Interferon‐gamma (IFN γ) induced epithelial barrier dysfunction in T84 human intestinal epithelial cells (IECs) occurs via phosphatidylinositol 3‐kinase (PI3‐K) mediated activation of adenosine monophosphate‐activated protein kinase (AMPK)

Abstract

IFNγ plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD) and some of the effects of IFNγ on intestinal epithelial barrier function are dependent on PI3‐K. Here we investigated the role of the cellular energy sensor, AMPK in the effects of IFNγ on epithelial barrier function.Protein analysis was performed by Western blotting and AMPK knock‐down by siRNA. Transepithelial resistance (TER) was assessed by voltohmeter and epithelial permeability as the flux rate of 10 kDa FITC‐dextran.IFNγ (1000 U/ml) induced activation of AMPK in a manner independent of changes in intracellular ATP (the classic trigger for AMPK activation). Inhibition of PI3‐K with LY294002 (20 µM) blocked IFNγ‐induced AMPK phosphorylation (n=9; p<0.05; 6h), and mitigated the IFNγ‐induced decrease in TER across T84 monolayers (n=9; p<0.05). Knock‐down of AMPK diminished the IFNγ‐induced increase in epithelial permeability (72 h; n=3; p<0.05) and prevented IFNγ‐induced downregulation of the tight junction proteins occludin and ZO‐1 (n=3; p<0.05).We conclude that IFNγ sequentially activates PI3‐K and AMPK to impair tight junction protein expression and thus barrier function in IECs. Our data indicate a novel role for AMPK as a mediator of cytokine signalling, independent of changes in cellular energy status, and thus may have implications for mucosal defense in the setting of inflammation.Supported by the CCFA and NIH.