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Title: Dysfunctional innate immunity in cystic fibrosis lung disease
Author: Law, Sheonagh MacPhail
ISNI:       0000 0004 8509 5394
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Cystic Fibrosis (CF) lung disease is characterised by dysfunctional innate immunity. Even before microbial colonisation is established, there is an accumulation of neutrophils within the airways. Neutrophil extracellular traps (NETs) are an evolutionarily conserved antimicrobial defence mechanism of neutrophils. However, NETs can also cause inflammation and damage when in excess and as such are implicated in the pathogenesis of CF lung disease. Within the airways, NETs are a source of proinflammatory proteins including myeloperoxidase, neutrophil elastase and calprotectin. Treatment of CF patients with DNase reduces airway inflammation and sputum viscosity, which may be due to the drug increasing clearance and/or decreasing the production of NETs. Our group has previously found that NETs are proinflammatory to monocyte-derived macrophages (MDM) in co-culture, and that this was more exaggerated in CF MDM, so it is likely that an interplay between the innate immune cells exacerbate airway damage. Several defects have been reported in CF MDM previously, which may exacerbate airway infection and inflammation. One such reported defect is failure of the macrophage phagolysosome to acidify, which impairs bacterial killing and may lead to more bacteria surviving in the airway as a driver of inflammation. Throughout this PhD, sputum samples from both CF and healthy control (HC) participants were collected and lung function measured to investigate whether NETs are associated with airways inflammation and increased severity of lung disease. Using ELISA quantification of NETs (by an in-house ELISA which measures histone-bound calprotectin) and proinflammatory cytokines, CF participants were found to produce higher levels of sputum NETs than HC. Those CF participants taking DNase had significantly decreased levels of sputum NETs relative to those not on the drug. Furthermore, positive associations were found between NETs and proinflammatory proteins, whilst negative correlations were demonstrated between these proteins and lung function. Furthermore, when forced expiratory volume in one second (FEV1) was predicted by multi-variate linear regression, the level of sputum NETs was a significant independent indicator of FEV1. A murine model of lipopolysaccharide (LPS)-induced acute lung injury was used to characterise how CFTR alters NET formation and associated inflammation following a sterile inflammatory challenge and whether DNase affected this. It was demonstrated that CFTR-/- mice have an exaggerated inflammatory response to LPS-induced acute lung injury relative to wild type (WT) littermates in terms of airway proinflammatory cytokine concentrations and histopathological scoring of acute lung inflammation. This was despite no acute increase in airway neutrophil or macrophage numbers, suggesting intrinsic defects exist within these innate immune cells due to absence of CFTR. The receptor for advanced glycation end-products (RAGE) had higher gene expression in CFTR-/- vs WT littermates, which leads us to speculate that up-regulation of NF-κB signalling could be responsible for increased inflammation in CF mice. Importantly, airway NETs were not significantly different between genotypes and DNase had no effect on inflammation, suggesting the CF murine model is imprecise in mimicking human disease. This is perhaps unsurprising given that people with CF have non-resolving airways inflammation, rather than an acute injury. Final experiments investigated the role of CFTR in regulating macrophage phagolysosomal pH. Using MDM from both HC and CF donors, we developed a novel technique compatible with real-time analysis of phagocytosis, which showed that surface-enhanced Raman spectroscopy-based nanosensors exhibit superiority over conventional fluorescence spectroscopy in measuring macrophage phagolysosomal pH in terms of sensitivity, ratiometric quantification, and both spatial and temporal resolution. Human MDM phagolysosomal acidification was found to be CFTR-independent and may not be critical in the pathophysiology of CF lung disease, although further experiments using alveolar macrophages would strengthen these conclusions. To summarise, NETs are associated with inflammation and disease severity in human CF lung disease. Our mouse work suggested that intrinsic defects of innate immune cells exist in CFTR-/- mice, manifest by an exaggerated response to sterile inflammation, possibly via RAGE-NFκB signalling pathways. We also demonstrated that macrophage phagolysosomal acidification was not impaired in human CF MDM. Further research investigating the underlying mechanisms causing innate immune cell dysfunction will help identify therapeutic targets for the treatment of inflammation in CF lung disease.
Supervisor: Gray, Robert ; Dransfield, Ian ; Whyte, Moira Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Cystic Fibrosis ; CFTR protein ; neutrophil extracellular traps ; NETs ; macrophages ; inflammation ; CF macrophage phagolysosomal acidification