The role of neutrophil-derived microvesicles in lung inflammation

During the pathogenesis of chronic obstructive pulmonary disease (COPD) and in subsequent exacerbations, neutrophilic inflammation predominates and contributes to disease progression. Levels of neutrophil-derived microvesicles (NMVs; small extracellular vesicles formed from membrane blebbing in resting and activated cells) have previously been shown to be elevated in COPD patient sputum and in the circulation during inflammation. Evidence of the pro-inflammatory activity of NMVs in several cell types, along with an ability to increase epithelial monolayer permeability, led to the hypothesis that NMVs are released upon neutrophil activation in the circulation and the inflamed airway environment and are subsequently internalised by lung epithelial cells, inducing their pro-inflammatory activation and dysfunction. The overall aim of my PhD was to investigate the role of NMVs in lung inflammation. This was firstly done by characterising circulating MVs in COPD patients as both potential disease biomarkers and as mediators of inflammation using stored plasma samples from two COPD patient cohorts. Subsequently, it was crucial to isolate human neutrophils from the peripheral blood of healthy volunteers so as to generate, isolate and characterise a pure population of NMVs from these cells and to investigate the functional effects of these vesicles using the bronchial epithelial cell line BEAS2B. Finally, to further understand the biological relevance of NMV effects in the lung, C57BL/6 mice were utilised in a series of experiments to explore lung inflammation and NMV fate. Key findings: 1. Plasma MV matrix metalloproteinase-9 (MMP-9) content is significantly increased in COPD patients compared with age-matched controls, however, numbers of circulating leukocytederived MVs are not differential between these groups. 2. NMVs from fMLP-stimulated healthy participant neutrophils contain active MMP-9 and can degrade extracellular matrix proteins, but activated and quiescent neutrophils do not produce differential NMV numbers. 3. NMVs are rapidly taken up by bronchial epithelial cells, likely via an endocytic mechanism, with functional effects of NMVs on epithelial activation, barrier integrity and proliferation. 4. In vivo in mice, airway administration of NMVs does not induce or augment lung inflammation, instead, mass uptake and clearance of these NMVs by alveolar macrophages occurs. In this thesis I have shown that MV-associated protein content is altered in COPD, and investigation of these differences will be important going forward in defining the role of NMVs in chronic lung diseases. Degradation of extracellular matrix shown here and epithelial cell activation and permeability induced by NMVs when applied directly to these cells define key pathological events in COPD and indicate a role of these vesicles in epithelial dysfunction. In contrast, NMV uptake by alveolar macrophages was identified as a mechanism of NMV clearance in healthy lung tissue. Together, these findings provide novel insight into neutrophil interactions and activity in the lungs. This work contributed to our understanding of the processes occurring in inflammation and identified several valuable avenues for future investigation to understand more about complex conditions like COPD.