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

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.A dengue é uma doença causada pelo vírus da dengue (DENV), composto por 4 sorotipos, sendo transmitida pela picada do mosquito Aedes aegypti, principalmente. Alguns pacientes podem desenvolver uma forma grave da doença, chamada de dengue grave, caracterizada por extravasamento vascular, hemorragia grave e falência dos órgãos. Inflamação exacerbada com concentrações elevadas de citocinas inflamatórias contribuem para o extravasamento vascular e a gravidade da dengue. Estudos recentes demonstraram altos níveis da proteína não estrutural 1 (NS1) do DENV no sangue dos pacientes com dengue grave. A NS1 está relacionada à interação com TLR4 em macrófagos, induzindo a secreção de mediadores inflamatórios, e à ruptura do endotélio vascular, contribuindo para o extravasamento vascular. Apesar de a interação da NS1 com macrófagos e células endoteliais já ter sido demonstrada, sua habilidade em ativar plaquetas ainda é desconhecida. Nesse estudo, plaquetas de voluntários saudáveis foram estimuladas com NS1 do DENV2 recombinante e a resposta plaquetária foi avaliada por citometria de fluxo e ELISA. Nós observamos que a NS1 do DENV2 recombinante produzida em E. coli aumentou a ativação plaquetária, indicada pela expressão da P-selectina (CD-62P) na superfície. Nossos resultados indicam que a NS1 aumenta a secreção das quimiocinas RANTES/CCL5 e PF4/CXCL4 e das citocinas pró-inflamatórias IL-1α e MIF. Contudo, não houve aumento nos níveis da citocina antinflamatória TGF-β. O pré-tratamento com polimixina B não afetou a habilidade da NS1 em induzir a translocação da P-selectina e secreção de citocinas, indicando ausência de contaminação por LPS. Além disso, pré-exposição a NS1 potencializou a ativação plaquetária e secreção de quimiocinas em resposta a uma concentração sub-ótima de trombina. As plaquetas também foram estimuladas com NS1 produzida em células HEK e os resultados obtidos demonstraram um aumento na expressão superficial de P-selectina, confirmando os dados anteriores de que a NS1 do DENV2 promove ativação plaquetária. Posteriormente, plaquetas de voluntários saudáveis pré-tratadas com anticorpos neutralizantes anti-TLR4 ou IgG inespecífica foram estimuladas com NS1 produzida em células HEK . Nossos resultados demonstram que a NS1 ativa plaquetas parcialmente através do receptor TLR4. Finalmente, nós demonstramos que plaquetas infectadas com DENV além de replicarem o RNA viral, são capazes de traduzir o genoma do vírus, sintetizando NS1. No entanto, nossos resultados mostram que as plaquetas não secretam partículas virais, indicando que o ciclo de replicação do vírus não é completo. Apesar dessa replicação abortiva, as plaquetas são capazes de secretar a NS1, que pode agir como um PAMP induzindo respostas imuno-inflamatórias. Nossos dados demonstram que NS1 do DENV2 é capaz de ativar plaquetas, induzindo a secreção de mediadores inflamatórios. Somado a isso, NS1 do DENV2 potencializa a ativação plaquetária pela trombina, sugerindo uma intensificação da resposta tromboinflamatória. Nossos resultados indicam que as plaquetas não só respondem a NS1 exógena como também são fontes de NS1 durante a infecção pelo DENV. Em conjunto, estes eventos podem potencialmente contribuir para a patogênese da dengue

Ativação plaquetária pela NS1 do DENV2: receptores, mecanismos e perfil de mediadores

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Platelets in dengue infection: more than a numbers game

Dengue virus (DENV) infection is responsible for the development of dengue illness, which can be either asymptomatic, present mild manifestations or evolve to severe dengue. Thrombocytopenia is an important characteristic during DENV infection, being observed both in mild and severe dengue, although the lowest platelet counts are encountered during severe cases. This review gathers information regarding several mechanisms that have been related to alterations in platelet number and function, leading to thrombocytopenia but also platelet-mediated immune and inflammatory response. On this regard, we highlight that the decrease in platelet counts may be due to bone marrow suppression or consumption of platelets at the periphery. We discuss the infection of hematopoietic progenitors and stromal cells as mechanisms involved in bone marrow suppression. Concerning peripheral consumption of platelets, we addressed the direct infection of platelets by DENV, adhesion of platelets to leukocytes and vascular endothelium and platelet clearance mediated by anti-platelet antibodies. We also focused on platelet involvement on the dengue immunity and pathogenesis through translation and secretion of viral and host factors and through platelet-leukocyte aggregates formation. Hence, the present review highlights important findings related to platelet activation and thrombocytopenia during dengue infection, and also exhibits different mechanisms associated with decreased platelet counts. Graphical abstract: Schematic mechanistic representation of platelet-mediated immune responses and thrombocytopenia during dengue infection. (A) DENV-infected platelets secrete cytokines and chemokines and also adhere to activated vascular endothelium. Platelets aggregate with leukocytes, inducing the secretion of NETs and inflammatory mediators by neutrophils and monocytes, respectively. (B) DENV directly infects stromal cells and hematopoietic precursors, including megakaryocytes, which compromises megakaryopoiesis. Both central and peripheric mechanisms contribute to DENV-associated thrombocytopenia

Platelet function in HIV plus dengue coinfection associates with reduced inflammation and milder dengue illness

Submitted by Sandra Infurna ([email protected]) on 2019-09-10T15:09:30Z No. of bitstreams: 1 PatriciaTBozza_FernandoBozza_etal_IOC_2019.pdf: 2796563 bytes, checksum: b6576c01a5b8037869f23b18a03de3f7 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-09-10T15:21:35Z (GMT) No. of bitstreams: 1 PatriciaTBozza_FernandoBozza_etal_IOC_2019.pdf: 2796563 bytes, checksum: b6576c01a5b8037869f23b18a03de3f7 (MD5)Made available in DSpace on 2019-09-10T15:21:35Z (GMT). No. of bitstreams: 1 PatriciaTBozza_FernandoBozza_etal_IOC_2019.pdf: 2796563 bytes, checksum: b6576c01a5b8037869f23b18a03de3f7 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil / Universidade Federal de Juiz de Fora. Instituto de Ciências Biológicas. Departamento de Bioquímica. Laboratório de Análise de Gliconjugados. Juiz de Fora, MG, Brasil.Universidade Federal de Juiz de Fora. Instituto de Ciências Biológicas. Departamento de Bioquímica. Laboratório de Análise de Gliconjugados. Juiz de Fora, MG, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil. Rio de Janeiro, RJ. Brasil.University of Utah. Department of Internal Medicine. Molecular Medicine Program. Salt Lake City, Utah, USA.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Medicina Intensiva. Rio de Janeiro, RJ, Brasil / Instituto D’Or de Pesquisa e Ensino. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil.HIV-infected subjects under virological control still exhibit a persistent proinflammatory state. Thus, chronic HIV infection changes the host homeostasis towards an adapted immune response that may affect the outcome of coinfections. However, little is known about the impact of HIV infection on inflammatory amplification and clinical presentation in dengue. Platelets have been shown to participate in immune response in dengue and HIV. We hypothesized that altered platelet responses in HIV-infected subjects may contribute to altered inflammatory milieu and disease progression in dengue. We prospectively followed a cohort of 84 DENV-infected patients of whom 29 were coinfected with HIV under virological control. We report that dengue and HIV coinfection progress with reduced inflammation and milder disease progression with lower risk of vascular instability. Even though the degree of thrombocytopenia and platelet activation were similar between dengue-infected and HIV plus dengue-coinfected patients, plasma levels of the platelet-derived chemokines RANTES/CCL5 and PF4/CXCL4 were lower in coinfection. Consistently, platelets from coinfected patients presented defective secretion of the stored-chemokines PF4 and RANTES, but not newly synthesized IL-1β, when cultured ex vivo. These data indicate that platelets from HIV-infected subjects release lower levels of chemokines during dengue illness, which may contribute to milder clinical presentation during coinfection

Platelet–leukocyte interactions in the pathogenesis of viral infections

Evolving evidence demonstrates that platelets have major roles in viral syndromes through previously unrecognized viral sensing and effector functions. Activated platelets and increased platelet-leukocyte aggregates are observed in clinical and experimental viral infections. The mechanisms and outcomes of platelet–leukocyte interactions depend on the interacting leukocyte as well as on the pathogen and pathological conditions. In this review, we discuss the mechanisms involved in platelet interactions with leukocytes and its functions during viral infections. We focus on the contributions of human platelet–leukocyte interactions to pathophysiological and protective responses during viral infections of major global health relevance, including acquired immunodeficiency syndrome (AIDS), dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), influenza pneumonia, and COVID-19

Purificação da nucleosídeo trifosfato difosfohidrolase 1 (NTPDase 1) antigênica de Leishmania infantum

As NTPDases (nucleosídeo trifosfato difosfohidrolases) são enzimas que hidrolisam nucleosídeos di- e trifosfatados sob ativação de íons bivalentes. Uma atividade NTPDásica foi caracterizada em promastigotas de Leishmania infantum, um protozoário causador de leishmaniose visceral.Neste trabalho, a NTPDase 1 foi purificada de preparação de promastigotas usando como estratégia eletroforese em gel não-desnaturante e os detergentes deoxicolato de sódio e Triton X-100, os quais preservam a atividade fosfohidrolítica desta enzima. Após eletroforese, incubação do gel com ATP e inibidor de ATPases do tipo P permitiu a visualização de uma única banda com depósito branco de fosfato de cálcio. Esta banda ativa foi isolada e por SDS-PAGE e coloração pela prata foi identificada como polipeptídeos de 50 e 47 kDa. Por Western blot, os dois polipeptídeos foram reconhecidos pelo soro imune anti-LbB1LJ, o qual reconhece especificamente a porção N-terminal do domínio B de NTPDases 1 de Leishmania. Por espectrometria de massas, a identidade da NTPDase 1 foi confirmada pela identificação de seis peptídeos trípticos coincidentes com a NTPDase 1 anotada em seu genoma (NCBI gi|134068433), uma cobertura de 19% de sua sequência primária. As atividades ATPásica (555 ± 93 nmol Pi x mg-1 x min-1) e ADPásica (644 ± 38 nmol Pi x mg-1 x min-1) revelaram um índice de purificação de aproximadamente 62 vezes e uma razão de atividade ATPase/ADPase de aproximadamente 1, confirmando a efetividade desta estratégia para o isolamento da NTPDase 1 de promastigotas de L. infantum