Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity

Dengue virus NS1 protein induces inflammatory responses via TLR4 and disrupts endothelial cell monolayer integrity. A leak in the dike Everyone knows how mosquitos can wreck an end-of-summer picnic. But in some climates, these pesky intruders persist and carry a variety of detrimental diseases—some with no preventative vaccines or targeted therapies. One such passenger is dengue virus (DENV), which infects up to 400 million people each year and comes in several serotypes (1 to 4) and disease presentations—from mild infection to severe disease and sometimes death. But to treat or prevent dengue requires that we have a more complete picture of the disease pathology. Now, Modhiran et al. and Beatty et al. describe the results of in vitro and in vivo experiments that point to circulating dengue virus non-structural protein 1 (NS1) and the innate immune Toll-like receptor 4 (TLR4) as a focus for basic scientists as well as vaccine and drug developers. DENV infection protects a patient from future reinfection with the same DENV serotype as well as producing temporary immune protection from severe dengue disease caused by a different DENV serotype. But unlike diamonds, this immune protection doesn’t last forever, and when the protected period passes, the patient becomes at increased risk of enhanced infection and progression to severe disease if he or she is infected with a second DENV serotype. This severe form of dengue infection is believed to result from immunopathogenic processes that induce cytokine storm and cause vascular leakage that leads to shock. Until now, no dengue viral proteins have been linked to vascular endothelium permeability (that is, vascular leakage). Beatty et al. show that inoculation of mice with DENV NS1 protein alone induces both vascular leak and secretion of inflammatory cytokines and that administration of NS1 with a sublethal dose of DENV2 leads to lethal vascular leak syndrome. In human endothelial cell monolayers in culture, NS1 from any of the four DENV serotypes triggered endothelial barrier permeability. NS1’s pathogenic effects were blocked by NS1-immune polyclonal mouse serum or monoclonal antibodies to NS1 (in vivo and in vitro), and immunization of mice with NS1 protected against lethal DENV2 challenge. In an independent study, Mondrian et al. explore the underlying mechanism of NS1’s effects. They show that highly purified NS1 acts as a pathogen-associated molecular pattern (PAMP) that activates mouse macrophages and human peripheral blood mononuclear cells (PBMCs) in culture via TLR4, resulting in release of inflammatory cytokines—an effect that was blocked by either a TLR4 antagonist or an anti-TLR4 antibody. Then, in an in vitro model of vascular leak, the authors found that NS1 fractured the integrity of endothelial cell monolayers through a TLR4-dependent pathway, a finding that was supported by the observation that a TLR4 antagonist quelled capillary leak in a mouse model of dengue virus infection. Together, these new findings highlight NS1 as an instigator of dengue-associated vascular leak and thus pinpoint a potential target for dengue drugs and component for dengue vaccines. Complications arising from dengue virus infection include potentially fatal vascular leak, and severe disease has been linked with excessive immune cell activation. An understanding of the triggers of this activation is critical for the development of appropriately targeted disease control strategies. We show here that the secreted form of the dengue virus nonstructural protein 1 (NS1) is a pathogen-associated molecular pattern (PAMP). Highly purified NS1 devoid of bacterial endotoxin activity directly activated mouse macrophages and human peripheral blood mononuclear cells (PBMCs) via Toll-like receptor 4 (TLR4), leading to the induction and release of proinflammatory cytokines and chemokines. In an in vitro model of vascular leak, treatment with NS1 alone resulted in the disruption of endothelial cell monolayer integrity. Both NS1-mediated activation of PBMCs and NS1-induced vascular leak in vitro were inhibited by a TLR4 antagonist and by anti-TLR4 antibody treatment. The importance of TLR4 activation in vivo was confirmed by the reduction in capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. These results pinpoint NS1 as a viral toxin counterpart of the bacterial endotoxin lipopolysaccharide (LPS). Similar to the role of LPS in septic shock, NS1 might contribute to vascular leak in dengue patients, which highlights TLR4 antagonists as a possible therapeutic option.