The JAK inhibitor, tofacitinib, rescues IFN‐γ‐induced barrier permeability in human colonoids and acts independent of tight junction protein expression changes in T84 epithelial monolayers
Authors: Sayoc Anica , Fan Shujun , Jimenez Jossue , Butt A. Grant , McCole Declan F. 
Published: June 2021

A new milestone was reached in IBD therapeutics with the FDA approval of the Janus kinase (JAK) inhibitor, tofacitinib, for the treatment of ulcerative colitis. However, the biological mechanisms by which tofacitinib alleviates IBD symptoms remain to be fully identified. We have shown that tofacitinib can reduce the increase in barrier permeability caused by the pro‐inflammatory cytokine interferon‐gamma (IFN‐γ) in intestinal epithelial cell (IEC) lines. The aims of this study were (1) to determine if the effects of tofacitinib on barrier function can be recapitulated in a three‐dimensional human colonoid model, and (2) to identify whether tofacitinib exerts its protective effects by modulating the expression of tight junction (TJ) proteins associated with restriction of macromolecule permeability.MethodsHuman colonoids were generated from crypts isolated from biopsy samples from patients undergoing elective colonoscopies. Written informed consent was obtained before specimen collection and studies were approved by the Health and Disabilities Ethics Committee (New Zealand, Ethics No.13/STH/155). After 12d in culture, colonoids were treated with IFN‐γ (50ng/ml) followed by tofacitinib (16.7μM) 6h later. Further addition of tofacitinib (16.7μM) was made at 24h and 48h post‐IFN‐γ administration. Colonoid permeability was measured by adding 4kDa FITC‐dextran (FD4, 0.1mg/ml; ‘basolateral’) to the growth media 48h post‐IFN‐γ treatment. After 24h, FD4 fluorescence intensity in the mid‐point of each colonoid lumen was measured and normalized to the surface area and volume of each colonoid. To determine how tofacitinib protects barrier integrity, protein lysates from T84 cells treated with tofacitinib or vehicle before or after IFN‐γ (1000U/ml) exposure were subjected to Western blotting. Expression of TJ proteins occludin, tricellulin and ZO‐1 was quantified by densitometry and normalized to β‐actin.ResultsIFN‐γ treatment resulted in a 3–4‐fold increase in FD4 flux into the lumen of human colonoids (P<0.001, n=4). Tofacitinib reduced the IFN‐γ‐induced increase in FD4 flux (P<0.01, n=4), while it had no effect on the basal FD4 flux of the colonoids. We have shown that tofacitinib prevented and rescued IFN‐γ‐induced FD4 permeability in T84 IECs, which was associated with normalization of the apical membrane localization of ZO‐1. We now show that IFN‐γ did not significantly alter protein expression of occludin, tricellulin or ZO‐1, and this was also unaffected by tofacitinib or vehicle (n=4).ConclusionsThese results suggest that tofacitinib rescues cytokine‐induced barrier dysfunction independent of effects on TJ protein expression and is likely mediated, in part, by restoring membrane localization of ZO‐1. These data also demonstrate that tofacitinib rescue of barrier integrity is consistent across different models of the intestinal epithelium including primary human colonoids.Support or Funding InformationNIH 2R01DK091281, ASPIRE‐Pfizer JAK‐STAT in IBD Research Awards 2016 (W1214999), 2017 (W1227049) (DFM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.