The JAK-Inhibitor Tofacitinib Rescues Human Intestinal Epithelial Cells and Colonoids from Cytokine-Induced Barrier Dysfunction

Abstract

Background: Alterations to epithelial tight junctions can compromise the ability of the epithelium to act as a barrier between luminal contents and the underlying tissues, thereby increasing intestinal permeability, an early critical event in inflammatory bowel disease (IBD). Tofacitinib (Xeljanz), an orally administered pan-Janus kinase (JAK) inhibitor, was recently approved for the treatment of moderate to severe ulcerative colitis. Nevertheless, the effects of tofacitinib on intestinal epithelial cell functions are largely unknown. The aim of this study was to determine if JAK inhibition by tofacitinib can rescue cytokine-induced barrier dysfunction in intestinal epithelial cells (IECs).

Methods: T84 IECs were used to evaluate the effects of tofacitinib on JAK-signal transducer and activator of transcription (STAT) activation, barrier permeability, and expression and localization of tight junction proteins. The impact of tofacitinib on claudin-2 promoter activity was assessed in HT-29 IECs. Tofacitinib rescue of barrier function was also tested in human colonic stem cell-derived organoids.

Results: Pretreatment with tofacitinib prevented IFN-γ-induced decreases in transepithelial electrical resistance (TER) and increases in 4 kDa FITC-dextran permeability (FD4), partly due to claudin-2 transcriptional regulation and restriction of ZO-1 rearrangement at tight junctions. Although tofacitinib administered after IFN-γ challenge only partially normalized TER and claudin-2 levels, FD4 permeability and ZO-1 localization were fully recovered. The IFN-γ-induced FD4 permeability in primary human colonoids was fully rescued by tofacitinib.

Conclusions: These data suggest differential therapeutic efficacy of tofacitinib in the rescue of pore vs leak-tight junction barrier defects and indicate a potential contribution of improved epithelial barrier function to the beneficial effects of tofacitinib in IBD patients.

Keywords: IFN-γ; STAT1; ZO-1; claudin-2; intestinal permeability; organoids.

FIGURE 1.

Tofacitinib pretreatment prevented IFN-γ-induced JAK1, STAT1, and STAT3 phosphorylation in IECs in vitro. A, Schematic of the acute prevention protocol for tofacitinib to determine JAK-STAT activation in IECs. T₈₄ IECs were treated with vehicle (DMSO) or tofacitinib (Tofa, 50 μM) for 1 hour followed by IFN-γ (1000 U/mL) treatment for either 30 minutes (for JAK activation) or 1 hour (for STAT activation). Cells were lysed and protein extracts were subjected to Western blotting for the proteins indicated. Densitometric analysis was performed and normalized to (B) pJAK1/JAK1, (C) pSTAT1/STAT1, and (D) pSTAT3/STAT3 levels of each replicate’s respective untreated (Unt.) controls (* P < 0.05, ### P < 0.001, n = 3-4).

FIGURE 2.

Tofacitinib prevents and rescues IECs against IFN-γ-induced barrier dysfunction. A, Schematic of the prevention protocol (pretreatment): T₈₄ IECs grown on transwells were treated with varying concentrations of tofacitinib apically for 1 hour followed by basolateral administration of IFN-γ (1000 U/mL) for 24 hours. DMSO was used as a vehicle control. (B) TER and (C) FD4 permeability were measured 24 hours post-IFN-γ. (**** P < 0.0001, ### P < 0.001, #### P < 0.0001, n = 3). D, Schematic of the rescue protocol (post-treatment): therapeutic effects of tofacitinib were studied by treating T₈₄ IECs post-IFN-γ treatment (1000 U/mL) with tofacitinib (16.7 μM) apically at 2 time points (6 h and 18 h) to mimic twice daily clinical administration. Again, DMSO was used as a vehicle control. E, TER and (F) FD4 permeability were measured 24 hours after IFN-γ treatment. (**** P < 0.0001, ** P < 0.01, #### P < 0.0001, ## P < 0.01, n = 3).

FIGURE 3.

Tofacitinib rescues the permeability increase induced by IFN-γ in human colonic organoids. A, Representative series of optical slices through a human colonoid bathed in growth media containing 0.1 mg/mL FD4. Scale bar = 100 μm. B, Following a rescue protocol, human colonoids cultured for 12 days were treated with IFN-γ (50 ng/mL, administered in the bathing media). Tofacitinib (16.7 μM, administered in the bathing media) was added 6 hours, 24 hours, and 48 hours post-IFN-γ treatment. FD4 flux across colonoid epithelium normalized to surface area of colonoids was measured 72 hours after IFN-γ administration. (*** P < 0.001, ## P < 0.01, n = 4). C, Colonoids were harvested, lysed, and subjected to Western blotting for ZO-1 and occludin. Representative blots and densitometric analysis for ZO-1 and occludin are shown.

FIGURE 4.

Tofacitinib does not alter protein expression of tight junction proteins involved in macromolecule permeability. Pretreatment: T₈₄ cells grown in transwells were pretreated with DMSO or tofacitinib (50 μM) for 1 hour before IFN-γ (1000 U/mL) treatment for 24 hours. Post-treatment: T₈₄ cells grown in transwells were first treated with IFN-γ (1000 U/mL) followed by 2 doses of DMSO or tofacitinib (16.7 μM) at 6 hours and 18 hours post-IFN-γ administration. Twenty-four hours after IFN-γ exposure, cells were lysed, processed for Western blotting, and probed for the proteins indicated. A, Representative blots probed for ZO-1, occludin, tricellulin, and βactin. B, Quantification of densitometric analysis normalized to untreated controls from 4 independent experiments (n = 4).

FIGURE 5.

Tofacitinib reduces the number of apical intercellular gaps via ZO-1 rearrangement/localization caused by IFN-γ. A, Pretreatment: T₈₄ cells grown on coverslips pretreated with tofacitinib or DMSO for 1 hour followed by IFN-γ (1000 U/mL, 24 h) treatment were stained for ZO-1 and visualized via immunofluorescence microscopy. B, Average number of intercellular gaps in 5 fields of view per condition from 3 independent pretreatment experiments (**** P < 0.0001, ### P < 0.001, ## P < 0.01, n = 3). C, Post-treatment: ZO-1 staining in cells treated with tofacitinib or DMSO at 6 hours and 18 hours post-IFN-γ (1000 U/mL, 24 h) treatment. D, Number of intercellular gaps taken in 10 fields of view per condition from 3 independent post-treatment experiments (**** P < 0.0001, ### P < 0.001, n = 3). White arrows indicate intercellular gaps.

FIGURE 6.

Tofacitinib restricts IFN-γ-induced claudin-2 protein expression and promoter activity. A, Pretreatment: claudin-2 protein expression was determined in T₈₄ IECs pretreated with tofacitinib or DMSO 1 hour before IFN-γ treatment for 24 hours and quantified using densitometric analysis and normalized to untreated controls. (*** P < 0.001, # P < 0.05, n = 4). B, Post-treatment: claudin-2 protein expression was determined in T₈₄ cells treated with tofacitinib or DMSO 6 hours and 18 hours post-IFN-γ exposure and quantified using densitometric analysis and normalized to untreated controls. (**** P < 0.0001, # P < 0.05, n = 3). C, Pretreatment: claudin-2 promoter activity was measured using a luciferase reporter assay on HT-29 IECs subjected to the treatment schedule outlined in Figure 1A. (*** P < 0.001, ### P < 0.001, n = 4).

FIGURE 7.

The protective effect of tofacitinib on IFN-γ-induced barrier dysfunction. During inflammation in which cytokines such as IFN-γ are released and bind to their receptors on epithelial cells, JAK1 proteins are brought in close proximity to each other and are activated. These in turn phosphorylate cytokine receptors that serve as docking sites for STAT proteins, which are then activated by JAK1 proteins. Phosphorylated STATs dimerize, translocate to the nucleus, and serve as transcription factors for specific genes, such as claudin-2. Upon IFN-γ treatment, claudin-2 expression is upregulated and ZO-1 mislocalizes to form intercellular gaps. As a small molecule inhibitor, when tofacitinib binds to the ATP-binding site of JAK proteins, the first step of the signaling cascade is stopped, therefore preventing and/or reducing the detrimental effects of IFN-γ on the intestinal epithelial barrier (summarized in right-hand panel; broken arrow reflects reduced paracellular permeability vs. inflammation alone shown in left-hand panel).