Avaliação da ativação plaquetária e secreção de citocinas em resposta à proteína não estrutural 1 (NS1) do vírus da Dengue-2

Abstract

Dengue is a disease caused by dengue virus (DENV), composed by 4 serotypes, and transmitted mostly by Aedes aegypti mosquitos. Some patients may develop a severe form of the disease, called severe dengue, characterized by vascular leakage, severe hemorrhage and organ failure. High concentrations of inflammatory cytokines in the blood of dengue patients contribute to vascular leakage and dengue severity. Recent studies have shown high plasma levels of the non-structural protein 1 (NS1) in the blood of dengue patients. NS1 has been related to interaction with TLR4 on macrophages, inducing the secretion of proinflammatory mediators, and to the disruption of the vascular endothelium, contributing to vascular leakage. Although interactions of NS1 with macrophages and endothelial cells have already been demonstrated, its ability to activate platelets is still unclear. Platelets from healthy volunteers were stimulated with recombinant DENV-2 NS1 produced in E. coli and platelet responses evaluated by flow cytometry and ELISA. We observed that DENV-2 NS1 increased platelet activation as indicated by P-selectin surface expression. Our results indicate that NS1 increases the secretion of the chemokines RANTES/CCL5 and PF4/CXCL4 and the proinflammatory cytokines IL-1α and MIF. However, there was no increase in the levels of the anti-inflammatory cytokine TGF-β. Treatment with polymixin B did not affect the ability of NS1 to induce P-selectin translocation and cytokine secretion, indicating the absence of LPS contamination. In addition, pre-exposure to NS1 potentiated platelet activation and chemokine secretion in response to a suboptimal concentration of thrombin. Platelets were also stimulated with recombinant NS1 produced in HEK cells and the results show a similar increase in the surface expression of P-selectin, confirming previous data in which DENV2 NS1 promotes platelet activation. Furthermore, platelets from healthy volunteers were incubated with anti-TLR4 neutralizing antibodies or control IgG and then stimulated with NS1 produced in HEK cells. Our data shows that NS1 activates platelets partially depending on TLR4. Finally, we demonstrated that DENV-infected platelets replicated viral RNA, as quantified by qPCR, and also expressed and secreted NS1, as detected by western blot of pellets and supernatants of infected platelets. However, although DENV2-infected platelets are not able to secrete viral particles, promoting abortive replication, they secrete NS1, which may act as a PAMP inducing immune-inflammatory responses. Our data demonstrate that DENV NS1 is capable of activating platelets inducing the release of pro-inflammatory mediators. Besides, DENV NS1 potentiates platelet activation by thrombin, suggesting an intensification of the thromboinflammatory response. Our data also indicate that platelets not only respond to exogenous NS1 but also are a source of NS1 during DENV infection.