Chronic non-healing wounds commonly present in patients with diabetes. These wounds are characterised by elevated numbers of immature leukocytes and M1 macrophages and reduced numbers of endothelial cells and M2 macrophages, impairing wound healing resolution. Topical treatment of murine diabetic wounds with a Hoxa3 gene expression vector redresses the balance of inflammatory and pro-healing cells within the lesion, reducing excessive inflammation and rescuing the wound healing phenotype. In this thesis I present experiments to further understanding of how diabetes alters the macrophage phenotype and how this may cause the decreased endothelial cell and M2 macrophage numbers in the diabetic wound. In vitro culture was used to characterize the intrinsic changes of diabetic macrophages isolated from the environmental effects of the diabetic wound milieu. These same systems were used to develop a cell culture system for the promotion of monocytic to endothelial transdifferentiation. Finally the in vitro macrophage culture system was used to assess the effects of Hoxa3 treatment upon diabetic macrophages and how Hoxa3 transcriptional activity in macrophages may contribute to the restoration of wound healing. In vitro cultured diabetic macrophages were observed to raise an increased response to classical and alternative activation signals that may contribute to the excessive inflammatory state of diabetic cutaneous wounds. Treatment of these macrophages for four days with a Hoxa3 conditioned medium protein transduction system upregulated the expression of the plasminogen activator urokinase receptor gene Plaur and enhanced the expression of macrophage maturation markers. These macrophages also exhibit an enhanced response to classical activation stimuli, a reduced alternative activation response. In an in vitro neovascularisation assay Hoxa3 treated macrophages inhibit vessel growth. These effects of Hoxa3 treatment of diabetic macrophages are unexpected based on the rescue of the inflammatory phenotype with Hoxa3 treatment of diabetic wounds. Non-diabetic macrophages were also treated for four days with a Hoxa3 conditioned medium and exhibited upregulation of macrophage maturation markers. These macrophages showed no difference in activation state polarisation compared to macrophages grown in a control conditioned medium but did upregulate activation markers in unstimulated cells. This may be indicative of a priming for response to low levels of activation stimuli. The Hoxa3 treated non-diabetic cells also promoted the formation of vessel networks in a neovascularisation co-culture assay, possibly through the promotion of angiogenesis. These results suggest that diabetes directly effects the maturation and inflammatory phenotype of macrophages and that Hoxa3 treatment rescues the impaired maturation phenotype and may stimulate macrophage populations to a pro-angiogenic state.

Innate immune cells, such as macrophages, are trained by the unique microenvironment of the tissue they occupy. This tissue influence can include the extracellular matrix, the presence of inflammatory stimuli or signals and, in some tissues, the microbiota. Most studies, however, have examined such tissue specific training in health whereas little is known about the possibility of immune re-training in the lung following acute inflammation. The lung extracellular matrix is important for mechanical stability and structural support, as well as influencing inflammation via altering cell adhesion, migration, survival, proliferation and differentiation. Matrix alterations are a feature of a number of significant chronic respiratory diseases that carry high clinical unmet need. These include idiopathic pulmonary fibrosis, cystic fibrosis and chronic obstructive pulmonary disease (COPD). On the other hand, the impact on matrix after acute inflammation and whether it is returned to its pre-infection state is relatively unexplored. In murine models, macroscopic examination of the lung following acute inflammation implies a return to a reasonable homeostatic state. However, using more sensitive techniques, we now show that this is not the case. In this thesis we test the premise that a more thorough interrogation of lung extracellular matrix by mass spectrometry will reveal long term alterations that are not visible by histology. After influenza virus infection, we demonstrate that heightened extracellular matrix persists in the lung tissue, often forming structures that were not present in health. Furthermore, basement membrane components, for example collagen IV and laminin, are reduced. In vitro investigations show that individual extracellular matrix components affect lung macrophage activity. For example, hyaluronan and fibronectin alter macrophage expression of microRNA species known to influence toll-like receptor responsiveness and fibrosis. We also describe an alteration in microRNA species in response to influenza virus infection as well as a non-infectious model of pulmonary inflammation using carbon nanotubes. Collectively, this implies that altered matrix composition impacts on the inflammatory tone of the lung innate immune system. It is therefore feasible that such changes following severe lung inflammation could be overcome by targeting abnormal matrix production or degradation.

Les macrophages sont une cible cellulaire du VIH-1, et jouent un rôle important dans la pathogenèse virale. Au cours de ma thèse, je me suis intéressée au rôle des macrophages dans la pathogenèse du VIH-1, mais aussi au cours de la co-infection avec Mycobacterium tuberculosis (Mtb), l'agent étiologique de la tuberculose. J'ai tout d'abord participé à une étude mettant en évidence que l'infection par le VIH-1 reprogramme la migration des macrophages, favorisant notamment le mode migratoire protéolytique. Cet effet est médié par l'interaction de la protéine virale Nef avec les protéines de l'hôte Hck et WASP, ce qui conduit à une modification de l'organisation et de la fonction des podosomes, structures impliquées dans la dégradation de la matrice extracellulaire et la migration dépendante des protéases. La meilleure capacité à migrer des macrophages infectés par le VIH-1 in vitro se traduit in vivo par une augmentation du recrutement des macrophages dans différents tissus de souris transgéniques qui expriment la protéine Nef. Ces travaux ont ainsi révélé un nouveau mécanisme par lequel le VIH-1 dissémine dans les tissus, via l'action de Nef dans les macrophages. L'association fréquente du VIH-1 avec Mtb complique le problème de santé publique posé par l'infection virale. En effet, Mtb aggrave la pathogenèse du VIH-1 chez les patients co-infectés. L'étude des mécanismes impliqués et le rôle des macrophages dans ce phénomène constituent les objectifs principaux de ma thèse. J'ai révélé que les macrophages infectés par Mtb génèrent un microenvironnement qui active les macrophages voisins vers un programme de polarisation anti-inflammatoire dit M(IL-10). J'ai mis en évidence que ces macrophages M(IL-10) sont particulièrement efficaces pour la production de VIH-1. J'ai démontré que le microenvironnement associé à la tuberculose entraîne la formation de nanotubes entre les macrophages, grâce à l'activation de la signalisation cellulaire médiée par l'axe IL-10/STAT3. Ces nanotubes, qui favorisent le transfert du virus d'un macrophage à un autre, sont à l'origine de la spectaculaire production de VIH-1 par les macrophages. Nous avons également constaté que ces cellules M(IL-10) s'accumulent dans la circulation sanguine des patients co-infectés ainsi que dans les poumons de primates non-humains co-infectés. Dans l'ensemble, mes travaux identifient les nanotubes comme des acteurs clés dans l'aggravation de la pathogenèse du VIH-1 lors de la co-infection avec Mtb. Ainsi, les nanotubes et la voie de signalisation IL-10/STAT3 pourraient représenter des cibles pour développer de nouvelles thérapies de lutte contre la comorbidité VIH/Mtb. Les résultats obtenus lors de ma thèse contribuent à une meilleure compréhension du rôle des macrophages dans la pathogenèse et la dissémination du VIH-1 dans un contexte de mono-infection, ou lors d'une co-infection avec Mtb. / Macrophages are both crucial host effector cells for HIV-1 and important leukocytes involved in viral pathogenesis. For my doctoral thesis, I was interested in further characterizing the role of macrophages in HIV-1 pathogenesis, and during co-infection with Mycobacterium tuberculosis (Mtb), the etiological agent for tuberculosis (TB). I first participated in a study that provided evidence that HIV-1 infection reprograms the migration of macrophages, particularly by triggering the protease-dependent migration mode. This effect was mediated by the interaction of the viral protein Nef with the host proteins Hck and WASP, which leads to modification in the organization and proteolytic activity of podosomes, important structures for protease-dependent migration. The higher migration capacity of HIV-1-infected macrophages translated in vivo by an increase in the recruitment of macrophages in several tissues of Nef-transgenic mice. This work revealed a novel mechanistic understanding of how HIV-1 infection drives macrophages into tissues, contributing to viral dissemination and possibly creating a hidden cellular reservoir of virus. Worsening this public health issue posed by the HIV-1 epidemic is the frequent association of the virus with Mtb. Indeed, Mtb aggravates HIV-1 pathogenesis in co-infected individuals. Yet, the mechanisms involved in this process are still poorly understood, including the contribution of macrophages. To investigate how Mtb exacerbates the HIV-1 infection in human macrophages was the main focus of my thesis. First, I revealed that Mtb-infected macrophages generate a microenvironment that drives bystander macrophages towards phenotypic and functional features of the so-called M(IL-10) anti-inflammatory program. I found that these M(IL-10) macrophages are highly efficient for HIV-1 production. I demonstrated that the TB-associated microenvironment induces the formation of macrophage-to-macrophage connecting tunneling nanotubes (TNTs) through the IL- 10/STAT3 axis, a phenomenon that is responsible for the dramatic increase of HIV-1 production in M(IL-10) macrophages. Moreover, I provided evidence that M(IL-10) cells are expanded in the peripheral blood of co-infected patients and accumulate in the lungs of co-infected non-human primates. Altogether, this central part of my PhD thesis sheds light to TNTs as key players in the aggravation of HIV-1 pathogenesis in human macrophages during co-infection with Mtb. Thus, this cellular mechanism (together with the IL- 10/STAT3 axis) could represent an unexpected target to develop novel therapeutics against AIDS/TB co-morbidity. Collectively, the results obtained during my thesis contribute to a better understanding of the role of macrophages during HIV-1 pathogenesis and their ability to disseminate the virus in a mono-infection context, or during co-infection with Mtb.

Breast cancer is the second leading cause of cancer-related death in women in the United States. Complications that lead to mortality of cancer patients are associated with tumor metastasis. Specifically, lymphatic metastasis in breast cancer patients strongly correlates with poor patient survival and this process is facilitated by the formation of new tumor lymphatic vessels termed lymphangiogenesis. Previously, our lab reported that lymphangiogenesis was promoted by a distinct subset of bone marrow (BM)-derived myeloid cells that co-express lymphatic-specific markers designated as myeloid-derived endothelial cell progenitors (M-LECPs). Furthermore, our lab has generated M-LECP in vitro from a mouse macrophage cell line (RAW264.7) by LPS stimulation. Taken together, these data suggest that chronically inflamed sites drive M-LECP differentiation and that these cells can contribute to the formation of new lymphatic vessels and promote lymph node metastasis. Evidence supporting this hypothesis was indicated by high levels of circulating M-LECP in peripheral blood of breast cancer patients but undetectable levels in healthy donors, cancer-free donors. Additionally, the generation of M-LECP was prompted through TLR4-signaling pathway, and de novo expression of VEGFR-3 and VEGF-C. This co-expression produces an autocrine loop essential for pro-lymphatic reprogramming in both primary human monocytes and the immature monocytic cell line, THP-1. Taken together, these data indicate the major regulatory role of TLR4 in inflammation-driven lymphangiogenesis involves the recruitment and differentiation of M-LECP, a process that may promote lymphatic metastasis.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-30T20:45:01Z No. of bitstreams: 1 José Geraldo Bomfim Lima Efeito da autofagia... 2009.pdf: 32365208 bytes, checksum: 34504be1e8f6483c263480600d8e1880 (MD5) / Made available in DSpace on 2012-11-30T20:45:01Z (GMT). No. of bitstreams: 1 José Geraldo Bomfim Lima Efeito da autofagia... 2009.pdf: 32365208 bytes, checksum: 34504be1e8f6483c263480600d8e1880 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A autofagia vem sendo alvo de estudos que demonstram sua participação em infecções por diversos patógenos intracelulares. A depender do patógeno, a autofagia pode facilitar a sobrevivência intracelular do patógeno ou pode funcionar como controle da infecção pela célula hospedeira. Pouco se sabe sobre a participação da autofagia na infecção por Leishmania. Foi demonstrado que o vacúolo parasitóforo induzido por L mexicana adquire nutrientes citosólicos por microautofagia. Além disso, recentemente foi demonstrado que a indução de autofagia promove aumento da carga parasitária de L. amazonensis em macrófagos infectados. Esses dados sugerem a participação do processo autofágico no estabelecimento da infecção por Leishmania, como um mecanismo que favorece a sobrevivência intracelular do parasito. Assim, o objetivo desse estudo foi determinar a influência da autofagia na infecção, in vitro, de macrófagos de camundongos CBA/J por L. amazonensis. Macrófagos foram induzidos à autofagia por duas formas, fisiológica ou farmacológica, após ou antes da infecção por L. amazonensis ou exposição a partículas de levedo ou zimosan. O percentual de infecção e de fagocitose foi estimado. Os resultados mostram que a indução de autofagia, após a infecção, não altera o percentual de macrófagos infectados, mas promove o aumento na carga parasitária de macrófagos infectados por L. amazonensis. Além disso, a prévia indução de autofagia promove a inibição da capacidade fagocítica do macrófago murino. Estudos adicionais serão realizados no intuito de esclarecer os mecanismos pelos quais a indução de autofagia favorece a infecção por L. amazonensis e altera a capacidade fagocítica do macrófago murino / Recently, studies to delineate the participation of autophagy in intracellular pathogen infections have been performed. Dependent on pathogen infection, autophagy can facilitate microorganism intracellular survival or can control pathogen infection by host cell. Few works evaluated the role of autophagy in Leishmania infection. Recently, it was demonstrated that L mexicana-'mduced parasitophorous vacuoles acquire cytosolic nutrients by microautophagy. Additionally, it was also demonstrated that autophagy promotes enhancement of L. amazonensis burden on infected cells. Taken together, these data suggest the involvement of autophagic process on Leishmania infection, as a mechanism that favors parasite survival. The present work intent to determine the influence of autophagy in L. amazonensis infection of CBA/J macrophages in vitro. Autophagy was induced after and before L. amazonensis infection or particle addition to macrophage cultures. The percentage of infection and particle phagocytosis was estimated. The results show that autophagy induction after infection does not influence the percentage of L. a/rjazonens/s-infected cells, but enhances L. amazonensis burden on infected cells. In addition, previous autophagy induction inhibited macrophage phagocytic capacity. Further studies will be performed to understand the mechanisms involved in autophagy effect on L. amazonensis infection and on macrophage phagocytic capacity.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-10-29T14:03:53Z No. of bitstreams: 1 Niara de Jesus Almeida.Avaliação...2014.pdf: 2142198 bytes, checksum: c0f1caa073f88305a34bffb0a94263ff (MD5) / Made available in DSpace on 2014-10-29T14:03:53Z (GMT). No. of bitstreams: 1 Niara de Jesus Almeida.Avaliação...2014.pdf: 2142198 bytes, checksum: c0f1caa073f88305a34bffb0a94263ff (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Camundongos CBA são resistentes à infecção por Leishmania major e permissivos à infecção por L. amazonensis. Adicionalmente, macrófagos de camundongos CBA controlam à infecção por L. major, mas não por L. mazonensis in vitro. Em estudo comparativo realizado por nosso grupo foi demonstrado que o receptor scavenger MARCO teve expressão aumentada em resposta à infecção por L. major, mas não na infecção por L. amazonensis. Ainda, o bloqueio do receptor com o anticorpo específico reduziu a infecção por L. major em 30%, indicando que esta proteína tem participação no reconhecimento de promastigotas de L. major em macrófagos de CBA. Assim, nossa hipótese é que o receptor MARCO participa do reconhecimento e fagocitose de L. major por macrófagos, direcionando o curso da infecção. O objetivo do presente estudo consistiu em evidenciar o papel do receptor MARCO na infecção de macrófagos por L. major. Inicialmente, células J774 foram transfectadas com os vetores pcDNA3.1-MARCO (J774-MARCO) ou pcDNA3.1 (J774-MOCK). Foi observado que a expressão do gene do MARCO foi cinco vezes maior nas células J774-MARCO em comparação às células J774-MOCK. Ao avaliarmos o efeito da superexpressão sobre o metabolismo de células J774-MARCO foi observado que a atividade metabólica mitocondrial foi maior nos clones J774-MARCO e J774-MOCK, 14% e 39% respectivamente, em comparação com as células J774 controle não transfectadas. Entretanto, a diferença no metabolismo não alterou a viabilidade celular dos clones transfectados. A superexpressão de MARCO não aumentou a ligação de L. major em células J774, mas favoreceu tanto o aumento no percentual de infecção de L. major como o número de parasitos/célula nos tempos iniciais até 24 h após a infecção (p ≤ 0,05). Adicionalmente, foi investigado se MARCO estaria induzindo modificações na membrana celular que favorecessem a entrada de L. major. Foi demonstrado que as células superexpressando MARCO (67%) apresentaram um maior espraiamento da membrana celular, com a formação de lamelipódios e estruturas semelhantes a filopódios, eventos observados em um número reduzido de células J774-MOCK (24%). Ao avaliarmos o efeito da superexpressão de MARCO na produção de quimiocinas e citocinas durante a infecção por L. major foi observado que a adição de L. major induziu níveis significativamente maiores de MCP-1 e TNF-α nos tempos de 24 e 48 h após a infecção em comparação com as células J774-MOCK (p < 0,01). Níveis maiores de IL-6 foram observados após 48 h de infecção por L. major nas células J774-MARCO em comparação às células controle (p < 0,05). Similarmente, em resposta à infecção por L. major, células J774-MARCO produziram maior quantidade de NO nos tempos de 24 (p < 0,001) e 48 h (p < 0,01) após a infecção. Todavia, a superexpressão de MARCO não teve efeito sobre a sobrevivência intracelular do parasito. Em suma, nossos achados sugerem que a superexpressão do receptor scavenger MARCO favorece a entrada de L. major em células J774, além de desencadear uma resposta imune efetora direcionando o curso da infecção por Leishmania. / CBA mice are resistant to Leishmania major yet permissive to L. amazonensis infection. In addition, CBA macrophages control L. major, but not L. amazonensis infection in vitro. In a comparative study performed by our group increase in expression of the scavenger receptor MARCO has been detected in response to L. major, but not to L. amazonensis infection. Moreover, ED31 monoclonal antibody against MARCO reduced by 30% L. major infection in CBA macrophages. These findings indicate that MARCO plays a role in L. major recognition by CBA macrophages. We hypothesized that MARCO receptor participates in the recognition and phagocytosis of L. major by macrophages, directing the outcome of infection. In the present study, we aimed to further disclose the role MARCO plays in L. major infection of murine macrophages. First J774 cells were transfected with pcDNA3.1-MARCO vector (MARCO-J774) or pcDNA3.1 vector (MOCK-J774). Expression of MARCO gene shown to be five times higher in MARCO-J774 cells compared to MOCK-J774 cells. Overexpression of MARCO enhanced mitochondrial metabolic activity in 14% and 39% of both MARCO-J774 and MOCK-J774, respectively, when compared to the control non-transfected J774 cells. However, enhancement in mitochondrial metabolic activity did not alter the cell viability of transfected clones. MARCO overexpression did not increase the binding of L. major in J774 cells, but increased both the percentage of infection of L. major and the number of parasites / cell until 24 h after infection (p ≤ 0,05). Additionally, we investigated whether MARCO be inducing changes in cell membrane that would favor L. major uptake by macrophages. We observed that MARCO-J774 cells (67%) showed a pronounced spread of cell membranes, with short microvilli and lamellipodia, events observed in a reduced number of MOCK-J774 (24%). Then, we evaluated production of pro-inflammatory chemokine and cytokine induced by L. major in MARCO-J774 cells. L. major addition to MARCO-J774 cells induced higher significantly levels of MCP-1 and TNF-α compared to MOCK-J774 cells at 24 and 48 h post infection (p < 0,01). Higher levels of IL-6 were also observed at 48 h of L. major infection in MARCO-J774 cells compared to control (p < 0,05). Similarly, NO production induced by L. major was much more higher in MARCO-J774 cells compared to MOCK-J774 at 24 and 48 h post infection (p ≤ 0,01). Although in MARCO-overexpressing cells, enhancement in pro-inflammatory chemokines and cytokines in response to L. major has been observed, no effect on parasite intracellular survival has been detected. In summary, our findings suggest that the scavenger receptor MARCO overexpression favors the entry of L. major in J774 cells, and trigger an effector immune response that directs outcome of Leishmania infection.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-27T17:39:55Z No. of bitstreams: 1 José Geraldo Bomfim Lima Estudo do efeito....pdf: 7893051 bytes, checksum: a043a24c2d08a6c835942fd381ead5a4 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-27T17:40:27Z (GMT) No. of bitstreams: 1 José Geraldo Bomfim Lima Estudo do efeito....pdf: 7893051 bytes, checksum: a043a24c2d08a6c835942fd381ead5a4 (MD5) / Made available in DSpace on 2016-04-27T17:40:27Z (GMT). No. of bitstreams: 1 José Geraldo Bomfim Lima Estudo do efeito....pdf: 7893051 bytes, checksum: a043a24c2d08a6c835942fd381ead5a4 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: A influência da autofagia em processos celulares que participam da homeostase celular, como a endocitose e a adesão celular, até o momento, foi pouco estudada. A endocitose consiste na internalização de material extracelular, quando as vesículas endocíticas são menores que 500nm é chamada de endocitose em microescala e quando as vesículas formadas são maiores que essa medida trata-se de endocitose em macroescala. Foi demonstrado que a conexão da via endocítica com a via autofágica é fundamental para a degradação de material citosólico e, subsequente, produção de energia e disponibilização de substrato para o metabolismo celular. Estudos controversos da literatura mostraram que a autofagia pode favorecer ou não interferir com a endocitose em macroescala. Além disso, alguns trabalhos demonstraram que o processo autofágico foi capaz de reduzir a reciclagem de integrinas para a membrana plasmática por alterar a endocitose em microescala envolvida na internalização desse tipo de proteína, reduzindo a capacidade de adesão e, consequentemente, a migração celular. Assim, em conjunto, esses achados evidenciam que a autofagia pode interagir e interferir com eventos celulares dependentes da participação da membrana plasmática como a endocitose e a adesão celular. OBJETIVO: No presente estudo, hipotetizamos que a prévia indução de autofagia em macrófagos é capaz de reduzir a endocitose em micro e macroescala, além de reduzir a capacidade de adesão celular. Desta forma, o objetivo desse estudo foi determinar o efeito da indução de autofagia, in vitro, sobre a endocitose e a adesão de macrófagos murino. MATERIAL E MÉTODOS: Macrófagos foram induzidos à autofagia por privação de nutrientes (starvation) ou pelo tratamento com um indutor farmacológico, a rapamicina, seguida da exposição a macromoléculas ou grandes partículas de diferentes naturezas. Além disso, após indução de autofagia, macrófagos em suspensão foram incubados em superfícies como o vidro ou uma matriz de colágeno e fibronectina para avaliação da capacidade de adesão. Os percentuais de endocitose em microescala, em macroescala e de adesão foram estimados. RESULTADOS: Mostramos que a indução de autofagia promoveu redução da capacidade fagocítica em cerca de 60% no percentual de macrófagos que internalizam grandes partículas, como levedo, sendo um mecanismo precoce e reversível. Ao passo que a indução de autofagia por privação de aminoácidos ou farmacológica não interferiu na endocitose em microescala. A indução de autofagia não alterou a endocitose de transferrina (endocitose mediada por receptores) e endocitose de BSA (endocitose de fase fluida). Em contraste, a indução de autofagia promoveu redução em aproximadamente 70% da quantidade de macrófagos que aderem a matriz de colágeno e fibronectina. Uma possível explicação para a redução da endocitose em macroescala pode estar relacionada à autofagia diminuir a disponibilidade de grandes extensões de membrana necessárias à internalização de partículas maiores que 500nm. Alternativamente, a indução de autofagia pode estar levando a célula a uma indisponibilidade de receptores na membrana plasmática que justificaria a redução da capacidade fagocítica e de adesão do 11 macrófago murino. CONCLUSÕES: A indução de autofagia diminui a capacidade fagocítica e a capacidade de adesão do macrófago murino. / INTRODUCTION: The influence of autophagy on cellular processes that participate in cellular homeostasis, such as endocytosis and cell adhesion has been poorly evaluated. Endocytosis consists in the internalization of extracellular material and includes microscale endocytosis, when endocytic vesicles are smaller than 500nm, and macroscale endocytosis, when the formed vesicles are larger than this measure. It has been shown that the connection between the endocytic and the autophagic pathways is essential for degradation of cytosolic material and, subsequently, power generation and provision of substrate for cellular metabolism. Controversial studies showed that autophagy can improve or do not interfere with macroscale endocytosis. Furthermore, some studies demonstrated that the autophagic process reduced integrin recycling to the plasma membrane through the modulation of microscale endocytosis involved in the internalization of this protein, reducing cell adhesion and migration. Taken together, these findings show that autophagy can interact and interfere with cellular events that depend on plasma membrane participation, such as endocytosis and cell adhesion. OBJECTIVES: In the present study, we hypothesized that prior autophagy induction in macrophage reduces micro and macroscale endocytosis, as well as cell adhesion. Thus, the aim of this study was to determine the effect of autophagy induction, in vitro, on endocytosis and adhesion of murine macrophages. MATERIAL AND METHODS: Autophagy by nutrient deprivation (starvation) or by treatment with an inducer drug, rapamycin, was induced in macrophages, followed by exposure to macromolecules or large particles of different natures. Furthermore, after autophagic induction, macrophages were plated on different surfaces like glass or collagen-fibronectin matrix to evaluate cell adhesiveness. After that, the percentage of endocytosis in micro and macroscale and adhesion were determined. RESULTS: We showed that autophagy induction decreases phagocytic ability to 60% in macrophages that internalized large particles like yeast. This is a reversible mechanism that occurs at early stages after autophagy induction. On the other hand, autophagy by amino acid deprivation or pharmacological induction does not interfere with the microscale endocytosis. The autophagy induction doesn’t alter transferrin endocytosis (receptor-mediated endocytosis) and BSA endocytosis (fluid-phase endocytosis). By contrast, the autophagy induction leads to a reduction of approximately 70% in macrophages adhesion on a collagen-fibronectin matrix. The reduction of macroscale endocytosis may be related to the decreased availability of large areas of membrane required for internalization of particles larger than 500nm caused by autophagy. Alternatively, autophagy induction may be leading to receptors unavailability in plasm membrane, which would explain the reduction of the phagocytic and adhesion ability. CONCLUSIONS: autophagy induction reduces

A disfunção do sistema imune inato e a neuroinflamação tem sido cada vez mais reconhecidas como elementos importantes na fisiopatologia do transtorno bipolar (TB). Como componentes essenciais da imunidade inata, os macrófagos tem múltiplas funções tanto na inibição como na promoção da proliferação celular e na reparação tecidual, sendo a diversidade e a plasticidade características marcantes deste tipo celular. A polarização M1 clássica e a polarização alternativa M2 de macrófagos representam dois extremos de um estado dinâmico na mudança da ativação dos mesmos. Os macrófagos do tipo M1 sintetizam citocinas próinflamatórias que inibem a proliferação de células circundantes e danificam tecidos, enquanto os macrófagos do fenótipo M2 liberam citocinas antiinflamatórias que podem promover reparo tecidual. Um desequilíbrio da polarização M1-M2 dos macrófagos é frequentemente associado a várias doenças ou condições inflamatórias. O objetivo desta tese foi, além de revisar a importância da inflamação sistêmica na modulação da resposta inflamatória da microglia/macrófagos e consequentemente seu potencial envolvimento na fisiopatologia do TB, avaliar o perfil de polarização M1/M2 em cultura de macrófagos de sujeitos com TB comparados a indivíduos saudáveis. Monócitos foram isolados a partir de sangue periférico de dez sujeitos com TB e dez indivíduos saudáveis e diferenciados em macrófagos através da adição de fator estimulante de colônia de macrófagos (MCSF) ao meio de cultura. Para induzir a polarização M1 ou M2, as culturas foram incubadas com IFN-y e LPS ou IL-4 respectivamente. Após a incubação, recolheram-se os sobrenadantes e mediram-se as citocinas (IL-1β, IL-6, IL-10 e TNF-α) por ensaio multiplex. A secreção das citocinas IL-1β, TNF-α e IL-6 características do protótipo M1 e citocinas IL-10 do protótipo M2 foram semelhantes entre os pacientes e os controles. Utilizou-se a razão TNF-α / IL-10 do fenótipo M1 para refletir o estado inflamatório dos participantes. Não foi observada diferença entre os grupos (p=0,627). Duas hipóteses diferentes poderiam explicar esses resultados: todos os pacientes incluídos neste estudo representam um estágio inicial da doença como evidenciado pela pontuação FAST total inferior a 11. De acordo com o modelo de estadiamento em TB, as alterações biológicas (incluindo a inflamação) parecem estar relacionadas com os episódios de humor e progressão da doença. Juntamente com estudos anteriores, os nossos dados sugerem que os pacientes nos estágios iniciais ainda preservam a função do sistema imunológico sem apresentar um desequilíbrio a favor do perfil de macrófagos M1 como tem sido observado em pacientes no estágio tardio, destacando a relevância da intervenção precoce no TB. Ainda, estes pacientes estavam em tratamento com estabilizadores de humor e é plausível especular que esses fármacos exerçam efeitos sobre a polarização de macrófagos. Estudos futuros em pacientes drug-free são essenciais para avaliar esta questão. Em conclusão, nossos achados sugerem que os pacientes TB não apresentam desequilíbrio na polarização dos macrófagos em favor do fenótipo pró-inflamatório M1. O fato de todos estes pacientes estarem em estágios iniciais da doença reforça os efeitos protetores da intervenção precoce no TB na prevenção de alterações do sistema imune e, consequentemente, na progressão da doença. / Innate immune system dysfunction and neuroinflammation have been recognized as important elements in the pathophysiology of bipolar disorder (BD). As essential players of innate immunity, macrophages have multiple roles in inhibition and promotion of cell proliferation and tissue repair. The classical M1 polarization and the M2 alternative polarization of macrophages represent two extremes of a dynamic state in their change of activation. M1 macrophages synthesize proinflammatory cytokines that inhibit the proliferation of surrounding cells and damage tissues, whereas macrophages of the M2 phenotype release antiinflammatory cytokines that may promote tissue repair. An imbalance of the M1-M2 polarization of macrophages is often associated with various diseases or inflammatory conditions. The aim of this thesis was to review the importance of systemic inflammation in modulating the inflammatory response of microglia/ macrophages and consequently their potential involvement in the pathophysiology of BD, and also evaluate the M1/M2 polarization profile in macrophages of patients with BD compared to healthy individuals. Blood monocytes were obtained from ten BD patients and ten healthy controls. These cells were activated/polarized into the M1 (IFNγ + LPS) or M2(IL-4) phenotype. Supernatants were collected and the cytokines (IL-1β, IL-6, IL-10 and TNF-α) were measured by multiplex assay. Secretion of the IL- 1β, TNF-α, IL-6 and IL-10 were similar between patients and controls. The TNF-α/IL- 10 ratio of the M1 phenotype was used to reflect the inflammatory state of the participants. There was no difference between groups (p = 0.627). Two hypotheses could explain these results: all patients included in this study represent an early stage of disease as evidenced by the FAST score below 11. According to the BD staging model, biological changes (including inflammation) appear to be related to mood episodes and disease progression. Together with previous studies, our data suggest that patients in early stages of BD still preserve immune system function without presenting an imbalance in favor of M1 macrophages as has been observed in latestage patients, highlighting the relevance of early intervention. Moreover, these patients were under treatment with mood stabilizers and it is plausible to speculate that these drugs have effects on macrophage polarization. Future studies in drug-free patients are essential to assess this issue. In conclusion, our findings suggest that BD patients do not present imbalance in macrophage polarization in favor of the M1 proinflammatory phenotype. The fact that all these patients are in the early stages of the disease reinforces the protective effects of early intervention in BD to prevent changes in the immune system and, consequently, prevent the progression of the disease.

Os antimoniais pentavalentes, tais como o Glucantime, são geralmente usados como fármacos de primeira escolha para o tratamento das leishmanioses, no entanto seu mecanismo de ação não é completamente esclarecido. Atua contra formas amastigotas intracelulares de Leishmania sp, comprometendo o potencial redox levando danos ao DNA do parasito. Alguns trabalhos sugerem que o Glucantime aumenta a capacidade fagocítica e a produção de TNF-alfa por fagócitos. O objetivo deste estudo foi avaliar a capacidade do Glucantime modular a atividade do macrófago, a principal célula hospedeira da Leishmania. Inicialmente, a toxicidade do Glucantime foi testada sobre macrófagos peritoneais de camundongos BALB/c, tratando as monocamadas in vitro por 48 horas. A viabilidade celular foi avaliada pelo método do MTT. A capacidade do Glucantime (0,1, 1 e 10 mg/ml) modular os macrófagos foi avaliada tratando as monocamadas de macrófagos peritoneais por 24 horas antes da infecção com Leishmania braziliensis. Após 48 horas de incubação com meio de cultura foi avaliado o índice de infecção por contagem. Antes e após a infecção foram analisados a produção de óxido nítrico (NO) pelo método de Griess, espécies reativas de oxigênio (EROS) por fluorimetria usando a sonda H2DCFDA e a produção de citocinas por ELISA. Para avaliar se o Glucantime seria capaz de modular macrófagos in vivo, camundongos suíços foram tratados por 5 dias consecutivos com 8 mg de Glucantime pela via intraperitoneal. Macrófagos peritôneais foram avaliados quanto a sua capacidade de controlar a infecção in vitro com L. braziliensis. Os resultados mostraram que nas concentrações até 10 mg/ml, o Glucantime não alterou a viabilidade dos macrófagos in vitro. O pré-tratamento dos macrófagos com Glucantime nas concentrações de 0.1mg/mL, 1mg/mL e 10mg/mL, foi capaz de reduzir o índice de infecção em 49%, 74% e 85%, respectivamente. Em macrófagos não infectados a produção de NO foi aumentada na concentração de 10mg/ml de Glucantime. O tratamento com 1 e 10 mg/ml de Glucantime foi capaz de aumentar significativamente a produção de EROs (p<0,05 e p<0.01, respectivamente) e a produção IL-12 (p<0,05), mas a IL-10 não foi alterada. Não houve alterações significativas desses parâmetros em relação ao controle após a infecção com L. braziliensis. Os macrófagos oriundos dos animais tratados com Glucantime foram capazes de reduzir o índice de infecção por L. braziliensis (p<0,05). Esses resultados sugerem que o Glucantime é capaz de ativar os macrófagos e esse efeito pode contribuir para o mecanismo de ação desse fármaco. / The pentavalent antimonial drugs, such as Glucantime, are generally used as first choice for the treatment of leishmaniasis, however its mechanism is not fully understood. It has activity against intracellular amastigotes of Leishmania sp, compromising the redox potential and causing damage to the DNA of the parasite. Some studies suggesting that Glucantime enhances phagocytosis and TNF-&#945; production by phagocytes. The aim of this study is to evaluate the modulation of Glucantime on macrophages, the major host cell of Leishmania. Initially, Glucantimes toxicity was tested on peritoneal macrophages from BALB/c mice, by treating the monolayers in vitro for 48 hours. Cell viability was evaluated by MTT method. The capacity of Glucantime (0,1, 1 and 10 mg/ml) of modulate macrophages was evaluated by treating the monolayers of peritoneal macrophages for 24 hours before the infection with Leishmania braziliensis. After 48 hours of incubation with culture medium the infection index was evaluated by counting. Before and after the infection were analyzed the production of nitric oxide (NO) by Griess method, reactive oxygen species (ROS) by fluorimetry using the H2DCFDA dye and cytokines by ELISA. To evaluate if Glucantime could modulate macrophages in vivo, Swiss Webster mice were treated for 5 consecutive days with 8 mg Glucantime by intraperitoneal route. Peritoneal macrophages were evaluated about its capacity of control the in vitro infection with L. braziliensis. Results showed that until the concentration of 10 mg/ml, Glucantime did not alter the macrophages viability in vitro. The pre-treatment of macrophages with Glucantime at 0.1mg/mL, 1mg/mL and 10mg/mL was able to reduce the infection index in 49%, 74% and 85%, respectively. On non-infected macrophages the NO production was increased at 10mg/ml of Glucantime. The treatment with 1 and 10 mg/ml de Glucantime was able to significantly increase the ROS production (p<0,05 and p<0.01, respectively) and IL-12 production (p<0,05), however the IL-10 production was not altered. There were no significant changes of these parameters comparing to control after the L. braziliensis infection. The macrophages from the treated mice were capable of reduce the infection index by L. braziliensis (p<0,05). These results suggest that Glucantime is capable of activate macrophages and this effect could contribute to the mechanism of action of this drug.

There is a growing literature of clinical studies of bone marrow (BM) cell therapy for liver cirrhosis. At present, the optimum choice of cell type(s) and the mechanism(s) of effect remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, I tested the therapeutic effects of these cells in the context of experimental murine liver fibrosis. The effects of unmanipulated, syngeneic macrophages, their specific BM precursors and unfractionated (whole) BM cells were examined in the iterative carbon tetrachloride model of liver fibrosis. BM-derived macrophage (BMM) delivery resulted in early chemokine upregulation with the hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9 respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the anti-inflammatory cytokine IL-10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Upregulation of the liver progenitor cell mitogen TWEAK preceded expansion of the progenitor cell compartment. BMM delivery increased hepatic expression of cytokines with reparative effects (including colony stimulating factor-1, insulin-like growth factor-1 and vascular endothelial growth factor). In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly improved by the application of macrophage precursors and was exacerbated by whole BM. BMMs did not affect liver fibrosis or regeneration in the 1% DDC model of biliary disease. These effects were only detected following the intraportal delivery of BM cells. The peripheral (tail) vein administration of BMMs, either singly or repeatedly did not recapitulate the therapeutic phenotype. This was investigated by in vivo tracking of BMMs constitutively expressing green fluorescent protein (GFP). The peripheral administration route resulted in the early (1 hour) accumulation of BMMs within the pulmonary system. This was followed by delayed hepatic engraftment, which was also numerically reduced (< 30%) compared with intraportal administration. Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signalling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.