Papel das espécies reativas de oxigênio durante a infecção por Leishmania infantum

Abstract

Reactive oxygen species (ROS) are a group of ions, radicals and highly reactive molecules derived from oxygen metabolism. They participate in many biological processes from hormone biosynthesis, cell signaling and killing of microbial pathogens. They are produced by phagocytes when properly stimulated. NADPH oxidase is the enzyme responsible for the conversion of O2 to O2•- using NADPH as electron donor. Several studies have shown different effects of ROS in infections caused by parasites of the genus Leishmania. In the present work we focus on the determination of the different oxygen species generated by macrophages when infected by Leishmania infantum and the influence of these radicals in the early inflammatory cell infiltrate after intradermal infection. We showed that L. infantum-infected macrophages produced high levels of O2•- and we detected H2O2 in the supernatant of cell cultures. Moreover, wild-type (WT)- and NADPH oxidase deficient (Phox-/-)-activated macrophages produced similar amount of •NO. Besides, when activated and infected, only WT macrophages produced peroxynitrite. Our data demonstrated that these different oxygen species have an important effector role since there was decrease in the percentage of infected cells. A difference was found when comparing the percentages of infection between WT, Phox-/- and nitric oxide sintase deficient (iNOS-/-) macrophages, suggesting an important role of peroxynitrite in the in vitro parasite killing. The oxygen species produced by phagocytes play roles in the modulation of protein expression and transcription factors, induction of cell death by apoptosis and killing of parasites in vivo. Initially, we showed that there were differences in parasite -/- -/- -/- and iNOS . Phox load in WT, Phox iNOS-/- and WT animals. We compared the cellular infiltrate influenced by the absence of expression of the enzyme NADPH oxidase and inducible nitric oxide synthase. The deficiency in the expression of NADPH oxidase induced larger neutrophil migration, peaking at 12 hours after infection. These cells were the main cell type harboring parasites at this time. The presence of the parasite induced apoptosis in neutrophils, but we did not find any differences between Phox-/- and WT mice. However, we found differences in neutrophil necrosis. Phox-/- mice showed more necrosis than WT in all time periods. Furthermore, neutrophil influx was coincident with higher levels of CXCL1and CXCL2 detected in Phox-/- but not in WT and iNOS-/- mice. We suggest that the neutrophil maintenance at the site of infection in the times between 24 and 72 hours -/- was due the higher chemokine production and necrosis in Phox . Our data suggest for the first time that reactive oxygen species directly influence the elimination of the parasite modulating chemokine production, differential cell migration and neutrophil necrosis regulation in the early stages of infection by L. infantum.