Estudio de la respuesta inmunitaria-inflamatoria y oxidativa en pacientes y en un modelo experimental "in vitro" de infección por el virus dengue

Resum

Dengue is an important arboviral disease in tropical countries caused by a flavivirus Dengue virus infection induces several immune disorders in the host that could be involved in the pathogenesis of the disease. It's not has been well established the role of monocyte/macrophage in increased expression of inflammatory mediators found in dengue infections. The aim of this study was to evaluate inflammatory mediators of the immune response in patients with dengue infection and its clinical significance, emphasizing the severity of the disease, the type of infection and the infecting viral serotype. In addition, we evaluated the effect of dengue virus on the function and viability of monocytes experimentally infected with dengue virus and under stimulation with LPS, from umbilical cord blood of newborns and peripheral blood of young adults and the elderly. In this regard, we determined the concentration of proinflammatory cytokines (TNF-α, IL-6 and IL-1β), complement proteins (C3 and C4), C-reactive protein (CRP) and nitric oxide (NO) in 70 patients with dengue in the acute phase. These patients were classified according to the severity of the disease according to WHO criteria in DF and DHF, and type of infection in primary infection (PI) and secondary infection (SI). The control group consisted of healthy individuals. The levels of circulating cytokines (TNF-α, IL-6 and IL-1β) and CRP were determined by ELISA, C3 and C4 by nephelometry, NO by Griess method and MDA by thiobarbituric acid method. We used virus serotypes isolated from patients with dengue and reference strains, which were propagated in C6/36 HT mosquito cells, and counting in VERO cells by the Plaque forming unit technique. Tests conducted in vitro, cultures of monocytes were infected with virus reference strains and stimulated with LPS for 1 and 3 days post-infection (PI). We determined the cytokines TNF-α, IL-6 and IL-1β, NO and MDA, using the culture supernatants and catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) in cell lysates. Apoptosis was determined by TUNEL. Our results show a significant increase in the concentrations of TNF-α (p <0.001), IL-6 (p <0.001), CRP (p <0.001) and NO (p <0.01) in patients with DF and DHF and consumption of the C3 complement protein (p <0.01). These findings were consistent with an increased production of TNF-α (p <0.001), IL-6 (p <0.01) and NO (p <0.001) at 1 and 3 post-infection days in cultured monocytes. The patients were infected with serotypes 2, 3 and 4 of DENV. TNF-α is related to the severity of the disease, but not with the infecting serotype. While IL-6, CRP and C3 were associated with both the severity of the disease, and DENV-4, DENV-2 and DENV-3 serotypes, respectively. There was a differential activation of antioxidant mechanisms according to the age and the infecting serotype, because the increased production in monocytes from adults to CAT and GSH, and the elderly in the case of SOD, while DENV-1 and 4 induced higher production was observed. In addition, the DENV induced an increased number of apoptotic cells to the 1st (p <0.0001) and 3rd (p <0.0001) days PI in cultured monocytes. These results show that increased production of inflammatory mediators, oxidation and apoptosis in dengue, may be in part mediated by virus-monocyte interactions.