Role of neutrophil extracellular vesicles in leukocyte-mediated pulmonary inflammation and injury during sepsis
File(s)
Author(s)
Sachouli, Eirini
Type
Thesis
Abstract
Elevated levels of circulating neutrophil-derived extracellular vesicles (N-EVs) during sepsis, alongside with indications of increased uptake of these vesicles within the pulmonary microvasculature during endotoxemia in mice, suggest an important role of N-EVs in the development of indirect acute lung injury during sepsis. However, it's uncertain whether N-EVs promote inflammation, as some studies have shown anti-inflammatory effects. Moreover, N-EV functional heterogeneity has been linked to different parameters centred around the activation status of the parental cells and the responses of tested target cell populations. Interestingly, most in vitro investigations of N-EVs’ activity focus on stimulating purified neutrophils with single agonists, which oversimplifies the complex in vivo interplay between different stimuli and other vascular cells contributing to neutrophil responses during sepsis.
Using in vitro modelling approaches with human primary leukocytes and primary microvascular endothelial cells, I showed that physiological-relevant models are required for the in vitro generation of pro-inflammatory N-EVs, highlighting the importance of other cells (i.e., RBCs and platelets) and mediators (i.e., PAF or LPS and fMLP) in modulating the inflammatory activity of N-EVs. Following this, I confirmed the N-EVs’ uptake by both neutrophils and monocytes and revealed a monocytes-dependent neutrophil activation, which in turn led to the development of a novel monocyte/neutrophil/HLMEC tri-culture model of pulmonary microvasculature. Moreover, using the tri-culture model, I demonstrated a central role for N-EVs as mediators of acute vascular inflammation, promoting direct leukocyte activation and the release of pro-inflammatory cytokines and pro-injurious mediators (such as neutrophil extracellular traps). Lastly, using the tri-culture model in combination with a macromolecular tracer assay (used to investigate endothelial cell monolayer permeability), I observed a N-EV mediated pulmonary endothelial damage that was leukocyte-dependent.
Using in vitro modelling approaches with human primary leukocytes and primary microvascular endothelial cells, I showed that physiological-relevant models are required for the in vitro generation of pro-inflammatory N-EVs, highlighting the importance of other cells (i.e., RBCs and platelets) and mediators (i.e., PAF or LPS and fMLP) in modulating the inflammatory activity of N-EVs. Following this, I confirmed the N-EVs’ uptake by both neutrophils and monocytes and revealed a monocytes-dependent neutrophil activation, which in turn led to the development of a novel monocyte/neutrophil/HLMEC tri-culture model of pulmonary microvasculature. Moreover, using the tri-culture model, I demonstrated a central role for N-EVs as mediators of acute vascular inflammation, promoting direct leukocyte activation and the release of pro-inflammatory cytokines and pro-injurious mediators (such as neutrophil extracellular traps). Lastly, using the tri-culture model in combination with a macromolecular tracer assay (used to investigate endothelial cell monolayer permeability), I observed a N-EV mediated pulmonary endothelial damage that was leukocyte-dependent.
Version
Open Access
Date Issued
2023-08
Date Awarded
2024-07
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
O'Dea, Kieran
Gordon, Anthony
Takata, Masao
Publisher Department
Department of Surgery & Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)