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DOP001. Epithelial polarity is disrupted by interleukin-22

L. Lebenheim1, C. May1, M. Munoz2, M.M. Heimesaat2, B. Siegmund1, J.-D. Schulzke1, M. Schumann1, 1Charité University Medicine, Gastroenterology Infectious Diseases and Rheumatology, Berlin, Germany, 2Charité University Medicine, Dept. for Microbiology, Berlin, Germany

Background

Interleukin-22 (IL-22), a cytokine secreted by TH17 cells, acts specifically on epithelia via the heterodimeric IL22R1/IL10 receptor. Depending on the context it is examined, IL-22 was shown to have either protective (wound healing) or deleterious effects (induction of ileitis) on intestinal epithelia. We have previously shown that it strongly disturbs the epithelial barrier by attacking the protein complex at tight junctions (TJ). Herein epithelial polarity is analyzed using the 3D cyst model as well as IL-22 knock-out mice.

Methods

Caco-2 cells, seeded in Matrigel, were grown to develop 3-dimensional cysts. These cysts were treated with IL-22 (10 ng/ml) and analyzed for the number of lumina, proliferation and apoptosis (active caspase-3). Moreover, fully developed cysts, as well as cysts in early phases of cyst formation were immunostained for apical junctional as well as polarity complex proteins and examined with confocal LSM. To examine TJ assembly, Caco2 cells on filter supports were mounted to Ussing chambers, to perform a calcium switch assay. Ilea of wild-type and IL-22 knock-out mice, previously been treated with T. gondii to induce ileitis, were mounted to Ussing chambers to measure the epithelial portion (Repi) of the transmural electrical resistance by impedance spectroscopy.

Results

IL-22-treated cysts revealed a multiple lumen phenotype as seen in stages of dyspolarity. Furthermore complex cysts with branched lumina were found. IL-22 treated cysts revealed a basolateral expression of the apical membrane marker Ezrin. The expression of polarity proteins Par-3 and Dlg-1 - normally expressed at the TJ and the basolateral membrane, respectively - were reduced and shifted to an intracellular vesicle pool.

Epithelial apoptosis in cysts was only modestly increased. Accordingly, the calcium switch assay of filter-grown Caco-2's revealed the functional outcome of a defective TJ assembly as expected in epithelia with defective cell polarity.

IL-22 knock-out mice and wild-type littermates were examined for Repi. IL-22−/− mice were protected from T. gondii-associated loss of Repi. On microscopy of intestinal mucosae, IL-22 dependent epithelial changes paralleled findings in 3D cysts.

Figure 1. Caco 3D cysts reveal changes in polarity protein expression when treated with IL-22 (10 ng/ml).

Figure 2. Calcium switch (Caco-2, grown on PCF filter support).

Conclusion

IL-22 affects epithelial polarity, which has important implications on its known role in inflammation-associated carcinogenesis.