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|Title:||Developing an in vitro co-culture Model to Investigate the Role of TRAIL in Lung Inflammation|
|Presented at:||University of Leicester|
|Abstract:||Lung inflammatory conditions such as asthma and Chronic Obstructive Pulmonary Disease (COPD) are on the rise and there is a need for improved treatments. Current therapies such as corticosteroids simply alleviate symptoms which can lead to further complications. Apoptosis has an important role in the clearance of infiltrating immune cells, thus the balance between pro-inflammatory and pro-apoptotic signals dictates the severity of the inflammation. TRAIL, a ligand of the TNF superfamily, can to signal to both apoptosis and NFκB activation and therefore could be of therapeutic benefit. However, its role in lung inflammation is yet to be fully understood. The aim of this thesis was to develop an in vitro model of lung inflammation to elucidate the potential role of the TRAIL/TRAIL-R1/R2 signalling axis in this setting. To implement the model, activated CD4+ and CD8+ T cells, important drivers of inflammation in asthma and COPD, were co-cultured with lung epithelial cells. Before developing a co-culture model, basal TRAIL/TRAIL-R1/R2 signalling was established in both cell types. NHBE cells were characterised for the first time for their basal TRAIL signalling and were found to be dependent on TRAIL-R2 for efficient TRAIL signalling. Several lines of evidence advocated the iHBEC cell line to be the most representative cell line of primary lung epithelial cells (NHBE cells). For example, downstream of a complete and active TRAIL Death Inducing Signalling Complex (DISC) in both iHBEC and NHBE cells, caspase 3 was insufficiently active to induce apoptosis and instead NFκB was found to be active as shown by phosphorylation of IκB and the translocation of p65 to the nucleus. ILZ TRAIL, a highly oligomerised form of TRAIL, was synthesised as it signals more efficiently via TRAIL-R2. Crucially, ILZ TRAIL proved to be an invaluable tool for distinguishing between TRAILR1/ TRAIL-R2 signalling in this project. The co-culture of activated T cells and lung epithelial cells provided a novel in vitro model of lung inflammation, as demonstrated by the secretion of pro-inflammatory cytokines. The inclusion of exogenous TRAIL/ILZ TRAIL in this model revealed a potential pro-inflammatory role for TRAIL/TRAIL-R1/2 signalling in this setting that was not evident when lung epithelial cells were cultured alone. These data suggest that TRAIL/ILZ TRAIL signalling is context dependent. Further evaluation of the role of TRAIL in lung inflammation could provide potential new insights in the pathogenesis of inflammatory conditions such as asthma or COPD.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Leicester Theses|
Theses, MRC Toxicology Unit
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