Neutrophil extracellular vesicles as mediators of acute pulmonary vascular inflammation during sepsis

Circulating neutrophil-derived extracellular vesicles (EVs) are acutely increased during sepsis and systemic inflammatory response syndrome. Although in vitro studies have shown that neutrophil-EVs have pro-inflammatory activities, little is known about their roles in the propagation of systemic inflammation. Recent studies indicate that the uptake of circulating EVs by the lungs increases dramatically during systemic inflammation, primarily through their interactions with pulmonary intravascular monocytes. In this study, we hypothesised that circulating neutrophil-EVs are potent long-range mediators of pulmonary vascular inflammation, contributing to the development of indirect acute lung injury. Using human cell culture-based models, my main aims were to: 1) characterise neutrophil-EV uptake by pulmonary vascular cells under resting and inflammatory conditions, 2) develop a whole blood model of lipopolysaccharide (LPS)-induced neutrophil-EV subtype production, 3) isolate and characterise the pro-inflammatory activity of these neutrophil-EVs. For the assessment of neutrophil-EV function, I developed a co-culture model of pulmonary vascular inflammation, consisting of peripheral blood mononuclear cells (PMBCs) or monocytes, co-cultured with human lung microvascular endothelial cells (HLMECs). Neutrophil-EVs were taken up by both HLMECs and monocytes and subject to dynamic changes under physiological flow and inflammatory conditions. LPS stimulation of whole blood revealed acute increases in neutrophil- and platelet-EVs. Using immunoaffinity isolation for EV subpopulations, neutrophil- but not platelet-EVs were found to be pro-inflammatory, inducing TNF-α-dependent activation of HLMECs in the presence of PBMCs or monocytes. Further investigations revealed that neutrophil-EVs activate monocytes via Toll-like receptor 4 (TLR4) signalling, in an EV surface protein-dependent manner. These findings indicate that neutrophil-EVs released under septic-like conditions in vitro are potent mediators of inflammation, with the potential to generate localised inflammation within 3 the monocyte-enriched environment of the pulmonary vasculature. Furthermore, the neutrophil-EV protein-dependent TLR4 signalling activity suggests a novel mechanism for propagation of inflammation within the circulation from local sites of infection.Open Acces

Circulating Myeloid Cell-derived Extracellular Vesicles as Mediators of Indirect Acute Lung Injury

Blood-borne myeloid cells, neutrophils and monocytes, play a central role in the development of indirect acute lung injury (ALI) during sepsis and non-infectious systemic inflammatory response syndrome (SIRS). By contrast, the contribution of circulating myeloid cell-derived extracellular vesicles (EVs) to ALI is unknown, despite acute increases in their numbers during sepsis and SIRS. Here, we investigated the direct role of circulating myeloid-EVs in ALI using a mouse isolated perfused lung system and a human cell coculture model of pulmonary vascular inflammation consisting of lung microvascular endothelial cells and peripheral blood mononuclear cells. Total and immunoaffinity-isolated myeloid (CD11b+) and platelet (CD41+) EVs were prepared from the plasma of i.v. LPS-injected endotoxemic donor mice and transferred directly into recipient lungs. Two-hour perfusion of lungs with unfractionated EVs from a single donor induced pulmonary edema formation and increased perfusate levels of receptor for advanced glycation end products (RAGE), consistent with lung injury. These responses were abolished in the lungs of monocyte-depleted mice. The isolated myeloid- but not platelet-EVs produced a similar injury response and the acute intravascular release of proinflammatory cytokines and endothelial injury markers. In the in vitro human coculture model, human myeloid (CD11b+) but not platelet (CD61+) EVs isolated from LPS-stimulated whole blood induced acute proinflammatory cytokine production and endothelial activation. These findings implicate circulating myeloid-EVs as acute mediators of pulmonary vascular inflammation and edema, suggesting an alternative therapeutic target for attenuation of indirect ALI