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TSLP is a cytokine implicated in the pathophysiology of asthma through a TSLP-OX40L-T cell axis and a TSLP-mast cell axis. Whether these pathways operate in human asthma is unknown. The objective was to investigate whether mucosal TSLP protein expression relates to asthma severity, and distinct immunological pathways. GMA-embedded bronchial biopsies from subjects and patients with mild, moderate and severe asthma were immunostained for TSLP, OX40/OX40L, CD83, IL-4, IL-13, and inflammatory cells. TSLP and IL-13 release were measured in supernatants from epithelial cells co-cultured with human lung mast cells (HLMC). There was considerable heterogeneity in TSLP, IL-13 and IL-4 immunostaining across the asthmatic subjects. TSLP protein expression was significantly increased in airway epithelium and lamina propria of asthmatic patients, in particular severe asthma which correlated with the severity of airflow obstruction. IL-13 staining was increased in non-epithelial cells within the epithelium in severe asthma of which lineage-negative CD45+ cells represented a substantial proportion. Asthmatic subjects with elevated IL-13 immunostaining in the lamina propria also had elevated levels of IL-4 and TSLP expression. Recombinant TSLP attenuated FcεRI-dependent HLMC degranulation and TSLP production, and HLMC rapidly degraded TSLP. In summary, TSLP expression is elevated in severe asthma despite high-dose corticosteroid therapy. Although the TSLP-OX40L-T cell pathway within asthmatic bronchial mucosa was not detected, it is possible that it operates in secondary lymphoid organs. The close approximation of airway stroma and mast cells suggests the TSLP-mast cell axis maybe active in asthmatic bronchial mucosa, but my in vitro data suggests that TSLP may inhibit HLMC activation. The interaction of TSLP with CD45+ lineage-negative innate lymphoid cells group 2 maybe the most important pathway contributing to increased IL-13 expression in subset of patients with asthma, including severe disease. Targeting TSLP may only be efficacious in the subset of asthma characterised by increased Th2 inflammation.

Severe asthma is a complex and heterogeneous disease characterised by chronic airway inflammation, disordered airway physiology and airway remodelling. Computed tomography (CT) has emerged as a non-invasive tool for assessment of airway structural changes. A critical gap in our understanding of severe asthma is the ability to relate structural changes to important clinical outcomes. This thesis examines the relationship between CT assessed airway structure, airway inflammation and airway physiology in severe asthma patients. I first present the largest qualitative study of CT findings in severe asthma patients. I have shown that airway structural changes such as bronchiectasis and bronchial wall thickening are common and demonstrate association with disease duration and airflow obstruction. I then present a study describing airway and densitometry phantom models that were developed to study errors associated with quantitative airway morphometry and lung densitometry and device validation and standardisation methods for quantitative CT indices. In the next quantitative cross-sectional study, I report for the first time that right upper lobe apical bronchus (RB1) percent wall area (%WA) was associated with the preceding burden of neutrophilic inflammation over time measured by repeated sputum analysis. RB1 dimensions were not significantly different in four severe asthma phenotypes determined based on clinical and physiological indices. I also present a study demonstrating a decrease in RB1 wall dimensions after 1 year of treatment with mepolizumab (anti-IL-5) compared to placebo providing strong evidence in favour of the eosinophils playing a key role in airway remodelling determined by CT. Finally, I report for the first time three distinct asthma phenotypes identified based on CT assessed proximal and distal airway remodelling. Temporal assessment in severe asthma subjects demonstrates increase in RB1 wall dimensions over time but no change in RB1 lumen dimensions. These findings underpin the role of CT in multi-dimensional phenotyping of severe asthma.

This thesis comprises three studies. The first investigates non-cystic fibrosis ( non-CF) bronchiectasis patients in a stable stage of their disease. The relationship between bacteria found on sputum culture and systemic and airway inflammatory mediators has been characterised here. It has been shown that, even in a clinically stable state, the airways of this heterogeneous group of patients have multiple different species of bacteria growing therein which elicit a low grade inflammation in the airways but little systemic response. The second looks at non-CF bronchiectasis patients and the relationship between bacteria and inflammatory markers during and after antibiotic treatment for an infective exacerbation. Results show that the bacterial load within the sputum does not change significantly following ng antibiotic therapy. The composition of the microbiome alters with a primary pathogen suppressed but total viable count remains similar with commensals increasing in number. Inflammation is transiently decreased, returning to pre-treatment levels b) 4 weeks after stopping antibiotics. The third study is a pilot study to investigate the pulsed-field gel electrophoresis genotype profiles of Pseudomonas aeruginosa isolates from the sputum of non-CF bronchiectasis patients. The results suggest that there is a clonal strain of Pseudomonas aeruginosa present in the sputum of the bronchiectasis population in Northern Ireland. Continued typing of the clinical population would be required to determine whether the strains are transmitted between patients.