Glucans are fungal cell wall PAMPs that promote survival in polymicrobial and candidal sepsis. Dectin-1 is the primary PRR for glucans. The goals of the present study were to characterize 1) the effects of fungal infection on Dectin-1; 2) the effects of polymicrobial sepsis in the presence and absence of glucan on Dectin-1; 3) the effects of systemic administration of glucans on Dectin-1; and 4) the intracellular trafficking of glucans. Mice were either systemically infected with Candida albicans, or made septic by CLP with and without glucan phosphate (GP) injection, or injected with GP. Flow cytometry was performed to assess cell surface Dectin-1 expression. C. albicans sepsis resulted in an increase in the percentage of Dectin-1 positive (Dectin+) blood and splenic leukocytes by increasing the percentage of neutrophils. C. albicans infection increased the percentage of Dectin+ splenic T cells. CLP decreased the percentage of highly Dectin-1 positive leukocytes in the blood by decreasing the percentage of Dectin+ neutrophils. GP treatment in sepsis further decreased the percentages of Dectinhigh blood leukocytes and Dectin+ neutrophils. CLP decreased the percentage of Dectin+ splenic leukocytes by decreasing the percentage of splenic macrophages. GP administration to CLP mice further decreased the percentage of Dectin+ splenocytes by decreasing the percentage of Dectin+ macrophages. Administration of GP resulted in a prolonged decrease in the percentage of Dectinhigh blood leukocytes. The changes in Dectin-1 expression with GP were because of decreases in the percentage of Dectin+ neutrophils and monocytes. In the trafficking studies, macrophages were incubated with fluorescent labeled glucans and then stained for intracellular organelles and signal transduction molecules. Cells were imaged using confocal microscopy. GP is internalized by clathrin and trafficked to the Golgi apparatus. GP internalization is regulated but not dependent on caveolin-1. GP co-localized with SRA, TLR2, and PI3K/p85. The trafficking of laminarin and particulate glucan is similar. We speculate that loss of cell surface Dectin-1 may be important in the protection conferred by glucans in sepsis. Additionally, intracellular trafficking and interaction with signaling components may be important steps in modulation of cellular function by glucan-pattern recognition receptor complexes.

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Orientador: Ronei Luciano Mamoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T05:56:25Z (GMT). No. of bitstreams: 1 Castro_LiviaFurquimde_M.pdf: 5966667 bytes, checksum: bd25c56ae25a8825069884bedd9ca8ce (MD5) Previous issue date: 2015 / Resumo: Diversos estudos demonstram que a resposta inflamatória é de extrema importância para o controle da Paracoccidioidomicose (PCM). Essa resposta inflamatória é iniciada pelo reconhecimento das células fúngicas por receptores expressos por células do sistema imunológico inato. Dentre esses receptores, o NLRP3 foi associado com o reconhecimento de fungos patogênicos em modelos experimentais, atuando em conjunto com o TLR2 e a dectina-1. O NLRP3 atua na formação de um complexo multiproteico denominado inflamassoma, o qual ativa a caspase-1, que é responsável pela produção das formas ativas de duas importantes citocinas inflamatórias: a IL-1? e a IL-18. Esse estudo teve por objetivo investigar o envolvimento do NLRP3 na ativação da resposta inflamatória de macrófagos e células dendríticas humanas (DCs) derivadas de monócitos em resposta ao Paracoccidioides brasiliensis (Pb), além de avaliar a participação do NLRP3 na indução da resposta imunológica adaptativa. Nossos resultados demonstraram que células de lesões de pacientes com PCM (mucosa oral ou linfonodos) apresentam produção de IL-1beta, IL-18 e IL-37 e que macrófagos dessas lesões são positivos para Caspase-1 e NLRP3. Também fomos capazes de demonstrar que o reconhecimento de células leveduriformes por DCs e macrófagos humanos leva à ativação do inflamassoma NLRP3 e consequente produção de IL-1 e IL-18. Esse reconhecimento envolve a participação de receptores de superfície (TLR2 e Dectina-1), sendo que a produção dessas citocinas é dependente da sinalização via dectina-1 e fosforilação da proteína Syk. Além disso, observamos que a ativação do inflamassoma NLRP3, após o reconhecimento do fungo, envolve como principais mecanismos a produção de ROS e o efluxo de K+. Nossos dados também demonstraram que o inflamassoma NLRP3 é essencial para a diferenciação de células Th17 e Th1 e que sua inibição leva à um aumento de células Th2 e Treg. Em conjunto nossos dados indicam que a ativação do NLRP3 desempenha um papel importante, tanto na indução de uma resposta inflamatória inicial, quanto no desenvolvimento de uma resposta adquirida que pode ser associada à resistência à infecção pelo P. brasiliensis / Abstract: Several studies have shown that the inflammatory response is crucial for the control of paracoccidioidomycosis (PCM). This inflammatory response is initiated by the recognition of fungal yeast cells by receptors expressed by cells of the innate immune system. Among these receptors, NLRP3 was associated with the recognition of pathogenic fungi in experimental models, working in conjunction with TLR2 and dectin-1. The NLRP3 acts forming a multiproteic complex called inflammasome, which activates caspase-1, and the production of the active forms of two important cytokines: IL-1? and IL-18. This study aimed to investigate the involvement of NLRP3 activation in the inflammatory response of macrophages and human dendritic cells (DCs) derived from monocytes, in response to Paracoccidioides brasiliensis (Pb), and to evaluate the participation of NLRP3 in the induction of the subsequent adaptive immune response. Our results demonstrated that cells of lesions from PCM patients (oral mucosa and lymph nodes) express IL-1beta, IL-18 and IL-37, and that macrophages in these lesions are positive for caspase-1 and NLRP3. We were also able to demonstrate that the recognition of Pb yeast cells by human macrophages and DCs leads to the NLRP3 inflammasome activation and production of IL-1 and IL-18. This recognition involves the participation of surface receptors (TLR2 and Dectin-1), and the production of these cytokines was dependent on signaling via dectin-1 and phosphorylation of Syk. In addition, we observed that the activation of the NLRP3 inflammasome, after recognition of the fungus, involves as main mechanisms the ROS production and the K+ efflux. Our data also demonstrate that the NLRP3 inflammasome are essential for the differentiation of Th1 and Th17 cells and its inhibition leads to an increased frequency of Th2 and Treg cells. Taken together our data indicated that activation of NLRP3 present an important role in both the induction of an initial inflammatory response, and in the development of an acquired immune response, which can be associated with the resistance to the P. brasiliensis infection / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.