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P004 IL-22 affects barrier function and cell polarity by MAPK/PI3 kinase signal transduction

D. Delbue da Silva*1, L. Lebenheim1, C. Heldt1, B. Siegmund1, M. Schumann1

1Charité Universitätsmedizin, Department of Gastroenterology, Berlin, Germany

Background

Polarity in intestinal epithelial cells (IECs) is crucial to the barrier function. IL-22 is a cytokine that has been related to directly affect the integrity of the epithelial layer. IL-22 receptor/signalling complex is found mainly in epithelial cells membranes. The activated complex leads to the activation of various cellular signalling pathways including STAT-3, MAPK and PI3K/AKT. The effect of IL-22 on epithelial cells concerning cell polarity and barrier defect is not clearly understood. Therefore, this study aimed to understand the mechanism underlying the development of dyspolar epithelia and barrier defect caused by IL-22.

Methods

To investigate the role of IL-22, we exposed various intestinal epithelial cell lines (Caco-2, T84 and HT29/B6) with IL-22. Single IECs implanted in Matrigel were grown to 3-dimensional cysts +/– IL-22 and analysed by confocal microscopy. The integrity of the barrier was monitored by measurements of transepithelial resistance (TER). Calcium switch experiments (Ussing chamber) was used to evaluate tight junction (TJ) assembly. To evaluate cell motility wound healing and invasion assays were performed. Intracellular localisation of immunostained proteins related to TJ (JAM and ZO-1) was investigated using confocal microscopy. Activated signal transduction pathways were identified in phosphoblots and inhibitors of STAT-3, MAPK/ERK, and PI3K pathways were applied to uncover the signal transduction of barrier and polarity effects.

Results

IL-22 treatment reduced TER, altered distribution of TJ proteins and caused multi-lumen cysts, suggesting disturbed cell polarity and secondary to that disturbance of barrier function of IECs. In addition, invasion and migration were increased after IL-22 treatment. It was, furthermore, observed that IL-22 treatment induced STAT-3, ERK, and AKT phosphorylation, which were associated with the observed IL-22 effects. Interestingly, only blocking of PI3K/AKT and MAPK pathways rescued barrier effects of IL-22 exposure, while STAT-3 primarily caused effects on cell viability.

Conclusion

IL-22 treatment alters cell polarity and has an effect in barrier function in IECs. Altogether, our data suggest that this effect is associated with the activation of PI3-kinase and ERK-pathways rather than STAT-3 pathways