Dengue virus–elicited tryptase induces endothelial permeability and shock
Abhay P.S. Rathore, Chinmay Kumar Mantri, Siti A.B. Aman, Ayesa Syenina, Justin Ooi, Cyril J. Jagaraj, Chi Ching Goh, Hasitha Tissera, Annelies Wilder-Smith, Lai Guan Ng, Duane J. Gubler, Ashley L. St. John
Abhay P.S. Rathore, Chinmay Kumar Mantri, Siti A.B. Aman, Ayesa Syenina, Justin Ooi, Cyril J. Jagaraj, Chi Ching Goh, Hasitha Tissera, Annelies Wilder-Smith, Lai Guan Ng, Duane J. Gubler, Ashley L. St. John
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Research Article Infectious disease Vascular biology

Dengue virus–elicited tryptase induces endothelial permeability and shock

Abstract

Dengue virus (DENV) infection causes a characteristic pathology in humans involving dysregulation of the vascular system. In some patients with dengue hemorrhagic fever (DHF), vascular pathology can become severe, resulting in extensive microvascular permeability and plasma leakage into tissues and organs. Mast cells (MCs), which line blood vessels and regulate vascular function, are able to detect DENV in vivo and promote vascular leakage. Here, we showed that an MC-derived protease, tryptase, is consequential for promoting vascular permeability during DENV infection through inducing breakdown of endothelial cell tight junctions. Injected tryptase alone was sufficient to induce plasma loss from the circulation and hypovolemic shock in animals. A potent tryptase inhibitor, nafamostat mesylate, blocked DENV-induced vascular leakage in vivo. Importantly, in 2 independent human dengue cohorts, tryptase levels correlated with the grade of DHF severity. This study defines an immune mechanism by which DENV can induce vascular pathology and shock.

Authors

Abhay P.S. Rathore, Chinmay Kumar Mantri, Siti A.B. Aman, Ayesa Syenina, Justin Ooi, Cyril J. Jagaraj, Chi Ching Goh, Hasitha Tissera, Annelies Wilder-Smith, Lai Guan Ng, Duane J. Gubler, Ashley L. St. John

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Figure 1

Tryptase and chymase break tight junctions to disrupt endothelial cell contact sites.

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Tryptase and chymase break tight junctions to disrupt endothelial cell c...
(A) DENV-stimulated MC supernatant was transferred onto huMEC monolayers or separated into soluble and particulate fractions, followed by transfer onto huMEC monolayers. For controls, DENV-alone or media-alone groups were used. TER of huMEC monolayers was measured 24 hours after treatment. Whole supernatant or isolated MC particles each significantly reduced the TER of huMEC monolayers (P < 0.05 by 1-way ANOVA with Dunnett’s post test). (B and C) TER of huMEC monolayers after treatment for 24 hours with either purified, recombinant tryptase (B) or chymase (C). *P < 0.05, decrease in TER over controls by 1-way ANOVA with Dunnett’s post test (A–C). (D–F) huMECs were treated with low (0.1 μM) or high (1 μM) concentrations of either tryptase or chymase for 24 hours, followed by fixation and staining against tubulin (green), nuclei using DAPI (blue), and tight junctions (ZO-1, red). (D) In control cells, tight junctions were intact in between cells, visualized by ZO-1 staining. (E) Tryptase induced a concentration-dependent reduction in ZO-1 staining that appeared disjunctive at low concentrations and absent at high concentrations. Lifting of cells forming gaps was also observed after high-concentration tryptase treatment. (F) Low-concentration chymase had no apparent effect on tight junctions, while staining grew more punctate at high concentrations. For A–F, data are representative of 3 independent repeats. Scale bars: 25 μm. (G and H) Levels of CD31 on endothelial cells were measured by flow cytometry on cells isolated from mouse footpads 6 hours after injection of 100 ng of tryptase, chymase, or saline vehicle control. (G) CD31+ cells showed reduced levels of staining after injection of tryptase (representative histogram plots). (H) Comparison of MFI of CD31 staining in mouse footpads (n = 5–6 each group) showed that tryptase, but not chymase, is sufficient to induce a significant decrease in CD31 staining in vivo (right panel, P < 0.05, 1-ANOVA with Dunnett’s post test). For graphs, error bars represent SEM.