Candidate Gene For Inflammatory Bowel Disease Research Articles

Myeloid Ptpn2 Deficiency in Mice Causes Functional Iron Deficiency

Background: Anemia is the most prevalent extraintestinal complication of inflammatory bowel disease (IBD) and is associated with the manifestation of Crohn’s disease. Anemia significantly reduces the quality of life, increases hospitalization visits, and increases medical care expenses of IBD patients. Iron deficiency is the leading cause of anemia in IBD; however, the physiological mechanisms involved are still poorly understood. Macrophages significantly contribute to iron homeostasis by mechanisms including iron recycling by phagocytosing senescent erythrocytes and damaged cells, and nurturing erythropoiesis in the bone marrow. In this study, we investigated the role of the IBD candidate gene, protein tyrosine phosphatase non-receptor type 2 ( PTPN2), in regulating macrophage iron homeostasis. Methods: Myeloid cell-specific knockout mice ( Ptpn2LysMCre) and control littermates ( Ptpn2fl/fl) were analyzed for serum iron content and liver and spleen non-heme iron. Duodenal epithelial cell (DEC) proteins were assayed by Western blotting. Localization of the brush border ferrous iron transporter, DMT1, and the coupled ferrous iron absorption driver, NHE3, in duodenal tissue was determined by immunohistochemistry (IHC). Gene expression profiles of macrophages were analyzed by Nanostring. Results: Ptpn2LysMCre mice had reduced serum iron ( p<0.0001; n=12) and reduced spleen non-heme iron (p=0.0017; n=6), whereas liver non-heme iron was unaltered (n=6), indicating iron deficiency in select tissues. DMT1 (n=8) and NHE3 (n=5) protein expression in DECs was unaltered and IHC confirmed the unaltered localization of DMT1 and NHE3 (n=6) in the duodenal epithelium of Ptpn2LysMCre mice, suggesting the reduction in serum iron was not due to an impairment in intestinal non-heme iron uptake. Nanostring analyses revealed that the gene expression of the iron transporter Dmt1, iron exporter Fpn, and iron importer Tfr1 were significantly downregulated (p<0.05; n=5) in macrophages from Ptpn2LysMCre mice, indicating an iron sequestering phenotype. Conclusions: Loss of myeloid Ptpn2 in mice causes functional iron deficiency. These findings indicate a major role of myeloid PTPN2 in regulating systemic iron homeostasis. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The IBD candidate gene, PTPN2, Restricts Epithelial and Macrophage Invasion of a Novel Reporter Adherent‐Invasive Escherichia coli

BackgroundDysbiosis of the gut microbiota and expansion of pathobionts such as adherent‐invasive Escherichia coli (AIEC) are associated with the pathogenesis of Inflammatory Bowel Disease (IBD). Mice deficient in expression of the IBD gene candidate, Ptpn2, exhibit pronounced expansion of a novel murine adherent‐invasive Escherichia coli (mAIEC). The aim of this study was to generate a fluorescent mAIEC to study host susceptibility to mAIEC invasion, and to identify how loss of PTPN2 expression in host cells modifies mAIEC invasiveness.MethodsmAIEC was isolated from the distal colon of a Ptpn2 knockout (KO) mouse. mAIEC was made chemically competent and then transformed with a plasmid expressing mCherry fluorescent protein and a chloramphenicol resistance cassette. For bacterial adherence and invasion studies, parental Caco‐2bbe cells were infected with mAIEC‐mCherry at multiplicity of infection (MOI) of 10 for 3 hours. Caco‐2bbe intestinal epithelial cells (IEC), stably expressing either scrambled shRNA or shRNA against Ptpn2 were infected with mAIEC‐mCherry (MOI=10) for 5 hours. Peritoneal macrophages from Ptpn2 wildtype, heterozygous and KO mice were infected with mAIEC‐mCherry for 5 hours.ResultsWe previously demonstrated that mAIEC can adhere to and invade IECs. We next validated that mAIEC‐mCherry adhered to (0.94 ±0.61 bacteria/cell compared to control E. coli K12; 0.14±0.03 bacteria/cell, n=4, P=0.0039) IECs. mAIEC‐mCherry had comparable adherence (n=4, P=0.35) to IECs as control (unlabeled) mAIEC. Bacterial invasion of mAIEC‐mCherry invasion of IECs was visually confirmed by fluorescence microscopy. To better understand if the loss of expression of PTPN2, which drives increased luminal abundance of mAIEC, also affected mAIEC pathogenicity, we next tested if Ptpn2 deficiency in host cells altered mAIEC‐mCherry invasion. We observed by immunofluorescence that Ptpn2 knockdown (KD) IECs were more susceptible to mAIEC‐mCherry invasion compared to control IECs (n=3). We also confirmed that macrophages from Ptpn2‐KO mice were more susceptible to mAIEC‐mCherry invasion compared to wildtype and Ptpn2‐heterozygous mouse macrophages (n=5, wildtype vs. KO, P=0.0044; KO vs. heterozygous, P=−0.8665). Ptpn2‐heterozygous macrophages were also more susceptible to invasion than wildtype, indicating a gene‐dosing effect of macrophage PTPN2 loss on bacterial invasion (wildtype vs. heterozygous, P=0.0117). These data identify that loss of PTPN2 expression increases susceptibility to mAIEC invasion.ConclusionHere, we validated a newly generated fluorescently tagged mAIEC to study interactions between this novel mouse AIEC with host cells. We further demonstrated that Ptpn2 deficiency promotes mAIEC of intestinal epithelial cells and macrophages. These data suggest that IBD patients with loss‐of‐function mutations in PTPN2 may have increased susceptibility to colonization by disease‐relevant AIEC.Support or Funding InformationNIH 2R01DK091281 (DFM)

Computational drug repurposing for inflammatory bowel disease using genetic information

As knowledge of the genetics behind inflammatory bowel disease (IBD) has continually improved, there has been a demand for methods that can use this data in a clinically significant way. Genome-wide association analyses for IBD have identified 232 risk genetic loci for the disorder. While identification of these risk loci enriches our understanding of the underlying biology of the disorder, their identification does not serve a clinical purpose. A potential use of this genetic information is to look for potential IBD drugs that target these loci in a procedure known as drug repurposing. The demand for new drug treatments for IBD is high due to the side effects and high costs of current treatments. We hypothesize that IBD genetic variants obtained from GWAS and the candidate genes prioritized from the variants have a causal relationship with IBD drug targets. A computational drug repositioning study was done due to its efficiency and inexpensiveness compared to traditional in vitro or biochemical approaches. Our approach for drug repurposing was multi-layered; it not only focused on the interactions between drugs and risk IBD genes, but also the interactions between drugs and all of the biological pathways the risk genes are involved in. We prioritized IBD candidate genes using identified genetic variants and identified potential drug targets and drugs that can be potentially repositioned or developed for IBD using the identified candidate genes. Our analysis strategy can be applied to repurpose drugs for other complex diseases using their risk genes identified from genetic analysis.

ERAP1 and HLA-C interaction in inflammatory bowel disease in the Spanish population.

Large genome-wide analysis studies (GWAS) and meta-analyses have dramatically increased our knowledge of the genetic risk factors of inflammatory bowel disease (IBD), identifying at least 163 loci. The endoplasmic reticulum aminopeptidase-2 ( ERAP2) gene has been reported as a potential candidate gene for IBD. GWAS have also shown the potential associations between ERAP single nucleotide polymorphisms (SNP) loci and susceptibility to several autoimmune diseases, and ERAP1 and ERAP2 polymorphisms are related to HLA class I-associated diseases, including ankylosing spondylitis and Behçet's disease. Interestingly, these associations were confined to individuals carrying HLA class I-risk alleles. The aim of this study was to investigate the association of ERAP1 and ERAP2 SNPs with IBD in a Spanish population, analysing their possible interaction with specific HLA-C alleles to IBD susceptibility. A total of 367 individuals were divided into 216 IBD cases and 151 controls. SNP genotyping was performed using TaqMan® genotyping assays, whereas HLA-C typing was analysed by sequence-specific oligonucleotide probing. Herein, we report an association of the ERAP1 SNP rs30187 with the HLA-C*07 allele. The existence of shared inflammatory pathways in immunologically related diseases together with the understanding of ERAP1 function may offer clues to novel treatment strategies.

VSL#3 Probiotic Stimulates T-cell Protein Tyrosine Phosphatase-mediated Recovery of IFN-γ-induced Intestinal Epithelial Barrier Defects.

VSL#3 is a probiotic compound that has been used in the treatment of inflammatory bowel disease. T-cell protein tyrosine phosphatase (TCPTP) is the protein product of the inflammatory bowel disease candidate gene, PTPN2, and we have previously shown that it protects epithelial barrier function. The aim of this study was to investigate whether VSL#3 improves intestinal epithelial barrier function against the effects of the inflammatory bowel disease-associated proinflammatory cytokine, interferon-gamma (IFN-γ) through activation of TCPTP. Polarized monolayers of T84 intestinal epithelial cells were treated with increasing concentrations of VSL#3 to determine effects on TCPTP expression and enzymatic activity. Therapeutic effects of VSL#3 against barrier disruption by IFN-γ were measured by transepithelial electrical resistance and fluorescein isothiocyanate-dextran permeability. A novel TCPTP-deficient HT-29 intestinal epithelial cell line was generated to study the role of TCPTP in mediating the effects of VSL#3. Tight junction protein distribution was assessed with confocal microscopy. VSL#3 increased TCPTP protein levels and enzymatic activity, correlating with a VSL#3-induced decrease in IFN-γ signaling. VSL#3 corrected the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ. Moreover, the restorative effect of VSL#3 against IFN-γ signaling, epithelial permeability defects, altered expression and localization of the tight junction proteins claudin-2, occludin, and zonula occludens-1, were not realized in stable TCPTP/(PTPN2)-deficient HT-29 intestinal epithelial cells. VSL#3 reduces IFN-γ signaling and IFN-γ-induced epithelial barrier defects in a TCPTP-dependent manner. These data point to a key role for TCPTP as a therapeutic target for restoration of barrier function using probiotics.

Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease.

BackgroundGenome-wide association studies (GWAS) have revealed many susceptibility loci for complex genetic diseases. For most loci, the causal genes have not been identified. Currently, the identification of candidate genes is predominantly based on genes that localize close to or within identified loci. We have recently shown that 92 of the 163 inflammatory bowel disease (IBD)-loci co-localize with non-coding DNA regulatory elements (DREs). Mutations in DREs can contribute to IBD pathogenesis through dysregulation of gene expression. Consequently, genes that are regulated by these 92 DREs are to be considered as candidate genes. This study uses circular chromosome conformation capture-sequencing (4C-seq) to systematically analyze chromatin-interactions at IBD susceptibility loci that localize to regulatory DNA.ResultsUsing 4C-seq, we identify genomic regions that physically interact with the 92 DRE that were found at IBD susceptibility loci. Since the activity of regulatory elements is cell-type specific, 4C-seq was performed in monocytes, lymphocytes, and intestinal epithelial cells. Altogether, we identified 902 novel IBD candidate genes. These include genes specific for IBD-subtypes and many noteworthy genes including ATG9A and IL10RA. We show that expression of many novel candidate genes is genotype-dependent and that these genes are upregulated during intestinal inflammation in IBD. Furthermore, we identify HNF4α as a potential key upstream regulator of IBD candidate genes.ConclusionsWe reveal many novel and relevant IBD candidate genes, pathways, and regulators. Our approach complements classical candidate gene identification, links novel genes to IBD and can be applied to any existing GWAS data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1100-3) contains supplementary material, which is available to authorized users.

Drug Repositioning in Inflammatory Bowel Disease Based on Genetic Information

Currently, 200 genetic risk loci have been identified for inflammatory bowel disease (IBD). Although these findings have significantly advanced our insight into IBD biology, there has been little progress in translating this knowledge toward clinical practice, like more cost-efficient drug development. Our aim was to use genetic knowledge to identify drugs that warrant further investigation in IBD treatment. We hypothesized that proteins encoded by IBD candidate genes are potential IBD drug targets because genetic information can increase successful drug identification. We identified drugs that target the proteins encoded by IBD candidate genes using the DrugBank. We included proteins that are in direct protein-protein interaction with proteins encoded by IBD risk genes. Promising potential IBD drugs were selected based on a manual literature search of all identified drugs (PubMed, ClinicalTrials.gov). We have identified 113 drugs that could potentially be used in IBD treatment. Fourteen are known IBD drugs, 48 drugs have been, or are being investigated in IBD, 19 are being used or being investigated in other inflammatory disorders treatment, and 32 are investigational new drugs that have not yet been registered for clinical use. We confirm that proteins encoded by IBD candidate genes are targeted by approved IBD therapies. Furthermore, we show that Food and Drug Administration-approved drugs could possibly be repositioned for IBD treatment. We also identify investigational new drugs that warrant further investigation for IBD treatment. Incorporating this process in IBD drug development will improve the utilization of genetic data and could lead to the improvement of IBD treatment.

Interleukin-27 as a Novel Therapy for Inflammatory Bowel Disease: A Critical Review of the Literature.

Inflammatory bowel disease (IBD) is an inflammatory disorder of the intestine that affects an estimated 329 per 100,000 people in the United States and is increasing in incidence within a number of cultures worldwide. Likely due to its incompletely understood pathophysiology and etiology, the standard treatments for IBD are only efficacious in subsets of patients and often do not induce lasting remission. As a result, novel therapies are needed. The success of anti-tumor necrosis factor-α treatment in a subset of patients with IBD demonstrated that therapy targeting a single cytokine could be efficacious in IBD, and clinical trials investigating the blockade of a variety of cytokines have commenced. Interleukin (IL) 27 is a relatively recently discovered type I cytokine with established roles in infectious disease, autoimmunity, and cancer in a variety of organs. IL-27 was identified as a candidate gene for IBD, and a number of studies in mouse models of IBD have demonstrated that IL-27 therapy is protective. However, in contrast to these investigations, genetic deletion of the IL-27 receptor has been shown to be protective in some mouse models of IBD. The purpose of this review is to highlight the recent literature investigating the role of IL-27 in IBD and to discuss the possible explanations for the sometimes conflicting results of these studies. Evidence supporting IL-27 therapy as a treatment for IBD will also be discussed.

Loss of PTPN2 in Intestinal Epithelial Cells Upregulates a Novel STAT‐Dependent Mechanism of Claudin‐2 Expression

Inflammatory bowel disease (IBD) is a multi‐factorial condition characterized by elevated levels of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Genome‐wide‐association‐studies (GWAS) revealed that individuals with single nucleotide polymorphic mutations in the locus of the protein tyrosine phosphatase non‐receptor type 2 (PTPN2) gene that encodes T‐cell protein tyrosine phosphatase (TCPTP), have an increased susceptibility to IBD. We have reported that reduced TCPTP expression increased effects of IFN‐γ on Signal Transducer and Activator of Transcription (STAT) signaling and led to a greater reduction in barrier function in intestinal epithelial cells (IEC). The protein composition of tight junctions (TJ) determines the tightness of the barrier. Claudin‐2 is a TJ protein that is upregulated in IBD and increases paracellular permeability to cations thus reducing transepithelial electrical resistance (TER). We have previously shown that transient knockdown of TCPTP permits increased expression of claudin‐2 through Janus Kinase (JAK)‐STAT signaling. The aim of the present study was to identify the molecular mechanisms underlying STAT regulation of claudin‐2 expression in PTPN2 (TCPTP)‐knockdown (PTPN2‐KD) HT‐29 IEC. Western blot and immunofluorescence studies revealed that basal STAT1 phosphorylation (Y701) and claudin‐2 expression were increased in unchallenged PTPN2‐KD cells compared to control shRNA transfected cells, and this effect was greatly enhanced by IFN‐γ treatment. Protein analysis of soluble and insoluble fractions showed increased claudin‐2 in the insoluble fraction in KD cells compared to control cells. siRNA silencing of STAT1 mRNA levels significantly decreased claudin‐2 protein and mRNA expression. To confirm STAT binding to the claudin‐2 promoter region, we used Chromatin immunoprecipitation (ChIP) assay to immunoprecipitate STAT1 from IFN‐γ‐treated PTPN2‐KD and control IEC with anti‐STAT1 antibody, and probed the associated DNA with primers against the claudin‐2 promoter. ChIP analysis showed increased STAT1 binding to the claudin‐2 promoter region in KD cells compared to control cells. Next we determined if STAT1 activates claudin‐2 promoter activity through the STAT binding site. We performed a luciferase reporter assay using a human claudin‐2 promoter‐luciferase construct expressed in PTPN2‐KD or Con‐shRNA IEC. Luciferase activity was significantly increased (214 ± 9%, p<0.0001; n=3) in PTPN2‐KD cells over control IEC. In further studies using two different constructs of the claudin‐2 promoter region with mutations of the STAT binding site, there was a significant decrease in promoter activity (43±15%, p<0.0001; n=3) when compared to full length claudin‐2 promoter activity in KD cells, but no significant difference with control cells. In summary, we have demonstrated that STAT1 regulates claudin‐2 expression through binding to its promoter region under conditions of decreased activity of the IBD candidate gene, PTPN2.

VSL#3 Probiotic Stimulates T‐Cell Protein Tyrosine Phosphatase‐Mediated Protection of Intestinal Epithelial Barrier Function

VSL#3 is a probiotic cocktail that has shown efficacy in treating murine colitis and patients with ulcerative colitis. VSL#3 can also exert a beneficial effect on epithelial barrier function which is dysregulated in inflammatory bowel disease (IBD). We have previously shown that the enzyme T-cell protein tyrosine phosphatase (TCPTP), encoded by the IBD candidate gene PTPN2, protects intestinal epithelial barrier function. The aim of this study was to identify if VSL#3 activates TCPTP and protects intestinal epithelial cells (IEC) from the effects of the IBD-associated pro-inflammatory cytokine interferon-gamma (IFN-γ). Administration of VSL#3 to T84 or HT-29 IEC polarized monolayers increased expression and enzymatic activity of TCPTP in a dose-dependent and time-dependent manner. VSL#3 reduced IFN-γ signaling, as reflected by decreased phosphorylation of STAT-1, in a TCPTP-dependent manner. In addition, VSL#3 alone increased IEC barrier integrity and promoted recovery of barrier function in IFN-γ pre-treated IEC monolayers. VSL#3 conditioned medium also promoted partial recovery from IFN-γ-induced barrier defects. Using a stable TCPTP-deficient HT-29 cell line, we observed that the recovery of TER induced by VSL#3 in IFN-γ treated cells was TCPTP-dependent. In conclusion, the probiotic VSL#3 increased activity and expression of TCPTP to reduce inflammatory signaling and correct epithelial barrier defects.

PTPN2 Gene Deficiency Leads to Increased Epithelial Permeability and Promotes STAT‐1‐dependent Claudin‐2 Expression

Interferon gamma (IFN‐γ) is an inflammatory cytokine involved in inflammatory bowel disease (IBD), and is known to compromise the intestinal barrier. siRNA silencing of the IBD candidate gene protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) in human intestinal epithelial cells (IEC) elevates expression of claudin‐2 causing increased cation permeability of tight junctions. The aim of this study was to identify the cellular and molecular mechanisms underlying claudin‐2 expression in the absence of PTPN2. We developed a HT‐29 IEC line stably expressing short hairpin RNA for PTPN2 using a lentiviral system to knockdown PTPN2 expression. RT‐PCR and densitometry analysis indicated that PTPN2 expression was significantly reduced (60 ± 4%, p<0.0001) in PTPN2‐depleted cells, paralleled with an increased expression in claudin‐2 protein (180 ± 4%, p<0.0005). Western blot and immunofluorescence studies revealed that basal phosphorylation of the PTPN2 substrate, STAT‐1, as well as claudin‐2 protein expression, were significantly increased in PTPN2 KD cells. This effect was potentiated following IFN‐γ treatment. siRNA silencing of STAT‐1 decreased claudin‐2 protein expression in PTPN2 KD cells. There was also a significant increase in in vivo intestinal permeability to FITC‐Dextran (4 kD) in 3‐week old Ptpn2 knockout (298 ± 5%, p<0.0001) and heterozygote mice (206 ± 12%, p<0.001) compared to wild‐type mice. STAT‐1 phosphorylation and claudin‐2 protein expression were increased in Ptpn2 KO mouse cecum vs. wild type. In summary, PTPN2 deficiency leads to increased intestinal permeability and elevated claudin‐2 expression through a STAT‐1‐dependent pathway.

Effects of GWAS-associated genetic variants on lncRNAs within IBD and T1D candidate loci.

Long non-coding RNAs are a new class of non-coding RNAs that are at the crosshairs in many human diseases such as cancers, cardiovascular disorders, inflammatory and autoimmune disease like Inflammatory Bowel Disease (IBD) and Type 1 Diabetes (T1D). Nearly 90% of the phenotype-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) lie outside of the protein coding regions, and map to the non-coding intervals. However, the relationship between phenotype-associated loci and the non-coding regions including the long non-coding RNAs (lncRNAs) is poorly understood. Here, we systemically identified all annotated IBD and T1D loci-associated lncRNAs, and mapped nominally significant GWAS/ImmunoChip SNPs for IBD and T1D within these lncRNAs. Additionally, we identified tissue-specific cis-eQTLs, and strong linkage disequilibrium (LD) signals associated with these SNPs. We explored sequence and structure based attributes of these lncRNAs, and also predicted the structural effects of mapped SNPs within them. We also identified lncRNAs in IBD and T1D that are under recent positive selection. Our analysis identified putative lncRNA secondary structure-disruptive SNPs within and in close proximity (+/−5 kb flanking regions) of IBD and T1D loci-associated candidate genes, suggesting that these RNA conformation-altering polymorphisms might be associated with diseased-phenotype. Disruption of lncRNA secondary structure due to presence of GWAS SNPs provides valuable information that could be potentially useful for future structure-function studies on lncRNAs.

IL-10 in humans: lessons from the gut, IL-10/IL-10 receptor deficiencies, and IL-10 polymorphisms.

Inflammatory bowel disease (IBD) represents a heterogeneous group of gastrointestinal disorders, where commensal gut flora provokes an either (a) insufficient or (b) uncontrolled immune response. This results either in a lack of or in excessive inflammation mainly manifesting as Crohn's disease or ulcerative colitis. IBD commonly presents in adolescence and adulthood and often follows a chronic relapsing course. Genetic and/or environmental factors contribute to the failure of gut immune homeostasis. Genome-wide association studies have identified over 160 susceptibility loci associated with IBD, including polymorphisms in interleukin-10 (IL-10). The anti-inflammatory cytokine IL-10 dampens intestinal inflammation and is therefore a good candidate gene for IBD. Polymorphisms in the IL-10 receptor are also associated with ulcerative colitis presenting in early childhood. Moreover, severe infantile enterocolitis resembling Crohn's disease, caused by loss-of-function mutations in IL-10 and IL-10 receptor, is characterised by a very early onset (usually within the first 3 months of life), unresponsiveness to standard treatment including immunosuppressive therapy, and severe perianal disease with abscesses and fistulas. In these patients, inflammation and polymorphic infiltrates are mainly confined to the colon with very little involvement of the small intestine. Ulceration and granulomas, bloody diarrhoea and failure to thrive also occur. Furthermore, patients may suffer from recurrent fever and respiratory infections. Individuals with IL-10 receptor mutations also experience cutaneous folliculitis and arthritis. Hematopoietic stem cell transplantation is currently the only curative therapy.

Polymorphisms in the IL2RA and IL2RB Genes in Inflammatory Bowel Disease Risk

Associations with different autoimmune diseases of polymorphisms in genes encoding the IL2RA and IL2RB subunits (located in 10p15 and 22q13, respectively), were identified through genome-wide studies. Polymorphisms in these two genes were studied in 107 inflammatory bowel disease (IBD) patients (39 Crohn's disease [CD] and 68 ulcerative colitis [UC]) and in 162 ethnically healthy controls from Tunisia (Sfax). Two of the 15 IL2RA single-nucleotide polymorphisms (SNPs) genotyped (rs4749924 and rs706778) were significantly associated with UC (pcorr=0.018 and 0.048, respectively), but no evidence of association with CD was observed. The IL2RA GTCT haplotype was also more frequent in UC patients compared to controls (2.6% vs. 0%; p=0.002). One of the 6 IL2RB SNPs genotyped (rs743776) was significantly associated with CD (pcorr= 0.039), but no evidence of association with UC was observed. No significant association between IL2RB haplotypes was observed among investigated groups. Our study identified markers in the IL2RA and IL2RB genes that are significantly associated with UC and CD, respectively. Our results supporting IL2RA and IL2RB as promising candidate genes for IBD and suggesting a potential role of IL2R in the pathogenesis of IBD, likely involves regulatory T cells.

Regulation of epithelial barrier function by the inflammatory bowel disease candidate gene, PTPN2

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) has been identified as an inflammatory bowel disease (IBD) candidate gene. However, the mechanism through which mutations in the PTPN2 gene contribute to the pathogenesis of IBD has not been identified. PTPN2 acts as a negative regulator of signaling induced by the proinflammatory cytokine, interferon-gamma (IFN-γ). IFN-γ is known not only to play an important role in the pathogenesis of Crohn's disease (CD), but also to increase permeability of the intestinal epithelial barrier. We have shown that PTPN2 protects epithelial barrier function by restricting the capacity of IFN-γ to increase epithelial permeability and prevent induction of expression of the pore-forming protein, claudin-2. These data identify an important functional role for PTPN2 as a protector of the intestinal epithelial barrier and provide clues as to how PTPN2 mutations may contribute to the pathophysiology of CD.

NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2

ObjectiveThe NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex...

A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents

Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens.

Genetic variation in the autophagy gene ULK1 and risk of Crohnʼs disease

The autophagy pathway has been linked with Crohn's disease (CD) through association of the ATG16L1 and IRGM genes with susceptibility for CD, and also to the Nod2 pathway, involved in CD. Our aim was to investigate polymorphisms in selected autophagy genes for their association with susceptibility to CD. We prioritized all known human homologs of yeast autophagy (Atg) genes according to their location in a known inflammatory bowel disease (IBD) locus or in a genomic region detected in a genome-wide association study (GWAS) or GWAS-meta-analysis. A total of 70 haplotype tagging single nucleotide polymorphisms (tSNPs) in 12 genes were genotyped in a cohort of CD patients (n = 947) and controls (n = 548). Transmission disequilibrium testing (TDT) was performed in an independent cohort of 335 parent-child CD-trios. The frequency of the T-allele of tSNP rs12303764 in ULK1 was significantly higher in CD (64%) versus controls (57%, corrected P-value 0.002). TDT demonstrated overtransmission of this allele to affected offspring (P = 0.02). Model-based multifactor dimensionality reduction (MB-MDR) interaction analysis confirmed a strong main effect for rs12303764. No interaction was found between ULK1 and CARD15, or between ULK1 genotypes and CD phenotypes. We report a genetic association with a tSNP in ULK1, an interesting candidate gene for IBD, given the role of ULK1 in autophagy initiation, and the interaction between Nod2 and autophagy pathways. To further clarify the role of ULK1 in CD, an in-depth investigation of the variation in the region and possible role for copy number variation in this region should be evaluated.

Polymorphisms in the gene encoding bovine interleukin-10 receptor alpha are associated with Mycobacterium avium ssp. paratuberculosis infection status

BackgroundJohne's disease is a chronic inflammatory bowel disease (IBD) of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Since this pathogen has been implicated in the pathogenesis of human IBDs, the goal of this study was to assess whether single nucleotide polymorphism (SNPs) in several well-known candidate genes for human IBD are associated with susceptibility to MAP infection in dairy cattle.MethodsThe bovine candidate genes, interleukin-10 (IL10), IL10 receptor alpha/beta (IL10RA/B), transforming growth factor beta 1 (TGFB1), TGFB receptor class I/II (TGFBR1/2), and natural resistance-associated macrophage protein 1 (SLC11A1) were sequenced for SNP discovery using pooled DNA samples, and the identified SNPs were genotyped in a case-control association study comprised of 242 MAP negative and 204 MAP positive Holstein dairy cattle. Logistic regression was used to determine the association of SNPs and reconstructed haplotypes with MAP infection status.ResultsA total of 13 SNPs were identified. Four SNPs in IL10RA (984G > A, 1098C > T, 1269T > C, and 1302A > G) were tightly linked, and showed a strong additive and dominance relationship with MAP infection status. Haplotypes AGC and AAT, containing the SNPs IL10RA 633C > A, 984G > A and 1185C > T, were associated with an elevated and reduced likelihood of positive diagnosis by serum ELISA, respectively.ConclusionsSNPs in IL10RA are associated with MAP infection status in dairy cattle. The functional significance of these SNPs warrants further investigation.

Anti‐Saccharomyces cerevisiae Antibodies Status Is Associated with Oral Involvement and Disease Severity in Crohn Disease

To determine anti-Saccharomyces cerevisiae antibodies (ASCA) status and its relation to disease phenotype in patients with inflammatory bowel disease (IBD). A total of 301 Scottish patients with early-onset IBD-197 Crohn disease (CD), 76 ulcerative colitis (UC), 28 indeterminate colitis (IC)-and 78 healthy control individuals were studied. ASCA status (IgA, IgG) was determined by enzyme-linked immunosorbent assay. ASCA status was then analyzed in relation to CD phenotype. Patients with CD had a higher prevalence of ASCA than patients with UC and healthy controls: 82/197 versus 12/76, odds ratio (OR) 3.80 (1.93-7.50) and 82/197 versus 6/78, OR 8.56 (3.55-20.62), respectively. Univariate analysis showed that positive ASCA status was associated with oral CD (17/25 vs 59/153, OR 3.39 [1.38-8.34]), perianal CD (39/77 vs 38/108, OR 1.89 [1.04-3.44]) and the presence of granulomata (63/132 vs 15/52, OR 2.25 [1.13-4.48]) and also with markers of disease severity: raised C-reactive protein (44/90 vs 12/49, OR 2.95[1.36-6.37]), hypoalbuminemia (44/85 vs 20/74, OR 2.28[1.19-4.37]), and surgery (27/49 vs 54/147, OR 2.11 [1.10-4.06]). From multivariate analysis, the presence of oral disease (adjusted P = 0.001, OR 22.22 [3.41-142.86]) and hypoalbuminemia (adjusted P = 0.01, OR 4.78 [1.40-16.39]) was found to be independently associated with ASCA status. No association was demonstrated between ASCA and IBD candidate genes. Patients with CD had a higher prevalence of ASCA than did other patients with IBD. ASCA status described patients with CD who had a specific phenotype, showing an association with markers of disease severity and oral CD involvement.