STAT6 deficiency ameliorates severity of oxazolone colitis by decreasing expression of claudin-2 and Th2-inducing cytokines

Mice

Wild type (WT) and STAT6^−/− C57BL/6 mice were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our facility under specific pathogen free conditions. Male 5–7 week old mice were used for colitis experiments. This study was carried out following recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Vanderbilt University Institutional Animal Care and Use Committee.

Oxazolone Colitis

Oxazolone colitis was induced as previously described (12). Briefly, anesthetized mice were sensitized by topically applying 3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich, Saint Louis, MO) in 100% ethanol (150 μl) on the shaved abdomen. Seven days later, 2% oxazolone in 50% ethanol (150 μl) was administered intrarectally with a 5 French plastic infant feeding tube (C. R. Bard, Covington, GA). Control mice were treated with ethanol vehicle alone. Mice were sacrificed 3 days after rectal oxazolone administration.

Colon Epithelial Cell and Lamina Propria Cell Isolation

Mouse colons were washed in cold PBS, and left on ice in buffer containing 0.5 mM DTT and 3mM EDTA for 60 minutes. Crypts were released into the supernatant by vigorous shaking in cold PBS, filtered through a 70 μm strainer, and centrifuged at 300xg for 10 minutes at 4°C. Whole cell protein lysates were prepared from pelleted epithelial cells. The remaining colon tissue was minced and agitated in DMEM with 2 mg/ml dispase (Roche, Indianapolis, IN) and 0.5 mg/ml collagenase (C5138, Sigma-Aldrich) at 37°C for 40 minutes. Released cells were filtered over a 70 μm strainer, with equal volume of DMEM containing 10% fetal bovine serum, and centrifuged at 300xg for flow cytometry. For identification of natural killer T cells (NKT) cells by flow cytometry, cells were further purified using a 40–70% discontinuous Percoll gradient.

Western Blot Analysis

Western blot analysis on cell lysates was performed as previously described (13). Briefly, membranes were blocked with 5% nonfat dry milk in TBST for 1 hour and incubated overnight at 4°C with primary Ab against total STAT6 (Millipore), pSTAT6 (Cell Signaling, Danvers, MA), claudin-2 (Abcam, Cambridge, MA), E-cadherin (BD Biosciences, San Jose, CA), or β-actin (Sigma-Aldrich). Membranes were incubated with secondary Ab, anti-rabbit- or anti-mouse horseradish peroxidase (Cell Signaling), and protein bands were detected by chemiluminescence using Western Lightning (Perkin Elmer, Waltham, MA). Densitometry was performed using ImageJ software (NIH, Bethesda, MD).

Intracellular pSTAT6 Detection

pSTAT6⁺ lamina propria T cells, macrophages, and NKT cells were quantified by flow cytometry. A fluorescently labeled α-GalCer-loaded murine CD1d tetramer was prepared from CD1d monomers (NIH Tetramer Facility) as described previously (14). Isolated lamina propria cells were surface stained with anti-CD3 (Biolegend, San Diego, CA), anti-F4/80 (Life Technologies, Carlsbad, CA), or a combination of anti-B220 (BD Biosciences), anti-TCR-β (Biolegend), and tetramer prior to intracellular staining with anti-pSTAT6 (Cell Signaling) and/or anti-IL-13 (eBioscience, San Diego, CA). Flow cytometric analysis was performed using a BD LSRII instrument and FlowJo software (Tree Star, Ashland, OR).

High-Resolution Colonoscopy

The colonic mucosa was visualized in live anesthetized mice using a veterinary endoscope (KARL STORZ Imaging, Goleta, CA) according to previously described methods (15).

Histopathology

Colon sections were stained with hematoxylin and eosin stain analyzed by light microscopy. A pathologist (M.K.W.) blinded to genotype and treatment scored each mouse using a previously described 15-point scale, with a maximum of 3 points each for enterocyte loss, crypt hyperplasia, crypt inflammation, neutrophil infiltrate, and mononuclear cell infiltrate (16).

Immunohistochemistry

Sections were deparaffinized, rehydrated, and antigen was unmasked in a citrate-containing buffer (Vector Labs, Burlingame, CA). Sections were stained with mouse anti-claudin-2 Ab (Invitrogen, Carlsbad, CA) and counterstained with hematoxylin. Sections were scored in a blinded fashion by averaging the percent of the crypt axis that was claudin-2 positive for at least 50 crypts per mouse.

Cell Culture

T84 cells were cultured in complete growth medium containing 1:1 DMEM/Ham’s F-12 medium, 5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cultures were incubated at 37°C in a 95% air/5% CO₂ atmosphere. Cells were exposed to IL-13 (10 ng/ml) for 24 hours.

shRNA Lentiviral Particle Transduction

T84 cells were grown to 50% confluence and transduced with lentiviral particles carrying STAT6-specific or non-targeting short hairpin (sh)RNA (Santa Cruz, Santa Cruz, CA) in medium containing polybrene 4 μg/ml (Santa Cruz) with a multiplicity of infection of 0.1. Following overnight transduction, shRNA expressing cells were expanded and selected using medium containing puromycin (8 μg/ml; Santa Cruz). Reduction of STAT6 expression was confirmed by Western blot analysis. Stably transfected cells were maintained with 4 μg/ml puromycin selection.

Transepithelial Resistance

For measurements of transepithelial resistance (TER), T84 cell monolayers were grown on standing mixed cellulose ester membrane inserts (Millipore, Billerica, MA). TER across each monolayer was assessed by using a voltmeter (WPI, Sarasota, FL). Measurements were calculated in Ω · cm² and expressed as a percentage of baseline. For all experiments, baseline TER was > 1000 Ω · cm².

Immunofluorescent Staining

Cells cultured in chamber slides were fixed in methanol at −20°C for 5 minutes, permeabilized in PBS plus 0.2% Triton-X 100 (Sigma-Aldrich), and blocked in 5% goat serum (Life Technologies). Cells were stained with 2.5 μg/ml anti-claudin-2 (Abcam) overnight at 4°C, followed by secondary staining with Dylight 488-conjugated goat-anti-rabbit (Jackson ImmumoResearch, West Grove, PA) and mounting with VectaShield containing DAPI (Vecta Labs, Burlingame, CA).

RNA Expression

RNA was isolated from tissue using the RNeasy Mini Kit (Qiagen, Valencia, CA) per the manufacturer’s instructions. RNA (1 μg) was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). SYBR Green I Master Mix (Roche) was used for real-time PCR reactions for IL-10, IL-17, IFN-γ, the membrane-bound and soluble isoforms of the IL-33 receptor (ST2L and sST2, respectively), and β-actin, and the primers are listed in Supplemental Table I. TaqMan Gene Expression Assays (Life Technologies) were used for IL-13 (assay ID: Mm00434204_m1), IL-33 (Mm00505403_m1), TSLP (Mm00498739_m1), and GAPDH (Mm99999915_g1). All reactions were performed in duplicate with 1 μl cDNA on a Light Cycler 480 II real-time PCR platform (Roche). Relative mRNA levels were determined using the 2^−ΔΔCT method with β-actin or GAPDH as the reference.

Ex Vivo Lymphocyte Cytokine Production

Single cell suspensions were prepared from mesenteric lymph nodes (MLNs) as previously described (17). 5 X 10⁵ cells were cultured in RPMI 1640 media containing 10 mM HEPES, 10 μg/mL gentamicin, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated FBS in round bottom 96-well plates with 5 μg/ml plate-bound anti-CD3 and 1 μg soluble anti-CD28 (Biolegend), with or without 10 ng/ml recombinant mouse IL-33 (Biolegend). Culture supernatants were stored at −80°C for cytokine quantification. Cytokines quantification was performed using a custom Milliplex MAP cytokine panel (Millipore, Billerica, MA) per the manufacturer’s protocol on a Luminex FlexMap 3D (Luminex Corporation, Austin, TX).

Statistical Analyses

For each set of experiments yielding continuous data in 3 or more groups, one-way ANOVA was applied as a global test for differences in the primary outcome variable. Log-transformation was applied if data exhibited a skewed distribution. Histopathologic scoring, as an ordinal variable, was compared globally amongst groups using the nonparametric Kruskal-Wallis test. Pre-determined pair-wise comparisons of interest were made using Student’s t-test or Mann Whitney test with Bonferroni correction only when an overall effect was detected. The relationship between tissue IL-33 relative mRNA expression and histopathology score was explored using Spearman’s correlation coefficient. Repeated measures two-way ANOVA with Bonferroni correction was used to compare measurements of mouse weight and epithelial membrane TER over time amongst experimental groups and to determine the effect of IL-33 treatment on activated MLN cytokine production across experimental groups. Statistical analyses were performed using Prism 5.0b (GraphPad Software, La Jolla, CA).

Increased STAT6 activation in oxazolone colitis

We have previously shown increased epithelial pSTAT6 in pediatric subjects with UC, and that IL-13-induced apoptosis and claudin-2 expression in vitro is STAT6-dependent (8). We hypothesized STAT6 would be activated in cell types acted on by increased IL-13 in oxazolone colitis. To determine whether epithelial pSTAT6 is induced in a murine model of UC, we assessed expression of total and pSTAT6 in colonic epithelial cells isolated from WT mice with oxazolone colitis by Western blot analysis. There was a 2.6-fold increase in epithelial STAT6 phosphorylation in WT mice with oxazolone colitis compared to WT ETOH-treated controls (p = 0.005), with no difference in total STAT6 expression (Figure 1A and1B). Important roles for STAT6 have also been established in lymphocytes and macrophages (18–21). To determine if increased lymphocyte or macrophage STAT6 activation is involved in oxazolone colitis, we performed intracellular staining for pSTAT6 on isolated colonic lamina propria cells from WT mice treated with oxazolone or ETOH vehicle and assessed by flow cytometry. We observed increased absolute numbers of total colon CD3⁺ T cells and pSTAT6⁺CD3⁺ T cells in WT mice treated with oxazolone compared to WT control mice (Figure 1C and 1D). Furthermore, we also observed increased absolute numbers of total colon F4/80⁺ macrophages and pSTAT6⁺F4/80⁺ macrophages in WT mice treated with oxazolone compared to WT control mice (Figure 1E and 1F). Since NKT cells have been proposed as the primary source of IL-13 in oxazolone colitis (10), we interrogated lamina propria cells for pSTAT6⁺IL13⁺ NKT cells (B220⁻TCR-β⁺Tetramer⁺) by flow cytometry. As expected, we found increased percentages of NKT cells in WT mice treated with oxazolone compared to WT control mice (Figure 2A). In addition, WT colitic mice exhibited an increased percentage of pSTAT6⁺ and pSTAT6⁺IL-13⁺ NKT cells (Figure 2B and 2C). These data suggest a role for STAT6 signaling in epithelial cells, T cells, macrophages, and IL-13-producing NKT cells in oxazolone colitis.

Oxazolone colitis is attenuated in STAT6 deficient mice

To determine the role of STAT6 in vivo we induced colitis with oxazolone (OXA) in WT and STAT6^−/− mice. STAT6^−/− OXA mice recovered more quickly from colitis as evidenced by more rapid weight gain between Day 1 and Day 3, with complete weight recovery by Day 3 (Days 2 and 3, p < 0.001) (Figure 3A). There were fewer deaths in STAT6^−/− OXA mice as compared to WT OXA mice; however, the difference in survival was not statistically significant given the overall low mortality rate (Figure 3B). By high-resolution endoscopy, WT OXA mice demonstrated qualitatively obscured vascular pattern, erythematous and granular mucosa, and loose stool consistency compared to WT ETOH mice. In STAT6^−/− OXA mice there was improved vascular pattern, less erythema and granularity, and more formed stool compared to WT OXA mice (Figure 3C). Upon dissection, there was reduced colon length in WT OXA versus WT ETOH mice, an indicator of colitis severity, but not in STAT6^−/− OXA mice (Figure 3D).

As anticipated, WT OXA mice demonstrated histologic features similar to human UC with crypt hyperplasia, crypt loss, cryptitis, and infliltrates of neutrophils and mononuclear cells into the lamina propria, which were less pronounced in STAT6^−/− OXA mice (Figure 3E). In line with these findings, histopathology scores were elevated in WT OXA mice as compared to WT ETOH mice (p < 0.001), and STAT6^−/− OXA mice scored significantly lower than WT OXA mice (p < 0.01) (Figure 3F).

IL-13-induced barrier dysfunction is mediated through STAT6

In colon epithelial cell monolayers, IL-13 impairs barrier function by inducing apoptosis and increasing expression of claudin-2 (22). Others and we have previously shown that suberoylanilide hydroxamic acid (SAHA), a protein-deacetylase inhibitor with STAT6 inhibitory properties, lessens IL-13-mediated reductions in TER in epithelial monolayers (8, 23). As SAHA is an established protein deacetylase inhibitor and has effects on other proteins beside STAT6, we developed a T84 colon epithelial cell line with stable reduction of STAT6 expression using shRNA delivered through a transduced lentiviral vector. STAT6 expression was reduced 83 ± 7% (p = 0.002) in STAT6 shRNA-transduced cells compared to cells transduced with control non-targeting (NT) shRNA. Cells were exposed to IL-13 (10 ng/ml) for 24 hours. As expected, induction of claudin-2 expression with IL-13 was reduced in STAT6 shRNA-transduced cells as demonstrated by Western blot analysis and immunoflourescent staining (Figure 4A and 4B). Transduced cells were plated on semi-permeable transwells and treated with IL-13 to examine whether the effect of IL-13 on TER is mediated through STAT6. Mean TER as a percentage of baseline was greater in the STAT6 shRNA-transduced cells compared to NT shRNA transduced cells after 12 and 24 hours of IL-13 exposure (p < 0.05 for 12 and 24 hours), confirming a role for STAT6 in IL-13-induced epithelial barrier dysfunction in vitro (Figure 4C).

STAT6 regulates claudin-2 induction in oxazolone colitis

Claudin-2 expression is increased in the colonic mucosa of patients with UC, and induction of claudin-2 is an important mechanism through which IL-13 impairs colon epithelial barrier dysfunction (22, 23). We sought to determine whether epithelial claudin-2 expression is induced is oxazolone colitis, and if the induction is mediated through STAT6. We performed immunohistochemical staining for claudin-2 in WT and STAT6^−/− mice treated with oxazolone or ETOH. In WT ETOH mice, claudin-2 staining was concentrated at the intercellular junctions in the lower 33.1 ± 2% of the crypt. Claudin-2 staining was increased in WT/OXA mice extending up to 45.5 ± 2% of the crypt axis (p < 0.001), but not in STAT6^−/− OXA mice, which only demonstrated staining of the bottom 34.5 ± 2% of the crypt (p < 0.001 vs. WT OXA) (Figure 5A and 5B).

STAT6 regulates expression of IL-33 and TSLP in oxazolone colitis

Oxazolone colitis was initially described as a Th2-driven model of colitis with increased lamina propria lymphocyte production of IL-4 and IL-13 (9, 10). Since STAT6 is an important regulator of Th2 lymphocyte differentiation (21), we hypothesized expression of IL-4 and IL-13 would be reduced or absent in STAT6^−/− compared to WT OXA mice. Interestingly, using real-time PCR analysis of colon tissue mRNA, we observed a mixed T-helper lymphocyte response with significantly increased expression of IL-13, IFN-γ, IL-17, and IL-10 (IL-4 was not detected by real-time PCR, data not shown) in WT OXA compared to WT ETOH mice. Furthermore, there was no detectable difference in expression of these cytokines between WT OXA and STAT6^−/− OXA mice (Figure 6A). In recent years, IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 have emerged as important cytokines for the initiation and amplification of Th2 immune responses (24). We observed a 5.6-fold increased relative expression of IL-33 in WT OXA mice as compared to WT ETOH mice, which was abrogated in STAT6^−/− OXA mice. We also observed a 2-fold increased relative expression of TSLP in WT OXA mice which was eliminated in STAT6^−/− OXA mice (Figure 6A). We did not observe any difference in IL-25 expression amongst mouse groups (data not shown). IL-33 mRNA expression strongly correlated with histopathologic severity (Figure 6B). These data suggest that STAT6 regulates mucosal expression of the Th2-inducing cytokines IL-33 and TSLP.

Deficiency of STAT6 reduces MLN cell cytokine secretion in oxazolone colitis

To determine the role of STAT6 in the potential for T cell cytokine production in oxazolone colitis, we compared cytokine secretion from MLN cells activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs amongst WT ETOH, WT OXA, and STAT6^−/− OXA mice. We observed increased IL-4, IL-5, IL-13, IFN-γ, and IL-17 secretion from cells from WT OXA compared to WT ETOH mice, and less secretion of each of these cytokines from cells from STAT6^−/− OXA mice (Figure 7). There was no difference in IL-6 secretion amongst groups. While there was no significant increase in IL-10 secretion from cells from WT OXA mice compared to WT ETOH mice, there was less IL-10 secretion from cells from STAT6^−/− OXA mice compared to WT OXA mice. Thus, STAT6 deficiency impaired T cell secretion of multiple pro-inflammatory cytokines increased in oxazolone colitis.

IL-33 augments MLN pro-inflammatory cytokine secretion

Several groups have implicated IL-33 in the pathogenesis of human UC (25–28). To determine the effect of IL-33 on lymphocyte function in oxazolone colitis, we assessed the cytokine secretion from IL-33-exposed activated MLN cells from WT and STAT6^−/− mice. In addition to activation with anti-CD3 and anti-CD28 mAbs alone, MLN cells were similarly activated in the presence of IL-33 (10 ng/ml). IL-33 augmented the secretion of the Th2 cytokines IL-13 and IL-5, but not IL-4 (Figure 8). IL-33 also augmented the secretion of IFN-γ and IL-6 (Figure 8). The overall effect of IL-33 on MLN cell cytokine secretion did not differ amongst WT ETOH, WT OXA, and STAT6^−/− OXA mice. In STAT6^−/− OXA mice, IL-33 increased MLN cell IL-13 and IL-5 secretion to levels seen in cells from WT OXA mice activated without IL-33, indicating that these cells are still capable of Th2 cytokine production. Others have observed increased mucosal expression of the IL-33 receptor, ST2 in intestinal inflammation (29). We assessed mRNA expression for both the soluble (sST2) and membrane-bound (ST2L) isoforms of the receptor in both colon tissue and MLN cells from these mice and found no differences in expression of either isoform amongst WT ETOH, WT OXA, and STAT6^−/− OXA mice (Supplemental Figure 1). Taken together, IL-33 augments T cell proinflammatory Th2 and Th1 cytokine secretion, even in STAT6^−/− mice.

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