que a molécula PGAM5 depende de IL-1βpara controlar o crescimento do parasita. Por fim, encontramos que essas proteínas participam do controle da replicação por Leishmania em um modelo experimental de Leishmaniose cutânea. Esses achados indicam um potencial deletério para a HO-1 na infecção por L. chagasi, e um papel protetor da necroptose na infecção porLeishmania. / Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to cell death pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania infantum chagasi infection, the main etiologic agent of human VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are involved in resistance to Leishmania infection. Our observations indicate that HO-1 is induced in macrophages infected with L. infantum chagasi and pharmacological induction for HO-1 by CoPP increases parasite load of infected macrophages and reduces production on inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that favors L. chagasi replication through a higher IL-10/TNF-α ratio in macrophages. We also observed that bone marrow derived macrophages knockout to HO-1 gene have a significant lower parasite load when infected by L. infantum chagasi than their wild type counterparts. Beyond this, we found that patients with VL presented higher systemic concentrations of HO- 1 and heme than healthy individuals. We found that heme is able to induce programmed necrosis “necroptosis” in human cells and that molecular players from necroptosis pathway contribute to resistance to Leishmania infection. RIPK1 controls Leishmania replication through a mechanism independent of IL-1β production, while PGAM5 requires IL-1β to control Leishmania replication. Finally, we found that RIPK1 and PGAM5 play an important role in controlling Leishmania replication in a cultaneous leishmaniasis experimental model. Our findings argue that HO-1 has a critical role in L. chagasi replication and necroptosis pathway is involved in resistance against Leishmania infection.

The interaction between epithelial cells and macrophages is integral to mucosal immune fate: determining the decision between tolerance and immune activation/inflammation. Endotoxin tolerisation (ET) is a circumstance where cells go through a hypo-responsive state, unable to respond to further endotoxin-LPS challenge. Mucosal macrophages (MΦs) have a dual functionality that determines tolerance to commensal organisms or immune response to entropathogens such as E. coli. In the case of mucosal inflammatory pathologies, such as Crohn’s disease, this state of tolerance is broken, resulting in destruction of gut mucosal tissue where the macrophage phenotype has been altered from a regulatory M2-like subset phenotype to an inflammatory M1-like subset phenotype, responding to both pathogenic and commensal bacteria. Chronic inflammation by bacterial pathogen related molecular patterns (PAMPs), such as LPS, is well established to induce tolerisation. The aims of this project were firstly, to characterise the control of macrophage differentiation in a mucosal setting by investigating the immunomodulatory effects of PAMPs, such as LPS in presence or absence of TNFα and to investigate ET mechanisms associated with MΦ subsets responding to the entropathogen E. coli K12-LPS. Secondly, to investigate the effect of epithelial cells on macrophage subsets behaviour upon inflammation and ET. M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively, whereas differentiated epithelial cells (Caco-2) were obtained by long term culturing for 21 days. A transwell co-culture system of Caco2 cells and MΦ subsets was developed to mimic the cell-to-cell cross-talk between epithelial cells and immune cells. Mono- and co-culture models were pre-treated with either LPS, TNFα or IL-1β prior to stimulation by PAMPs. TNFα, IL-1β, IL-18, IL-6 and IL-10 were qualified by ELISA. Cytokines, PRRs and endogenous negative regulatory molecules were detected by RT-PCR and WB and epithelial barrier function was measured by trans epithelial electrical resistance (TEER). ET induced by K12-LPS failed to demonstrate a differential subset-specific response in MΦ mono-culture system whereas, LPS differentially suppress LPS induced cytokine expression in MΦ co-culture system. Tolerised M1- and M2-like MΦs exhibited a significant reduction in expression and secretion of pro-inflammatory cytokines and comparable levels of anti-inflammatory cytokine, IL-10. The suppression of pro-inflammatory cytokine in these MΦs appeared to be linked to the differential TLR4 expression and up-regulation of negative regulators, such as IRAK-M and Tollip. In addition, MΦ subsets differentially responded to inflammation induced by pro-inflammatory cytokines, TNFα and IL-1β in mono- and co-culture models. In conclusion, tolerisation induced in MΦs is presented by the suppression of pro-inflammatory cytokine, which is associated with corresponding up-regulation of IL-10, TLR4 receptor and the negative regulators, in a subset-independent manner. In the case of cross-talk between epithelial cells and macrophages however, a differential sensitivities to ET was displayed. These findings allow more understanding of MΦ subsets functions and ET mechanisms, which may be beneficial for the development of in-vitro models of MΦ subsets and therapeutics targeting Crohn’s diseases.