O complexo NADPH oxidase e espécies reativas de oxigênio favorecem a infecção de macrófagos por Trypanosoma cruzi

• Autor(a) principal: Paula, Jéssica Isis Oliveira de
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14388
• topic: Trypanosoma cruzi; Infection; Reactive oxygen species; Amastigogenesis; Trypanosoma cruzi; Infecção. Espécies reativas de oxigênio. Amastigogênese; Trypanosoma cruzi; NADPH Oxidases; Macrófagos; Espécies Reativas de Oxigênio; CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA::PROTOZOOLOGIA DE PARASITOS

Trypanosoma cruzi is an obligate intracellular parasite, able to infect and replicate in macrophages, which responds by activating the NADPH oxidase (Nox) enzyme complex. This enzyme generates superoxide anion (O2 -) that maybe dismutate to other reactive oxygen species (ROS), hydrogen peroxide (H2O2). However, is still unclear how the parasite survives and develops this adverse environment. Thus, we investigated the crosstalk between Nox and ROS during macrophage infection by T. cruzi. For this end, we used two Nox inhibitors, diphenyl iodonium (DPI) at 5 µM and apocynin at 10 µM, the antioxidant, N-acetylcysteine (NAC) and urate both 1 mM, the pro-oxidant hydrogen peroxide (H2O2) at 10,30 or 50 µM, menadiona at 5 µM and paraquat at 20 or 40 µM, the PKC inhibitor, BIS at 0,05 µM and PP2A inhibitor, okadaic acid (AO) at 0,1 µM. First of all, we evaluated the Nox and ROS influence in the intracellular infection of macrophages by T. cruzi. For this, the macrophages were incubated with DPI, apocynin, NAC, H2O2, or menadione and subsequently infected with trypomastigotes in the ratio 10:1. After 72 h, the infection was quantified by amastigote counting inside the macrophages. It was possible to observe a significant reduction in the infection of cells treated with DPI, apocynin and NAC whereas it incresead by H2O2. Menadione reduced the percentage of infected macrophage but did not alter the parasites per cell and endocytic index. From these results, we investigated the involvement of Nox and ROS in the intracellular T. cruzi proliferation. So, macrophages were previously infected for 3 h with trypomastigotes and incubated with DMEM supplemented with 10% FCS overnight for complete differentiation into amastigotes. After, the cells were treated with DPI, NAC, apocynin, H2O2 or menadiona for 48 h, and then intracellular amastigotes were quantified. Only DPI reduced the infection parameters. Among pro-oxidant, just H2O2 increasead amastigotes inside macrophage. In addition, we evaluate the effect of antioxidant, NAC and urate, as well as H2O2, menadiona and paraquat in the amastigogenesis in vitro. Curiously, only H2O2 influenced the differentiation in vitro, raising 19% the percentage of amastigotes after 2 h. The other side, menadiona reduced 15% of differentiation after 4 h. Thereat, we evaluated the PKC and PP2A involvement in amastigogenesis in vitro induced by H2O2. For this, we used BIS, PKC inhibitor, and AO, PP2A inhibitor. The amastigogenesis was not altered by BIS. Although AO alone did not affect the differentiation, it reversed the effect of H2O2. Collectively, our results suggest new role to ROS and Nox activation during the macrophage infection by T. cruzi. In this scenario, ROS produced by Nox helps intracelular infection, since it is impaired enzyme inhibiton or the presence of antioxidant. On the other hand, H2O2 promotes the differentiation via PP2A activation and intracelular replication of T. cruzi.