Tofacitinib Inhibits Intestinal Epithelial JAK‐STAT Signaling and Prevents Barrier Dysfunction Induced by Interferon‐Gamma (IFN‐γ)

The orally‐administered Janus kinase (JAK)‐1,3 inhibitor, tofacitinib, is effective as an induction and maintenance therapy for ulcerative colitis (UC). Although tofacitinib promotes mucosal healing and remission in patients with UC, little is known about how it directly affects intestinal epithelial cells (IECs) and barrier function. The UC and Crohn's disease susceptibility gene protein tyrosine phosphatase non‐receptor type 2 ( PTPN2 ) encodes the T cell protein tyrosine phosphatase (TCPTP), which dephosphorylates JAK‐STAT signaling molecules such as JAK1/3, STAT1/3. The aims of this study were to determine: (1) if tofacitinib reduces JAK‐STAT activation in IECs to prevent IFN‐γ‐induced barrier dysfunction; and (2) if tofacitinib corrects the JAK‐STAT signaling defects in IECs lacking TCPTP. Methods T 84 IECs were treated with tofacitinib at a concentration (50 μM) determined in a previous dose‐response study, or vehicle (0.5% DMSO), for 1 hr prior to treatment with IBD‐associated inflammatory cytokine IFN‐γ (1000 U/ml) for 1 hr. Effects of tofacitinib were also determined in HT29 IECs stably expressing control shRNA (CTL) or shRNA targeted against PTPN2 ( PTPN2 KD) treated with tofacitinib (50 μM) or vehicle (0.5% DMSO) for 1 hr +/‐subsequent treatment of IFN‐γ (1000 U/ml) for 1 hr. Luciferase reporter assay was conducted in HT29 IECs to assess the claudin 2 gene promoter activity after treatment with tofacitinib (50 μM) or vehicle (0.5% DMSO) 1 hr prior to IFN‐γ (1000 U/ml; 24 hrs) treatment. Tight junction protein expression was analyzed in T 84 IECs grown on transwells for 11–12 days followed by tofacitinib treatment as above or with tofacitinib or vehicle 6 hrs and 18 hrs post‐IFN‐γ challenge. Protein expression was determined by Western blotting and densitometry. Results Tofacitinib pre‐treatment inhibited IFN‐γ‐induced JAK1 (n=4, p<0.05) and STAT1 (n=3, p<0.001) phosphorylation in T 84 IECs. Tofacitinib reduced basal and IFN‐γ‐stimulated levels of phospho‐JAK1 (n=3, p<0.05; n=3, p<0.05, respectively) and phospho‐STAT1 (n=4, p<0.05; n=3, p<0.001, respectively) in HT29 PTPN2 KD cells to levels comparable to untreated or vehicle‐treated HT29 CTL cells. We previously showed that tofacitinib prevented decreases in TER caused by IFN‐γ (n=3, p<0.001), and that IFN‐γ increases claudin‐2 expression by JAK‐STAT activation. Here, we observed that tofacitinib pre‐treatment inhibited IFN‐γ induction of claudin 2 promoter activity (n=4, p<0.001) and protein expression (n=5, p<0.05). Tofacitinib also partially decreased claudin 2 protein levels after IFN‐γ induction (n=3, p<0.05). These data suggest that tofacitinib directly inhibits the JAK‐STAT signaling pathway in IECs to restrict intestinal barrier defects induced by IFN‐γ through claudin 2 regulation. This indicates the therapeutic potential of using tofacitinib to correct the consequences of PTPN2 genetic defects as a personalized medicine approach for patients harboring TCPTP loss‐of‐function mutations. Support or Funding Information NIH 2R01DK091281 (DFM) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .