Anti–Dengue Virus Nonstructural Protein 1 Antibodies Cause NO-Mediated Endothelial Cell Apoptosis via Ceramide-Regulated Glycogen Synthase Kinase-3β and NF-κB Activation

Abstract

Immunopathogenetic mechanisms of dengue virus (DENV) infection are involved in hemorrhagic syndrome resulting from thrombocytopenia, coagulopathy, and vasculopathy. We have proposed a mechanism of molecular mimicry in which Abs against DENV nonstructural protein 1 (NS1) cross-react with human endothelial cells and cause NF-κB–regulated immune activation and NO-mediated apoptosis. However, the signaling pathway leading to NF-κB activation after the binding of anti-DENV NS1 Abs to endothelial cells is unresolved. In this study, we found that anti-DENV NS1 Abs caused the formation of lipid raftlike structures, and that disrupting lipid raft formation by methyl-β-cyclodextrin decreased NO production and apoptosis. Treatment with anti-DENV NS1 Abs elevated ceramide generation in lipid rafts. Pharmacological inhibition of acid sphingomyelinase (aSMase) decreased anti-DENV NS1 Ab-mediated ceramide and NO production, as well as apoptosis. Exogenous ceramide treatment induced biogenesis of inducible NO synthase (iNOS)/NO and apoptosis through an NF-κB–regulated manner. Furthermore, activation of glycogen synthase kinase-3β (GSK-3β) was required for ceramide-induced NF-κB activation and iNOS expression. Notably, anti-DENV NS1 Abs caused GSK-3β–mediated NF-κB activation and iNOS expression, which were regulated by aSMase. Moreover, pharmacological inhibition of GSK-3β reduced hepatic endothelial cell apoptosis in mice passively administered anti-DENV NS1 Abs. These results suggest that anti-DENV NS1 Abs bind to the endothelial cell membrane and cause NO production and apoptosis via a mechanism involving the aSMase/ceramide/GSK-3β/NF-κB/iNOS/NO signaling pathway.

Role of nitric oxide in immune responses against viruses: beyond microbicidal activity

“…Studies have shown that this cross-linking leads to high NO production, which increases the production of pro-apoptotic proteins, such as p53 and Bax, and decreases Bcl-2 and Bcl-xL. This increased apoptosis of endothelial cells was correlated directly with the severity of infection [37,38].…”

Antibodies Against Modified NS1 Wing Domain Peptide Protect Against Dengue Virus Infection

“…Passive transfer of anti-DENV NS1 Abs or active immunization with NS1 can protect mice from DENV challenge 25 , 27 – 29 . However, several Abs against NS1 show molecular mimicry with host proteins and can cross-react with platelets, endothelial cells and coagulation factors, causing platelet dysfunction, endothelial cell damage and hemorrhage 22 , 30 – 32 . These factors hinder the development of a potential NS1-based vaccine.…”

Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

“…GSK-3β has been reported as a pro-apoptotic protein in various cell types343536. A genetic approach was used to investigate the essential role of GSK-3β in ZA-induced apoptosis.…”