Neutrophil Extracellular Traps in Dengue Are Mainly Generated NOX-Independently

Abstract

Neutrophil extracellular traps (NETs) are increasingly recognized to play a role in the pathogenesis of viral infections, including dengue. NETs can be formed NADPH oxidase (NOX)-dependently or NOX-independently. NOX-independent NETs can be induced by activated platelets and are very potent in activating the endothelium. Platelet activation with thrombocytopenia and endothelial dysfunction are prominent features of dengue virus infection. We postulated that dengue infection is associated with NOX-independent NET formation, which is related to platelet activation, endothelial perturbation and increased vascular permeability. Using our specific NET assays, we investigated the time course of NET formation in a cohort of Indonesian dengue patients. We found that plasma levels of NETs were profoundly elevated and that these NETs were predominantly NOX-independent NETs. During early recovery phase (7-13 days from fever onset), total NETs correlated negatively with platelet number and positively with platelet P-selectin expression, the binding of von Willebrand factor to platelets and levels of Syndecan-1. Patients with gall bladder wall thickening, an early marker of plasma leakage, had a higher median level of total NETs. Ex vivo, platelets induced NOX-independent NET formation in a dengue virus non-structural protein 1 (NS1)-dependent manner. We conclude that NOX-independent NET formation is enhanced in dengue, which is most likely mediated by NS1 and activated platelets.

Keywords: NADPH-oxidase independent; NET formation; dengue; neutrophil extracellular traps; plasma leakage; platelets.

Figure 1

Circulating NETs in dengue patients are mainly NOX-independently generated. (A) Time course of total NETs (μg/mL) in plasma of dengue patients according to day since fever onset and in a group of healthy controls (HC). Depicted are individual data together with a box plot showing median with interquartile range. Differences in the dengue patients over time were analyzed using the Wilcoxon signed rank test; differences with the HC group were analyzed using Mann-Whitney U test; *p < 0.05, **p < 0.01, ***p < 0.001. (B) NET formation (ng/ml) after incubation of neutrophils with 10% plasma of dengue patients according to day since fever onset and of a group of HC. Depicted are individual data of five plasmas per group tested on 3 neutrophil donors, together with a box plot showing median with interquartile range. Differences with the HC group were analyzed using Mann-Whitney U test;, ***p < 0.001. (C) Scatterplot quadrant analysis to differentiate NOX-dependent and NOX-independent NET formation on the basis of results of the total NETs (X-axis) and MPO-H4K8Ac, K12Ac, K16Ac assay (Y-axis). Cut-off points were determined from mean values and standard deviations from HC samples (orange and blue-dotted lines). Samples positive to both assays were classified as NETs derived NOX-independently, while samples only positive for MPO-DNA were classified as NETs derived from NOX-dependently. HC, healthy controls; NOX, NADPH-oxidase.

Figure 2

Associations of total NETs with platelet parameters. Depicted are individual data points with the Spearman correlation between total NETs (μg/mL) and (A) platelet number (x10⁹/L), (B) platelet P-selectin expression (MFI) and (C) binding of von Willebrand factor (VWF) to platelets (MFI). Platelet P-selectin expression and binding of VWF to platelets were measured by flow cytometry and are expressed as mean fluorescence intensity (MFI).

Figure 3

Platelets stimulate NOX-independent NET formation in a DENV NS1-dependent manner. Polymorphonuclear neutrophils (PMNs) of healthy volunteers were co-incubated with autologous platelets in the presence or absence of 10 µg/mL DENV2 NS1 or 156μM platelet agonist TRAP-6. Experiments were performed in triplicate with 2 donors for each condition. Depicted are the relative values to medium (unstimulated samples) for (A) Total NETs and (B) NOX-independent NETs. (C) Heat-inactivated DENV2 does not induce NET formation. PMNs isolated from three healthy donors were incubated with 0.5x10⁷ TCID/mL heat-inactivated DENV2 or medium control (mock) in the presence or absence of autologus washed platelets (Plt). Depicted is the total NET formation (ng/mL). Data are presented as mean with standard error mean (SEM) and analyzed using repeated measure a one-way ANOVA and Bonferroni’s multiple comparisons test; *p < 0.05, **p < 0.01. TCID, Tissue culture infectious dose.

Figure 4

NET formation is associated with syndecan-1, as a surrogate marker for glycocalyx damage, and markers of plasma leakage. (A) Depicted is the Spearman correlation between total NETs (μg/mL) and plasma syndecan-1 concentrations (ng/mL) at different time points. (B) Total NETs (μg/mL) of dengue patients from either acute or critical phase were grouped according to the presence of hemoconcentration, pleural effusion/ascites or a thickened (>3 mm) gall bladder wall. Hemoconcentration was defined as an increase in hematocrit of ≥ 20%, or single hematocrit value of >50% for male or >44% for female patients. Differences were assessed using Mann-Whitney U test. **p < 0.01, ***p < 0.001, ****p < 0.0001. HC, healthy controls.