Investigating the role of neutrophils in Dengue virus infection

Abstract

Dengue virus (DENV), a flavivirus endemic in tropical/sub-tropical regions, threatens four billion people globally. Clinical outcomes range from asymptomatic to Severe Dengue (SD), a potentially fatal condition characterised by increased endothelial permeability, resulting in plasma leakage. Neutrophils, the most numerous immune cells, contribute to vascular leakage in inflammatory diseases. This thesis investigates the hypothesis that neutrophils are activated by DENV infection, that an exaggerated neutrophil response in SD enhances endothelial permeability and that DENV infection impacts neutrophil differentiation. Both the effect of DENV on healthy donor-derived neutrophils ex vivo, and the cellular and protein profiles of DENV-infected patient blood samples were analysed.

No evidence was found that DENV infects neutrophils, or neutrophil precursors, unlike other myeloid cells. High levels of neutrophil activation were identified by flow cytometry in DENV-infected patients with both mild disease and SD, with an increased immature neutrophil population present in SD patients.

Proteins predicted to be released by DENV-activated neutrophils or other closely related myeloid cells, were identified using mass spectrometry of patient serum samples; 35 such proteins were significantly elevated in SD compared to mild disease, including some associated with endothelial dysfunction in other pathologies. Trans-endothelial electrical resistance assays demonstrated that key proteins negatively modulate permeability of a vein endothelial cell monolayer, mimicking an SD hallmark.

In neutrophil precursors generated from stem cell differentiations, DENV caused immune activation and altered proliferation with limited alteration in cell surface marker expression. Transcriptomic analysis identified a substantial innate immune signalling response to DENV, alongside alterations in neutrophil differentiation pathways.

This thesis furthers knowledge of the role of neutrophils in Dengue disease by demonstrating that DENV infection activates neutrophils, affects neutrophil development and induces an innate immune response. These findings indicate that differential neutrophil responses should be considered significant for future investigations into treating SD.