Papel fisiológico das microvesículas derivadas de macrófagos M2 na doença renal aguda
Nenhuma Miniatura disponível
Data
2019
Autores
Henao Agudelo, Juan Sebastian [UNIFESP]
Orientadores
Pacheco-Silva, Alvaro [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A renoproteção induzida por macrófagos (Mɸs) M2 é principalmente mediada por fatores parácrinos. Recentemente foi demonstrado que Mɸs liberam vesículas extracelulares. Nesse sentido, foi demonstrado que Mɸs-M2 secretam vesículas com material genético associado à polarização do tipo M2. Desse modo, nós hipotetizamos, que exossomos e microvesículas derivadas de Mɸs-M2 possam conter microRNAs e proteínas bioativas que converteriam Mɸs pró-inflamatórios (Mɸs-M1) em Mɸs anti-inflamatórios M2, os quais potencializariam processos renoprotetores na LRA através da geração de um microambiente anti-inflamatório. Nesse contexto, objetivou-se avaliar o papel imunoregulador das vesículas de Mɸs-M2 (MVs-M2) em Mɸs com perfil M1 e verificou-se o seu papel no desenvolvimento e progressão da doença renal aguda. Métodos: O meio condicionado de Mɸs-M2 livre de recombinante foi ultracentrifugado em 100.000g para extração de vesículas. Em seguida, Mɸs derivados da medula óssea foram tratados com MVs-M2 e subsequentemente, ativados por LPS/IFN- por 24 horas. Complementarmente, o efeito das MVs-M2 foi avaliado num modelo experimental de glomeruloesclerose segmentar e focal (GESF) induzida por adriamicina e na lesão renal aguda promovida por cisplatina em camundongos machos C57BL6. O material coletado nos experimentos, foi avaliado por qPCR, Citometria de Fluxo, PCR array, Western Blott, array de citocinas (CBA), NanoSitgh, e finalmente microscopias de: fluorescência, de transmissão e de varredura. Resultados: Inicialmente documentamos a capacidade de Mɸs-M2 de produzir uma população heterogênea de microvesículas e exossomos, possuindo alta variabilidade em marcadores de superfície associados à: vesículas, macrófagos, ativação M2, monócitos e células mieloides. Neste contexto, as vesículas exibiram elevada expressão de CD9, F4/80, CD206, CD11b e ly6C respectivamente. A análise de NanoSight revelou que as vesículas de Mɸs-M2 têm um tamanho médio de ~144,8 nm e uma concentração de ~9,00 x109 partículas/mL. Também foi verificada a capacidade de incorporação das MVs-M2 nos Mɸs-M1. Nos experimentos in vitro, as MVs-M2 reduziu CD86, IL-1, TNF-, MCP-1 e NLRP3 nos Mɸs-M1. Por outro lado, elas aumentaram o receptor de manose, expressão de Arg-1 e atividade enzimática da arginase nesses mesmos macrófagos tratados. Adicionalmente, análises intracelulares revelou que MVs-M2 inibe a secreção de IL-6 em Mɸs-M1. Complementarmente, analises de PCR array mostrou que 21 miRNAs estavam regulados positivamente e 6 estavam regulados negativamente. Curiosamente, os miRNAs hiper-regulados foram particularmente associados à polarização M2. Assim, quando realizados analises de bioinformática com estes miRNAs alterados, foi apontado que a via de sinalização PI3K/AKT poderia estar sendo regulada. Posteriormente, foi confirmado que AKT fosforilado se encontrava significativamente reduzida em Mɸs-M1 tratados com MVs-M2. Finalmente, camundongos tratados com MVs-M2 e expostos a GESFs foram protegidos contra a taxa de perda de peso corporal e proteinúria, ao mesmo tempo que apresentaram a expressão elevada de genes associados com funções podocitarias. Em relação à LRA induzida por cisplatina observamos que MVs-M2 aumentou a expressão de Mɸs com funções anti-inflamatórias e reduziu a presença de neutrófilos infiltrantes. Conclusões: Esses resultados sugerem que as MVs-M2 têm propriedades imunoreguladoras em Mɸs-M1 via modulação de miRNAs, os quais atuam na sinalização AKT, fenômeno este que promove propriedades anti-inflamatórias nos modelos de lesão renal aguda.
Introduction: The renoprotection induced by M2 macrophages (Mɸs) is mainly mediated by paracrine factors. Recently have been demonstrated that Mɸs release extracellular vesicles. These Mɸ-M2 vesicles present genetic material associated with M2 polarization. Thus, we hypothesize that exosomes and microvesicles derived from Mɸ-M2 (MVs-M2) may contain microRNAs and bioactive proteins that would convert proinflammatory Mɸs (Mɸs-M1) into anti-inflammatory Mɸs, and therefore, modulated renoprotective processes in the AKI through the generation of an anti-inflammatory microenvironment. In this context, our objective was evaluating the immunoregulatory properties of MV-M2 in M1Mɸs and verify its role in the development and progression of acute renal disease. Methods: The Mɸ-M2 conditioned medium was ultracentrifuged at 100,000g for vesicle extraction, then, Mɸs derived from bone marrow were treated with MV-M2 and subsequently activated by LPS/IFN- for 24 hours. In addition, the effect of MV-M2 was evaluated in an experimental model of adriamycin-induced Focal Segmental Glomerulosclerosis (FSGS) and other with cisplatin-promoted acute kidney injury (AKI), both, in C57BL6 male mice. The material collected in the experiments was evaluated by qPCR, Flow Cytometry, PCR array, Western Blott, cytokine array (CBA), NanoSitgh, and finally fluorescence, transmission and scanning microscopy were utilized. Results: Initially, we verified that Mɸ-M2 produced a heterogeneous population of microvesicles and exosomes with variable expression of surface markers associated with vesicles, macrophages, M2 activation, monocytes and myeloid cells. In this context, they exhibited high expression of CD9, F4/80, CD206, CD11b and ly6C respectively. During characterization steps, NanoSight analysis revealed that the Mɸ-M2 vesicles have a mean size of ~144.8 nm and a concentration of ~ 9.00 x 109 particles/mL. In addition, the incorporation capacity of MV-M2 into Mɸ-M1 was verified. In the in vitro experiments, MVs-M2 reduced CD86, IL-1b, TNF-, MCP-1 and NLRP3 in Mɸs-M1 and, on the other hand, they also increased the expression of mannose receptor, Arg-1 and, its enzymatic activity, in the treated macrophages. Also, intracellular analyzes revealed that MVs-M2 inhibit IL-6 secretion in Mɸs-M1. Complementarily, PCR array analyzes showed that 21 miRNAs were upregulated and 6 were down-regulated. Interestingly, the overexpressed miRNAs were particularly associated with M2 polarization. Thus, after bioinformatics analyzes, it was pointed out that the PI3K/AKT signaling pathway could be regulated. Therefore, it was confirmed that phosphorylated AKT was significantly reduced in Mɸs-M1 treated with MVs-M2. Finally, mice treated with MVs-M2 and exposed to FSGS, were protected against weight loss, proteinuria and had an improvement of genes associated with podocyte functions. In cisplatin-induced AKI, we observed that MVs-M2 increased anti-inflammatory functions in Mɸs and reduced the presence of infiltrating neutrophils. Conclusions: These results suggest that MV-M2 have immunoregulatory properties in Mɸ-M1 via modulation of miRNAs which are associated with AKT signaling pathways, promoting anti-inflammatory properties in experimental models of AKI.
Introduction: The renoprotection induced by M2 macrophages (Mɸs) is mainly mediated by paracrine factors. Recently have been demonstrated that Mɸs release extracellular vesicles. These Mɸ-M2 vesicles present genetic material associated with M2 polarization. Thus, we hypothesize that exosomes and microvesicles derived from Mɸ-M2 (MVs-M2) may contain microRNAs and bioactive proteins that would convert proinflammatory Mɸs (Mɸs-M1) into anti-inflammatory Mɸs, and therefore, modulated renoprotective processes in the AKI through the generation of an anti-inflammatory microenvironment. In this context, our objective was evaluating the immunoregulatory properties of MV-M2 in M1Mɸs and verify its role in the development and progression of acute renal disease. Methods: The Mɸ-M2 conditioned medium was ultracentrifuged at 100,000g for vesicle extraction, then, Mɸs derived from bone marrow were treated with MV-M2 and subsequently activated by LPS/IFN- for 24 hours. In addition, the effect of MV-M2 was evaluated in an experimental model of adriamycin-induced Focal Segmental Glomerulosclerosis (FSGS) and other with cisplatin-promoted acute kidney injury (AKI), both, in C57BL6 male mice. The material collected in the experiments was evaluated by qPCR, Flow Cytometry, PCR array, Western Blott, cytokine array (CBA), NanoSitgh, and finally fluorescence, transmission and scanning microscopy were utilized. Results: Initially, we verified that Mɸ-M2 produced a heterogeneous population of microvesicles and exosomes with variable expression of surface markers associated with vesicles, macrophages, M2 activation, monocytes and myeloid cells. In this context, they exhibited high expression of CD9, F4/80, CD206, CD11b and ly6C respectively. During characterization steps, NanoSight analysis revealed that the Mɸ-M2 vesicles have a mean size of ~144.8 nm and a concentration of ~ 9.00 x 109 particles/mL. In addition, the incorporation capacity of MV-M2 into Mɸ-M1 was verified. In the in vitro experiments, MVs-M2 reduced CD86, IL-1b, TNF-, MCP-1 and NLRP3 in Mɸs-M1 and, on the other hand, they also increased the expression of mannose receptor, Arg-1 and, its enzymatic activity, in the treated macrophages. Also, intracellular analyzes revealed that MVs-M2 inhibit IL-6 secretion in Mɸs-M1. Complementarily, PCR array analyzes showed that 21 miRNAs were upregulated and 6 were down-regulated. Interestingly, the overexpressed miRNAs were particularly associated with M2 polarization. Thus, after bioinformatics analyzes, it was pointed out that the PI3K/AKT signaling pathway could be regulated. Therefore, it was confirmed that phosphorylated AKT was significantly reduced in Mɸs-M1 treated with MVs-M2. Finally, mice treated with MVs-M2 and exposed to FSGS, were protected against weight loss, proteinuria and had an improvement of genes associated with podocyte functions. In cisplatin-induced AKI, we observed that MVs-M2 increased anti-inflammatory functions in Mɸs and reduced the presence of infiltrating neutrophils. Conclusions: These results suggest that MV-M2 have immunoregulatory properties in Mɸ-M1 via modulation of miRNAs which are associated with AKT signaling pathways, promoting anti-inflammatory properties in experimental models of AKI.
Descrição
Citação
HENAO AGUDELO, Juan Sebastian. Papel fisiológico das microvesículas derivadas de macrófagos M2 na doença renal aguda. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2019.