The passage of fluid, ions and macromolecules across the epithelium is controlled primarily by epithelial tight junctions. Altered epithelial permeability is associated with lung disease, and barrier function is impaired by the Th2 cytokine IL-13. This thesis investigates the signalling pathways involved in the modulation of the epithelial barrier by IL-13 stimulation.Initial experiments demonstrated that the human sub-bronchial epithelial cell line Calu-3 could be easily manipulated when grown using an air-liquid culture system. Expression of various key tight junction proteins was demonstrated, as well as a high trans-epithelial resistance (TER) for up to 7 days. Stimulation with IL-13 resulted in a decrease in TER compared with controls and this decrease was shown to be prevented with the PI3K inhibitor ZSTK474. IL-13 did not increase paracellular permeability of the epithelial monolayer to FITC-dextran from the apical to the basolateral chamber and ZSTK474 did not influence FITC-dextran flux. Immunocytochemistry showed that the expression of the tight junction protein claudin 2 was increased by IL-13 stimulation and this change in expression was shown to be PI3K dependent with the PI3K inhibitor ZSKT474 preventing the increase.Further studies were carried out in an attempt to uncover the PI3K isoform responsible for the effects seen on both the TER and the TJ expression. It was shown that inhibition of the p110α isoform with PIK75 mimicked the result observed with the pan-PI3K inhibitor ZSTK474 and prevented the IL-13-induced claudin 2 upregulation. However none of the PI3K isoform inhibitiors showed the prevention of TER, as shown by the pan PI3K inhibitor ZSTK474. The role of STAT6 in TJ modulation was shown to be similar to that of PI3K, in that inhibition of STAT6 had a positive effect on the epithelial barrier by preventing the IL-13-induced TER decrease and the increase in the expression of claudin 2. In addition, both PI3K inhibition and STAT6 inhibition demonstrated effects on basal TER and claudin 2 expression, indicating that both pathways are involved in maintenance of epithelial barrier integrity.
|Date of Award||19 Jul 2013|
|Supervisor||Malcolm Watson (Supervisor)|