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Effects of Alpha-1 Antitrypsin on the Neutrophil Plasma Membrane Proteome

thesis
posted on 02.12.2020, 10:09 by Thomas McEnery

Alpha-1 antitrypsin (AAT) deficiency (AATD) results in early onset emphysema and liver disease and represents a common indication for lung transplant. Patients homozygous for the mutant Z allele (Pi*ZZ) are the most commonly affected by AATD-related disease. Treatment with weekly infusion of pooled human AAT has been shown to slow the progression of emphysema in these individuals. AAT is an important anti-protease and the primary mechanism for lung disease in AATD is via unopposed action of the serine protease neutrophil elastase. Our hypothesis is that AAT also functions as a key regulator of neutrophil activation and function. Our aim is to elucidate the effects of AAT on the circulating neutrophil in vivo by performing the first proteomic analysis of the neutrophil plasma membrane in AATD, both preand post-augmentation therapy, as compared to healthy controls.

Neutrophils were isolated from Pi*ZZ AATD individuals and from healthy controls (n=10 per group). Neutrophils were also isolated from AATD patients on day 0 and day 2 of AAT augmentation therapy (nadir and peak AAT levels respectively, n=6 per group) and from FEV1-matched COPD patients without AATD (n=6). A plasma membrane fraction was prepared by sucrose density ultracentrifugation after lysis by sonication. Proteomic analysis was performed using liquid chromatography mass spectrometry following digestion by trypsin. Resultant proteomic data was analysed for differential expression of proteins between cohorts and between time-points of AAT augmentation.

Results demonstrated that the neutrophil plasma membrane proteome is significantly altered in AATD compared to HC. Furthermore, these differences persisted when controlling for the presence of lung disease. Exogenous AAT altered the neutrophil proteome, with protein expression on day 2 of augmentation therapy reverting towards that of COPD inflammatory control. Exogenous AAT in vivo was shown to modify expression on the neutrophil surface of key proteins associated with degranulation and integrin-mediated signalling, in keeping with previously established effects of AAT on neutrophil function.

This high-throughput methodology has generated further hypotheses that will guide investigation of altered neutrophil function in AATD, as well as the effects of exogenous AAT. This may have clinical implications, both for individuals with AATD and for the potential use of AAT as a treatment for inflammatory lung disease.

Funding

Health Research Board Ireland

History

First Supervisor

Professor Noel G. McElvaney

Second Supervisor

Dr Emer Reeves

Comments

A thesis submitted for the degree of Doctor of Medicine from the Royal College of Surgeons in Ireland in 2019.

Published Citation

McEnery T. Effects of Alpha-1 Antitrypsin on the Neutrophil Plasma Membrane Proteome [MD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2019.

Degree Name

Doctor of Medicine (MD)

Date of award

30/11/2019

Programme

  • Doctor of Medicine (MD)

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