Dengue induces iNOS expression and nitric oxide synthesis in platelets through IL-1R.

Introduction: Dengue is an arthropod-born disease caused by dengue virus (DENV), that may manifest as a mild illness or severe form, characterized by hemorrhagic fever and shock. Nitric oxide (NO) is a vasodilator signaling molecule and an inhibitor of platelet aggregation known to be increased in platelets from dengue patients. However, the mechanisms underlying NO synthesis by platelets during dengue are not yet elucidated. IL-1ß is a pro-inflammatory cytokine able to induce iNOS expression in leukocytes and present in dengue patients at high levels. Nevertheless, the role of IL-1ß in platelet activation, especially regarding iNOS expression, are not clear. Methods: We prospectively followed a cohort of 28 dengue-infected patients to study NO synthesis in platelets and its relationship with disease outcomes. We used in vitro infection and stimulation models to gain insights on the mechanisms. Results and Discussion: We confirmed that platelets from dengue patients express iNOS and produce higher levels of NO during the acute phase compared to healthy volunteers, returning to normal levels after recovery. Platelet NO production during acute dengue infection was associated with the presence of warning signs, hypoalbuminemia and hemorrhagic manifestations, suggesting a role in dengue pathophysiology. By investigating the mechanisms, we evidenced increased iNOS expression in platelets stimulated with dengue patients´ plasma, indicating induction by circulating inflammatory mediators. We then investigated possible factors able to induce platelet iNOS expression and observed higher levels of IL-1ß in plasma from patients with dengue, which were correlated with NO production by platelets. Since platelets can synthesize and respond to IL-1ß, we investigated whether IL-1ß induces iNOS expression and NO synthesis in platelets. We observed that recombinant human IL-1ß enhanced iNOS expression and dose-dependently increased NO synthesis by platelets. Finally, platelet infection with DENV in vitro induced iNOS expression and NO production, besides the secretion of both IL-1α and IL-1ß. Importantly, treatment with IL-1 receptor antagonist or a combination of anti-IL-1α and anti-IL-1ß antibodies prevented DENV-induced iNOS expression and NO synthesis. Our data show that DENV induces iNOS expression and NO production in platelets through mechanisms depending on IL-1 receptor signaling.

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-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.

Innate immune receptors in platelets and platelet-leukocyte interactions.

Platelets are chief cells in hemostasis. Apart from their hemostatic roles, platelets are major inflammatory effector cells that can influence both innate and adaptive immune responses. Activated platelets have thromboinflammatory functions linking hemostatic and immune responses in several physiological and pathological conditions. Among many ways in which platelets exert these functions, platelet expression of pattern recognition receptors (PRRs), including TLR, Nod-like receptor, and C-type lectin receptor families, plays major roles in sensing and responding to pathogen-associated or damage-associated molecular patterns (PAMPs and DAMPs, respectively). In this review, an increasing body of evidence is compiled showing the participation of platelet innate immune receptors, including PRRs, in infectious diseases, sterile inflammation, and cancer. How platelet recognition of endogenous DAMPs participates in sterile inflammatory diseases and thrombosis is discussed. In addition, platelet recognition of both PAMPs and DAMPs initiates platelet-mediated inflammation and vascular thrombosis in infectious diseases, including viral, bacterial, and parasite infections. The study also focuses on the involvement of innate immune receptors in platelet activation during cancer, and their contribution to tumor microenvironment development and metastasis. Finally, how innate immune receptors participate in platelet communication with leukocytes, modulating leukocyte-mediated inflammation and immune functions, is highlighted. These cell communication processes, including platelet-induced release of neutrophil extracellular traps, platelet Ag presentation to T-cells and platelet modulation of monocyte cytokine secretion are discussed in the context of infectious and sterile diseases of major concern in human health, including cardiovascular diseases, dengue, HIV infection, sepsis, and cancer.

Inflammatory signaling in dengue-infected platelets requires translation and secretion of nonstructural protein 1.

Emerging evidence identifies major contributions of platelets to inflammatory amplification in dengue, but the mechanisms of infection-driven platelet activation are not completely understood. Dengue virus nonstructural protein-1 (DENV NS1) is a viral protein secreted by infected cells with recognized roles in dengue pathogenesis, but it remains unknown whether NS1 contributes to the inflammatory phenotype of infected platelets. This study shows that recombinant DENV NS1 activated platelets toward an inflammatory phenotype that partially reproduced DENV infection. NS1 stimulation induced translocation of α-granules and release of stored factors, but not of newly synthesized interleukin-1ß (IL-1ß). Even though both NS1 and DENV were able to induce pro-IL-1ß synthesis, only DENV infection triggered caspase-1 activation and IL-1ß release by platelets. A more complete thromboinflammatory phenotype was achieved by synergistic activation of NS1 with classic platelet agonists, enhancing α-granule translocation and inducing thromboxane A2 synthesis (thrombin and platelet-activating factor), or activating caspase-1 for IL-1ß processing and secretion (adenosine triphosphate). Also, platelet activation by NS1 partially depended on toll-like receptor-4 (TLR-4), but not TLR-2/6. Finally, the platelets sustained viral genome translation and replication, but did not support the release of viral progeny to the extracellular milieu, characterizing an abortive viral infection. Although DENV infection was not productive, translation of the DENV genome led to NS1 expression and release by platelets, contributing to the activation of infected platelets through an autocrine loop. These data reveal distinct, new mechanisms for platelet activation in dengue, involving DENV genome translation and NS1-induced platelet activation via platelet TLR4.

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

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-1ß, 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.