The JAK Inhibitor Tofacitinib Rescues Intestinal Barrier Defects Caused by Disrupted Epithelial-macrophage Interactions

Abstract

Background and aims: Loss-of-function variants in protein tyrosine phosphatase non-receptor type-2 [PTPN2] promote susceptibility to inflammatory bowel diseases [IBD]. PTPN2 regulates Janus-kinase [JAK] and signal transducer and activator of transcription [STAT] signalling, while protecting the intestinal epithelium from inflammation-induced barrier disruption. The pan-JAK inhibitor tofacitinib is approved to treat ulcerative colitis, but its effects on intestinal epithelial cell-macrophage interactions and on barrier properties are unknown. We aimed to determine if tofacitinib can rescue disrupted epithelial-macrophage interaction and barrier function upon loss of PTPN2.

Methods: Human Caco-2BBe intestinal epithelial cells [IECs] and THP-1 macrophages expressing control or PTPN2-specific shRNA were co-cultured with tofacitinib or vehicle. Transepithelial electrical resistance and 4 kDa fluorescein-dextran flux were measured to assess barrier function. Ptpn2fl/fl and Ptpn2-LysMCre mice, which lack Ptpn2 in myeloid cells, were treated orally with tofacitinib citrate twice daily to assess the in vivo effect on the intestinal epithelial barrier. Colitis was induced via administration of 1.5% dextran sulphate sodium [DSS] in drinking water.

Results: Tofacitinib corrected compromised barrier function upon PTPN2 loss in macrophages and/or IECs via normalisation of: [i] tight junction protein expression; [ii] excessive STAT3 signalling; and [iii] IL-6 and IL-22 secretion. In Ptpn2-LysMCre mice, tofacitinib reduced colonic pro-inflammatory macrophages, corrected underlying permeability defects, and prevented the increased susceptibility to DSS colitis.

Conclusions: PTPN2 loss in IECs or macrophages compromises IEC-macrophage interactions and reduces epithelial barrier integrity. Both of these events were corrected by tofacitinib in vitro and in vivo. Tofacitinib may have greater therapeutic efficacy in IBD patients harbouring PTPN2 loss-of-function mutations.

Keywords: IBD; JAK-STAT; TCPTP; epithelial cells; macrophage; permeability; tight junction.

Figure 1.

Tofacitinib corrects barrier defects caused by loss of PTPN2 in intestinal epithelial cells and macrophages. A] Transepithelial electrical resistance [TEER] and B] 4 kDa fluorescein-dextran [FD4] flux across Caco-2BBe monolayers pre-treated with vehicle [0.5% DMSO] or tofacitinib [Tofa, 50 μM] for 1 h before co-culture with control [Ctr] or PTPN2 knockdown [KD] THP-1 macrophages for 24 h; *p ≤0.05 compared with [cf.] vehicle-treated control Caco-2BBe without co-culture; #p ≤0.05 cf. vehicle-treated Ctr Caco-2BBe with Ctr macrophages; §p ≤0.05 cf. vehicle Ctr Caco-2BBe with KD macrophages; ^xp ≤0.05 cf. vehicle-treated KD Caco-2BBe without macrophages; ¶p ≤0.05 cf. vehicle-treated KD Caco-2BBe with control macrophages; ^op ≤0.05 cf. vehicle-treated Ctr Caco-2BBe with KD macrophages; n = 3. The “−” symbol indicates no co-culture.

Figure 2.

Tofacitinib normalises tight junction protein expression in Caco-2BBe cells mediated by PTPN2 loss in intestinal epithelial cells [IECs] and/or macrophages. Caco-2BBe monolayers pre-treated with vehicle [0.5% DMSO] or tofacitinib [Tofa, 50 μM] for 1 h before co-culture with control [Ctr] or PTPN2 knockdown [KD] THP-1 cells for 24 h, lysed and subjected to western blotting. A] Representative western blot images of the indicated proteins from Caco-2BBe whole-cell lysates. Densitometric analyses of B] claudin-2, C] claudin-4, D] JAM-A, E] occludin, F] tricellulin, and G] TCPTP expression, all normalised to β-actin. H] Caco-2 cells were grown on cover slides and pre-treated with tofacitinib before addition of conditioned medium from Ctr or KD THP-1 cells for 24 h and subsequent immunofluorescence staining for ZO-1. Arrows point to regions with ZO-1 internalisation. Scale bar: 10 μm; *p ≤0.05, **p ≤0.01, ****p ≤ 0.0001 cf. vehicle-treated Ctr Caco-2BBe without co-culture; #p ≤0.05, ##p ≤0.01, ###p ≤0.001, ####p ≤ 0.0001 cf. #p ≤0.05 cf. vehicle-treated Ctr Caco-2BBe with Ctr macrophages; §p ≤0.05, §§p ≤0.01 cf. vehicle Ctr Caco-2BBe with KD macrophages; ^xp ≤0.05 cf. vehicle-treated KD Caco-2BBe without macrophages; ¶p ≤0.05 cf. vehicle-treated KD Caco-2BBe with Ctr macrophages; ^op ≤0.05 cf. vehicle-treated Ctr Caco-2BBe with KD macrophages; n = 3. The “−” symbol indicates no co-culture.

Figure 3.

Functional inhibition of STAT signalling by tofacitinib in Caco-2BBe cells co-cultured with macrophages. Caco-2BBe monolayers pre-treated with vehicle [0.5% DMSO] or tofacitinib [Tofa, 50 μM] for 1 h before co-culture with control [Ctr] or PTPN2 knockdown [KD] THP-1 cells for 24 h. Representative western blot images and densitometric analyses of A] phospho-STAT1 [pSTAT1] and B] phospho-STAT3 [pSTAT3] normalised to their respective total forms [tSTAT1, tSTAT3] in Caco-2BBe whole cell lysates; *p ≤0.05, ****p ≤0.0001 cf. vehicle-treated Ctr Caco-2BBe without co-culture; ##p ≤ 0.01 cf. vehicle-treated Ctr Caco-2BBe with Ctr macrophages; §§p ≤ 0.01, §§§ p ≤0.001 cf. vehicle Ctr Caco-2BBe with KD macrophages; ^xxp ≤0.01, ^xxxp  0.01 cf. vehicle-treated KD Caco-2BBe without macrophages; ¶¶p ≤0.01, ¶¶¶p ≤0.001 cf. vehicle-treated KD Caco-2BBe with Ctr macrophages; ^oop ≤0.01, ^ooop ≤0.001 cf. vehicle-treated Ctr Caco-2BBe with KD macrophages; n = 3. The “−” symbol indicates no co-culture.

Figure 4.

Tofacitinib reduces STAT signalling in THP-1 macrophages and cytokine IL-6 secretion in co-culture studies. THP-1 macrophages co-cultured with tofacitinib-treated Caco-2BBe cells for 24 h were lysed and subjected to western blotting. Representative western blot images and densitometric analyses of A] phospho-JAK1 [pJAK1], B] phospho-STAT1 [pSTAT1], and C] phospho-STAT3 [pSTAT3] normalised to their respective total forms [tJAK1, tSTAT1, tSTAT3] in THP-1 whole-cell lysates. D] IL-6, IL-22, and IL-10 concentrations present in the culture media after co-culture experiments, measured by ELISA; *p ≤0.05, *** p ≤ 0.001 cf. vehicle-treated Ctr THP-1 without co-culture; §p ≤0.05, §§p ≤0.01, §§§p ≤0.001 cf. vehicle-treated Ctr THP-1 with KD Caco-2BBe; ^xp ≤0.05, ^xxp ≤0.01, ^xxxp ≤0.01 cf. vehicle-treated KD THP-1 without co-culture; ¶p ≤0.05, ¶¶¶p ≤0.001 cf. vehicle-treated KD THP-1 without co-culture; ^op ≤0.05, ^ooop ≤0.001 cf. vehicle-treated Ctr THP-1 with Ctr Caco-2BBe; n = 3. The “−” symbol indicates no co-culture.

Figure 5.

Tofacitinib citrate treatment reduced barrier permeability to FD4 in vivo. Intestinal permeability to creatinine, FD4, and RD70 was measured in Ptpn2^fl/fl and Ptpn2-LysMCre mice subjected to twice daily treatment with vehicle [1% methylcellulose in PBS] or tofacitinib citrate [50 mg/kg] for 7 days. Serum concentrations of A] FD4, B] creatinine, and C] RD70 were measured 5 h after gavage; *p ≤ 0.05; n = 6 per group, representative results of two independent experiments.

Figure 6.

Aberrant tight junction protein patterns and elevated STAT3 phosphorylation in Ptpn2-LysMCre mice were reversed upon tofacitinib citrate treatment. Whole-cell lysates of proximal colon IECs isolated from Ptpn2^fl/fl and Ptpn2-LysMCre mice treated with vehicle or tofacitinib citrate [Tofa] for 7 days were subjected to western blot analysis. A] Representative western blot images and respective densitometric analyses of B] claudin-2, C] claudin-4, D] JAM-A, E] occludin, F] tricellulin, normalised to β-actin. G] Immunofluorescence staining for ZO-1 [white], E-cadherin [red], and claudin-2 [green] in colon sections from Ptpn2^fl/fl and Ptpn2-LysMCre mice treated with vehicle or tofacitinib citrate for 7 days. White arrows indicate cell border localisation of ZO-1, green arrows indicate cell border localisation of claudin-2. See also Supplementary Figure S2, available as Supplementary data at ECCO-JCC online. Densitometric analysis of H] phospho-STAT3 [pSTAT3] normalised to total STAT3 [tSTAT3] and I] TCPTP normalised to β-actin; *p ≤0.05, **p ≤0.01, ***p ≤0.001; n = 6 per group, representative results of two independent experiments.

Figure 7.

Cytokine production and macrophage polarisation are normalised by tofacitinib citrate treatment in Ptpn2-LysMCre mice. A] mRNA expression of Il6, Il10, and Il22 normalised to Gapdh in whole proximal colons of mice treated with vehicle or tofacitinib citrate twice daily for 7 days. B] Protein expression of IL-6, IL-10, and IL-22 as determined by ELISA. C] Proportions of total macrophages [CD11b+, Ly6G-, F4/80+, CD64 + cells], M1 [CD86 high, CD206 low], and M2 [CD86 low, CD206 high] macrophages in whole proximal colons of mice as determined by flow cytometry [all cells were pre-gated on live, single, CD45 + cells]; *p ≤0.05, **p ≤0.01, ***p ≤0.001, **** p ≤0.0001; n = 6 per group, representative results of two independent experiments.

Figure 8.

Tofacitinib citrate treatment prevents colitis in Ptpn2-LysMCre mice. Ptpn2-LysMCre and Ptpn2^fl/fl mice were treated twice daily with tofacitinib citrate starting 3 days before exposure to 1.5% dextran sulphate sodium [DSS] in the drinking water for 7 days. A] FD4 permeability at the start of the experiment [Day 3], at the day of DSS start [Day 0], and at the end of the experiment [Day 7]. B] Weight development, C] disease activity score, D] colon length, E] representative pictures from haematoxylin and eosin [H&E] stained terminal colon sections, and F] respective histological scoring of colitis severity. Scale bars: 100 μm. *p ≤0.05, **p ≤0.01; n = 4 per group.