STAT3 Genotypic Variation and Cellular STAT3 Activation and Colon Leukocyte Recruitment in Pediatric Crohn Disease

Abstract

Objectives: 

Genotypic variation in signal transducer and activator of transcription 3 (STAT3) increases risk for inflammatory bowel disease (IBD), and STAT3-dependent inflammatory networks are induced in the colon in these patients. We hypothesized that STAT3 “A” risk allele carriage would be associated with increased cellular STAT3 activation and colon leukocyte recruitment.

Methods: 

Colonic expression of genes regulating STAT3 signaling and leukocyte recruitment and function was measured in pediatric patients with Crohn disease (CD) stratified by STAT3 genotype. The frequency of colonic pSTAT3⁺ and CXCR2⁺ neutrophils was determined using immunohistochemistry. STAT3 tyrosine phosphorylation (pSTAT3) was measured in circulating leukocytes by flow cytometry, and mechanisms regulating STAT3 activation were tested in IBD Epstein-Barr virus (EBV)–transformed lymphocytes (EBL).

Results: 

Colonic expression of interleukin 6 (IL-6), the STAT3 target gene SOCS3, the neutrophil chemoattractants IL-8, CXCL1, and CXCL3, and the neutrophil products S100A8, S100A9, and S100A12 were increased in patients carrying the STAT3 “A” risk allele. The frequency of neutrophils expressing the cognate receptor for IL-8, CXCR2, was increased in colonic biopsies from patients carrying the risk allele, and the frequency of pSTAT3⁺ or CXCR2⁺ neutrophils correlated with histologic severity. The frequency of CD4⁺ lymphocytes and granulocytes expressing pSTAT3 was increased in patients carrying the STAT3 “A” risk allele. EBLs from patients carrying the STAT3 “A” risk allele exhibited increased basal and IL-6–stimulated STAT3 tyrosine phosphorylation, increased transcription of STAT3 and SOCS3 after IL-6 stimulation, and increased membrane localization of the IL-6 receptor, GP130, and Janus-associated kinase 2.

Conclusions: 

The STAT3 “A” risk allele is associated with increased cellular STAT3 activation and upregulation of pathways that promote recruitment of CXCR2⁺ neutrophils to the gut.

Crohn disease (CD) and ulcerative colitis (UC) are chronic relapsing and remitting inflammatory disorders of the gastrointestinal tract commonly referred to as the inflammatory bowel diseases (IBDs). IBD pathogenesis is complex involving the gut flora, epithelial barrier function, and innate and adaptive immunity. The precise etiology remains unclear, but evidence suggests that it involves dysregulation of the host immune response to luminal flora. Genome-wide association studies (GWASs) have identified multiple genetic factors that confer IBD susceptibility, with some unique to CD or UC, and some shared ^(1–3). Genotypic variation in signal transducer and activator of transcription 3 (STAT3) has been linked to risk for both CD and UC ⁽¹⁾. Newly described permutations to build protein–protein interaction (PPI) networks from GWAS identified risk-associated genetic loci revealed that in CD the core candidate network involved Janus-associated kinase 2 (JAK2) and STAT3 ⁽⁴⁾.

In IBD, STAT3 activation has been well documented, with several potential roles. In animal models, STAT3 activation in intestinal epithelial cells is required for acute wound-healing responses but also promotes development of colitis-associated cancer during chronic inflammation ^(5,6). STAT3 activation in myeloid cells mediates anti-inflammatory effects of interleukin (IL)-10; targeted deletion of STAT3 in this cell type leads to severe spontaneous enterocolitis ⁽⁷⁾. Conversely, STAT3 activation in CD4⁺ T cells is required for differentiation of T_H17 effector lymphocytes, and blockade of IL-6:STAT3 signaling ameliorates both ileitis and colitis in animal models ^(8,9). Nguyen et al ⁽¹⁰⁾ have shown that STAT3 is essential for the neutrophil migratory response to CXCR2 ligands such as CXCL2 via activation of granulocyte-colony stimulating factor–induced CXCR2 expression and modulation of CXCR2 signal transduction. We have shown that STAT3 activation was increased in peripheral blood (PB) granulocytes, IL-6–stimulated CD3⁺/CD4⁺ lymphocytes, and affected colon biopsies of pediatric patients with IBD at diagnosis and during therapy ⁽¹¹⁾. Furthermore, we identified an IL-6:STAT3 biologic network that drives leukocyte recruitment and thereby mucosal inflammation in this setting.

We sought to define the functional consequences of the G>A intronic single nucleotide polymorphism (SNP) (rs744166) within the STAT3 gene in pediatric patients with CD. We hypothesized that the STAT3 risk allele “A” would be associated with increased cellular STAT3 activation and differences in colonic expression of chemokines driving leukocyte recruitment. We found that carriage of the STAT3 “A” risk allele is associated with increased cellular STAT3 activation and upregulation of chemokines expressed on 4q12-13, which promote CXCR2⁺ neutrophil recruitment to the gut.

METHODS

Materials

Human IL-6 and soluble IL-6 receptor (sIL-6R) were from R&D Systems (Minneapolis, MN). Tyrosine phosphorylation state specific STAT3 (pSTAT3) (SC-7993; Santa Cruz Biotechnology, Santa Cruz, CA), CXCR2 (CD182; BD Biosciences, San Jose, CA) and secondary antibodies (Vector Laboratories, West Grove, PA) were used for immunohistochemical (IHC) analysis. pSTAT3 antibodies and antibodies for CD3, CD4, IL-6 receptor (IL6R), and GP130 for flow cytometry and ImageStream (Amnis, Seattle, WA) analysis were from BD Biosciences. Western blot antibodies to pSTAT3 (SC-7993), STAT3, pSTAT1 (SC-135648), STAT1, GP130, IL-6 receptor-α, JAK2, and SOCS3 were from Santa Cruz Biotechnology.

Study Subjects for Microarray and Interrogation of Colonic Biopsies

Colon biopsies were obtained from an area of active disease in the ascending colon in pediatric patients with CD, and from the same segment of normal colon in healthy controls. The diagnosis of CD was made using established clinical, radiologic, and histologic criteria. The Pediatric Crohn's Disease Activity Index (PCDAI) was used to measure clinical severity, and the Crohn's Disease Histological Index of Severity (CDHIS) was used to measure mucosal severity ^(12,13). The Montreal system was used to classify disease location and behavior ⁽¹⁴⁾. The mean age (range) for the healthy controls for the colon biopsy studies was 10 (6–18) years; 57% were boys.

Genotyping

Genomic DNA was extracted from whole blood using the PureGene Kit (Gentra System, Minneapolis, MN). Patients were genotyped for the STAT3 G>A (rs744166) SNP using the TaqMan system ⁽¹⁾.

Gene Array Analysis

Two colon biopsies from the ascending colon were placed in RNAlater (Qiagen, Valencia, CA) at 4°C. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). Samples were submitted to the Cincinnati Children's Hospital Medical Center Digestive Health Center Microarray Core in which the quality and concentration of RNA was measured and the global pattern of gene expression was determined using Affymetrix GeneChip Human Genome HG-U133 Plus 2.0 arrays as previously reported ⁽¹¹⁾. These data were published in a study that examined overall biological networks induced in the colon in pediatric IBD, without stratification by STAT3 genotype ⁽¹¹⁾. Data were normalized to allow for array-to-array comparisons, and differences between groups were detected in Genespring with a significance at the 0.05 level and mean fold change relative to healthy control samples. The complete dataset is available at the NCBI gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo), accession number GSE9686.

Gene Expression by Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) or an RNeasy Plus Kit (Qiagen) and reverse transcribed using Accuscript High Fidelity First-Strand Synthesis System (Stratagene, Cedar Creek, TX). Brilliant SYBR Green-based detection (Stratgene) utilizing the Stratagene Mx3000P polymerase chain reaction (PCR) machine was used to determine gene expression. The mRNA levels of the gene of interest and that of the internal standard, hypoxanthine phosphoribosyltransferase (HPRT) or glyceraldehyde phosphate dehydrogenase (GAPDH), were measured and expressed as a ratio to HPRT or GAPDH. Primer sequences are as follows: STAT3 forward 5′-ATG GAA GAA TCC AAC AAC GGC AGC-3′; STAT3 reverse 5′-AGG TCA ATC TTG AGG CCT TGG TGA-3′; CXCL3 forward 5′-AGC ACC AAC TGA CAG GAG AGA AGT-3′; CXCL3 reverse 5′-AGT CCT TTC CAG CTG TCC CTA GAA-3′; IL-8 forward 5′-AGA AAC CAC CGG AAG GAA CCA TCT-3′; IL-8 reverse 5′-AGA GCT GCA GAA ATC AGG AAG GCT-3′; SOCS3 forward 5′-ATT CGC CTT AAA TGC TCC CTG TCC-3′; SOCS3 reverse 5′-TGG CCA ATA CTT ACT GGG CTG ACA-3′.

Colon Histology and IHC

Paraffin-embedded hematoxylin-stained colon biopsies were scored in a blinded manner by a pediatric pathologist (M.C.) using the CDHIS ⁽¹³⁾. For IHC, paraffin-embedded slides were deparaffinized and antigen unmasking was done by boiling for 10 minutes 10 mmol/L sodium citrate (pH 6) for CXCR2 and 1 mmol/L EDTA (pH 8) for pSTAT3. Endogenous peroxide was quenched with 3% hydrogen peroxide for 15 minutes at reverse transcription (RT) and tissue was permeabilized with 0.3% Triton X-100 (Sigma, St Louis, Mo) for 15 minutes at room temperature. Slides were subsequently blocked with 3% serum and then incubated overnight at 4°C with primary antibodies. Detection and visualization of stained cells was achieved using the RTU kit (Vector Laboratories) with DAB (diaminobenzidine) as the chromogen.

Enzyme-linked Immunosorbent Assay for Measurement of Serum IL-6

Sera of patient with IBD were tested using sandwich enzyme-linked immunosorbent assays (ELISAs) for the presence of human IL-6 as previously reported ^(11,15). Media from Epstein-Barr virus (EBV)–transformed lymphocyte (EBL) cultures was analyzed for IL-6 concentration by ELISA at baseline before stimulation.

Flow Cytometry

Whole blood was collected in sodium heparin tubes at the time of colonoscopy and placed directly on ice. Samples were stimulated with IL-6/sIL-6R and surface and intracellular staining was performed as described ⁽¹¹⁾.

Epstein-Barr Virus–transformed Lymphoblastoid Cell Lines Culture and Stimulation

Peripheral blood samples were obtained from 18 patients with IBD, and cells were isolated by gradient centrifugation. These cells were then transfected with EBV to create immortalized EBLs. EBLs were cultured overnight in serum-free media with 1 × 10⁶ cells per milliliter and then stimulated with 100 ng/mL IL-6 and 50 ng/mL IL-6 receptor for 10 minutes before protein isolation and for 3 hours before RNA isolation.

Preparation of EBL Cytosolic, Membrane, and Nuclear Proteins

Nuclear and cytosolic protein fractions were obtained using the NE-PER Nuclear and Cytoplasmic Extraction kit according to the manufacturer's recommendations (Thermo Scientific, Waltham, MA). Membrane protein fraction was obtained using MEM-PER Eukaryotic Membrane Protein Extraction Reagent kit according to the manufacturer's recommendations (Thermo Scientific).

Immunoblot Analysis

Forty micrograms of cytoplasmic protein were separated by NuPAGE Novex 4% to 12% Bis-Tris gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were independently stained for rabbit-anti-human antibodies against STAT3, STAT1, IL-6 receptor, GP130, JAK2, SOCS3, and β-actin loading control. Twenty micrograms of nuclear protein were separated by gel electrophoresis in a similar fashion and stained for rabbit-anti-human antibodies against pSTAT3, pSTAT1, and TFIIβ loading control. Forty micrograms of membrane protein were separated by gel electrophoresis in a similar fashion and stained for rabbit-anti-human antibodies against IL-6 receptor, GP130, JAK2, and β-tubulin loading control. Protein bands were quantified by normalized chemoluminescent arbitrary units (AU) via LAS Image Reader and MultiGauge Software (Fujifilm, Edison, NJ).

ImageStream Analysis

EBLs were stimulated as described above and then stained with antibodies for pSTAT3, IL-6 receptor, and DRAQ5 nuclear marker. A total of 5 × 10⁴ cells were analyzed by IDEAS application (version 4.0.779.0) for nuclear colocalization of pSTAT3.

Statistical Analysis

Statistical analyses were performed using GraphPad PRISM (version 4.01). Continuous variables were analyzed using unpaired t test, 2 sample t test with Welch correction or Kruskal-Wallis with Dunn test for multiple comparisons. Discrete variables were analyzed using the Fisher exact test. A P value <0.05 was considered significant.

Ethical Considerations

The patient-based studies were approved by the Cincinnati Children's Hospital Medical Center and University of Utah institutional review boards, and consent was obtained from parents and assent from subjects ages 11 years and older.

Results

Clinical and Demographic Characteristics

The clinical and demographic data for the patient with CD cohort stratified by STAT3 “A” risk allele used for the fluorescent activated cell sorter (FACS) and colon biopsy studies are provided in Table 1. There were no differences for age, sex, disease location, medication exposure, or clinical or histologic disease activity in patients stratified by the STAT3 “A” risk allele.

TABLE 1: Clinical and demographic characteristics

Colon Expression of Genes Regulating Leukocyte Recruitment and Function Is Upregulated in Patients With CD Carrying the STAT3 “A” Risk Allele

We first asked whether the STAT3 “A” risk allele would be associated with differences in colonic expression of genes we had previously reported to be upregulated in active colonic IBD and involved in leukocyte recruitment. We stratified gene expression determined by microarray as a function of STAT3 “A” risk allele (Table 2). We found that patients carrying the STAT3 “A” risk allele exhibited a significant increase in colonic expression of IL-6, the IL-6:STAT3 target gene SOCS3, leukocyte recruitment genes expressed on 4q12-q13, and S100A8, S100A9, and S100A12. Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated in children with active IBD, have enhanced expression in pathologic conditions of chronic inflammation, and are involved in phagocyte chemotaxis and function ⁽¹⁶⁾. By comparison, expression of CXCL9-11 or CCL11 did not differ between the groups. Importantly, differences in colon gene expression were not accounted for by differences in overall histologic severity, epithelial injury, or lamina propria cellularity (mononuclear or polymorphonuclear cells), as measured by the CDHIS subscores (Table 1).

TABLE 2: Colonic expression of JAK/STAT signaling and leukocyte recruitment genes stratified by STAT3 genotype

Because SOCS3 expression is an indicator of STAT3 activity and IL-8 and CXCL3 play a critical role in neutrophil chemotaxis, we elected to confirm their upregulation by quantitative real-time PCR. SOCS3 expression was induced 5-fold in patients with CD carrying the STAT3 “A” risk allele (P = 0.03, Fig. 1A). IL-8 expression trended toward a 3-fold higher value in patients with CD carrying the STAT3 “A” risk allele (P = 0.07; Fig. 1B). CXCL3 mRNA expression was induced 12-fold in patients with CD carrying the STAT3 “A” risk allele (P = 0.01; Fig. 1C). Collectively, these results demonstrated that the STAT3 “A” risk allele is associated with increased expression of genes mediating leukocyte recruitment located on 4q12-q13 and phagocyte S100 products.

FIGURE 1: Colon expression of the STAT3 target gene SOCS3 and chemokines expressed on 4q12-q13 are increased in patients with Crohn disease carrying the STAT3 “A” risk allele. Colon mRNA expression was measured by quantitative real-time polymerase chain reaction for (A) SOCS3, (B) interleukin (IL)-8, and (C) CXCL3 in biopsies taken from active areas of disease. Data are expressed relative to hypoxanthine phosphoribosyltransferase expression as the mean ± standard error of the mean. GG (n = 4) and AG/AA (n = 10): Crohn disease at diagnosis null, heterozygous, or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t test with Welch correction.

Frequency of Neutrophils Expressing CXCR2 Is Increased in Colon of Patients With CD Carrying STAT3 “A” Risk Allele

The frequency of total neutrophils in nonrisk allele patients was 5.5 + 1.3 per high-powered field (HPF) compared with 7.5 + 1.9 per HPF in patients with CD carrying the STAT3 “A” risk allele, which was not significantly different. Both were, however, significantly increased compared with 2.1 + 0.3 neutrophils per HPF in controls (P < 0.05; data not shown). We then asked whether there would be an increase in the frequency of neutrophils expressing activated STAT3 (pSTAT3) or the cognate receptor for CXCL3 and IL-8, CXCR2, a STAT3 target gene, within colon samples stratified by STAT3 “A” risk allele. In disease controls lacking the risk allele, the frequency of pSTAT3⁺ neutrophils was equal to 1.8 + 0.5 per HPF, compared with 3.6 + 0.8 per HPF in patients with CD carrying the STAT3 “A” risk allele (P = 0.09; Fig. 2A and B). The frequency of pSTAT3-positive neutrophils per HPF was positively associated with the overall histologic index of severity (r = 0.65, P = 0.02; Fig. 2C). Patients with CD carrying the STAT3 “A” risk allele exhibited an increased frequency of neutrophils expressing CXCR2 in the colon compared with disease controls (P = 0.03; Fig. 2D and E). The frequency of CXCR2⁺ cells per HPF was highly related to the overall histologic index of severity within the colon biopsy (r = 0.68, P = 0.01; Fig. 2F). Peripheral blood lymphocyte and granulocyte STAT3 tyrosine phosphorylation are increased in patients with CD carrying the STAT3 “A” risk allele.

FIGURE 2: The frequency of CXCR2 ⁺ neutrophils is increased in colon biopsies from patients with Crohn disease (CD) carrying the STAT3 “A” risk allele and the frequency of pSTAT3⁺ and CXCR2 ⁺ neutrophils correlates with histologic severity. Immunohistochemistry for (A) pSTAT3 was performed in colon biopsies taken at diagnosis from active areas of disease and (B) the frequency of pSTAT3⁺ neutrophils in 10 random high-powered fields (HPFs; original magnification ×400) was determined. CTR: healthy control (n = 6); GG (n = 3), AG/AA (n = 12): CD at diagnosis null, heterozygous, or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t test with Welch correction. C, Pearson regression analysis was used to test for an association between the frequency of pSTAT3⁺ neutrophils per HPF and histologic severity measured by the Crohn's Disease Histological Index of Severity (r = 0.65; P < 0.02). Thin arrows depict pSTAT3-positive neutrophils; fat arrows depict nonstained neutrophils. Immunohistochemistry for (D) CXCR2 was performed in colon biopsies taken at diagnosis from active areas of disease and the frequency of CXCR2 ⁺ (E) neutrophils in 10 random (HPFs; original magnification ×400) was determined. CTR: healthy control (n = 6); GG (n = 3), AG/AA (n = 14): CD at diagnosis null, heterozygous, or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t test with Welch correction. F, Pearson regression analysis was used to test for an association between the frequency of CXCR2 ⁺ neutrophils per HPF and histologic severity measured by the CDHIS (r = 0.68; P < 0.01). Thin arrows depict CXCR2-positive neutrophils; fat arrows depict nonstained neutrophils.

We then asked whether differences in the frequency of pSTAT3 neutrophils observed in the mucosa would be reflected in differences in circulating granulocytes. We have shown that STAT3 activation was increased in PB granulocytes, IL-6–stimulated CD3⁺/CD4⁺ lymphocytes, and affected colon biopsies of pediatric patients with IBD ⁽¹¹⁾. We therefore asked whether carriage of the STAT3 “A” risk allele would be associated with increased cellular STAT3 tyrosine phosphorylation. We measured intracellular CD3⁺/CD4⁺ lymphocyte and granulocyte STAT3 tyrosine phosphorylation (pSTAT3) before and after stimulation with IL-6/IL-6R by flow cytometry ⁽¹⁷⁾. The cell surface markers CD3 and CD4 were used to identify the lymphocyte population and granulocytes were identified based upon scatter properties (Fig. 3A). Patients carrying the STAT3 “A” risk allele exhibited a significantly higher basal frequency of pSTAT3⁺CD3⁺/CD4⁺ lymphocytes (P = 0.01; Fig. 3B) and pSTAT3⁺ granulocytes (P = 0.0004; Fig. 3C) compared with nonrisk allele patients. Moreover, patients carrying the STAT3 “A” risk allele also exhibited a significantly higher frequency of pSTAT3⁺ granulocytes after IL-6/IL-6R stimulation (P = 0.001; Fig. 3C) compared with nonrisk allele patients. Comparison of unstimulated and stimulated cells within the same genotype revealed that only samples from patients carrying the risk allele exhibited a significantly higher frequency of pSTAT3⁺ lymphocytes (P = 0.003; Fig. 3B) or granulocytes (P = 0.02; Fig. 3C) following IL-6/IL-6R stimulation. This was specific because the frequency of pSTAT5⁺CD3⁺/CD4⁺ lymphocytes was not different in STAT3 “A” risk allele patients at 13 + 8 (n = 21) compared with 9.4 + 8 (n = 5) in nonrisk allele patients. Similarly, the frequency of pSTAT5⁺ granulocytes was not different in STAT3 “A” risk allele patients at 48 + 34 (n = 21) compared with 36 + 22 (n = 5) in nonrisk allele patients. We also measured the circulating concentration of the STAT3 activating cytokine IL-6 and found that there was significantly less in patients carrying the STAT3 “A” risk allele at 34 + 5 pg/mL (n = 22) compared with nonrisk allele patients 86 + 25 pg/mL (n = 3; P < 0.05). Collectively, these data demonstrated that the STAT3 “A” risk allele is associated with increased STAT3 activation in primary peripheral blood leukocytes (PBLs).

FIGURE 3: Peripheral blood lymphocyte and granulocyte STAT3 tyrosine phosphorylation is increased in patients with Crohn disease (CD) carrying the STAT3 “A” risk allele. Intracellular STAT3 tyrosine phosphorylation (pSTAT3) was measured by flow cytometry in washed peripheral blood leukocytes before and after stimulation with interleukin (IL)-6/IL-6R (PBLs). T helper lymphocytes were identified as the CD3⁺CD4⁺ population and granulocytes were identified based upon forward and side scatter properties. A, Representative scattergrams for the frequency of CD3⁺CD4⁺ lymphocytes or granulocytes expressing pSTAT3 are shown, with and without IL-6/IL-6R stimulation. Frequency of (B) CD3⁺CD4⁺ lymphocytes and (C) granulocytes expressing pSTAT3 are shown as the mean ± standard error of the mean. GG (n = 5), AG/AA (n = 20–22): CD at diagnosis null, heterozygous, or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t test with Welch correction.

IL-6–Stimulated STAT3 Activation and Membrane Localization of the IL-6–Signaling Complex Are Increased in Immortalized B-cell Lines From Patients With IBD Carrying the STAT3 Risk Allele

To investigate the mechanism by which the STAT3 “A” risk allele could be mediating differences in cellular STAT3 activation, we used EBV-transformed B-cell lines (EBL) from patients with IBD genotyped for the STAT3 risk allele. Table 3 provides the clinical and demographic data for the EBL patient cohort stratified by STAT3 “A” risk allele carriage, whereas Table 4 provides B-cell phenotyping stratified by the STAT3 “A” risk allele. There were no differences by genotype for age at diagnosis, sex, IBD phenotype, or medication exposures at the time of sample collection. Moreover, the B-cell phenotype did not vary by IBD phenotype, so results were stratified by STAT3 “A” risk allele within the entire IBD cohort.

TABLE 3: Epstein-Barr virus cell line patient demographics and medication exposures

TABLE 4: B-cell surface markers

We first tested whether the STAT3 “A” risk allele would be associated with differences in cytosolic abundance of the IL-6:STAT3-signaling complex, or STAT3 itself. Neither cytosolic protein abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor SOCS3 varied by STAT3 “A” risk allele (Fig. 4). Nuclear protein abundance of tyrosine phosphorylated STAT3 was, however, significantly increased at baseline in EBLs from STAT3 “A” risk allele patients to 4 ± 1 normalized chemoluminescent AU, compared with 1 ± 0.4 AU in nonrisk allele patients (P = 0.04; Fig. 5A and B). Following IL-6 stimulation, the nuclear protein abundance of phosphorylated STAT3 was also significantly increased to 32 ± 4 AU in EBLs from STAT3 “A” risk allele patients compared with 19 ± 4 AU in nonrisk allele patients (P = 0.04). Conversely, analysis of the alternative IL-6:STAT1 pathway determined that the nuclear protein abundance of phosphorylated STAT1 was significantly decreased following IL-6 stimulation in EBLs from STAT3 “A” risk allele patients compared with nonrisk allele patients (P = 0.001; Fig. 5C and D). Because nuclear STAT3 accumulation was increased, we then asked whether transcription of STAT3 target genes including STAT3 and SOCS3 would also be increased. Neither STAT3 nor SOCS3 basal mRNA expression differed by STAT3 risk allele carriage; however, following IL-6 stimulation, STAT3 and SOCS3 mRNA expression were significantly increased in EBLs from STAT3 “A” risk allele patients compared with nonrisk allele patients (P = 0.003 and P = 0.04; Fig. 5E and F).

FIGURE 4: Cytosolic protein abundance of STAT3, STAT1, interleukin (IL)-6 receptor, JAK2, GP130, nor SOCS3 varied by STAT “A” risk allele. Epstein-Barr virus–transformed lymphocyte (EBL) cytosolic protein abundance of (A) STAT3, (B) STAT1, (C) JAK2, (D) IL-6 receptor, (E) GP130, and (F) SOCS3 were measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units to loading control β-actin via LAS Image Reader and MultiGauge Software (Fujifilm). Representative immunoblots for each are shown. GG (n = 6) and AA (n = 9): EBLs null or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t test.

FIGURE 5: Interleukin (IL)-6–stimulated STAT3 activation and expression of STAT3 and SOCS3 are increased in immortalized B-cell lines from patients with inflammatory bowel disease carrying the STAT3 “A” risk allele. Epstein-Barr virus–transformed lymphocyte (EBL) nuclear protein abundance of (A) tyrosine phosphorylated STAT3 and (C) tyrosine phosphorylated STAT1 before and after IL-6/IL-6R stimulation was measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units to loading control TFIIβ via LAS Image Reader and MultiGauge Software (Fujifilm). Representative immunoblot for (B) pSTAT3 and (D) pSTAT1. EBL mRNA expression was measured by quantitative real-time PCR for (E) STAT3 and (F) SOCS3 before and after stimulation with IL-6/ IL-6R. Data are expressed relative to glyceraldehyde phosphate dehydrogenase as mean ± standard error of the mean. GG (n = 6) and AA (n = 6–9): EBLs null or homozygous for STAT3 “A” risk allele. Differences were tested by (A), (C), and (E) unpaired t test with Welch correction or (F) unpaired t test.

Membrane protein abundance of the IL-6 receptor under basal conditions was increased 2-fold in EBLs from STAT3 “A” risk allele patients compared with nonrisk allele patients (P = 0.04; Fig. 6A and B). The membrane protein abundance of GP130 was increased 2-fold in EBLs from STAT3 “A” risk allele patients compared with nonrisk allele patients (P = 0.003; Fig. 6C and D). The membrane protein abundance of JAK2 was increased 3-fold in EBLs from STAT3 “A” risk allele patients compared with nonrisk allele patients (P = 0.008; Fig. 6E and F). IL-6 did not appear to be acting in an autocrine fashion to regulate the IL-6:STAT3-signaling complex because EBL supernatants measured by ELISA demonstrated no difference in IL-6 concentration between STAT3 “A” risk allele patients at 22 pg/mL compared with nonrisk allele patients at 20 pg/mL. Additionally, analysis of EBLs by ImageStream (Amnis) demonstrated increased nuclear colocalization of pSTAT3 following IL-6 stimulation in the STAT3 “A” risk allele EBL (10.8-fold increase) compared with the nonrisk allele EBL (Fig. 7). Collectively, these data demonstrated that targeting of the IL-6:STAT3-signaling complex to the membrane, and IL-6:STAT3 signaling was enhanced in EBLs from patients carrying the STAT3 “A” risk allele.

FIGURE 6: Membrane localization of the interleukin (IL)-6 receptor, GP130, and JAK2 are increased in immortalized B-cell lines from patients with IBD carrying the STAT3 “A” risk allele. Epstein-Barr virus–transformed lymphocyte (EBL) membrane protein abundance of (A) IL-6 receptor (C) GP130 and (E) JAK2 were measured by immunoblot analysis. Data are expressed as normalized chemoluminescent arbitrary units to loading control β-tubulin via LAS Image Reader and MultiGauge Software (Fujifilm). Representative immunoblot for (B) IL-6 receptor, (D) GP130, and (F) JAK2. GG (n = 7) and AA (n = 10): EBLs null or homozygous for the STAT3 “A” risk allele. Differences were tested by unpaired t test.

FIGURE 7: Interleukin (IL)-6–stimulated nuclear localization of STAT3 is increased in Epstein-Barr virus–transformed lymphocyte (EBL) carrying the STAT3 “A” risk allele. EBLs from patients with IBD carrying the STAT3 “A” risk allele or disease controls carrying the “G” nonrisk allele were examined under basal conditions and following IL-6 stimulation. ImageStream (Amnis) analysis demonstrated increased nuclear colocalization of pSTAT3 following IL-6 stimulation in the STAT3 “A” risk allele EBL (10.8-fold increase) compared with no change in the nonrisk “G” allele EBL.

DISCUSSION

Recently identified IBD risk loci encode candidate genes involved in the maintenance of the epithelial barrier, innate responses to microbial products, and differentiation and function of effector and regulatory lymphocytes. STAT3 activation has been well documented in these processes in both human and murine colitis in which transient activation induces protective mechanisms, but persistent activation furthers disease progression and ultimately malignant transformation. The aim of the present study was to delineate how the recently identified intronic G >A STAT3 SNP (rs744166) is associated with specific pathways involved in the pathogenesis of CD. We found that the STAT3 “A” risk allele is associated with increased cellular STAT3 activation and induction of pathways regulating leukocyte recruitment and function in the affected colon in the patient subgroup with this genotype.

The mechanism of enhanced cellular STAT3 responsiveness was not known, and to dissect the biochemical pathway, we used EBLs created from patients with IBD. We demonstrated increased IL-6–dependent STAT3 tyrosine phosphorylation in EBLs from STAT3 “A” risk allele patients compared with nonrisk allele patients. This mirrored our findings regarding increased PBL STAT3 tyrosine phosphorylation in patients carrying the STAT3 “A” risk allele. We found that EBLs carrying the STAT3 “A” risk allele possess increased membrane protein abundance of the IL-6:STAT3 receptor complex (IL-6 receptor, GP130, and JAK2), in the absence of a difference in autocrine IL-6 exposure. The enhanced membrane accumulation of the IL6R-signaling complex likely accounts for the increased cellular responses to IL-6 via STAT3 activation. We believe this is specific to the STAT3 pathway in that we found a decrease in STAT1 activation in EBLs carrying the STAT3 “A” risk allele. These differences in cell signaling may drive disease in the subgroup of patients who carry the STAT3 risk allele via STAT3-dependent effects upon T-lymphocyte and granulocyte differentiation, activation, and survival.

The STAT3 risk SNP (rs744166) is located within the intron between exon 1 and exon 2 and may be predicted to regulate gene expression; however, we did not observe associations between the STAT3 “A” risk allele and mRNA expression. Future work will require sequencing of the entire gene and its surrounding genomic sequence to delineate the genetic basis for differences in cellular STAT3 responses. The IL-6:STAT3 biological network was the focus for our work, and we realize our limitation in that we did not interpret responses to other STAT3 IBD effector cytokines such as IL-10, IL-11, IL-17, IL-22, IL-23, and IL-27 or the expression level of their cognate receptors. We used the EBV-transformed B-cell lines as a model system for testing the effect of STAT3 genotype upon JAK:STAT signaling. Although the B-cell–signaling responses may have been influenced by the EBV transformation, we observed a similar enhancement in lymphocyte STAT3 activation in primary cells from patients with IBD. Future studies will be required to characterize the mechanistic basis for differences in cell signaling in IL-6–stimulated T cells and granulocytes from the peripheral blood of patients with IBD.

Patients carrying the STAT3 “A” risk allele exhibited increased colonic expression of chemokines located on 4q12-q13 (IL-8, CXCL2, and CXCL3) and S100A8, S100A9, and S100A12. Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, known as damage-associated molecular patterns, are found at high concentrations in inflamed tissue and have been shown to be involved in neutrophil chemotaxis ⁽¹⁹⁾. Increased mucosal release correlates with fecal markers of IBD disease activity, and in myeloid progenitor cells upregulation of S100A8 and S100A9 was shown by direct binding of STAT3 to the gene promoter via chromatin immunoprecipitation ^(18,20). Here we demonstrate that S100A8, S100A9, and S100A12 are upregulated in patients with CD carrying the STAT3 “A” risk allele, in the absence of an overall difference in clinical or mucosal disease activity. Furthermore, we did not find differences in the expression of genes classified in other pathways such as immune and inflammatory mediators, cancer and cell proliferation, extracellular matrix tissue remodeling, or metabolism. This suggests that patients carrying the STAT3 “A” risk allele may have underlying biology that involves increased neutrophil chemotaxis and activation. Future studies that directly measure neutrophil chemotaxis will, however, be required to test this theory.

We did investigate pathways that are involved in neutrophil mobilization because they are closely associated with the outcome of inflammation ^(21,22). It has been shown that STAT3 regulates CXCR2 expression during mobilization responses and CXCR2 binds IL-8 and CXCL6 to promote neutrophil migration, whereas CXCL1, -2, -3, and -5 enhance neutrophil chemoattractant activity ^(10,23,24). In murine models of chemically induced colitis, small molecule antagonism of the CXCR2 receptor or genetic deletion reduces myeloperoxidase (neutrophil) activity, colonic damage, and clinical symptoms ^(25,26). Thus, we evaluated the frequency of neutrophils expressing pSTAT3, and the cognate receptor for IL-8, CXCR2⁺, and found them to be increased in colonic biopsies from patients with CD carrying the STAT3 “A” risk allele. Consistent with the murine studies, we found that the frequency of pSTAT3⁺ or CXCR2⁺ neutrophils was highly correlated with histologic severity. Overall lamina propria cellularity measured within the same biopsies from which we scored the above parameters did not vary for mononuclear or polymorphonuclear subscores. These data demonstrate that the existing clinical scoring systems are not able to distinguish between these differential pathways driving disease and also suggest the utility of fecal calprotectin as a plausible biomarker for this subpopulation of CD. We did not observe an association between STAT3 risk allele carriage and disease location or severity within out cohort. Recent work from Ferguson et al, however, confirmed increased risk for CD associated with the STAT3 “A” risk allele and demonstrated associations with clinical phenotypes ⁽²⁷⁾. In that study the frequency of patients with CD with STAT3 “GG” homozygous allele carriage was equal to 12.3%. They reported a significantly increased frequency of extraintestinal manifestations, inflammatory disease behavior, and colonic involvement in individuals who have the STAT3 “A” risk allele. This is consistent with our studies regarding mechanisms of colonic disease in patients carrying the STAT3 “A” risk allele, although patients homozygous for the “G” allele were at extremely low numbers in our patient population, ultimately limited the power of our analyses. Future studies with greater power to detect clinical associations are needed to elucidate associations with disease behavior, response to therapy, and rates of colorectal cancer and surgery.

Data from human and murine models of colitis indicate that STAT3 may be an important target for the treatment of IBD. Recent clinical trials reported in abstract form have shown that the oral JAK inhibitor, CP-690,550 (CP), is effective in moderate to severe patients with UC in a dose-dependent manner with improvements in clinical response and remission rates ⁽²⁸⁾. CP was, however, not effective in CD ⁽²⁹⁾. The specificity of CP is for JAK1 and JAK3 over JAK2, and JAK3 is restricted to hematopoietic cells, whereas JAK1 and JAK2 are ubiquitously expressed ^(30,31). The divergent result in CD versus UC may reflect differences in the underlying pathogenesis and supports further study of specific JAK:STAT-signaling pathways in these disorders.

It is likely that there are several immunogenetic forms of IBD, with CD and UC representing the broadest clinical classifications. Although therapeutic options have increased during the last decade, our ability to target newer biological therapies to specific subgroups of patients has lagged behind. Our data suggest that inhibition of JAK:STAT3 signaling warrants further clinical investigation, and that stratification of patients with CD by the STAT3 “A” risk allele may define patient populations that have varying clinical efficacy to investigational agents including the oral JAK inhibitor, CP-690,550. Furthermore, activation of STAT3 occurs during innate and acquired immune responses in multiple cell types having both pro- and anti-inflammatory functions ^(7,32–35). Thus, STAT3 activation has been referred to as a double-edged sword, and investigating factors that mediate inflammation downstream of STAT3 may lead to more targeted approaches. Collectively, our studies demonstrate that the STAT3 IBD “A” risk allele (rs744166) is associated with increased cellular STAT3 activation and upregulation of chemokines that promote CXCR2⁺ neutrophil recruitment to the gut in a newly described subpopulation of patients with CD.

Acknowledgments

Ramona Bezold, Kathleen Lake, and Ann Rutherford provided outstanding support with subject recruitment.