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.

Keywords: Nlrp3 inflammasome; apoptosis; dengue hemorrhage fever; envelope protein domain III; ferroptosis; necroptosis; platelet; pyroptosis.

Figure 1

Platelet-binding property of DENV and different recombinant DENV envelope protein (rE) fragments. Flow cytometry analysis of the platelet-binding properties of biotinylated DENV and recombinant DENV envelope proteins (rEs) through PE/Cy5-avidin labeling. (A) Platelet PE/Cy5-avidin binding signal served as the background level. The respective blots and histograms on the platelet-binding of (B) DENV, (C) rE (domain III), (D) rE (domain I + II + III), (E) rE (domain I + II), and (F) rE (domain II + III) were indicated, and (G) quantified. n = 6, *P < 0.05, **P < 0.01 vs. avidin groups; ^## P < 0.01 vs. rEIII groups. DENV2 PL046 viral particles were used in this and the following figures.

Figure 2

Challenges of rEIII-induced thrombocytopenia and hypercoagulation in mice. (A) Experiment outline. (B) RBC counts, (C) platelet counts, and (D) aPTT measures 1-3 d after mice were exposed to DENV particles (1.2 × 10⁷ PFU/kg; approximately 3 × 10⁵ PFU/25 g mouse) and rEIII (2 mg/kg). Compared with rGST treatments, injections of both DENV and rEIII induced (C) thrombocytopenia and (D) hypercoagulation (shortened aPTT plasma clotting time) in mice. *P < 0.05, **P < 0.01 vs. respective rGST groups; n = 6 (three experiments with two mice per group). The mouse drawing used in this and following figures was originally published in the Blood journal: Huang, H. S., Sun, D. S., Lien, T. S. and Chang, H. H. Dendritic cells modulate platelet activity in IVIg-mediated amelioration of ITP in mice. Blood, 2010; 116: 5002–5009. ^© the American Society of Hematology.

Figure 3

DENV- and rEIII-induced RCD in platelets. (A) Washed mouse platelets treated with vehicle and various doses of DENV and rEIII; the live and dead cell populations were estimated using Zombie-NIR Kit labeling and flow cytometry analysis. [rEIII: 1× = 0.3 μM, 2× = 0.6 μM, 4× = 1.2 μM; DENV e(rEIII 1×) is a DENV level equivalent to 0.3 μM rEIII, as indicated by the methods described in Figure S8 ]. (B) RCD inducers, doxorubicin (DOX; apoptosis) (2.5 μg/ml), rapamycin (autophagy) (0.5 μM), erastin (ferroptosis) (10 μM), TNF-α (necroptosis) (2.5 ng/ml), and nigericin (pyroptosis) (3.5 μM) induced relatively simple RCD patterns. By contrast, DENV and rEIII induced multiple RCD pathways, in which pyroptosis is the major RCD response, with counts approximately 60% of total RCD (B). If we normalize the respective RCD by the population of death cells (dead cell population normalized to 100%), we can obtained a more similar RCD pattern in DENV and rEIII groups (C). *P < 0.05, **P < 0.01 vs. vehicle groups.

Figure 4

Treatment with Nlrp3-inflammasome inhibitors protects wild type (WT) mouse platelets (PLT) from DENV- (equivalent to 0.6 μM rEIII) and rEIII (0.6 μM)-induced pyroptosis. Treatment with Nlrp3 inhibitor OLT1177 (100 μM) and caspase 1 inhibitor Z-WHED-FMK (100 μM) rescued rEIII-induced platelet cell death (A). Treatments with both OLT1177 and Z-WHED-FMK rescued % of RCDs (B, C, F, G, J, K). Intriguingly, if we normalize the respective RCD % by the population of death cells (D; dead cell population normalized to 100%), we found that OLT1177 and Z-WHED-FMK preferentially rescued (E) pyroptosis, (H, I, L, M) but not the other tested RCDs. n = 6, *P < 0.05, **P < 0.01 vs. vehicle groups.

Figure 5

Nlrp3 and caspase-1 deficiencies protect platelets from DENV (equivalent to 0.6 μM rEIII) - and rEIII (0.6 μM)-induced RCD. (A) Compared with wild type (WT) controls, platelets from Nlrp3 (Nlrp3^−/−) and caspase 1 (Casp1^−/−) deficient mice displayed less cell death levels in response to DENV and rEIII exposures. (B) Similar to the cell death analysis, platelets from Nlrp3 (Nlrp3^−/−) and caspase 1 (Casp1^−/−) deficient mice displayed less pyroptosis levels in response to DENV and rEIII challenges. n = 6, *P < 0.05, **P < 0.01 vs. WT groups.

Figure 6

Nlrp3 and caspase-1 inhibitors protect platelets from DENV- and rEIII-induced activation. DENV- and rEIII-induced platelet activation, including (A, B) platelet aggregation, (C, D) platelet surface P-selectin expression, and (E, F) mitochondrial superoxide levels, could be suppressed with the treatments with Nlrp3 and caspase 1 inhibitors Z-WHED-FMK (10 μM) and OLT1177 (10 μM), respectively (B, D, F). n = 6, *P < 0.05, **P < 0.01 vs. respective vehicle groups.

Figure 7

Nlrp3 inhibitor treatments ameliorate DENV- and rEIII-induced thrombocytopenia in mice. Treatments with Nlrp3 inhibitor OLT1177 (50 mg/kg) ameliorated DENV (3 × 10⁵ PFU/mouse; DHF viral load)- and rEIII (2 mg/kg)-induced thrombocytopenia in C57BL/6J mice. n = 6, ^## P < 0.01 vs. untreated groups; **P < 0.01 vs. respective vehicle groups.