Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice

Abstract

Typically occurring during secondary dengue virus (DENV) infections, dengue hemorrhagic fever (DHF) causes abnormal immune responses, as well as endothelial vascular dysfunction, for which the responsible viral factor remains unclear. During peak viremia, the plasma levels of virion-associated envelope protein domain III (EIII) increases to a point at which cell death is sufficiently induced in megakaryocytes in vitro. Thus, EIII may constitute a virulence factor for endothelial damage. In this study, we examined endothelial cell death induced by treatment with DENV and EIII in vitro. Notably, pyroptosis, the major type of endothelial cell death observed, was attenuated through treatment with Nlrp3 inflammasome inhibitors. EIII injection effectively induced endothelial abnormalities, and sequential injection of EIII and DENV-NS1 autoantibodies induced further vascular damage, liver dysfunction, thrombocytopenia, and hemorrhage, which are typical manifestations in DHF. Under the same treatments, pathophysiological changes in the Nlrp3 inflammasome–deficient mice were notably reduced compared with those in the wild-type mice. These results suggest that the Nlrp3 inflammasome constitutes a potential therapeutic target for treating DENV-induced hemorrhage in DHF.

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

“…However, data obtained in this present study suggested that virion-surface EIII is a candidate virulence factor that contributes to dengue-elicited thrombocytopenia. In addition, similar to soluble NS1, virion-surface EIII is a virulence factor that contributes to the first-hit in our two-hit DHF model ( 13 , 16 ).…”

“…Here we found that in agreement with platelet activation data ( Figures 1 and S3 – S5 ), treatments with DENV and rEIII induced platelet cell death in a dose-dependent manner ( Figure 3A ). Various cell death inducers, including nigericin (pyroptosis), doxorubicin (apoptosis), rapamycin (autophagy), TNF- α (necroptosis), and erastin (ferroptosis) roughly induced the respective cell death pathway of tested platelets ( Figure 3B , RCD % of total cells; 3C, RCD % of total dead cells; Figure S9 , flow cytometry gating and calculations) ( 13 , 14 ). Intriguingly, when compared with these cell-death agonists, treatments with DENV and rEIII induced considerable pyroptosis, necroptosis, and ferroptosis responses of platelets but only minor levels of apoptosis ( Figures 3B, C ) .…”

“…In this present report, we found that DENV and EIII but not the other cell death inducers, induced a similar RCD pattern in platelets ( Figures 3B, C ) . Although further investigations are needed, this platelet cell-type-specific RCD patterns (CTS-RCDPs) ( 13 , 14 ) may be also useful on the characterization of specific pathway inhibitors; as inflammasome inhibitors OLT1177 and Z-WHED-FMK preferentially blocked pyroptosis, but not ferroptosis, apoptosis, and autophagy ( Figure 4E , vs. Figures 4I, L, M ) .…”

“…Pieces of evidence have shown that DENV-induces activation of inflammasome complexes ( 11 , 12 ). Our previous study revealed that challenges with the DHF-viral-load-equivalent levels of EIII can induce cell death in endothelial cells ( 13 ) and neutrophils ( 14 ), and suppress megakaryopoiesis through autophagy impairment and apoptosis ( 15 ). These results suggest that EIII binding to platelets could be cytotoxic, particularly when circulating virion-surface EIII reaches the peak of the viremia stage.…”

“…These results suggest that EIII binding to platelets could be cytotoxic, particularly when circulating virion-surface EIII reaches the peak of the viremia stage. In addition, because gene deficiencies in Nlrp3 inflammasome components display protective effects on the hemorrhage fever mouse model ( 13 , 16 ), we further investigated whether Nlrp3 inflammasome contributes to EIII-induced platelet defects. In this study, EIII treatments directly caused the activation and death of platelets in vitro and thrombocytopenia in mice.…”