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Elevated levels of the cysteine protease cathepsin S (cat S) are found in Cystic fibrosis (CF) lung secretions, however, the role of cat S in CF lung disease is unclear. Cat S is capable of maintaining its activity at a neutral pH allowing it to remain active outside of the cell. Consequently, cat S has the capacity to promote remodelling of the extracellular matrix via its potent elastolytic activity. In addition, cat S can cleave and inactivate key antimicrobials in the CF airways. On the basis of findings to date, we hypothesise that active cat S contributes to the pathogenesis of CF lung disease and represents a viable therapeutic target for the treatment of chronic lung disease. The βENaC transgenic mouse model recapitulates essential features of chronic CF lung disease such as airway mucus obstruction, inflammatory lung damage and increased levels of cat S activity in the lungs of the βENaC transgenic mouse compared to wild-type controls. Pharmacological knockdown of cat S activity was achieved in the βENaC mouse using the cat S inhibitor VBY-999. In this thesis we have demonstrated that inhibition of cat S reduces inflammatory cell infiltration into the lung as well as levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid in both the early and late stage lung disease in the βENaC mouse model. Furthermore, concomitant reductions in mucus plugging were also observed. Late treatment with the cat S inhibitor had no effect on lung tissue damage, however, mucus plugging was reduced. We generated a βENaC mouse in which cat S was genetically deleted, the results from which supported our findings from the inhibitor study. These results support the hypothesis that active cat S plays a role in the pathogenesis of chronic lung disease and may be a viable and promising target in the treatment of chronic lung diseases such as CF and COPD.

In the current era, there are no effective therapies for the treatment of acute respiratory distress syndrome (ARDS) despite numerous clinical trials. Current strategies are aimed at improving pulmonary perfusion, recruitment of atelectatic alveoli and reducing iatrogenic injuries to the lung. The human models of ARDS give important information when testing potential drug therapies and serve as a bridge between experimental studies and phase 11/111 clinical trials. I was able to establish the ex vivo lung perfusion model and studied the effects of aspirin in reducing pulmonary inflammation produced by lipopolysaccharide (LPS). To translate the beneficial effect of aspirin on pulmonary inflammation seen in experimental models of ARDS into a phase I clinical trial, the healthy volunteer model of LPS inhalation was used. Aspirin in these human models of ARDS was shown to reduce the pulmonary makers of inflammation due to its anti-inflammatory properties, however further clinical studies will be required to establish its role as a potential drug therapy for ARDS.

Background: Eosinophilic airway inflammation (>3% sputum eosinophils) is a feature of subgroup of subjects with chronic obstructive pulmonary disease (COPD). My objectives were to investigate the clinical characteristics of eosinophilic COPD, its stability over time and extent in bronchial tissue, whether it is related to parasite exposure or atopy and whether its persistence is due to abnormal clearance by macrophages. Methods: Subjects were studied that had participated in previous observational studies. The repeatability of sputum eosinophils was measured between 3 monthly visits for 1 year. The extent of eosinophilic inflammation in bronchial tissue was assessed using immunohistology on bronchial tissue from COPD and control subjects. Positive serology for parasites was tested in serum samples for 4 helminth species. Atopy was assessed in the subjects using serum total Ig-E and skin prick test. Eosinophil efferocytosis by macrophages was investigated in vivo using cytoplasmic area of red hue of macrophages and in vitro using apoptotic eosinophils fed to monocyte-derived macrophages from COPD and healthy controls. The dynamics of eosinophil clearance during exacerbations was explored using the red hue technique. Results: Eosinophilic and non-eosinophilic COPD have similar lung function and exacerbation frequency. In eosinophilic COPD the health status is better and bacterial colonisation is lower. This phenotype is stable over time. Airway tissue eosinophilis are increased in COPD subjects with high blood eosinophils and are positively correlated with features of remodelling. Eosinophilic COPD is not associated with helminth exposure, but is related to elevated total Ig-E. Macrophage efferocytosis of eosinophils is impaired in COPD and is associated with the severity and frequency of COPD exacerbations. Efferocytosis of eosinophils by macrophages is increased following oral corticosteroid therapy at exacerbation. Conclusion: Eosinophilic COPD is a distinct and stable phenotype that persists in the blood, bronchus and sputum. Its persistence is partly related to atopy and impaired clearance by macrophages with the latter associated with COPD exacerbation severity and frequency.

Members of the Bcc, previously described as non-mucoid, produced copious amounts of EPS when onion extract was provided as a sole nutrient. When investigating the onion extract content, it was found that fructose, sucrose and alcohol sugars were able to induce this change in phenotype. Interestingly, none of the virulent<i> B. cenocepacia</i> ET-12 isolates was able to produce EPS on any medium tested. This loss of EPS phenotype correlated with an 11 bp deletion in <i>bceB</i>, which is part of the <i>bce</i> gene cluster associated with the biosynthesis of cepacian, a previously characterised <i>Burkholderia</i> EPS. It has been proposed that instead of a non-mucoid to mucoid conversion, which is strikingly characteristic of <i>P. aeruginosa </i>infections in CF patients, members of the Bcc revert from a mucoid to a non-mucoid phenotype, suggesting a role for EPS in initial persistence, and that a loss of mucoidy may lead to increased virulence. Bcc LPS has previously been shown to be constitutively modified with 4-amino-4-deoxy-<i>β</i>-L-arbinoase (L-Ara4N). The enzymes involve din L-Ara4N biosynthesis were recently shown to be essential for <i>B. cenocepacia </i>viability. In this study, UDP-glucose dehydrogenase (Ugd) and 4-amino-4-deoxy-<i>β</i>-L-arbinose transferase (ArnT), the first and last enzymes of the biosynthetic pathway, were investigated. The L-Ara4N biosynthetic enzymes may be an Achilles’ heel of <i>B. cenocepacia.</i> Inhibitors to any of the enzymes in the L-Ara4N biosynthetic pathway could potentially be used alone, or in combination with other antimicrobial agents that would, under normal conditions, not be able to penetrate the outer membrane.

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