Exposure of Platelets to Dengue Virus and Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Platelet Cell Death and Thrombocytopenia in Mice

Abstract

In tropical and subtropical regions, mosquito-borne dengue virus (DENV) infections can lead to severe dengue, also known as dengue hemorrhage fever, which causes bleeding, thrombocytopenia, and blood plasma leakage and increases mortality. Although DENV-induced platelet cell death was linked to disease severity, the role of responsible viral factors and the elicitation mechanism of abnormal platelet activation and cell death remain unclear. DENV and virion-surface envelope protein domain III (EIII), a cellular binding moiety of the virus particle, highly increase during the viremia stage. Our previous report suggested that exposure to such viremia EIII levels can lead to cell death of endothelial cells, neutrophils, and megakaryocytes. Here we found that both DENV and EIII could induce abnormal platelet activation and predominantly necrotic cell death pyroptosis. Blockages of EIII-induced platelet signaling using the competitive inhibitor chondroitin sulfate B or selective Nlrp3 inflammasome inhibitors OLT1177 and Z-WHED-FMK markedly ameliorated DENV- and EIII-induced thrombocytopenia, platelet activation, and cell death. These results suggest that EIII could be considered as a virulence factor of DENV, and that Nlrp3 inflammasome is a feasible target for developing therapeutic approaches against dengue-induced platelet defects.

Nanodiamond-Induced Thrombocytopenia in Mice Involve P-Selectin-Dependent Nlrp3 Inflammasome-Mediated Platelet Aggregation, Pyroptosis and Apoptosis

“…Collected mouse blood samples were transferred into polypropylene tubes containing anticoagulant acid-citrate-dextrose solution (38 mM citric acid, 75 mM sodium citrate, and 100 mM dextrose) ( 64 , 65 ). Washed platelets were prepared as previously described ( 19 , 38 ). Platelet counts of mice were measured using a hematology analyzer (KX-21N; Sysmex, Kobe, Japan) ( 64 – 66 ).…”

“…To distinguish populations of platelets, NDs and platelet-ND aggregates, CellTracker Blue Dye (ThermoFisher Scientific) labeled mouse platelets, and red fluorescent 50 nm NDs (brFND-50, FND Biotech) were used in this experiment. The counts of platelet aggregates per field (> 400 pixels) and the total platelet aggregate area (pixels) per field were analyzed using ImageJ software (version 1.32; National Institutes of Health, USA) ( 38 , 68 ).…”

“…In addition, platelet inflammasome activation has been revealed in sepsis ( 36 ), thrombosis formation ( 37 ) and hindlimb ischemia ( 32 ) models. Despite the detailed mechanism remains to be further elucidated, activation of inflammasome by dengue virus has been associated with induction of platelet pyroptosis ( 38 ). Pyroptosis belongs to the family of regulated cell deaths (RCDs), which include additional cell death pathways such as apoptosis, necroptosis, ferroptosis and autophagy ( 39 – 41 ).…”

Activating Transcription Factor 3 Protects against Restraint Stress-Induced Gastrointestinal Injury in Mice

“…Evans blue (1.2 g/kg, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was fed to the mice using a steel feeding tube 4 h after the commencement of stress challenge. Their blood plasma was isolated by collecting blood in an Eppendorf tube and mixing it with an equal proportion of anticoagulant citrate dextrose solution to prevent coagulation [ 41 , 42 , 43 ]. The collected plasma was transferred to 96-well plates, in which the concentration of Evans blue was determined using a full-spectrum analyzer (Multiskan Spectrum, Thermo Fisher Scientific, Waltham, MA, USA) at 620 nm.…”

Platelet Innate Immune Receptors and TLRs: A Double-Edged Sword

“…Moreover, NLRP3 inflammasome activation has also been evidenced in platelets from dengue-infected patients and from healthy controls exposed in vitro to the virus, which results from the increased ROS production by platelet mitochondria (via the RIP-1/RIP-3 kinase pathway) and leads to vascular leakage and monocyte activation [ 7 , 223 ]. Very recently, NLRP3 activation was confirmed in platelets upon infection with dengue virus, and the involvement of the envelope protein domain III has been evidenced as an important inducer of platelet pyroptosis related to NLRP3 activation [ 224 ]. In addition to DC-SIGN, CLEC-2 has been demonstrated to participate in platelet activation by dengue virus, as reflected not only by the expression of activation markers, but also by the release of extracellular vesicles capable of activating neutrophils, causing the formation of NETs, and increasing the rate of mouse death.…”