Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn's Disease.

BACKGROUND & AIMS: Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). METHODS: Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. RESULTS: We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1 × 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. CONCLUSIONS: We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

IFNG rs1861494 polymorphism is associated with IBD disease severity and functional changes in both IFNG methylation and protein secretion.

BACKGROUND: Mucosal expression of interferon (IFN)-γ plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD) and IBD risk regions flank IFNG. The conserved IFNG rs1861494 T/C introduces a new CpG methylation site, is associated with disease severity and lack of therapeutic response in other infectious and immune-mediated disorders, and is in linkage disequilibrium with a ulcerative colitis (UC) disease severity region. It seems likely that CpG-altering single nucleotide polymorphisms modify methylation and gene expression. This study evaluated the association between rs1861494 and clinical, serologic, and methylation patterns in patients with IBD. METHODS: Peripheral T cells of UC and Crohn's disease (CD) patients were genotyped for rs1861494 and analyzed for allele-specific and IFNG promoter methylation. Serum antineutrophil cytoplasmic autoantibodies and IFN-γ secretion were measured by enzyme-linked immunosorbent assay and nucleoprotein complex formation by electrophoretic mobility shift assay. RESULTS: IFNG rs1861494 T allele carriage in patients with IBD was associated with enhanced secretion of IFN-γ. T allele carriage was associated in UC with high levels of antineutrophil cytoplasmic autoantibodies and faster progression to colectomy. In CD, it was associated with complicated disease involving a stricturing/penetrating phenotype. Likewise, IFNG rs1861494 displayed genotype-specific modulation of DNA methylation and transcription factor complex formation. CONCLUSIONS: This study reports the first association of IFNG rs1861494 T allele with enhanced IFN-γ secretion and known IBD clinical parameters indicative of more aggressive disease and serological markers associated with treatment resistance to anti-tumor necrosis factor therapy in patients with IBD. These data may be useful prognostically as predictors of early response to anti-tumor necrosis factor therapy to identify patients with IBD for improved personalized therapeutics.

Multiple activating and repressive cis-promoter regions regulate TNFSF15 expression in human primary mononuclear cells.

TL1A/TNFSF15 has been associated with IBD (inflammatory bowel disease) in GWAS (genome-wide association study) and plays a role mediating mucosal inflammation in IBD. Higher TL1A expression is associated with disease severity in both patients and mouse models. Although TL1A has been studied extensively for IBD-associated SNPs, the cis/trans-regulatory regions are poorly defined. Herein we identify response elements regulating TNFSF15 in primary human myeloid cells. Peripheral mononuclear cells transfected with TNFSF15 promoter constructs displayed 30-fold enhanced promoter activity in a minimal -74 bp region. Transactivation was mediated partly by AP-1, since mutation of the AP-1 site resulting in loss of promoter activity. Monocytes transfected with c-Jun siRNA or treated with TAT-TI-JIP (JNK Inhibitor VII TAT-TI-JIP) demonstrated reduced TL1A mRNA and protein levels. Surprisingly, constructs larger than -74 bp did not increase promoter expression (expression of -1275 bp construct was 25% of -74 bp activity), suggesting the presence of both activating and repressing TL1A promoter elements. In fact, mutation of the -210 bp NFκB site enhanced promoter activity (60-fold) suggesting a repressive role for this site. DNA-protein binding to the TL1A AP-1 and NFκB elements was inhibited by excess consensus or TL1A oligonucleotides and binding and confirmed by chromatin immuno-precipitation analysis. Yet, despite the fact that the -210 bp NFκB site acts as a suppressor element, overall mRNA and protein expression were inhibited in monocytes treated with MG132 (NFκB/proteasome inhibitor) or SN50 (NFκB-p50 blocking peptide), suggesting that NFκB acts as both an activator and silencer of TL1A expression. These data suggest that modulation of TL1A expression involves a complex interplay between positive and negative signals, binding to distinct regulatory regions.

Distinct IFNG methylation in a subset of ulcerative colitis patients based on reactivity to microbial antigens.

BACKGROUND: High antibody reactivity toward microbial antigens in Crohn's disease (CD) patients is predictive of a more aggressive disease course. However, few ulcerative colitis (UC) patients exhibit serologic reactivity toward microbial antigens. Mucosal expression of IFN-γ plays a pivotal role in inflammatory bowel disease (IBD) pathogenesis. Recent genome-wide association studies (GWAS) surprisingly link UC, but not CD, risk loci to IFNG. We recently demonstrated that mucosal T cells from IBD patients exhibit distinct patterns of IFNG methylation compared to controls. This study evaluated the relationship between IFNG methylation and serologic and clinical profiles in peripheral T cells from IBD patients. METHODS: DNA from peripheral T cells of 163 IBD patients (91 CD and 64 UC) and 42 controls was analyzed for methylation of eight IFNG sites. Serum markers ASCA, OmpC, I2, CBir, and pANCA were measured by enzyme-linked immunosorbent assay (ELISA). IFN-γ secretion was measured by ELISA. RESULTS: IBD patients requiring surgery exhibited reduced IFNG methylation compared to nonsurgical patients (P < 0.02). Enhancement of IFN-γ secretion (P < 0.003), along with high antibody responses toward multiple microbial antigens (P < 0.017) in UC, but not CD, patients was correlated with decreased IFNG methylation. pANCA levels were not correlated with IFNG methylation. CONCLUSIONS: Levels of IFNG methylation were correlated with immune response to microbial components and expression of IFN-γ in UC patients. Serological and epigenetic markers identify a subset of UC patients with an expression profile of a key TH1 pathogenic cytokine. These data may provide a useful tool to classify a more homogeneous subset of UC patients, allowing for improved diagnostics and targeted therapeutics.

Distinct Methylation of IFNG in the Gut.

Mucosal expression of proinflammatory cytokines plays a pivotal role in inflammatory bowel disease (IBD) pathogenesis. Epigenetic remodeling of chromatin via DNA methylation regulates gene expression. In this study, IFNG DNA methylation was analyzed within the mucosal compartment in both normal and IBD populations and compared to its peripheral counterparts. Overall IFNG methylation (across eight CpG sites) was significantly lower in lamina propria (LP) T cells compared to peripheral blood (PB) T cells. No methylation differences were detected when comparing PB T derived from normal to IBD patients. However, LP T-cell DNA derived from IBD patients displayed different levels of IFNG methylation of the upstream regulatory regions compared to DNA from normal controls. In fact, IFNG DNA promoter methylation levels functionally correlate with IFNG mRNA expression in unstimulated T cells, using quantitative real-time PCR. A 5% decrease in promoter methylation status is associated with nearly a 3-fold increase in IFNG expression. Likewise, methylation of the single -54 bp IFNG SnaB1 site strongly inhibited IFNG promoter expression. These results suggest that the epigenetic methylation status of IFNG may play a mechanistic role in the modulation of cytokine secretion in the mucosa.

Enhancer role of STAT5 in CD2 activation of IFN-gamma gene expression.

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.

CD2 mediates activation of the IFN-gamma intronic STAT binding region in mucosal T cells.

The pathways leading to activation of mucosal lamina propria (LP) T cells differ from those of peripheral T cells. LP T cells exhibit enhanced IFN-gamma secretion when activated through the CD2 pathway. This study demonstrates CD2 signaling is followed by activation of STAT proteins in both peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC), although, distinct differences exist in regulation of IFN-gamma promoter gene expression. Both PBMC and LPMC exhibit enhanced secretion and transactivation of the -2.7 kb IFN-gamma promoter region following CD2 signaling, but the IFN-gamma STAT-binding region (within the first intron) serves as an orientation-independent enhancer of promoter activity only in LPMC. Mutation of the STAT site impairs enhancer activity. In LPMC, but not PBMC, CD2 mediates binding of STAT1 and STAT4 to the IFN-gamma intronic element. Unstimulated LMPC exhibit low levels of phosphotyrosine-STAT4 and STAT1 and phosphoserine-STAT1, which increase substantially following CD2 activation. In PBMC, CD2-mediated phosphorylation is primarily restricted to enhanced levels of phosphotyrosine-STAT1. Thus, these results indicate that both common as well as unique molecular mechanisms are involved in CD2 signaling and activation of the STAT pathway in LP T cells which are critical for regulation of IFN-gamma expression in the gut.