Gomez, Nancy
(2024)
The effect of cigarette smoke extract and e-cigarette vape extract on extracellular vesicle production by human immune cells.
PhD thesis, University of Nottingham.
Abstract
Background:
Chronic Obstructive Pulmonary Disease (COPD) is a common inflammatory airway disease, affecting the airways, lung parenchyma and vasculature, and is characterized by irreversible airflow limitation. Cigarette smoking is a significant risk factor associated with various diseases, including COPD, cardiovascular disease and cancers. E-cigarettes are promoted as a safer alternative to cigarettes and are being used as substitutes to quit cigarette smoking to reduce risk to health. Extracellular vesicles (EVs) are important intercellular communication mediators released by cells into the extracellular environment, with the capacity to transfer biological signals and information between cells and as such, influence the recipient cell function. Both cigarette smoke and e-cigarettes have been linked to increased release of EVs from a number of cellular sources. Therefore, the aim of this thesis was to investigate the effect of cigarette smoke extract (CSE) and e-cigarette vapour extract (ECVE) on the production of EVs by cells of the immune system.
Methods:
Peripheral blood mononuclear cells (PBMCs) from healthy individuals were exposed to CSE and ECVE. EVs produced by PBMCs were observed using various techniques, including transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), nanoparticle flow cytometry (nFCM), imaging flow cytometry (IFC) and flow cytometry. Membrane profile of EVs, focusing on tetraspanins and cell specific markers, was also investigated. PBMC phenotypes were analysed by spectral flow cytometry.
Results:
In this study, a protocol was optimised to analyse immune cell-derived EVs, focusing on high-throughput flow-based methods, including IFC that incorporates a novel gating strategy (using the spot count feature) and a rapid staining protocol that allows for quantification of EVs in culture and following isolation. CSE or ECVE did not significantly alter the number of EVs produced by PBMCs. However, the tetraspanin profile of EVs did change between conditions, particularly the expression of CD9 and CD63. There were also significant differences in the membrane profile of EVs between treatment and control, specifically of CD40, CD41b, CD42a, CD69 and CD31. Finally, spectral flow cytometry analysis showed that CSE and ECVE do influence some cell populations. Clustering analysis identified 15 clusters present but showed no significant differences between the clusters
Conclusion:
Overall, this study has outlined a method to study extracellular vesicles derived from immune cells in an inflammatory disease model. Although the number of EVs did not significantly change, the results of this study suggest that CSE and ECVE may modify EV profiles in humans, and exposure to these components can result in changes within the cells producing EVs, consequently affecting EV formation.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Fairclough, Lucy
Onion, David
James, Victoria |
| Keywords: |
Extracellular vesicles; Inflammatory disease model; Peripheral blood mononuclear cells |
| Subjects: |
Q Science > QP Physiology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
77660 |
| Depositing User: |
Gomez, Nancy
|
| Date Deposited: |
16 Jul 2024 04:40 |
| Last Modified: |
16 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/77660 |
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