Perfil da atividade de macrófagos in vitro, frente às amostras virulenta, atenuada e saprófita de leptospira spp
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Data
2018-12-11
Autores
Araujo Junior, Erivelto Corrêa de
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo do presente trabalho foi verificar a dinâmica da resposta imune celular in vitro utilizando-se cultivos de macrófagos de camundongos (J774A.1) quando expostos às amostras virulenta, atenuada e saprófita de Leptospira, observados nos períodos de 1h, 3h, 6h, 12h, 24h, 36, 48h e 72h após a exposição. Foram realizados ensaios que determinaram a produção de intermediários reativos do nitrogênio (NO), expressão de genes para citocinas e interleucinas, pela técnica de reação em cadeia de polimerase (PCR) e a presença e quantificação das mesmas no sobrenadante do cultivo celular pelo teste ensaio imunoenzimático (ELISA). Avaliam-se as diferenças na modulação da expressão gênica do hospedeiro por cepas de Leptospira com variados graus de virulência, pelo método do microarranjo. Os resultados demonstraram que as amostras virulenta e atenuada proporcionaram um estímulo maior aos macrófagos em comparação à amostra saprófita, principalmente na fase tardia da infecção, considerando-se os valores expressos por óxido nítrico (NO), óxido nítrico sintase endotelial (eNOS) e induzido (iNOS). Os valores, embora estatisticamente não significativos, apresentaram uma dinâmica maior à resposta de citocinas (TNF-α, IL-1β) pela amostra virulenta em comparação à atenuada e saprófita. Outro fator relevante foi o encontro de Caspase-3 e -8 na fase inicial da infecção, sugerindo que a apoptose dos macrófagos ocorreu no começo do processo, logo após a internalização das Leptospiras, atingindo níveis elevados às 24 horas, com pico máximo, às 72 horas. Após análise transcriptômica, os resultados mostraram um alto número de genes diferencialmente expressos após 6h de infecção por L. interrogans virulenta e atenuada, e em menor proporção com a linhagem saprófita L. biflexa. Isto sugere que RNAs são modulados após a infecção por Leptospira em macrófagos, independente do grau de virulência. Os dados produzidos neste estudo, contribuirão para melhor compreensão da patogênese da leptospirose.
The objective of the present work was verify the dynamic of the cellular immune response in vitro using macrophages cultures of mice (J774A.1) when exposed to virulent, attenuated and saprophytic Leptospira samples, observed in eight time intervals after exposure: 1h, 3h, 6h, 12h, 24h, 36, 48h and 72h. The assays that determined the production of reactive nitrogen intermediates (NO), the expression of genes for cytokines and interleukins by polymerase chain reaction (PCR) and the presence and quantification of them in the supernatant of the cell culture were performed by the assay ELISA. In this context, we aimed to evaluate the differences in modulation of host gene expression by Leptospira strains with varied virulence degrees, using the microarray method. The results demonstrated that the virulent and attenuated samples provided a greater stimulus to the macrophages compared to the saprophyte sample, especially in the late phase of the infection, considering the values expressed by nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and induced (iNOS). The values, although statistically insignificant, presented a greater dynamics to the cytokine response (TNF-α, IL-1β) by the virulent sample compared to the attenuated and saprophyte. Another important factor was the finding of Caspase-3 and -8 in the initial phase of the infection, suggesting that macrophage apoptosis occurred early in the process, soon after Leptospiras internalization, reaching high levels at 24 hours, with a maximum peak at 72 hours. After transcriptomic analysis, the results showed a high number of differentially expressed genes after 6h of infection by virulent and attenuated L. interrogans, and to a lesser extent with L. biflexa saprophytic lineage. This suggests that RNAs are modulated after infection by Leptospira in macrophages, regardless of the degree of virulence. It is hoped that the data produced will contribute to the understanding of the pathogenesis of leptospirosis.
The objective of the present work was verify the dynamic of the cellular immune response in vitro using macrophages cultures of mice (J774A.1) when exposed to virulent, attenuated and saprophytic Leptospira samples, observed in eight time intervals after exposure: 1h, 3h, 6h, 12h, 24h, 36, 48h and 72h. The assays that determined the production of reactive nitrogen intermediates (NO), the expression of genes for cytokines and interleukins by polymerase chain reaction (PCR) and the presence and quantification of them in the supernatant of the cell culture were performed by the assay ELISA. In this context, we aimed to evaluate the differences in modulation of host gene expression by Leptospira strains with varied virulence degrees, using the microarray method. The results demonstrated that the virulent and attenuated samples provided a greater stimulus to the macrophages compared to the saprophyte sample, especially in the late phase of the infection, considering the values expressed by nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and induced (iNOS). The values, although statistically insignificant, presented a greater dynamics to the cytokine response (TNF-α, IL-1β) by the virulent sample compared to the attenuated and saprophyte. Another important factor was the finding of Caspase-3 and -8 in the initial phase of the infection, suggesting that macrophage apoptosis occurred early in the process, soon after Leptospiras internalization, reaching high levels at 24 hours, with a maximum peak at 72 hours. After transcriptomic analysis, the results showed a high number of differentially expressed genes after 6h of infection by virulent and attenuated L. interrogans, and to a lesser extent with L. biflexa saprophytic lineage. This suggests that RNAs are modulated after infection by Leptospira in macrophages, regardless of the degree of virulence. It is hoped that the data produced will contribute to the understanding of the pathogenesis of leptospirosis.
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Palavras-chave
Leptospiras, Resposta Imune, Citocinas, Apoptose, Trancriptoma, Immune Response, Cytokines, Apoptosis, Transcriptome