Efecto inmunomodulador de la melatonina en modelos experimentales de la infección por el virus de encefalitis equina venezolana

Laburpena

The Venezuelan equine encephalitis (VEE) virus has been involved in epidemic outbreaks in Venezuela and America, affecting equids and humans. Melatonin (MLT) has been shown to improve some immunodeficient status caused by viral infections. In this regard, it was proposed an evaluation of the in vivo and in vitro immunomodulatory effect of MLT on experimental VEE virus infection. In vivo assays were performed in albino NMRI mice that were pretreated with 250, 500, 1000 and 5000 ug of MLT /Kg body weight. Mice were inoculated with the Guajira strain of the virus. Serum and brain samples were extracted to determine the viral titers, gamma interferon (IFNy), Interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 1beta (IL-1β), Tumor Necrosis Factor alpha (TNFa), Interleukin 10 (IL-10), nitrite-nitrate (Nitric oxide: NO) and malondialdehyde (MDA) on days 1, 3 and 5 post viral infection (p.i.). In addition, splenocytes obtained from VEE virus infected mice untreated or treated with MLT were used to determine their proliferative status to mitogens (PHA). In vitro experiments were performed using murine neuroblastoma (Na2) or splenocytes. In this regard, Na2 cells or splenocytes were cocultured with VEE virus untreated and treated with MLT. Supernatants and cells from Na2 cultures were used to determine NO, MDA and inducible nitric oxide synthase (iNOS) expression at 6, 12, 24, 36 and 48 hours p.i. and NO in splenocyte cultures. Luzindole was used to block MLT receptors. Cytokines and IgM antibodies against VEE virus were determined by ELISA. Viral titers were determined using chicken embryo fibroblast cultures and lymphocyte proliferation by incorporation of tritiated thimidine. NO was determined by the content of nitrite-nitrate and MDA by the thiobarbituric acid assay. iNOS expression was determined by indirect immunofluorescence. The results showed that MLT has a protective effect in mice infected with the VEE virus, retarding the beginning of the disease and extending the life of the infected mice. MLT significantly increased serum levels of IFNy in noninfected mice from the first to the sixth day of treatment. IL-1β and TNFa increased their values from days 1 to 6 p.i. when compared to the remainder groups. No differences in IL-4 and IL-2 levels were found in the studied groups. Blocking of IL-1β resulted in increased mortality rate of infected mice rising to 100% and suggesting that IL-1β inducer effect of MLT could be related to the beneficial effect of MLT during VEE virus infection. Likewise, MLT reduced the cytopatic effect of the virus in cell cultures. The increased nitrite-nitrate content and lipid peroxidation in infected cultures was blocked by MLT, suggesting a possible antioxidant effect of MLT as an additional protective mechanism against the VEE infection, besides the humoral immune response, since increased IgM antibodies anti VEE virus and IL-10 were also observed. The administration of Luzindole blocked the protective effect of MLT against the VEE infection. Luzindole is a competitive antagonist of different subtypes of MLT receptors. Splenocyte cultures infected with the VEE virus generated important amounts of NO and MLT reduced that production. In addition, treatment of splenocyte cultures with MLT induced increased levels of IL2 and IL-1β in the absence or the presence of PHA, and increased the proliferativeresponse when the PHA was added to the cultures, suggesting an important effect of MLT on the biology of immune cells which are probably involved in the protective effect of the indole. Likewise, MLT decreased NO production, iNOS expression and lipid peroxidation induced by VEE virus in neuroblastoma cell cultures. In conclusion, MLT has a beneficial effect in experimental VEE virus infection, that could be related to the induction of IL-1β, IL-2, IFNy and IL-10 productions and to its antioxidant properties.