Dissecting the roles of the Dectin-1R in the bladder innate defences

Abstract

Previous work using immortalised cells transfected with a NF-κB reporter and challenged with zymosan, to mimic a fungal infection, suggested possible co-operation between Dectin-1 and TLR5 receptors in urogenital tissues. These data were interesting as research describing Dectin-1R functioning relates specifically to myeloid derived cells and TLR5, classically, is known to function as a homodimer. The aim of the research reported in this thesis was therefore to focus on the Dectin-1 and TLR5 receptors in the bladder urothelium, with the objective of investigating Dectin-1 receptor functioning, Dectin1/TLR5 receptor co-operation and cell signalling events following a zymosan (β-glucan) challenge. Western analyses of cell lysates from a bladder biopsy and immortalised RT4 bladder cells using a Dectin-1R antibody (R&D systems, AF1859) revealed the synthesis of proteins representing full-length and truncated (stalk-less) Dectin-1 receptors. These data supported the use of the immortalised RT4 cells in all subsequent analyses exploring Dectin-1R functioning in the urothelial tissues. Following challenge of the cells with the yeast cell wall extract zymosan (50ug/ml) increased synthesis of an array of host defence agents including hBD2, IL-8 and LCN2 were observed (p<0.05). Furthermore IL-8 effector synthesis was significantly reduced following CLEC7A (Dectin-1R) gene knockdown (p<0.05) and antibody blocking of the Dectin-1R (p<0.01). These data supported synthesis of the Dectin-1R in urothelial cells and a role in defending the bladder against a fungal challenge. Knocking-down the TLR5 gene in RT4 cells (80% knockdown) and challenging the cells with zymosan, resulted in a significant reduction in hBD2, IL8 and LCN2 synthesis (p<0.05). These data suggested co-operation between the Dectin-1 and TLR5 receptors in bladder cells, which was supported by immunostaining and a proximity ligation assay approach. In addition these analyses supported clustering of the Dectin-1 and TLR5 receptors following activation. Experiments to explore roles for the encoded Dectin-1 receptor isoforms, 1, 2 and 4, in the TLR5 co-operation events involved engineering a suite of stable cell lines each over-expressing a Dectin-1 receptor isoform and either a TLR5 full-length or TLR5 truncated receptor. This approach exploited the pVitro2-neo-mcs vector, which is able to co-express two cDNA sequences simultaneously. However, the resultant cell lines did not over-express the Dectin-1 and TLR5 receptors. It was hypothesised that the increased number of receptors synthesised by the cells over-loaded the urothelial cell membranes causing gene silencing and/or cell death. The signalling mechanisms activating the transcription of host effector genes following a zymosan challenge of RT4 bladder cells were explored using western analyses and an antibody to Syk-P (Cell Signalling, C87C1), SYK gene knockdown and piceatannol (100 ug/ml) a Syk inhibitor followed by ELISA to measure LCN2 and IL-8 media concentrations. No phosphorylation of Syk was observed and the knock-down/inhibition approaches had no significant effects on the media concentrations of either IL-8 or LCN2. However, knockdown of the RAF1 gene resulted in decreased secretion of the host defence molecules LCN2 and IL-8 (p<0.01). Additionally western blot analyses showed phosphorylation and degradation of the NF-κB inhibitor IκBα. These data suggested that Dectin-1R activation in response to zymosan in bladder cells involved Raf-1 signalling, which supported a non-canonical signalling pathway. In summary, Dectin-1 receptors were shown to be synthesized in RT4 bladder epithelial cells and were activated in response to zymosan (fungal) challenge resulting in the production of host defence effector molecules. Data showed potential co-operation between Dectin-1 and TLR5 receptors in mediating the bladder cell response. Dectin-1R signalling in urothelial cells was also orchestrated via a non-canonical signalling pathway involving Raf-1. These data support a novel host defence mechanism, involving Dectin-1 and TLR5 receptors functioning co-operatively, to defend urothelial from fungal infections.