Research Article: Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability

Date Published: July 14, 2016

Publisher: Public Library of Science

Author(s): Henry Puerta-Guardo, Dustin R. Glasner, Eva Harris, Richard J. Kuhn.


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.

Partial Text

The four dengue virus serotypes (DENV1-4) are mosquito-borne flaviviruses that are responsible for ~390 million infections per year worldwide [1]. Of these, up to 96 million manifest in clinical disease. The majority of these cases are dengue fever (DF), the uncomplicated form of disease. However, a subset develop severe dengue disease, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), characterized by increased vascular leak, leading to shock and potentially death [2]. Pleural effusion resulting in respiratory distress is one of the most common signs of plasma leakage in DHF/DSS cases [3].

Secondary DENV infection with a serotype different from primary infection is considered an epidemiological risk factor for severe disease. Immune responses after primary DENV infection lead to protective immunity against homologous re-infection but may either protect against or cause increased disease severity in a subsequent DENV infection with a different serotype [34]. The latter is thought to be mediated by serotype cross-reactive T cells or antibody-dependent enhancement that triggers an exaggerated and skewed immune response to a previously infecting serotype, resulting in a “cytokine storm”, including TNF-α and IL-6, that leads to endothelial permeability and vascular leak [7]. New evidence has demonstrated the ability of DENV NS1 to directly induce release of vasoactive cytokines via TLR4 stimulation of PBMCs, leading to the disruption of endothelial barrier function in vitro and increased vascular leakage in vivo [9, 10]. However, NS1-mediated mechanisms specific to the endothelial barrier itself have yet to be defined. Here, we show that binding of DENV NS1 to endothelial cells triggers endothelial barrier dysfunction through alterations to the EGL. DENV NS1 induces the degradation of Sia, a major constituent of the EGL, an effect that is mediated by cellular sialidases. Further, DENV NS1 increases the activity of cathepsin L, which subsequently increases expression and activation of heparanase in endothelial cells, leading to shedding of heparan sulfate proteoglycans from the EGL, thus altering its integrity. Inhibition of sialidases or the cathepsin L-heparanase pathway prevents DENV NS1-mediated disruption of the EGL as well as endothelial hyperpermeability. These results were observed during treatment with amounts of DENV NS1 similar to levels reported in DHF/DSS patients [14, 15] and suggest a novel mechanism whereby soluble NS1 directly interacts with endothelial cells, inducing the activation of endothelial cell-intrinsic pathways that lead to hyperpermeability. A model summarizing these findings is shown in Fig 8.




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