Dengue Virus Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent NETosis-Mediated Inflammation in Mice

Abstract

Abnormal immune responses and cytokine storm are involved in the development of severe dengue, a life-threatening disease with high mortality. Dengue virus-induced neutrophil NETosis response is associated with cytokine storm; while the role of viral factors on the elicitation of excessive inflammation mains unclear. Here we found that treatments of dengue virus envelope protein domain III (EIII), cellular binding moiety of virion, is sufficient to induce neutrophil NETosis processes in vitro and in vivo. Challenges of EIII in inflammasome Nlrp3−/− and Casp1−/− mutant mice resulted in less inflammation and NETosis responses, as compared to the wild type controls. Blockages of EIII-neutrophil interaction using cell-binding competitive inhibitor or selective Nlrp3 inflammasome inhibitors OLT1177 and Z-WHED-FMK can suppress EIII-induced NETosis response. These results collectively suggest that Nlrp3 inflammsome is a molecular target for treating dengue-elicited inflammatory pathogenesis.

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

“…To verify these pyroptosis results using different methods, we performed additional caspase-1 colorimetric assay, detections of cleaved GSDMD and cell-surface GSDMD levels, and the releases of IL-1β and LDH. Consistently, inflammasome inhibitors Z-WHED-FMK, OLT1177, and pyroptosis/GSDMD inhibitor DMF ( 14 , 50 ) markedly rescued rEIII-induced pyroptosis and damages of platelets ( Figures S11 , S12 ). These results collectively suggest that EIII induced an Nlrp3 inflammasome-dependent pyroptosis in platelets.…”

“…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.…”

Complex Roles of Neutrophils during Arboviral Infections

“…This NET formation was alleviated in neutrophils from NLRP3 inflammasome-deficient mice, decreasing inflammation. Blocking EIII-neutrophil interactions also suppressed the NETosis [83].…”