Pharmacology and Therapeutics - Theses
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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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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.
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ItemTransforming growth factor-beta (TGF-ß) impairs glucocorticoid responses in A549 lung epithelial cells( 2012)Background: Airway and lung epithelial cells form a defence barrier to pathogens, allergens and foreign particles. They also orchestrate the inflammatory process and serve as a target of the anti-inflammatory actions of glucocorticoids. Glucocorticoid resistance occurs in many inflammatory diseases such as, asthma. This resistance has been attributed to a multitude of mechanisms, including oxidative inactivation of histone deacetylase-2, downregulation of glucocorticoid receptor-α (GR-α) or an upregulation of glucocorticoid receptor-β (GR-β). However, these mechanisms are neither exclusive of alternative explanations nor are they universal in distinct cell types. Aims: Glucocorticoid resistance is extensively investigated in inflammatory cell types, but not in epithelial cells. Transforming growth factor beta (TGF-β) is released by the epithelial cells and its levels are increased in inflammatory conditions. TGF-β initiates a signalling cascade of cytoplasmic kinases, including members of the mitogen-activated protein kinase (MAPK) family, phosphoinositol-3 kinase (PI3K) and glycogen synthase kinase-3 beta (GSK-3β) that have been linked to glucocorticoid resistance. The aim of this thesis is to investigate the role of TGF-β in the mechanism of glucocorticoid resistance in lung epithelial cells. Methods and results: In studies using A549 type II human epithelial cell line, glucocorticoid responsiveness was assessed by measuring dexamethasone regulation of interleukin-1α (IL-1α)-induced interleukin-8 (IL-8) and IL-6 production. Pre-incubation of A549 cells with TGF-β impaired dexamethasone regulation of both cytokines. This impairment was observed with TGF-β concentrations as low as 10 pM, and with budesonide as an alternative glucocorticoid to dexamethasone. TGF-β-induced insensitivity to IL-8 and IL-6 inhibition by dexamethasone was not dependent on TGF-β-mediated trans-differentiation of the epithelial cells to a mesenchymal phenotype, as it was observed in pre-incubation of 4 hours that is a too brief period to induce epithelial to mesenchymal cell transition (EMT). The central airway epithelial cell line, BEAS-2B, showed similar TGF-β-induced resistance to glucocorticoid regulation of granulocyte-macrophage colony stimulating factor (GM-CSF). Immunofluorescence and western blotting studies showed that dexamethasone-induced GR-α translocation in A549 cells was markedly reduced by 40 pM TGF-β. GR-α mRNA levels were II decreased by 40 pM TGF-β. However, GR-β expression was negligible compared to GR-α, excluding GR-β role in TGF-β-induced glucocorticoid insensitivity. In A549 cells transfected with a glucocorticoid response element (GRE)-controlled secretory alkaline phosphatase (SEAP), it was evident that TGF-β (40 pM) inhibited the GRE response to dexamethasone by more than 80 %. The impaired GRE activity was associated with failure to activate transcription of GRE-dependent genes, including glucocorticoid-induced leucine zipper (GILZ), inhibitor of kappa-B alpha (IκB-α) and SCNN1A. Treatment of cells with SB431542 (1 μM), a TGF-β receptor type I kinase inhibitor, restored dexamethasone activation of GRE. However, TGF-β-induced glucocorticoid insensitivity was unaffected by inhibitors of p38MAPK, extra-cellular regulated kinase (ERK), cJun N-terminal kinase (JNK), TGF-β-activated kinase-1 (TAK-1), PI3K, GSK-3β or proteasomal activity. Conclusion: We conclude that TGF-β is a candidate mediator of glucocorticoid resistance, acting by reducing nuclear localisation of GR-α with a consequent failure to activate the GRE and the transcription of GRE-dependent genes. Identification of the specific signal downstream of TGF-β receptor type I that induces glucocorticoid resistance may provide new strategies to develop glucocorticoid sensitising agents.