Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability

Abstract

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease. Dengue is the most prevalent mosquito-borne disease in humans and represents a major public health problem worldwide. Leakage of fluids and molecules from the bloodstream into tissues can lead to shock and potentially death and is a critical determinant of dengue disease severity. Recently, we showed that a secreted protein from dengue virus (DENV)-infected cells, non-structural protein 1 (NS1), can trigger increased leakage both in human cell culture and mouse models. It has been shown that NS1 can activate toll-like receptor 4 on peripheral blood mononuclear cells, leading to secretion of pro-inflammatory cytokines that can result in vascular leak. However, the mechanism by which NS1 triggers hyperpermeability directly in human endothelial cells remained undefined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound molecules that lines endothelial cells on the inside of blood vessels, helping to regulate proper vascular function. Here, we show that DENV NS1 can disrupt the integrity of the EGL, inducing breakdown and shedding of key components. This is mediated by NS1 induction of cellular enzymes (e.g., sialidases, heparanase, and cathepsin L) that contribute to EGL alterations. Inhibitors that block these enzymes prevent both EGL disruption and endothelial permeability. These effects were all demonstrated to be specific to NS1 from DENV serotypes 1–4, as NS1 from the related West Nile Virus did not produce EGL alterations or increased leakage. Our study suggests a novel role for DENV NS1 in inducing EGL disruption to increase fluid leakage during severe dengue disease.