Date Published: April 22, 2019
Publisher: Public Library of Science
Author(s): Chiao-Hsuan Chao, Wei-Chueh Wu, Yen-Chung Lai, Pei-Jane Tsai, Guey-Chuen Perng, Yee-Shin Lin, Trai-Ming Yeh, Richard J. Kuhn.
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.
Dengue is the most widespread mosquito-borne viral infection, infecting approximately 390 million people and causing 500,000 hospitalizations every year [1, 2]. Most infected patients have a mild, self-limited disease known as dengue without warning signs, but some can develop severe dengue, which is characterized by plasma leakage, fluid accumulation, severe bleeding, and organ impairment . The underlying mechanisms by which mild dengue progresses to severe dengue are still unclear [4–6]. Thrombocytopenia, a common feature observed in both mild and severe dengue disease, is correlated with disease severity and is considered a predictive biomarker of severe dengue [7, 8]. However, the mechanisms that cause thrombocytopenia during dengue infection are not fully understood.
In this study, we demonstrated that DENV NS1 in the concentration, which is within the range of NS1 in dengue patients’ sera (0.01–50 μg/ml), could activate platelets and induce apoptosis in a dose-dependent manner through TLR4 signal transduction. In addition, the aggregation of NS1-activated platelets in response to subthreshold concentrations of ADP was enhanced. The adhesion of NS1-activated platelets to endothelial cells and the phagocytosis of these platelets by THP-1 cells were also increased. Because NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release, all these effects induced by NS1 may contribute to platelet activation and thrombocytopenia during DENV infection (Fig 10).