Análise do perfil de células CD4+ e avaliação genotóxica em células mononucleares humanas infectadas in vitro com diferentes espécies de fungos do gênero Cryptococcus spp
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Data
2017-02-22
Autores
Andrade, Jéssica Cristina Bilizario Noguerol [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A Criptococose é uma micose sistêmica, cuja incidência tem aumentado nas últimas décadas. É causada pelos fungos Cryptococcus neoformans e Cryptococcus gattii que se apresentam sob a forma de levedura capsulada. Uma importante característica da infecção com as duas espécies do fungo é que enquanto o C.neoformans está associado à doença em pacientes imunossuprimidos, o C.gattii é capaz de induzir a micose em individuos imunocompetentes. O entendimento dos mecanismos envolvidos nestas diferenças tem sido um desafio para os pesquisadores da área. No que se refere à resposta imune do hospedeiro contra o Cryptococcus, está bem estabelecido que a resistência/suscetibilidade a infecção envolve a participação de diferentes subpopulações de células CD4. No entanto, os estudos sobre o papel das subpopulações TH17 e Treg são escassos. Além disso, são raros os trabalhos que de uma forma sistemática objetivaram comparar o perfil de células CD4 induzido por uma ou outra espécie do fungo. Diferencas nesse perfil poderiam explicar a indução da doença em diferentes populações. Assim, um primeiro objetivo do presente estudo foi avaliar o perfil de células CD4 efetoras induzido pelo C.neoformans e C.gatti . A abordagem experimental foi a incubação de células mononucleares do sangue periférico humano (PBMCs) in vitro com leveduras vivas de C. neoformans e C.gattii, seguida da avaliação da expressão intracitoplasmática das citocinas IFN-(TH1), IL-4 (TH2), IL-17 (TH17) e o fator de transcrição Foxp3 (células Tregs) , por citometria de fluxo, bem como a expressão de mRNA para Foxp3 e RORyt (fator de transcrição para TH17). A análise global dos resultados mostrou que o C.neoformans não é capaz de induzir a diferenciação de TH1 e TH2, mas sim de células Treg e TH17. Por outro lado, o C.gattii induz apenas a diferenciação de células Treg. Outro aspecto da relação hospedeiro/ Cryptococcus que merece ser avaliado, refere-se ao achado de que vários microrganismos podem causar genotoxicidade em células do hospedeiro como uma forma de evasão dos mecanismos de defesa. Assim, um segundo objetivo do presente estudo foi avaliar se as diferentes espécies do Cryptococcus são capazes de induzir danos no DNA de PBMCs e se este processo está relacionado aos níveis de óxido nitrico (NO) produzidos por essas células. Detectamos que ambas as espécies são igualmente capazes de induzir genotoxicidade em PBMCs. No entanto, uma associação entre danos no DNA e altos níveis de NO foi detectada apenas em relação a C. gattii. Os resultados apontam para a possibilidade de que os pacientes com criptococose sejam mais suscetíveis ao desenvolvimento de outras doenças.
Cryptococcosis is a systemic mycosis, whose incidence has increased in the last decades. It is caused by the fungi Cryptococcus neoformans and Cryptococcus gattii which present as capsulated yeasts. An important feature of infection with both fungus species is that while C.neoformans is associated with the disease in immunosuppressed patients, C.gattii is able to induce mycosis in immunocompetent individuals. To understanding the mechanisms involved in these differences has been a challenge for researchers. Regarding the host immune response against Cryptococcus, it is well established that resistance/ susceptibility to infection involves the participation of different subpopulations of CD4 cells. However, studies on the role of TH17 and Treg subpopulations are scarce. In addition, there are few studies that systematically aimed to compare the CD4 cell subpopulations profile induced by one or another species of the fungus. Differences in this profile could explain the induction of the disease in different populations. Thus, a first objective of the present study was to evaluate the effector CD4 cells subpopulations profile induced by C.neoformans and C.gattii. The experimental approach was the incubation of human peripheral blood mononuclear cells (PBMCs) in vitro with living yeasts of C. neoformans and C.gattii, followed by evaluation of the intracytoplasmic expression of the cytokines IFN-γ (TH1), IL-4 (TH2), IL-17(TH17) and Foxp3 transcription factor (Tregs cells) by flow cytometry, as well as mRNA expression for Foxp3 and RORyt (transcription factor for TH17). Overall, the analysis of the results showed that C.neoformans is not able to induce the differentiation of TH1 and TH2, but induces Treg and TH17 cells. On the other hand, C.gattii only induces Treg cells differentiation. Another aspect of the host / Cryptococcus interaction that deserves to be evaluated relates to the finding that various microorganisms may cause genotoxicity in host cells as a mechanism of evading defense mechanisms. Thus, a second objective of the present study was to evaluate if the different species of Cryptococcus are able to induce DNA damage of PBMCs and whether this process is related to the levels of nitric oxide (NO) produced by these cells. We have detected that both species are equally capable of inducing genotoxicity in PBMCs. However, an association between DNA damage and high NO levels was only detected in relation to C. gattii. The results point to the possibility that patients with cryptococcosis are more susceptible to the development of other diseases.
Cryptococcosis is a systemic mycosis, whose incidence has increased in the last decades. It is caused by the fungi Cryptococcus neoformans and Cryptococcus gattii which present as capsulated yeasts. An important feature of infection with both fungus species is that while C.neoformans is associated with the disease in immunosuppressed patients, C.gattii is able to induce mycosis in immunocompetent individuals. To understanding the mechanisms involved in these differences has been a challenge for researchers. Regarding the host immune response against Cryptococcus, it is well established that resistance/ susceptibility to infection involves the participation of different subpopulations of CD4 cells. However, studies on the role of TH17 and Treg subpopulations are scarce. In addition, there are few studies that systematically aimed to compare the CD4 cell subpopulations profile induced by one or another species of the fungus. Differences in this profile could explain the induction of the disease in different populations. Thus, a first objective of the present study was to evaluate the effector CD4 cells subpopulations profile induced by C.neoformans and C.gattii. The experimental approach was the incubation of human peripheral blood mononuclear cells (PBMCs) in vitro with living yeasts of C. neoformans and C.gattii, followed by evaluation of the intracytoplasmic expression of the cytokines IFN-γ (TH1), IL-4 (TH2), IL-17(TH17) and Foxp3 transcription factor (Tregs cells) by flow cytometry, as well as mRNA expression for Foxp3 and RORyt (transcription factor for TH17). Overall, the analysis of the results showed that C.neoformans is not able to induce the differentiation of TH1 and TH2, but induces Treg and TH17 cells. On the other hand, C.gattii only induces Treg cells differentiation. Another aspect of the host / Cryptococcus interaction that deserves to be evaluated relates to the finding that various microorganisms may cause genotoxicity in host cells as a mechanism of evading defense mechanisms. Thus, a second objective of the present study was to evaluate if the different species of Cryptococcus are able to induce DNA damage of PBMCs and whether this process is related to the levels of nitric oxide (NO) produced by these cells. We have detected that both species are equally capable of inducing genotoxicity in PBMCs. However, an association between DNA damage and high NO levels was only detected in relation to C. gattii. The results point to the possibility that patients with cryptococcosis are more susceptible to the development of other diseases.
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Palavras-chave
Criptococose, Cryptococcus gatti, Cryptococcus neoformans, Genotóxicidade, Resposta imune