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. Ptpn2^fl/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.

1. Introduction

Inflammatory bowel diseases [IBD] are chronic inflammatory conditions of the digestive tract, which manifest as diarrhoea, abdominal pain, ulcers, and in severe cases, anaemia and micronutrient deficiency. IBD encompasses ulcerative colitis [UC] and Crohn’s disease [CD], which differ in their histopathology and the affected intestinal regions.^1–3 IBD is multifactorial, with genetic, environmental, immunological and microbial factors contributing to disease manifestation.⁴ Due to the complex pathogenesis of IBD, most current therapeutic strategies alleviate IBD symptoms, rather than target specific disease-driving molecular pathways.^5,6 Biologics, such as anti-tumour necrosis factor [TNF] treatment, emerged as an effective treatment for IBD, but 30–50% of patients lose clinical response to anti-TNF treatment,^7,8 partially due to production of anti-drug antibodies.⁹ Thus alternative treatment strategies are being developed and/or tested for use in IBD.

Tofacitinib [Xeljanz®] is a small-molecule pan-Janus kinase [JAK] inhibitor approved for the treatment of moderate-to-severe ulcerative colitis. JAK family molecules are mediators of pro-inflammatory cytokine signalling, and tofacitinib has proven effective in treating chronic diseases, including IBD.^10–13 However, only 40.6% of UC patients showed sustained clinical remission under tofacitinib treatment, thus indicating that only a proportion of patients are responsive.¹² Preclinical animal studies have investigated the effects of tofacitinib on models of intestinal inflammation, but studies of specific cell types in the gut [ie, intestinal epithelial cells and macrophages] targeted by tofacitinib are limited in number.^14–16

The barrier function of the intestinal epithelium ensures separation of luminal contents from underlying tissues while allowing regulated passage of fluid and electrolytes. Tight junctions, which consist of several transmembrane proteins, selectively seal the paracellular space between intestinal epithelial cells [IECs] and control the passage of water and ions.^17,18 Epithelial barrier disruption allows bacteria and bacterial components to penetrate the epithelium and to initiate immune responses, which in turn have detrimental effects on the tight junction complex.¹⁹ Increased intestinal permeability is an early feature of IBD²⁰ that precedes intestinal inflammation and is a prognostic factor for relapse in Crohn’s disease.^21–25 Moreover, a recent prospective study of a large cohort of asymptomatic first-degree relatives of IBD patients identified that increased intestinal permeability is significantly associated with increased future risk of developing Crohn’s disease, and thus represents a separate pre-disease risk factor for later onset of Crohn’s disease independent of potential subclinical inflammation²⁶.

Over 240 single nucleotide polymorphisms [SNPs] have been associated with increased risk for IBD, including protein tyrosine phosphatase non-receptor type 2 [PTPN2], which encodes the T cell protein tyrosine phosphatase [TCPTP].^4,27–30 IBD-associated single nucleotide polymorphisms [SNPs] within the PTPN2 gene locus have been reported to result in decreased PTPN2 expression³¹ and TCPTP phosphatase activity³², and a recent report showed that a loss-of-function mutation in the phosphatase domain encoding region of PTPN2 results in loss of TCPTP activity and very early-onset intestinal inflammation ³³. The most studied PTPN2 variants in the context of IBD are SNP rs1893217 and SNP rs2542151. These two SNPs are in linkage disequilibrium³² and have a minor allele frequency of approximately 16% in the general population and 20% in IBD patients²⁷, although geographical regional differences exist²⁷. Substrates of TCPTP include members of the JAK-signal transducer and activator of transcription [STAT] signalling pathway.^34–36 Constitutive Ptpn2^-/- mice exhibit intestinal inflammation and succumb to systemic inflammation between 3–5 weeks of age.³⁷ Knockdown of PTPN2 in human IEC cultures promotes STAT activation and enhances barrier defects in the presence of the pro-inflammatory cytokine IFN-γ ³⁸ In a model of naïve T cell transfer-mediated colitis, RAG2 knockout mice injected with Ptpn2-deficient T cells showed increased STAT1 and STAT3 levels.³⁹ Further, Ptpn2-LysMCre mice, which lack TCPTP primarily in macrophages and monocytes, are more susceptible to acute dextran sulphate sodium [DSS]-induced colitis.⁴⁰ Recently, we have shown that TCPTP loss in macrophages affects IEC properties [ie, barrier function], and in turn, TCPTP-deficient IECs alter macrophage functions and cytokine secretion⁴¹. Further, JAK inhibition by tofacitinib reduced IFN-γ-induced barrier dysfunction in IEC lines and human colon organoids through modulation of tight junction molecules.⁴² Here, we translate earlier findings in individual cell types to relevant co-culture and in vivo models to show that tofacitinib directly affects IEC and macrophage cell function. Moreover, tofacitinib also corrects defects in the interactions between these cell types, culminating in increased intestinal permeability, which arise from TCPTP deficiency and elevated JAK-STAT signalling.

2. Methods

2.1. Cell culture

Human Caco-2BBe intestinal epithelial cells [IECs] were cultured in Dulbecco’s Modification of Eagle’s Medium [DMEM, #15-017-CV, Corning, Corning, NY] supplemented with 10% heat-inactivated fetal bovine serum [Thermo Fisher Scientific, Waltham, MA] and 1% L-glutamine [Lonza, 17-605E, Walkersville, MD]. Human THP-1 cells were cultured in RPMI 1640 medium [#10-040-CV, Thermo Fisher Scientific] with 10% heat-inactivated fetal bovine serum [Thermo Fisher Scientific]. All cells were grown in standard 12-well cell culture plates [#130,185, ThermoScientific, Rochester, NY] or transwell membranes [#3460, Corning, Kennebunk, ME] in a humidified incubator at 37°C with 5% CO₂.

2.2. Generating stable shRNA PTPN2-deficient cell lines

Cell lines stably expressing PTPN2 shRNA were generated as previously described.^43,44 Briefly, Caco-2BBe and THP-1 cells were infected with lentiviral constructs containing either PTPN2-specific, or non-targeting scrambled shRNA. After 72 h of infection, stable cell lines were selected using puromycin [400-128P, Gemini, New York, NY]. Stable Caco-2BBe and THP-1 control [Ctr] and PTPN2 knockdown [KD] cell lines were maintained at 4 μg/mL and 0.5 μg/ml puromycin, respectively. Knockdown of PTPN2/TCPTP was confirmed by quantitative polymerase chain reaction [qPCR] and western blotting⁴¹.

2.3. Measurement of transepithelial electrical resistance and 4 kDa FITC-dextran permeability in vitro

Transepithelial electrical resistance [TEER] of Caco-2BBe cells grown on transwells was measured using an EVOM2 Epithelial Voltohmmeter [World Precision Instruments, Sarasota, FL]. Values from an empty well were subtracted, and readings were multiplied by the transwell surface area and expressed as ohms/cm². After measurement of TEER, cells were washed twice and equilibrated in 1X PBS with CaCl₂ and MgCl₂ [D8662, Sigma-Aldrich, St Louis, MO] for 30 min at 37°C. Fluorescein isothiocynate [FITC]-4 kDa dextran [FD4, 1mg/ml, Sigma-Aldrich] was added to the apical side, and after 2 h at 37°C, fluorescence of duplicate samples from the basolateral medium was measured using a microplate reader [Promega, Madison, WI]. FD4 concentrations were calculated against a standard curve.

2.4. Co-culture experiments and tofacitinib treatments in vitro

Caco-2BBe IECs were seeded on transwell membranes at 0.5 × 10⁶ cells per well and cultured for 8 days. Cells were then switched to serum-free media overnight before co-culture with THP-1 cells and/or treated with tofacitinib. Prior to co-cultures with IECs, 0.25 × 10⁶ THP-1 monocytes were pulsed for 3 h with PMA [Sigma-Aldrich] to induce macrophage differentiation, seeded on 12-well standard cell culture plates for 48 h, and then switched to serum-free DMEM the day before the experiment. Prior the start of the co-culture, Caco-2BBe cells were pre-treated apically with vehicle [dimethyl sulphoxide, DMSO, 0.5%, Sigma-Aldrich] or tofacitinib [50 μM, MedChemExpress, Monmouth Junction, NJ] for 1 h, followed by placing the transwells into the THP-1 cell-containing 12-well plates. After 24 h, TEER and FD4 permeability were measured, the basolateral media collected, and protein from both Caco-2BBe and THP-1 cells harvested.

2.5. Harvesting and preparation of whole cell protein lysates

For protein isolation, cells were washed twice with ice-cold 1X PBS with CaCl₂ and MgCl₂ [D8662, Sigma-Aldrich] before lysis with RIPA lysis buffer [50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] supplemented with 1X protease inhibitor [Roche, South San Francisco, CA], 2 mM sodium fluoride, 2 mM PMSF, and phosphatase inhibitors [2 mM sodium orthovanadate, 1X Phosphatase Inhibitor Cocktail 2 and 3, Sigma-Aldrich] for 15 min at 4°C. Cells were scraped, transferred into microcentrifuge tubes and homogenised on ice using the Q125 Sonicator [QSonica Sonicators, Newtown, CT]. Cell lysates were centrifuged at 16 200 × g at 4°C for 10 min and the supernatants collected into new microcentrifuge tubes. Loading samples were prepared by mixing the same amount of total protein from each sample with Laemmli loading buffer [60 mM Tris-Cl pH 6.8, 2% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue, and 10% glycerol], and boiling at 95°C for 10 min.

2.6. Western blot

Proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene difluoride membranes [EMD Millipore, Temecula, CA]. Membranes were blocked with 3% milk, 1% BSA in TBS-T [Tris-buffered saline with 0.1% Tween-20] for 1 h at room temperature, followed by overnight incubation with primary antibodies [Supplementary Table 1, available as Supplementary data at ECCO-JCC online] at 4°C overnight. The following day, membranes were rinsed with TBS-T [x3], subjected to three 5-min washes with TBS-T, and then incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies [Supplementary Table 1, available as Supplementary data at ECCO-JCC online] in 3% milk, 1% BSA in TBS-T, for 1 h at room temperature, and again washed with TBS-T three times. Finally, membranes were incubated with SuperSignal^TM West Pico PLUS Chemiluminescent Substrate solution [Thermo Fisher Scientific] and immunoreactive proteins were detected by exposing the membranes to X-ray film [LabScientific Inc., Highlands, NJ]. Densitometric analysis of the blots was performed using ImageJ software.⁴⁵

2.7. Immunofluorescnce staining

Caco-2 cells were grown on cover slides and cultured for 24 h with conditioned medium from macrophages as previously described.⁴¹ Cells were washed three times in PBS, fixed in 100% methanol [20 min at -20°C], washed three times in PBS, permeabilised with Triton X [1% Triton X-100 in PBS] for 30 min, washed three times in PBS containing 0.1% Tween [PBST], and incubated with 10% normal donkey serum in PBST for 1 h before incubation with anti ZO-1 antibody [1:200, Thermo Fisher Scientific] overnight at 4°C. Slides were washed three times with PBST, incubated with secondary antibody [anti-Rabbit AlexaFluor 488, 1:200, Jackson ImmunoResearch] for 1 h at room temperature, washed three times in PBST, and mounted using ProlongGold Antifade Reagent with DAPI [Thermo Fisher Scientific].

For in vivo evaluation of tight junction molecules, OCT-embedded frozen sections were cut into 5-μm sections and the slides brought to room temperature, rinsed twice with PBST, and fixed in methanol [10 min at -20°C]. Slides were incubated in blocking buffer [PBS + 2% donkey serum, 1% BSA, 1% Triton X, 0.05% Tween-20] for 1 h before overnight incubation with F’ab donkey-anti-mouse antibody [Jackson ImmunoSearch Inc, West Grove, PA]. The slides were rinsed with PBST, incubated with mouse-anti-claudin-2, rabbit anti-ZO-1, and goat anti-E-cadherin antibody [all from Thermo Fisher Scientific, diluted in PBS + 5% normal donkey serum] for 30 min at room temperature, rinsed again with PBS before incubation with a biotinylated secondary anti-mouse antibody [Jackson ImmunoSearch Inc.] for 20 min, followed by an additional wash in PBST and subsequent incubation with Alexa Fluor 488-steptavidin, Alexa Fluor 647-anti-rabbit secondary antibody and Cy3-anti-goat secondary antibody [Jackson ImmunoSearch] for 30 min, and finally mounted with ProlongGold Antifade Reagent with DAPI [Thermo Fisher Scientific].

2.8. Mice

C57Bl/6 male and female mice homozygous for a floxed Ptpn2 gene and heterozygous for the LysMCre construct [Ptpn2-LysMCre mice, originally generated at the University Hospital Zurich, Zurich, Switzerland] and their LysMCre-negative littermates [Ptpn2^fl/fl mice] were used for all studies.^40,46 All animal experiments were conducted with approval from the Institutional Animal Care and Use Committee at the University of California Riverside, in accordance with the National Institute of Health guidelines for the use of live animals. For all studies, 8–14 week old male and female mice were used, housed in a specific pathogen-free [SPF] facility under a 12-h light/dark cycle and with food/water ad libitum .

2.9. Tofacitinib citrate treatment in vivo

Vehicle solution was prepared by dissolving methylcellulose [M7027, Sigma-Aldrich] at 1% w/v in PBS. Tofacitinib citrate gavage solution was prepared fresh every day by resuspending tofacitinib citrate powder [Selleckchem, Houston, TX] in vehicle solution. Gavage solutions were sonicated using the Q125 Sonicator [QSonica Sonicators] at 30% amplitude for 2 min at 10-s ON/OFF intervals at room temperature. Mice were orally gavaged with vehicle [5 μL per g body weight] or tofacitinib citrate [50 mg/kg body weight, in 5 μL/g body weight, concentration based on a previous study¹⁴] twice daily for 7 days [barrier measurements without colitis induction] or 10 days [experiments with colitis induction]. The amount of gavage solution was adjusted on a daily basis according to the weight of each individual and ranged 80–120 μL.

2.10. Measurement of intestinal permeability in vivo

To assess barrier permeability in vivo, mice were food-deprived for 2 h before oral administration [200 μL] of gavage solution containing 100 mg/mL creatinine [#4255, Sigma-Aldrich], 80 mg/mL FD4 [#46,944, Sigma-Aldrich], and 20 mg/mL rhodamine B-dextran 70kDa [RD70, #R9379, Sigma-Aldrich] dissolved in MilliQ H₂O and filtered through a 0.2-μM syringe filter. A water-gavaged mouse was used as control. After 5 h, blood samples were collected and serum concentrations of the three probes determined as previously described.^41,47 At this point, animals were given access to food.

2.11. Colitis induction

For 10 days, 12–14 week old Ptpn2-LysMCre mice and their Ptpn2^fl/fl littermates were treated with tofacitinib citrate as described above, starting 3 days before DSS exposure for 7 days [1.5% in the drinking water; MP Biomedicals, Irvine, CA]. Body weight and disease scores [based on activity level, stool consistency, weight loss, blood in stool scores] were recorded daily. At day 7 of DSS administration, the mice were euthanised, colon length measured, and the most distal 2 cm of the colon collected for histological assessment of colitis severity as described previously.⁴⁸ The most distal section of the colon was used in order to assess the beneficial effect of tofacitinib on the intestinal region that is most affected by inflammation in the DSS model.

2.12. Tissue collection

Intestinal tissues were snap-frozen in liquid nitrogen for protein expression and enzyme-linked immunosorbent assay [ELISA] analyses. To isolate IECs, proximal colons were everted, incubated in Cell Recovery Solution [#354,253, Corning] on ice for 2 h, and then vigorously shaken to release IECs. IECs were washed twice with ice-cold PBS and proteins isolated as described above.

2.13. Flow cytometry

After removal of IECs, intestinal tissues were cut into 0.5-cm pieces and digested with Collagenase type IV [0.6 mg/ml; Sigma-Aldrich, #C5138] for 15 min. Digested tissue pieces were then passed three times through a 18.5 G needle and homogenates were filtered through a 7-μm cell strainer [BD Biosciences; Franklin Lakes, NJ]. Cells were washed once with PBS before resuspension in 20 μL PBS containing Fc-receptor blocking antibodies for 10 min, before adding 20 μL of fluorescence-labelled antibodies [see Supplementary Table 2, available as Supplementary data at ECCO-JCC online] and incubation on ice for 30 min. Samples were washed twice with ice-cold PBS, resuspended in 200 μL FACS buffer [PBS, 2% FBS], and acquired on an LSR-II flow cytometer [BD Biosciences].

2.14. RNA isolation, real-time PCR, and quantitative PCR

Total RNA was isolated from whole proximal colons using RNeasy Mini Kit [Qiagen] per manufacturer’s directions. RNA concentration was measured by absorbance at 260 nm and 280 nm using Nanodrop 2000c [Thermo Scientific, Rockford, IL] and reverse transcribed into complementary DNA [cDNA] using qScript cDNA SuperMix [Quantabio, Beverly, MA]. qPCR was performed using iQ SYBR Green Supermix [Bio-Rad, Hercules, CA] and an IQ5 Real-Time PCR Thermal Cycler [Bio-Rad]. Primers used for murine Il6, Il10, Il22, and Gapdh are listed in Supplementary Table 3, available as Supplementary data at ECCO-JCC online. Gene expression was calculated using the average C_T values of triplicate readings normalised to the average of housekeeping gene Gapdh C_T values. Gapdh was chosen as the housekeeping gene based on a previous evaluation of several housekeeping genes, where Gapdh was found the most stable in our experimental settings [data not shown]. Results were analysed using the ΔΔC_T method.

2.15. Statistical analysis

Data are presented as mean ± standard deviation [SD] for n number of biological replicates. Animal data are presented as mean ± SD for one of two independent experiments, each with four to six mice as indicated in the figure legends. Statistical analysis was performed using analysis of variance [ANOVA] followed by Tukey’s or Holm‐Sidak post-test; P-values of ≤0.05 were considered significant.

3. Results

3.1. Tofacitinib corrects elevated permeability in IECs lacking PTPN2, and abrogates the effects of PTPN2-deficient macrophages on barrier function

To determine how macrophages influence IEC barrier function, and whether tofacitinib influences this cross-regulation, THP-1 cells were co-cultured with polarised Caco-2BBe cells that were treated with either vehicle or tofacitinib. As we have recently shown⁴¹, co-culture with control [Ctr] THP-1 macrophages tightened the epithelial barrier with increased TEER and reduced FD4 permeability, whereas co-culture with PTPN2 KD THP-1 cells resulted in a leaky barrier, ie, reduced TEER and increased FD4 flux [Figure 1A + B]. Treatment with tofacitinib for 24 h prevented both of these effects [Figure 1]. As previously shown,^43,44 TEER was significantly lower in resting PTPN2-deficient IECs compared with control IECs, and co-culture with macrophages did not promote barrier tightness [ie, no induction of TEER or reduced FD4 flux], whereas co-culture with PTPN2 KD THP-1 further decreased TEER and increased FD4 permeability in PTPN2-deficient IECs [Figure 1].⁴¹ Of note, tofacitinib treatment completely abrogated these effects [Figure 1]. Moreover, tofacitinib rescued the TEER of PTPN2-deficient Caco-2BBe cells to levels comparable to those of control Caco-2BBe cells [Figure 1A]. These results show that the presence of control THP-1 macrophages induces tightening of the epithelial barrier, whereas PTPN2-deficient macrophages promote paracellular passage of ions and macromolecules. Both of these effects on the IEC monolayer were abrogated by tofacitinib. These results demonstrate that JAK inhibition is sufficient to rescue the barrier defects observed in PTPN2-deficient IECs alone, or in IECs cultured in the presence of PTPN2-deficient macrophages.

3.2. Tofacitinib prevents macrophage-induced changes in the expression of tight junction proteins

Whereas the paracellular flux of macromolecules is mainly dictated by the presence and/or localisation of tight junction proteins [eg, occludin, tricellulin, JAM-A, and ZO-1], the major influence on TEER, which fluctuates due to paracellular passage of ions, is mediated by changes in the charge- and size-selective family of claudin proteins.^49,50 Consistent with previous findings⁴¹ and with the reduced TEER and increased macromolecular passage upon loss of TCPTP in macrophages and/or IECs, we observed increased expression of the pore-forming molecule claudin-2 and decreased expression of the barrier-sealing proteins junctional adhesion molecule [JAM-A], occludin, tricellulin, and claudin-4 in those cells. Consistent with the normalisation of TEER, tofacitinib treatment normalised claudin-2 expression in co-culture groups and partly restored expression of claudin-4 in PTPN2 KD Caco2-BBe cells, though these still remained lower than control Caco2-BBe cells [Figure 2A‐C]. In addition, we observed molecule-specific changes in the expression of barrier-sealing proteins: whereas occludin and JAM-A were both reduced in PTPN2-deficient IECs and in Ctr IECs co-cultured with PTPN2-deficient macrophages, JAM-A but not occludin was further decreased upon loss of PTPN2 in both cell types. Notably, only the suppressive effect on occludin was rescued by tofacitinib; tofacitinib treatment was not able to restore JAM-A levels. In contrast, tricellulin levels were only reduced when PTPN2 was absent in macrophages, and this effect was only rescued by tofacitinib treatment in Ctr IECs and not in PTPN2-deficient IECs [Figure 2A, D, F]. Of note, tofacitinib was able to normalise the expression of claudin-2 and occludin [Figure 2A, E], but it also suppressed the induction of tricellulin and JAM-A observed upon co-culture with Ctr macrophages [Figure 2A, D, F]. In addition to these changes in the expression of tight junction molecules, co-culture of Caco-2BBe cells with KD macrophages, or deletion of PTPN2 in Caco-2BBe cells, also promoted internalisation of the tight junction molecule ZO-1 and gap formation between neighbouring IECs, effects completely normalised upon treatment with tofacitinib. TCPTP protein expression in IECs remained unchanged by co-culture with macrophages, tofacitinib treatment, or in combination [Figure 2A, G]. Collectively, these results suggest that changes in the expression patterns of tight junction proteins are individual and distinct, and that tofacitinib rescues the underlying barrier defect of PTPN2-deficient IECs, at least in part, through effects on claudin-2, claudin-4, and occludin.

3.3. Elevated STAT1 and STAT3 phosphorylation in PTPN2-deficient IECs or macrophages are normalised by tofacitinib

TCPTP, the protein product of PTPN2, dephosphorylates several members of the JAK-STAT signalling pathway, including JAK1 and JAK3 that are targeted by tofacitinib. Caco-2BBe cells lacking PTPN2 had significantly higher STAT1 and STAT3 phosphorylation [Figure 3A + B], which were both effectively reduced upon treatment with tofacitinib for 24 h. These data show that tofacitinib can correct the elevated JAK-STAT activation in IECs with PTPN2 loss. Furthermore, co-culture with macrophages significantly increased STAT1 and STAT3 phosphorylation in control Caco-2BBe, both of which were normalised upon tofacitinib treatment [Figure 3A + B].

Similarly, and consistent with findings among other PTPN2-deficient models, macrophages lacking PTPN2 had elevated JAK1 and STAT1 phosphorylation, which was further enhanced upon co-culture with either control or PTPN2-KD IECs, and again fully reversed by tofacitinib [Figure 4A + B]. Co-culture of control THP-1 cells with PTPN2-deficient Caco-2BBe cells also induced JAK1 and STAT1 phosphorylation, and this was completely negated by tofacitinib treatment [Figure 4A + B]. STAT3 phosphorylation levels followed the same pattern in PTPN2-deficient macrophages, but in general, levels of phosphorylated STAT3 were rather low [Figure 4C]. These results demonstrate that in control macrophages, JAK1 is activated in the presence of PTPN2-deficient IECs, but not in the presence of IECs that express normal PTPN2 levels. However, co-culture with IECs of either genotype induced JAK1 and STAT1 phosphorylation in both control and PTPN2-deficient THP-1 cells, though to a much larger extent in PTPN2 KD THP-1 cells, perhaps due to the elevated basal JAK1/STAT1 phosphorylation. Overall, tofacitinib inhibited JAK-STAT signalling in macrophages co-cultured with IECs and reduced the significantly elevated level of JAK-STAT activation in PTPN2 KD macrophages to background levels.

3.4. IL-6, IL-22, and TNF-α secretion is induced by PTPN2 loss but corrected by tofacitinib

A major feature of IBD is altered cytokine secretion, and especially levels of the inflammatory cytokines TNF-α, IFN-γ, and IL-6, as well as levels of IL-22, are highly increased, whereas the production of or the response to the regulatory cytokine IL-10 is compromised in IBD patients.⁵¹ Co-culture of PTPN2-deficient macrophages with control Caco-2BBe cells resulted in secretion of IL-6, IL-22, and TNF-α, an effect further enhanced in PTPN2-deficient Caco-2BBe cells [Figure 4D; Supplementary Figure 1, available as Supplementary data at ECCO-JCC online]. Notably, tofacitinib treatment significantly reduced the robust increases in IL-6 and IL-22 [Figure 4D], suggesting that the aberrant secretion of IL-6 and IL-22 upon loss of PTPN2 in IECs or macrophages may be due to elevated JAK signalling. Loss of PTPN2 in either cell type, however, had no statistically significant effect on IL-10 secretion and was unaltered by tofacitinib treatment [Figure 4D].

3.5. Tofacitinib citrate corrected barrier dysfunction observed in Ptpn2-LysMCre mice in vivo

Building on our observations with in vitro co-culture models, we next determined whether the restoration of barrier function by tofacitinib translated to an in vivo setting using Ptpn2-LysMCre mice [lacking Ptpn2 primarily in monocytes and macrophages⁴⁰] and their Ptpn2^fl/fl littermates. Consistent with previous findings,⁴¹Ptpn2-LysMCre mice exhibited higher in vivo FD4 permeability compared with their Ptpn2^fl/fl counterparts, which was substantially decreased upon twice daily gavage with tofacitinib citrate for 7 days [Figure 5A]. In contrast, tofacitinib treatment had no significant effect on FD4 flux in Ptpn2^fl/fl animals [Figure 5A], suggesting no negative impact on the epithelial barrier upon tofacitinib treatment. Conversely, permeability to creatinine and RD70, which can detect effects on the paracellular pores or the unrestricted pathways, respectively, were not affected by genotype or tofacitinib citrate treatment [Figure 5B + C]. Similar to our in vitro findings, these data suggest that intestinal permeability is elevated by PTPN2 loss in macrophages in vivo, and this effect can be corrected with JAK inhibition by tofacitinib.

3.6. Tofacitinib rescues the molecular remodelling of tight junctions in IECs from Ptpn2-LysMCre mice

To delineate the mechanism behind changes in barrier function in vivo, the abundance of tight junction proteins in proximal colon IECs was analysed. Consistent with previous findings,⁴¹ the expression of claudin-2 was significantly increased in IECs from Ptpn2-LysMCre mice [Figure 6A + B], whereas claudin-4, JAM-A, and occludin were all decreased when compared with Ptpn2^fl/fl controls [Figure 6A‐E]. These effects were corrected by tofacitinib citrate treatment, and expression patterns of these tight junction proteins were all reversed [Figure 6A‐E]. Of note is the induction of occludin expression in Ptpn2^fl/fl mice by tofacitinib [Figure 6E], though this did not result in changes in FD4 permeability. In contrast, no changes in tricellulin expression were seen as a result of loss of Ptpn2 in macrophages or tofacitinib citrate treatment [Figure 6F]. As described previously, and in line with our in vitro data, loss of Ptpn2 in macrophages promoted the expression of claudin-2 in the crypt regions of Ptpn2-LysMCre mice and resulted in reduced and more diffuse E-cadherin and ZO-1 staining [Figure 6G; and Supplementary Figure 2, available as Supplementary data at ECCO-JCC online]. These effects were normalised upon treatment with tofacitinib [Figure 6G; and Supplementary Figure 2].

3.7. STAT signalling is elevated in IECs of Ptpn2-LysMCre mice, which is effectively reduced by tofacitinib citrate administration

Similar to our cell culture data, Ptpn2 deficiency in macrophages also increased STAT3 phosphorylation in IECs in vivo, an effect significantly reduced with tofacitinib citrate treatment [Figure 6H]. Notably, TCPTP levels remained unchanged in IECs of Ptpn2^fl/fl vs Ptpn2-LysMCre mice and were unaffected by tofacitinib treatment [Figure 6I]. IECs from Ptpn2-LysMCre mice did not exhibit elevated STAT1 phosphorylation, suggesting that specific STAT family members are differentially regulated in these animals [Supplementary Figure 3, available as Supplementary data at ECCO-JCC online].

3.8. Gene and protein expression profiles of cytokines Il6, Il10, and Il22 in Ptpn2-LysMCre colons are reversed by tofacitinib citrate treatment

The local cytokine milieu critically influences epithelial and immune cell processes in the gut. To determine whether loss of Ptpn2 in macrophages affected the local cytokine milieu in the intestine, Il6, Il10, and Il22 gene and protein expression in proximal colon of Ptpn2^fl/fl and Ptpn2-LysMCre mice, treated with vehicle or tofacitinib citrate, were determined by qPCR [Figure 7A] and ELISA [Figure 7B]. Il6/Il22 mRNA and IL-6/IL-22 protein expressions were significantly elevated, whereas Il10 mRNA and IL-10 protein expressions were decreased, in vehicle-treated Ptpn2-LysMCre mice compared with Ptpn2^fl/fl mice [Figure 7A + B]. With tofacitinib citrate treatment, expression patterns of Il6/IL-6, Il10/IL-10, and Il22/IL-22 mRNA and protein levels were reversed, though differences in IL-10 protein expression between vehicle- and tofacitinib citrate-treated Ptpn2-LysMCre mice did not reach statistical significance [Figure 7A + B].

3.9. Tofacitinib normalises macrophage polarisation profiles in Ptpn2-LysMCre mice

In line with these findings of altered cytokine production, increased proportions of M1 macrophages were observed in Ptpn2-LysMCre mice compared with their Ptpn2^fl/fl counterparts, whereas M2 macrophage proportions were reduced, with no change in overall macrophage numbers [Figure 7C]. Tofacitinib treatment had no effect on M1/M2 macrophage populations in Ptpn2^fl/fl mice, but reversed the higher M1/lower M2 proportions in Ptpn2-LysMCre mice back to approximate ratios found in control mice [Figure 7C]. These results confirm that loss of Ptpn2 affects macrophage polarisation, which can be normalised by tofacitinib administration.

3.10. Barrier normalisation by tofacitinib prevents increased susceptibility to DSS-induced colitis in Ptpn2-LysMCre mice

Finally, we assessed whether correction of barrier defects in Ptpn2-LysMCre mice using tofacitinib has consequences for the development of intestinal inflammation. Therefore, Ptpn2-LysMCre mice were treated with tofacitinib starting 3 days before induction of colitis using 1.5% DSS. Pre-treatment with tofacitinib corrected the underlying increase in FD4 permeability in Ptpn2-LysMCre mice before the start of DSS administration [Day 0; Figure 8A]. Moreover, tofacitinib pre-treatment prevented the DSS-induced increase in FD4 permeability in Ptpn2^fl/fl and Ptpn2-LysMCre mice [Day 7; Figure 8A]. Consistent with previous reports,⁴⁰ vehicle-treated Ptpn2-LysMCre mice showed increased colitis severity, as measured by enhanced weight loss, increased disease activity scores, more pronounced shortening of the colon, and elevated histological colitis scores [Figure 8B‐F]. Notably, these effects were fully prevented by tofacitinib treatment [Figure 8B‐D]. Furthermore, tofacitinib administration starting 3 days before DSS administration also prevented the development of colitis in Ptpn2^fl/fl mice, as indicated by reduced histological colitis scores [Figure 8E + F].

4. Discussion

In this study, we identified that tofacitinib corrects the disrupted homeostatic interaction between macrophages and intestinal epithelial cells lacking the IBD risk gene PTPN2. Our findings show that tofacitinib normalised barrier dysfunction and changes in tight junction protein expression following loss of PTPN2 in macrophages and IECs. This intestinal barrier-rescuing effect of tofacitinib was confirmed in mice with myeloid cell-specific knockout of Ptpn2. These mice have an underlying, subclinical barrier defect, characterised by increased FD4 permeability associated with reduced expression of tight junction-sealing proteins, elevated epithelial STAT3 activation, altered production of barrier-modulating cytokines, and increased levels of the pore-forming tight junction molecule claudin-2, and they are more susceptible to DSS-induced colitis.⁴⁰ All these effects were rescued upon treatment with tofacitinib. This demonstrates the clinical relevance of our findings and suggests that loss of functional PTPN2 in IBD patients, as observed in PTPN2 loss-of-function variant carriers,^32,52 might render them more responsive to tofacitinib treatment.

In agreement with previous data,⁴³PTPN2-deficient Caco-2BBe monolayers display much lower TEER and higher FD4 permeability compared with controls, and our data in Figure 1 show that tofacitinib rescues this effect. Here, we went a step further to demonstrate that tofacitinib rescues intestinal barrier defects caused by disruption of macrophage-IEC interactions upon loss of PTPN2 in either macrophages or IECs. In our co-culture studies, tofacitinib reversed the expression of some, but not all, tight junction proteins screened, implying that junctional proteins are differentially regulated by the presence of macrophages and/or JAK-STAT signalling in our co-culture system. In addition, since the effects of control and PTPN2-deficient THP-1 cells on permeability of Caco-2BBe control monolayers were negated by tofacitinib treatment, this suggests that tofacitinib-sensitive pathways [presumably JAK-driven] are involved in both the enhancing and the detrimental effects of macrophages on IEC barrier function in vitro. Of note, there was no adverse effect of tofacitinib citrate treatment on the barrier function of Ptpn2^fl/fl [wild-type] mice, which implies that in vivo tofacitinib has no detrimental effect on intestinal permeability.

STAT3 and STAT1 phosphorylation in Caco-2BBe cells correlated well with the changes seen in claudin-2 and occludin expression patterns. Disruption of the STAT-binding motif within the CLDN2 gene promoter prevents aberrantly high CLDN2 expression observed in PTPN2 KD IECs,⁴⁴ and increased STAT3 activation in PTPN2-deficient IECs promotes claudin-2 expression.⁴¹ Whether occludin expression is regulated through JAK-STAT signalling is also not yet known. Surprisingly, the elevated expression of occludin in tofacitinib-treated Ptpn2^fl/fl mice did not yield a functional decrease in FD4 permeability in vivo. The net effect on occludin versus JAM-A [slightly decreased] and tricellulin [no change] expression may thus contribute to the lack of change in macromolecular permeability.

We observed profound changes in the cytokine profile upon loss of PTPN2/TCPTP in macrophages and/or epithelial cells, which were corrected by tofacitinib treatment. The strong upregulation of inflammatory cytokines [ie, IL-6 and TNFα] is well in line with previous reports⁵³ and indicates that TCPTP exerts a critical regulatory function on JAK/STAT signalling to maintain cytokine balance even in the absence of additional stimulation. Specifically, we observed increased IL-6 secretion upon PTPN2 deletion in macrophages, IECs, and in combination. Notably, STAT3 is a major signalling transducer upon IL-6 receptor ligation,⁵⁴ and represents a key downstream target of tofacitinib. Many of the observed effects downstream of PTPN2 loss can be explained by elevated IL-6 and increased STAT3 activation, but the overall effect of IL-6 on epithelial barrier function is still under debate.⁵⁵ Whereas 72 h of IL-6 exposure has been shown to decrease TEER and increase mannitol permeability in Caco-2 monolayers,⁵⁶ 4 kDa-dextran permeability was increased in IL-6 knockout mice when compared with controls.⁵⁷ Thus IL-6 has pleiotropic functions and, whereas some of the observed effects might result from elevated IL-6 in Ptpn2-LysMCre mice,⁴¹ there are likely other factors that contribute to the decreased TEER/barrier function upon loss of PTPN2 in IEC or macrophages. In line with this, we observed that the secretion pattern of TNF-α in our co-culture studies was similar to that of IL-6. TNF-α can induce IL-6 production in Caco-2 cells⁵⁸ and, in combination with IFN-γ, in THP-1 cells,⁵⁹ and thus might be responsible for the elevated IL-6 levels observed here. Epithelial and immune cells produce IL-6,⁶⁰ and therefore the changes we observed in tight junction proteins and JAK-STAT activation could result from autocrine or paracrine signalling. In our co-culture experiments, tofacitinib reduced IL-6 and TNF-α secretion, which agrees with recent findings that tofacitinib pre-treatment reduced both IL-6 and TNF-α secretion in human peripheral monocytes.¹⁶

The profound changes in the expression profiles of IL-6, IL-22, and IL-10 upon loss of TCPTP, which were partially rescued upon tofacitinib treatment, yield insight into which mediators may be causing the changes observed in mice treated with tofacitinib citrate. The elevated expression of the pro-inflammatory cytokine IL-6 and reduced expression of anti-inflammatory IL-10 in proximal colons of vehicle-treated Ptpn2-LysMCre mice suggest that at basal levels, the intestines of these mice are primed towards a pro-inflammatory phenotype. Furthermore, and in agreement with findings that IL-22 upregulates claudin-2 to increase water efflux for bacterial clearance,⁴⁷ both IL-6 and IL-22 protein secretion and mRNA expression correlated with claudin-2 levels. IL22 mRNA expression is also elevated in active CD⁶¹ and UC,⁶² and has been shown to promote epithelial restitution in wound-healing assays⁶¹ and to attenuate intestinal inflammation in vivo.⁶³

Oral gavage of molecular probes such as FD4 and RD70, and assessment of their serum levels, allows for functional analysis of intestinal permeability in vivo.⁴⁷ In this approach, although permeability to RD70 confirmed the absence of mucosal damage in Ptpn2-LysMCre mice, increased FD4 flux demonstrated an increase in the leak pathway, which allows smaller macromolecules, such as bacterial products, to pass through the epithelial paracellular barrier and elicit an immune response. Importantly, this effect was reversed upon tofacitinib citrate treatment, indicating that this drug can normalise barrier defects elicited by macrophages with a pro-inflammatory phenotype. Furthermore, and in agreement with a recent study demonstrating that tofacitinib pre-treatment skews murine bone marrow-derived macrophages [BMDMs towards a regulatory, M2-like phenotype,¹⁴ tofacitinib reversed the enhanced M1 macrophage levels in Ptpn2-LysMCre mice and restored M2 macrophage proportions. Together with our co-culture studies, this effect of tofacitinib on macrophage polarisation indicates a novel mechanism by which JAK inhibition protects the intestinal epithelium. Of note, this protective effect on barrier permeability and correction of macrophage phenotype had direct consequences for the enhanced susceptibility to intestinal inflammation observed in Ptpn2-LysMCre mice, which was completely rescued upon tofacitinib administration. One notable difference from the De Vries study¹⁴ is that we observed inhibition of onset of DSS colitis symptoms by tofacitinib citrate pre-treatment, albeit starting 3 days prior to DSS vs concurrent administration. Our results not only suggest that the detrimental effects of PTPN2-deficient macrophages on IEC barrier function are relevant for the development of intestinal inflammation, but also indicate that tofacitinib can restore barrier function and revert the enhanced colitis susceptibility of mice lacking PTPN2 in macrophages. Moreover, the protective effect when administered before disease induction indicates that tofacitinib treatment might be a promising candidate to maintain remission in UC patients and especially in those with PTPN2 loss-of-function variants.

The genetic profiles of IBD patients included in tofacitinib clinical studies were not reported and thus it is still unknown whether genetic factors determine treatment success. Our results with macrophages and IECs lacking the IBD risk gene PTPN2, suggest that analysing subgroups of patients with distinct genetic profiles for their response to tofacitinib might be a promising approach to detect patients who are more responsive for tofacitinib treatment. Adverse events such as increased rates of overall infections, herpes zoster infections, cardiovascular events, and increased serum lipid levels were observed in IBD patients who received tofacitinib treatment for 52 weeks,¹² and higher rates of pulmonary embolisms and mortality have occurred in rheumatoid arthritis patients receiving high [10 mg] amounts of tofacitinib.⁶⁴ Given the potential for side effects, specifically selecting patients who might be genetically predisposed to respond more favourably to tofacitinib treatment would represent a crucial advance in treating IBD and other autoimmune diseases.

In summary, our results demonstrate that tofacitinib treatment corrected barrier function and tight junction regulation in a co-culture system of IECs with PTPN2-deficient inflammatory macrophages, as well as in an in vivo model system reflecting disrupted macrophage-IEC cross-talk upon loss of Ptpn2 in macrophages, and was able to revert enhanced colitis susceptibility in mice with macrophage-specific Ptpn2 deletion. Our work integrates studies demonstrating effects of tofacitinib on macrophages alone with a major physiological role for these innate immune cells in the regulation of intestinal epithelial barrier function. Our findings indicate that tofacitinib treatment might be an especially effective treatment to restore barrier function and maintain remission in patients harbouring PTPN2 loss-of-function mutations. In addition, the presence of PTPN2 variants might be a good predictor for treatment response to tofacitinib not only in approved conditions like ulcerative colitis, but also in patients not currently indicated for treatment with tofacitinib, such as those with Crohn’s disease. Given the recent important findings demonstrating that increased intestinal permeability is significantly associated with the future risk of developing Crohn’s disease, our data make a potentially significant contribution to the discovery of new agents that can be used to prevent IBD onset by promoting and maintaining a healthy gut barrier.²⁶ Since PTPN2 variants are not only associated with an increased risk to develop IBD, but also with a number of other inflammatory disorders, our findings might not only be relevant for patients suffering from intestinal inflammation, but also for patients suffering from other disease entities that are associated with PTPN2 variants in which tofacitinib represents an established therapy [eg, rheumatoid arthritis], or where it might be considered as a therapeutic strategy in the future.

Funding

This work was supported by a postdoctoral research stipend from the Swiss National Science Foundation [project number P300PB_177932] to MRS, UC Riverside School of Medicine Medical Student Externship Scholarship to CO, research support from the NIH [R21AI137830] to MGN, research support from the NIH [2R01DK091281, 1R01AI153314] to DFM, an ASPIRE-Pfizer 2017 IBD Research Award [W1227049] to DFM, and a Pfizer Global Medical Grants 2020 Research Award [59444033] to DFM.

Conflict of Interest

DFM received research grants from Pfizer (ASPIRE-Pfizer IBD Research Award, Pfizer Global Medical Research Grant). Pfizer had no role in study design, data acquisition, interpretation of the results or approval of the manuscript. All other authors declare no conflict of interest related to this study.

Author Contributions

MRS, AS-B: experiment design, data acquisition, analysis, and interpretation, writing of the manuscript; CO, VC, ANS, SK: data acquisition and analysis; MK: generation of lentiviral constructs for PTPN2-specific and scrambled shRNA; MGN: critical input on flow cytometry; MS: data interpretation and critical intellectual input; DFM, MRS: data interpretation, study design, funding, editing of the manuscript. All authors wrote, revised. and approved the manuscript.

References

Author notes