Pharmacology and Therapeutics - Theses
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ItemExpression and function of the novel membrane-spanning protein, MS4A8B, in the human airways( 2018)The airway epithelium is an important barrier interface that protects the lungs against environmental insults and pathogens. Airway epithelial cells are increasingly recognized as key regulators of lung immune homeostasis by orchestrating various aspects of both innate and adaptive immunity. Dysfunction of airway epithelial cell functions have been strongly associated with the pathogenesis of various airway disorders. In this body of work, I identify the expression of a novel membrane-spanning protein, MS4A8B, on the motile cilia of airway epithelial cells. Predictive protein sequence analysis reveals the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular C- terminal domain. MS4A8B is a member of the MS4A protein family, which comprises a group of 4-domain membrane-spanning proteins. Whilst the core functions of MS4A proteins have not been established, they have been suggested to play active roles in cellular signaling, cellular proliferation and differentiation. Examination of MS4A8B expression in the human airways reveals a highly restricted pattern of expression that is induced only upon mucociliary differentiation of airway epithelial cells. MS4A8B protein is localized to the motile cilia of the airway epithelium in human airway biopsies and in differentiated primary bronchial airway epithelial cell (PBEC) cultures. The expression of MS4A8B decreased in various airway inflammatory disorders including asthma and chronic obstructive pulmonary disease (COPD). In vitro assessment of MS4A8B function reveal that it possesses immune-regulatory functions, with the putative ITIM motif predicted to be a primary effector. In vitro phosphorylation studies show that the putative ITIM motif is capable of being tyrosine phosphorylated, with site-directed mutagenesis of a key tyrosine residue contained in the motif significantly impairing this ability. Combined findings suggest that MS4A8B may exert an important ‘braking’ mechanism on proinflammatory cytokine production in airway epithelial cells, with its observed loss in various airway disorders likely resulting in dysregulated expression of proinflammatory cytokines and the exacerbation of disease symptoms.
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ItemNovel insights into mechanisms of glucocorticoid actions and sensitivity in the airway epithelium( 2018)Glucocorticoids (GCs) remain the frontline treatment in the management of chronic inflammatory diseases, as they are the most potent and effective anti-inflammatory agents available so far. However, impaired responses to glucocorticoid therapy in some patients with severe disease remain a challenging clinical problem. The airway epithelial function influences inflammation in chronic respiratory diseases. Epithelium, as the site of deposition of inhaled glucocorticoids (ICS), is a key target of GC action. Synthetic GCs, including ICS, exert anti-inflammatory effects in airway epithelium by transactivation of genes and by inhibition of release of pro-inflammatory cytokines. Emerging evidence suggests that physiological GC, cortisol, might act as a partial agonist at the glucocorticoid receptor (GR) in the airway epithelium. However, whether cortisol can be a limiting factor to beneficial effects of synthetic GCs, remains to be established. Therefore, through a better understanding of the impact of cortisol on the effects of synthetic GCs in vitro and in vivo, as well as of novel individual mediators of GC actions in the airway epithelium, new strategies may arise for restoring GC responsiveness. Data presented within this thesis has provided evidence that cortisol acts like a partial agonist at the glucocorticoid receptor, limiting GC-induced GC Receptor-dependent transcription in the BEAS-2B human bronchial epithelial cell line. Cortisol also limited the inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF) release by synthetic GCs in TNFα-activated BEAS-2B cells. The relevance of these findings is supported by observations on tracheal epithelium obtained from mice treated for 5 days with systemic GC, showing limitations in selected GC effects, including inhibition of pro-inflammatory cytokine IL-6. Moreover, gene transactivation by synthetic GCs was compromised by standard air-liquid interface (ALI) growth medium cortisol concentration of 1.4 µM in the ALI differentiated organotypic culture of primary human airway epithelial cells. These findings suggest that endogenous corticosteroids may limit certain actions of synthetic pharmacological GCs and contribute to GC insensitivity, particularly when corticosteroid levels are elevated by stress. Data obtained during these thesis studies also highlight the potential of the transcriptional repressor, promyelocytic leukaemia zinc finger (PLZF) to mediate selected glucocorticoid effects in the airway epithelium, including the induction of targets important in mediating physiological effects on the normal lung development and of ones with relevance to the distinct glucocorticoid effects on the epithelial restitution following inflammation and injury. This thesis has provided novel insights into mechanisms of glucocorticoid action and insensitivity in the airway epithelium, allowing the development of strategies for improved treatment of chronic airway inflammatory diseases.
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ItemExploring the role of early life respiratory infection in asthma( 2017)The underlying cause of asthma is yet to be determined. Asthma is presently the most common chronic disease affecting children, and it is becoming clear that disease originates in early life and as a result of complex synergistic interactions of various environmental exposures. There has been increasing interest in the role of neonatal infections; with evidence emerging that asymptomatic pneumococcal colonisation is a strong predictor of future asthma, which can be exacerbated by viral infection. It is therefore paramount that appropriate models of disease are developed in order to elucidate these complex immunological interactions that have profound pathogenic potential during this window of lung development. Importantly, small airways dysfunction is present in the majority of asthmatics, and remains relatively undertreated by existing therapy, contributing considerably to the severity of disease. The lung slice technique is a powerful in vitro tool that can be used to explore airway and vascular pharmacology, offering a unique experimental link between cell-based assays and in vivo experimentation. Methodological studies compared airway reactivity in lung slices in varying experimental conditions, and application of the lung slice technique to characterize reactivity following acute respiratory infection in vivo, permitting application of this technique within the rest of this thesis. Utilising novel mouse models of asthma, parameters of bronchial reactivity, lung immunity and structural changes were assessed in adulthood following exposures in infancy, elucidating key mechanisms driving the deleterious effects caused by early life exposures. Using a mouse model of neonatal respiratory co-infection, the effect of early life co-infection with Streptococcus pneumoniae (SP) and influenza A virus (IAV) on mouse lung health, immunity and structure in adulthood was investigated. Co-infection caused a significant increase in central airway resistance that was not associated with conventional airway remodelling such as mucus overproduction, smooth muscle thickening or epithelial leakage. Airways hyperresponsiveness (AHR) was not maintained in lung slices in vitro. An increase in hysteresivity was observed in both IAV and co-infected mice. Hence, it is possible that co-infection in infancy is causing distinct structural changes that contribute to ventilation heterogeneity, which persists into adulthood. A novel model of early life co-infection combined with house dust mite (HDM) aeroallergen challenge was developed and characterised. Neonatal co-infection in the background of HDM sensitisation resulted in chronic lung colonisation, exacerbated neutrophilic inflammation, increased mucin production and increased AHR. Hence, pneumococcal colonisation does not protect against allergic airways disease which is predominately driven by mechanisms that are independent of TH2 immunity. This study identified SAA, IL-17A and G-CSF as molecular markers for this phenotype, which parallels features of more severe asthma. Taken together, the strong neutrophilic/TH17/SAA signal, accompanied by AHR and mucus hyper-secretion generated in the present study in early life co-infection plus HDM group suggests this model may be capturing key features of severe, steroid insensitive asthma, providing novel insight into clinically relevant pathology. Ultimately, this model presents a unique opportunity to develop new treatment strategies to circumvent a highly relevant cause of childhood asthma.
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ItemThe impact of cyclic AMP elevating agents on steroid resistance( 2016)Glucocorticoids are the most effective treatment for long-term control of asthma. However, a subset of patients require a much higher dose of glucocorticoids to achieve the effect of of treatment, and even the ceiling glucocorticoid response in those patients is lower. The insensitivity to glucocorticoid is termed glucocorticoid resistance. Many factors have been discovered to impair glucocorticoid response, including transforming growth factor beta (TGF-β). The research aimed to elucidate the effect of cAMP-elevating agents on TGF-β impaired glucocorticoid response. The experiments were mostly conducted in cells. GRE-SEAP assay was used to measure agonist activity and real-time polymerase chain reaction was used to measure the expression level of related genes. The results showed that compounds which induced higher cAMP level enhanced glucocorticoid response; whereas the repression caused by TGF-β and whether the repression can be attenuated by cAMP-elevating agents varied between genes and cell types. We attempted to investigate the effect of cAMP-elevating agents on glucocorticoid receptor (GR) translocation in airway epithelial cells, but GR predominantly distributed in the nucleus at the basal level and thus how cAMP influence GR translocation was not confirmed yet. A pilot study of glucocorticoid resistance in human nasal polyps is also included.
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ItemMolecular mechanisms underlying glucocorticoid insensitivity in the airways( 2014)Glucocorticoids are the most effective anti-inflammatory drugs available for the treatment of chronic inflammatory diseases, such as asthma. However, intrinsic or acquired resistance to the anti-inflammatory actions of glucocorticoids limits their utility. Different tissues from the same patient may even differ in sensitivity to glucocorticoids, suggesting that it is the chronic inflammatory fibrotic microenvironment that is responsible for localised resistance to glucocorticoid action. Importantly, most research to date examining glucocorticoid resistance mechanisms has focused on mechanisms in cells with a primary immune and/or inflammatory function. However, much of the efficacy of glucocorticoid treatments derives from actions on resident structural cell types in the airways. The epithelium in particular, as the site of deposition of inhaled glucocorticoids, is a key target of glucocorticoid action. Novel therapeutic targets may therefore emerge from understanding mechanisms of glucocorticoid resistance in epithelial cells. Data presented within this thesis has provided the first evidence that TGF-β induces resistance to glucocorticoid transactivation mechanisms in air-liquid interface differentiated primary bronchial epithelial cells. Similarly, the combination of TNFα, IL-4 and IL-13, each a known inducer of glucocorticoid insensitivity in inflammatory cell types, was also shown to impair glucocorticoid transactivation mechanisms in ALI-differentiated primary bronchial epithelial cells. Initial evidence indicating that the composition of the sub-epithelial extracellular matrix affects the glucocorticoid responsiveness of the over-lying epithelial cells is also presented. Data within this thesis particularly implicates novel TGF-β-inducible mechanisms as targets to restore glucocorticoid sensitivity, since TGF-β appears to induce glucocorticoid impairment more potently, more rapidly, and to a greater extent than the combination of TNFα, IL-4 and IL-13. Within this thesis, it was demonstrated that glucocorticoid impairment occurs downstream from the TGF-β receptor (ALK5). However, none of the known canonical or non-canonical pathways appear to mediate this effect. This study therefore implicates novel TGF-β-inducible mechanisms as targets to restore glucocorticoid sensitivity. It is important to identify the downstream signalling pathways responsible, as broadly blocking TGF-β signalling is associated with the development of widespread inflammation and autoimmunity defects. However, the fact that TGF-β-induced glucocorticoid resistance appears to occur through a novel downstream signalling mechanism renders this an attractive therapeutic target, since it may be selectively targeted to restore glucocorticoid activity, whilst avoiding the multitude of side effects known to arise from comprehensive inhibition of TGF-β. This study has contributed to the growing body of evidence that glucocorticoid resistance occurs in epithelial cells. Furthermore, through the identification of mediators that induce glucocorticoid insensitivity in epithelial cells, and the systematic investigation into the molecular mechanisms through which this insensitivity is induced, this study has provided new insight into novel signalling pathways that may be targeted therapeutically to restore therapeutic sensitivity to glucocorticoids.