Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

Abstract

Dengue virus (DENV) infection, the most common mosquito-transmitted viral infection, can cause a range of diseases from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and shock syndrome. Thrombocytopenia is a major characteristic observed in both mild and severe dengue disease and is significantly correlated with the progression of dengue severity. Previous studies have shown that DENV nonstructural protein 1 (NS1), which can be secreted into patients’ blood, can stimulate immune cells via Toll-like receptor 4 (TLR4) and can cause endothelial leakage. However, it is unclear whether DENV NS1 can directly induce platelet activation or cause thrombocytopenia during DENV infection. In this study, we first demonstrated that DENV but not Zika virus cell culture supernatant could induce P-selectin expression and phosphatidylserine (PS) exposure in human platelets, both of which were abolished when NS1 was depleted from the DENV supernatant. Similar results were found using recombinant NS1 from all four serotypes of DENV, and those effects were blocked in the presence of anti-NS1 F(ab’) ₂, anti-TLR4 antibody, a TLR4 antagonist ( Rhodobacter sphaeroides lipopolysaccharide, LPS-Rs) and a TLR4 signaling inhibitor (TAK242), but not polymyxin B (an LPS inhibitor). Moreover, the activation of platelets by DENV NS1 promoted subthreshold concentrations of adenosine diphosphate (ADP)-induced platelet aggregation and enhanced platelet adhesion to endothelial cells and phagocytosis by macrophages. Finally, we demonstrated that DENV-induced thrombocytopenia and hemorrhage were attenuated in TLR4 knockout and wild-type mice when NS1 was depleted from DENV supernatant. Taken together, these results suggest that the binding of DENV NS1 to TLR4 on platelets can trigger its activation, which may contribute to thrombocytopenia and hemorrhage during dengue infection.

Author summary

Over the past 50 years, dengue has been a continuing global threat, with no effective vaccine or specific antiviral drug. Dengue infection causes a wide range of outcomes, from fever-like symptoms to severe dengue hemorrhagic fever. Thrombocytopenia, a reduction in platelet count, is a common feature observed in both mild and severe dengue and is correlated with disease severity. In this study, we used dengue viral supernatant or DENV recombinant NS1 protein to stimulate human-isolated platelets. We found that DENV NS1 could directly activate platelets through TLR4 and could further enhance platelet aggregation, adhesion to endothelial cells and phagocytosis by macrophages, which could lead to thrombocytopenia. We also proved that both NS1 and TLR4 are critical for DENV-induced thrombocytopenia and hemorrhage using a DENV-induced hemorrhagic mouse model. Our study reveals a new pathogenic role of NS1 during dengue infection and highlights that NS1 should be a topic of attention in the development of therapeutic drugs and vaccines against dengue infection.