Date of Award

3-2017

Document type

Thesis

Degree Name

MD (Medical Doctor)

First Supervisor

Professor Noel G McElvaney

Second Supervisor

Dr Emer Reeves

Abstract

Cystic fibrosis (CF) is an autosomal recessive multi-system disorder which primarily affects respiratory epithelia and results in abnormal mucus secretions, chronic pulmonary infection culminating in respiratory failure and premature death. One of the hallmarks of CF is neutrophil derived inflammation. CF neutrophils release excessive amounts of primary granule proteins possibly due to sustained infection and the continuous presence of inflammatory mediators. However, the cause of increased primary granule release in CF is unknown. Neutrophil exocytosis is under the tight control of small GTP-binding proteins, including Rasrelated C3 botulinum toxin substrate 2 (Rac2). The aim of this study was to investigate the cellular basis for aberrant neutrophil degranulation in CF.

In this study, we examined degranulation by CF neutrophils using Western blotting. This revealed excessive release of the primary granule proteins myeloperoxidase (MPO) (p=0.03) and bactericidal permeability increasing protein (BPI) (p=0.02). In addition, fluorescence resonance energy transfer (FRET) analysis confirmed increased extracellular activity of the neutrophil serine protease neutrophil elastase (NE) post activation with TNFα and fMLP (p=0.03 and 0.03 respectively). Similarly, circulating CF neutrophils exhibited greater levels of extracellular cathepsin G (Cath G) activity compared to control cells (p=0.02). Excessive Rac2 activity was confirmed in CF neutrophils using a Rac2 activation assay both at rest and post TNFα/fMLP activation (p=0.03 and p=0.004 respectively).

Furthermore, control neutrophils treated with the CFTR inhibitor drug- CFTRinh- 172 also displayed increased primary granule degranulation with excessive MPO (p=0.01) and BPI levels recorded (p=0.03), as well as enhanced Rac2 activation (p=0.03), mirroring the defects observed in CF neutrophils. These results indicate that the observed increased primary granule release is an intrinsic defect due to impaired CFTR function.

In vivo, increased primary granule release resulted in elevated plasma (p=0.01) and bronchoalveolar lavage fluid (BALF) levels of BPI (p=0.008) and corresponding circulating autoantibodies in patients with CF.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

10.4 MB

Comments

A thesis submitted for the degree of Doctor of Medicine from the Royal College of Surgeons in Ireland in 2017.

Available for download on Sunday, March 31, 2019

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