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Cristopher Capaldo, Attila Farkas, Michael Koval, Charkes Parkos, Asma Nusrat, O-028 Pro-inflammatory Cytokines Interferon Gamma and Tumor Necrosis Factor Alpha Disrupt the Epithelial Barrier by Restricting Claudin 4 Tight Junction Association, Inflammatory Bowel Diseases, Volume 19, Issue suppl_1, 1 December 2013, Page S16, https://doi.org/10.1097/01.MIB.0000438579.73162.74
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Tight Junctions (TJs) are transmembrane structures that act as a paracellular barrier in epithelial tissues. The proinflammatory cytokines Interferon gamma (IFN-gamma) and Tumor Necrosis Factor alpha (TNF-alpha are increased in inflamed intestinal mucosa and are known to disrupt intestinal epithelial cell (IEC) barrier properties by modulating TJ function. Our aim is to understand the molecular mechanisms by which IFN-gamma and TNF-alpha modulate epithelial Tight Junctions. We focus on the TJ protein claudin 4, which enhances IEC barrier function and is diminished during inflammation. We therefore hypothesized that IEC barrier is compromised through cytokine-induced increases in claudin 4 mobility within the TJ and that highly mobile claudins are deficient at forming tight paracellular seals.
To test this hypothesis, model IECs that express acGFP tagged claudin 4 were assessed for claudin 4 TJ dynamics by Fluorescence Recovery after Photobleaching (FRAP). Live cell confocal imaging and FRAP analysis were mathematically modeled to investigate claudin 4 kinetics.
Exposure of IECs to IFN-gamma and TNF-alpha dramatically increased the mobile fraction of TJ localized acGFP-claudin 4. Mathematical modeling of FRAP kinetics predicted that IFN-gamma/TNF-alpha signaling does not significantly alter acGFP-claudin 4 mobile pool exchange dynamics, which was subsequently confirmed as claudin 4 dynamics were unaffected by IFN-gamma and TNF-alpha in IECs with disassembled TJs. Interestingly, increased claudin 4 protein levels rescue IFN-gamma/TNF-alpha-induced barrier defects, claudin 4 mobility defects, and cytokine induced changes in claudin 2. Lastly, we observed claudin 4 long term mobility using photo-switchable Dendra-claudin 4.
In conclusion, IFN-gamma and TNF-alpha increase long-term claudin 4 mobility through a novel mechanism involving heterotypic incompatibility. This data supports a model of TJ restructuring by proinflammatory cytokines, through decreased long-term kinetics of claudin 4 assembly into TJs, thereby impairing IEC barrier function.
