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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Pediatr Gastroenterol Nutr. Author manuscript; available in PMC 2014 April 23.
Published in final edited form as:
PMCID: PMC3996847
NIHMSID: NIHMS350322

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

Abstract

Objectives

Genotypic variation in STAT3 increases risk for 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 CD patients 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 EBV-transformed lymphocytes (EBL).

Results

Colonic expression of IL-6, the STAT3 target gene SOCS3, the neutrophil chemo-attractants 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. EBL's 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 JAK2.

Conclusions

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

Keywords: Pediatric Crohn's disease, Signal Transducer and Activator of Transcription 3 (STAT3), neutrophil recruitment, CXCR2

Introduction

Crohn's 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 now identified multiple genetic factors that confer IBD susceptibility, with some unique to CD or UC, and some shared.[13]. Genotypic variation in STAT3 has been linked to risk for both CD and UC [1]. Recently, 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 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 IL-10; targeted deletion of Stat3 in this cell type leads to severe spontaneous entero-colitis [7]. Conversely, Stat3 activation in CD4+ T cells is required for differentiation of Th17 effector lymphocytes, and blockade of IL-6:Stat3 signaling ameliorates both ileitis and colitis in animal models [8, 9]. Recent work by Nguyen et al. has shown that Stat3 is essential for the neutrophil migratory response to CXCR2 ligands such as CXCL2 via activation of G-CSF induced CXCR2 expression and modulation of CXCR2 signal transduction [10]. We have previously shown that STAT3 activation was increased in PB granulocytes, IL-6-stimulated CD3+/CD4+ lymphocytes, and affected colon biopsies of pediatric IBD patients at diagnosis and during therapy [11]. Furthermore, we identified an IL-6:STAT3 biological network that drives leukocyte recruitment and thereby mucosal inflammation in this setting.

We sought to define the functional consequences of the recently identified G>A intronic SNP (rs744166) within the STAT3 gene in pediatric CD patients. 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 up-regulation of chemokines expressed on 4q12–13 which promote CXCR2+ neutrophil recruitment to the gut.

Materials and 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) and secondary antibodies (Vector Laboratories, West Grove, PA) were used for immunohistochemical (IHC) analysis. pSTAT3 antibodies and antibodies for CD3, CD4, IL6R, and GP130 for flow cytometry and ImageStreamx (amnis® Seattle, WA) analysis were from BD Biosciences (San Jose, CA). Western blot antibodies to pSTAT3 (SC-7993), STAT3, pSTAT1 (SC-135648), STAT1, GP130, IL-6 receptor alpha, JAK2, and SOCS3 were from Santa Cruz Biotechnology (Santa Cruz, CA).

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 CD patients, and from the same segment of normal colon in healthy controls. The diagnosis of CD was made using established clinical, radiological, and histological 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 male.

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) single nucleotide polymorphism (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, Valencia, CA). Samples were submitted to the CCHMC Digestive Health Center Microarray Core where 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 previously published in a study which examined overall biologic 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- PCR (RT-PCR)

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) or an RNeasy Plus Kit (Qiagen, Valencia, CA) and reverse transcribed using Accuscript High Fidelity First-Strand Synthesis System (Stratagene, Cedar Creek, TX). Brillant SYBR Green based detection (Stratgene) utilizing the Stratagene Mx3000P 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 reverse5'-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 Immunohistochemistry (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 10mM sodium citrate (pH 6) for CXCR2 and 1mM EDTA (pH 8) for pSTAT3. Endogenous peroxide was quenched with 3% hydrogen peroxide for 15min at RT and tissue was permeabilized with .3% Triton X-100 for 15 min at RT. 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 R.T.U kit (Vector Laboratories, West Grove, PA) with DAB (diaminobenzidine) as the chromogen.

Enzyme-Linked Immunosorbent Assay (ELISA) for measurement of serum IL-6

IBD patient sera were tested using sandwich ELISAs for the presence of human IL-6 as previously reported [11, 15]. Media from EBL cultures was analyzed for IL-6 concentration by ELISA at baseline prior to 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].

EBV-transformed lymphoblastoid cell lines (EBLs) culture and stimulation

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

Preparation of EBL Cytosolic, Membrane, and Nuclear Proteins

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

Immunoblot analysis

40 μg cytoplasmic protein was separated by NuPAGE® Novex® 4–12% 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. 20 μg of nuclear protein was separated by gel electrophoresis in a similar fashion and stained for rabbit-anti-human antibodies against pSTAT3, pSTAT1, and TFIIβ loading control. 40 μg membrane protein was 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®).

ImageStreamx analysis

EBLs were stimulated as described above and then stained with antibodies for pSTAT3, IL-6 receptor, and DRAQ5 nuclear marker. 5×104 cells were analyzed by IDEAS application © Version 4.0.779.0 for nuclear co-localization of pSTAT3.

Statistical analysis

Statistical analyses were performed using GraphPad PRISM© Version 4.01. Continuous variables were analyzed using unpaired t test, two sample t test with Welch's correction, or Kruskal-Wallis with Dunn's test for multiple comparisons. Discrete variables were analyzed using Fisher's exact test. A p-value <0.05 was considered significant.

Ethical Considerations

The patient-based studies were approved by the CCHMC and University of Utah Institutional Review Boards, and consent was obtained from parents and assent from subjects age 11 and above.

Results

Clinical and Demographic Characteristics

The clinical and demographic data for the CD patient cohort stratified by STAT3 “A” risk allele utilized for the FACS and colon biopsy studies are provided in Table 1. There were no differences for age, gender, 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 Up-regulated in CD Patients Carrying the STAT3 “A” Risk Allele

We first asked if the STAT3 “A” risk allele would be associated with differences in colonic expression of genes we had previously reported to be up-regulated 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 pathological 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 sub-scores (Table 1).

Table 2
Colonic Expression of JAK/STAT Signaling and Leukocyte Recruitment Genes Stratified by STAT3 Genotype

Since 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 up-regulation by quantitative real time PCR. SOCS3 expression was induced five-fold in CD patients carrying the STAT3 “A” risk allele (p = 0.03, Figure 1A). IL-8 expression trended towards a three-fold higher value in CD patients carrying the STAT3 ”A” risk allele (p = 0.07, Figure 1B). CXCL3 mRNA expression was induced twelve-fold in CD patients carrying the STAT3”A” risk allele (p = 0.01, Figure 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 CD patients Carrying the STAT3 “A” Risk Allele

The Frequency of Neutrophils Expressing CXCR2 is Increased in the Colon of CD Patients Carrying the STAT3 ”A” Risk Allele

The frequency of total neutrophils in non-risk allele patients was 5.5+1.3 per hpf compared to 7.5+1.9 per hpf in CD patients carrying the STAT3 “A” risk allele which was not significantly different. However, both were significantly increased compared to 2.1+.3 neutrophils per hpf in controls (p<0.05, data not shown). We then asked if 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 to 3.6 ± 0.8 per hpf in CD patients carrying the STAT3 “A” risk allele (p=0.09, Figure 2A and 2B). The frequency of pSTAT3-positive neutrophils per hpf was positively associated with the overall histologic index of severity (r=0.65, p= 0.02; Figure 2C). CD patients carrying the STAT3 ”A” risk allele exhibited an increased frequency of neutrophils expressing CXCR2 in the colon compared to disease controls (p=0.03; Figure 2D & 2E). 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; Figure 2F).

Figure 2
The Frequency of CXCR2+Neutrophils is Increased in Colon Biopsies from CD Patients Carrying the STAT3”A” Risk Allele and the Frequency of pSTAT3+ and CXCR2+ Neutrophils Correlates with Histological Severity

Peripheral Blood Lymphocyte and Granulocyte STAT3 Tyrosine Phosphorylation are increased in CD Patients Carrying the STAT3”A” Risk Allele

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 previously shown that STAT3 activation was increased in PB granulocytes, IL-6-stimulated CD3+/CD4+ lymphocytes, and affected colon biopsies of pediatric IBD patients [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 (Figure 3A). Patients carrying the STAT3 “A” risk allele exhibited a significantly higher basal frequency of pSTAT3+CD3+/CD4+ lymphocytes (p=0.01, Figure 3B) and pSTAT3+granulocytes (p=0.0004, Figure 3C) compared to non-risk 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, Figure 3C) compared to non-risk allele patients. Comparison of un-stimulated 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=.003, Figure 3B) or granulocytes (p=.02, Figure 3C) following IL-6/IL-6R stimulation. This was specific as the frequency of pSTAT5+CD3+/CD4+ lymphocytes was not different in STAT3 “A” risk allele patients at 13+8 (n=21) compared to 9.4+8 (n=5) in non-risk allele patients. Similarly, the frequency of pSTAT5+ granulocytes was not different in STAT3 “A” risk allele patients at 48+34 (n=21) compared to 36+22 (n=5) in non-risk 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±5pg/ml (n=22) compared to non-risk allele patients 86±25pg/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.

Figure 3
Peripheral Blood Lymphocyte and Granulocyte STAT3 Tyrosine Phosphorylation is Increased in CD Patients Carrying the STAT3 ”A” Risk Allele

IL-6 stimulated STAT3 activation and membrane localization of the IL-6 Signaling Complex are increased in Immortalized B-cell lines from IBD patients 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 utilized EBV transformed B-cell lines (EBL) from IBD patients 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, while Table 4 provides B cell phenotyping stratified by the STAT3”A” risk allele. There were no differences by genotype for age at diagnosis, gender, 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
EBV 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 (Figure 4). However, nuclear protein abundance of tyrosine phosphorylated STAT3 was significantly increased at baseline in EBLs from STAT3 “A” risk allele patients to 4±1 normalized chemoluminescent arbitrary units (AU), compared to 1±0.4 AU in non-risk allele patients (p=0.04, Figure 5A and 5B). 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 to 19±4 AU in non-risk 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 to non-risk allele patients (p=0.001, Figure 5C and 5D). Since nuclear STAT3 accumulation was increased we then asked if 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 to non-risk allele patients (p=0.003 and p=0.04, Figure 5E and 5F).

Figure 4
Cytosolic protein abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor SOCS3 varied by STAT ”A” risk allele
Figure 5
IL-6 Stimulated STAT3 Activation and Expression of STAT3 and SOCS3 are Increased in Immortalized B-cell lines from IBD Patients Carrying the STAT3”A” Risk Allele

Membrane protein abundance of the IL-6 receptor under basal conditions was increased two-fold in EBLs from STAT3 “A” risk allele patients compared to non-risk allele patients (p=0.04, Figure 6A and 6B). The membrane protein abundance of GP130 was increased two-fold in EBLs from STAT3 “A” risk allele patients compared to non-risk allele patients (p=0.003, Figure 6C and 6D). The membrane protein abundance of JAK2 was increased three-fold in EBLs from STAT3 “A” risk allele patients compared to non-risk allele patients (p=0.008, Figure 6E and 6F). Importantly, IL-6 did not appear to be acting in an autocrine fashion to regulate the IL-6:STAT3 signaling complex as EBL supernatants measured by ELISA demonstrated no difference in IL-6 concentration between STAT3 “A” risk allele patients at 22pg/ml compared to non-risk allele patients at 20 pg/ml. Additionally, analysis of EBLs by ImageStreamx (Amnis®) demonstrated increased nuclear co-localization of pSTAT3 following IL-6 stimulation in the STAT3 “A” risk allele EBL (10.8 fold increase) compared to the non-risk allele EBL (Figure 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 IL-6 receptor, GP130, and JAK2 are Increased in Immortalized B-cell lines from IBD Patients Carrying the STAT3”A” Risk Allele
Figure 7
IL-6 Stimulated Nuclear Localization of STAT3 is Increased in EBL Carrying the STAT3”A” Risk Allele

Discussion

Recently identified IBD risk loci encode candidate genes involved in 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 where transient activation induces protective mechanisms but persistent activation furthers disease progression and ultimately malignant transformation. The aim of the current 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 sub-group with this genotype.

The mechanism of enhanced cellular STAT3 responsiveness was not known and to dissect the biochemical pathway we utilized EBLs created from IBD patients. We demonstrated increased IL-6 dependent STAT3 tyrosine phosphorylation in EBLs from STAT3 “A” risk allele patients compared to non-risk allele patients. This mirrored our findings regarding increased peripheral blood leukocyte 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 feel 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 sub-group 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 might be predicted to regulate gene expression; however, we did not observe association between the STAT3”A” risk allele and mRNA expression. Future work will ultimately 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 biologic 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 utilized the EBV-transformed B cell lines as a model system for testing the effect of STAT3 genotype upon JAK:STAT signaling. While 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 IBD patients. 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 IBD patients.

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 up-regulation 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 up-regulated in CD patients 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, ECM 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. However, future studies which directly measure neutrophil chemotaxis will be required to test this.

We did investigate pathways which are involved in neutrophil mobilization since 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 while 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 MPO (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 CD patients 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. Importantly, overall lamina propria cellularity measured within the same biopsies from which we scored the above parameters did not vary for mononuclear or polymorphonuclear sub-scores. These data demonstrate that current 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 sub-population of CD.

We did not observe an association between STAT3 risk allele carriage and disease location or severity within out cohort. However, recent work from Ferguson et. al confirmed increased risk for CD associated with the STAT3 “A” risk allele and demonstrated associations with clinical phenotypes{Ferguson, 2010 #1236}. In that study the frequency of CD patients with STAT3 “GG” homozygous allele carriage was equal to 12.3%. They reported a significantly increased frequency of extra-intestinal 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 very low numbers in our patient population, ultimately limited the power of our analyses. Future studies with greater power to detect clinical associations will be needed to elucidate associations with disease behavior, response to therapy, and rates of colorectal cancer and surgery.

Lastly, 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 Janus Kinase inhibitor, CP-690,550 (CP), is effective in moderate-to-severe UC patients in a dose-dependent manner with improvements in clinical response and remission rates[28]. However CP was 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. While therapeutic options have increased over the past decade, our ability to target newer biologic therapies to specific subgroups of patients has lagged behind. Our data suggests that inhibition of JAK:STAT3 signaling warrants further clinical investigation, and that stratification of CD patients by the STAT3 “A” risk allele may define patient populations that have varying clinical efficacy to investigational agents including the oral Janus Kinase 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, 3235]. Thus STAT3 activation has been referred to as a double-edged sword and investigating factors which 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 up-regulation of chemokines which promote CXCR2+ neutrophil recruitment to the gut in a newly described sub-population of CD patients.

Acknowledgment

This work was supported by the Bioinformatics, Gene Expression, Integrative Morphology and Flow Cytometry cores of the National Institutes of Health (NIH)-supported Cincinnati Children's Hospital Research Foundation Digestive Health Center (1P30DK078392-01), and NIH grants R01 DK078683 (LAD), R01 DK068164 (LAD), T32 DK007727 (BK & RC), and DK069513 and the Primary Children's Medical Center Foundation (SLG). This investigation was supported by Public Health Service research grant UL1-RR025764 and CO6-RR11234 from the National Center for Research Resources. Ramona Bezold, Kathleen Lake, and Ann Rutherford provided outstanding support with subject recruitment.

Grant Support: This work was supported by the Bioinformatics, Gene Expression, Integrative Morphology and Flow Cytometry cores of the National Institutes of Health (NIH)-supported Cincinnati Children's Hospital Research Foundation Digestive Health Center (1P30DK078392-01), and NIH grants R01 DK078683 (LAD), R01 DK068164 (LAD), T32 DK007727 (BK & RC), and DK069513 and the Primary Children's Medical Center Foundation (SLG). This investigation was supported by Public Health Service research grant UL1-RR025764 and CO6-RR11234 from the National Center for Research Resources.

Abbreviations

(CDHIS)
Crohn's Disease Histologic Index of Severity
(EBL)
Epstein Barr Virus transformed lymphocytes
(GM-CS Ab)
Granulocyte-Macrophage Stimulating Factor auto-antibodies
(IBD)
Inflammatory Bowel Disease
(IL-6)
Interleukin 6
(IL6R)
IL-6 receptor
(JAK2)
Janus-associated Kinase 2
(PCDAI)
Pediatric Crohn's Disease Activity Index
(SNP)
Single Nucleotide Polymorphism
(STAT3)
Signal Transducer and Activator of Transcription 3
(UC)
Ulcerative Colitis

Footnotes

Financial disclosures: The authors have no financial arrangement(s) with a company whose product figures prominently in the submitted manuscript or with a company making a competing product.

Transcript Profiling: NCBI gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) accession number GSE9686.

Competing Interest: None to declare

Author Contributions: Study concept and design: TW, BK, SG, LD

Acquisition of data: TW, BK, IJ, SG, EB, RC, MC

Analysis and interpretation of data: TW, BK, HX, AJ, SG, TD

Drafting of the manuscript: TW, BK, LD

Critical revision of the manuscript for important intellectual content: TW, SG, LD

Statistical analysis: TW, BK, SG, LD

Obtained funding: SG, LD

Administrative, technical, or material support: IJ, SG, EB, RC, HX

Study supervision: SG, LD

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