Polymorphisms in the Toll-Like Receptor and the IL-23/IL-17 Pathways Were Associated with Susceptibility to Inflammatory Bowel Disease in a Danish Cohort

Abstract

Background

The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), result from the combined effects of susceptibility genes and environmental factors. Previous studies have shown that polymorphisms in the Toll-like receptor (TLR), the apoptosis, the IL-23/IL-17 and the interferon gamma (IFNG) pathways are associated with risk of both CD and UC.

Methods

Using a candidate gene approach, 21 functional single nucleotide polymorphisms (SNPs) in 15 genes were assessed in a clinical homogeneous group of severely diseased ethnic Danish patients consisting of 624 patients with CD, 411 patients with UC and 795 controls. The results were analysed using logistic regression.

Results

The polymorphisms TLR5 (rs5744174) and IL12B (rs6887695) were associated with risk of CD, and TLR1 (rs4833095) and IL18 (rs187238) were associated with risk of both CD and UC (p<0.05). After Bonferroni correction for multiple testing, the homozygous variant genotype of TLR1 743 T>C (rs4833095) was associated with increased risk CD (OR: 3.15, 95% CI: 1.59–6.26, p = 0.02) and CD and UC combined (OR: 2.96, 95% CI: 1.64–5.32, p = 0.005).

Conclusion

Our results suggest that genetically determined high activity of TLR1 and TLR5 was associated with increased risk of both CD and UC and CD, respectively. This supports that the host microbial composition or environmental factors in the gut are involved in risk of IBD. Furthermore, genetically determined high activity of the IL-23/IL-17 pathway was associated with increased risk of CD and UC. Overall, our results support that genetically determined high inflammatory response was associated with increased risk of both CD and UC.

About the Authors:

Steffen Bank

* E-mail: [email protected]

Affiliations Medical Department, Viborg Regional Hospital, Viborg, Denmark, Biomedicine, University of Aarhus, Aarhus, Denmark

Paal Skytt Andersen

Affiliations Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark, Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark

Johan Burisch

Affiliation: Department of Gastroenterology, Herlev Hospital, Herlev, Denmark

Natalia Pedersen

Affiliation: Department of Gastroenterology, Herlev Hospital, Herlev, Denmark

Stine Roug

Affiliation: Department of Gastroenterology, Hvidovre Hospital, Hvidovre, Denmark

Julied Galsgaard

Affiliation: Medical Department, Køge Hospital, Køge, Denmark

Stine Ydegaard Turino

Affiliation: Medical Department, Hillerød Hospital, Hillerød, Denmark

Jacob Broder Brodersen

Affiliations Medical Department, Sydvestjysk Hospital, Esbjerg, Denmark, Department of medical Gastroenterology, Odense University Hospital, Odense, Denmark

Shaista Rashid

Affiliation: Medical Department, Bispebjerg Hospital, Bispebjerg, Denmark

Britt Kaiser Rasmussen

Affiliation: Medical Department, Nykøbing Falster Hospital, Nykøbing Falster, Denmark

Sara Avlund

Affiliation: Medical Department V, Aarhus University Hospital, Aarhus, Denmark

Thomas Bastholm Olesen

Affiliation: Medical Department, Slagelse Hospital, Slagelse, Denmark

Hans Jürgen Hoffmann

Affiliation: Department of Respiratory Diseases B, Institute for Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark

Bjørn Andersen Nexø

Affiliation: Biomedicine, University of Aarhus, Aarhus, Denmark

Jacob Sode

Affiliations Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark, Department of Autoimmunology and Biomarkers, Statens Serum Institut, Copenhagen, Denmark, Department of Rheumatology, Frederiksberg Hospital, Frederiksberg, Denmark

Ulla Vogel

Affiliation: National Research Centre for the Working Environment, Copenhagen, Denmark

Vibeke Andersen

Affiliations Medical Department, Viborg Regional Hospital, Viborg, Denmark, Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark, Research unit for Molecular Diagnostic, Hospital of Southern Jutland Aabenraa, Aabenraa, Denmark, OPEN Odense Patient data Explorative Network, Odense University Hospital, Odense, Denmark

Introduction

Chronic inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are complex diseases that result from the interaction of numerous genetic and environmental factors [1]. Genetic association studies have identified polymorphisms in the Toll-like receptor (TLR) [2,3], the apoptosis [4–6], the IL-23/IL-17 [4–6] and the interferon gamma (IFNG) [4–6] pathways associated with susceptibility of CD and UC. As described below the inflammasome is connected to all of these pathways and it could be speculated that polymorphisms in the inflammasome pathway could be associated with CD or UC.

These pathways are involved in the autoimmune response to Pathogen-Associated Molecular Patterns (PAMPs) such as bacterial or viral DNA, flagellin or lipopolysaccharide (LPS). PAMPs can be bound by the membrane bound TLRs and induce inflammation by activating a number of pro- and anti-inflammatory cytokines [7]. PAMPs can also be recognized by Nod-like receptors (NLRs), which are intracellular receptors. NLRP1 and NLRP3 are members of the inflammasome protein complexes, which can activate the pro-protein caspase-1. The activation of caspase-1 can be suppressed by CARD8 [8]. Caspase-1 activates the pro-inflammatory cytokines IL-1β and IL-18, which are synthesized as pro-proteins [9]. In turn, IL-1β and IL-18 can induce the production of IL-17 [10].

NLRP1, NLRP3 and CARD8 have also been described to be involved in the apoptotic pathway, where overexpression of NLRP1 and NLRP3 stimulates apoptosis through activation of caspases [11,12] and CARD8 inhibit apoptosis by inhibiting the activation of caspase-1 [13].

In the IL-23/IL-17 pathway, IL-23 enhances the secretion of the pro-inflammatory cytokine IL-17, which in turn enhances the production of pro-inflammatory mediators such as IL-1β, IL-6, IL-8 and TNF-α [14]. IL-23 is a heterodimer composed of IL-12p40 and IL-23p19. IL-12p40 also acts as a subunit of the IL-12p35/IL-12p40 heterodimer cytokine IL-12. IL-12 can act pro-inflammatory by activating IFN-γ and anti-inflammatory by activating the anti-inflammatory cytokine IL-10 [15] and by inhibiting the pro-inflammatory cytokine IL-17 [14]. The IL-12 and IL-23 pathways also share the receptor subunit IL12R-β1, which is part of the IL12 receptor (IL12R-β1/IL12R-β2) and IL23 receptor (IL12R-β1/IL23R) heterodimer complex. The signalling through the IL12 and IL23 receptor also share intracellular mechanisms, where both receptors bind the Jak2 kinase, which in turn activates the STAT4 transcription factor. However, the resulting DNA binding STAT transcription complexes differ and involve the STAT3/STAT4 heterodimer in IL23 signalling rather than the STAT4 homodimer in IL12 signalling [14].

IL-18, which can be activated by the inflammasome, can feedback activate the synthesis of IFN-γ. IFN-γ binds to the membrane bound IFNG receptor which is comprised of two ligand-binding IFNGR1 chains and two signal-transducing IFNGR2 chains. Binding of IFN-γ to the IFNG receptor recruits the Jak2 kinase. Jak2 initiates a kinase cascade, which ultimately activates the transcription factor STAT1 [16]. Another important activator of IFN-γ is the transcription factor T-bet (TBX21) [17].

In this study, we wanted to investigate whether functional single nucleotide polymorphisms (SNPs) in genes involved in the Toll-like receptor (TLR1, TLR5 and TIRAP), the inflammasome or apoptotic (NLRP1, NLRP3 and CARD8), the IL-23/IL-17 (IL12B, IL12RB1, IL12RB2, IL17A, IL18 and JAK2) and the IFNG (IFNGR1, IFNGR2 and TBX21) pathways were associated with risk of CD or UC in a Danish cohort of severely diseased patients. Knowing the biological effects of the studied polymorphisms allows a biological interpretation of the associations based on increased or decreased gene activity [3,18,19].

Materials and Methods

Cohort

As described by Bank et al. an ethnic Danish cohort consisting of 624 patients with CD and 411 patients with UC was established [3]. The patients either received or were considered candidates to anti-tumor necrosis factor-α (anti-TNF) therapy (infliximab or adalimumab) and were thus considered to be a homogenous group of moderately to severely ill IBD patients. The control group consisted of 795 healthy blood donors recruited from Viborg, Denmark [20].

Genotyping

For the patients, DNA was extracted from cryopreserved blood clots by using the Maxwell 16 Blood purification kit (Promega, Madison, Wisconsin, USA) according to the manufacturers`instructions [21]. For the healthy controls, DNA was extracted from EDTA-stabilized peripheral blood by either PureGene (Qiagen, Hilden, Germany) or Wizard Genomic (Promega, Madison, Wisconsin, USA) DNA purification kit according to the manufacturers`instructions [20]. Competitive Allele-Specific Polymerase chain reaction (KASP^™), an end-point PCR technology, was used by LGC Genomics for genotyping (LGC Genomics, Hoddesdon, United Kingdom) (http://www.lgcgenomics.com/).

The SNPs studied were TLR1 (rs4833095), TLR5 (rs5744174 and rs2072493), TIRAP (rs8177374), CARD8 (rs2043211), NLRP1 (rs878329 and rs2670660), NLRP3 (rs10754558), IL12B (rs3212217 and rs6887695), IL12RB1 (rs401502), IL12RB2 (rs11810249), IL17A (rs8193036), IL18 (rs1946518 and rs187238), IFNGR1 (rs2234711), IFNGR2 (rs8134145, rs8126756 and rs17882748), TBX21 (rs17250932) and JAK2 (rs12343867).

Genotyping of TLR5 (rs2072493), IFNGR2 (rs8134145) and IL17A (rs8193036) failed. The 18 SNPs were replicated in 94 randomly selected samples yielding >99% identical genotypes. The studied SNPs had minor allele frequencies of 0.09 to 0.50.

Linkage disequilibrium for the assessed SNPs in the same gene was calculated using the SNP Annotation and Proxy Search (SNAP) software [22]. In addition, linkage disequilibrium between the assessed SNPs in our study and known susceptibility loci in the same gene was calculated.

Power calculations

The Genetic Power Calculator was utilized for power analysis of discrete traits [23]. The lowest minor allele frequency (MAF) of the studied SNPs was 0.14. The ‘high-risk allele frequency’ was set to 0.14, the ‘prevalence’ was set to 0.00241 (CD), 0.00263 (UC) and 0.00504 (IBD) [24], D-prime was set to 1, type I error rate was set to 0.05 and number of cases and control:case ratio was based on Table 1. This cohort study had more than 80% chance of detecting a dominant effect with an odds ratio (OR) of 1.4 for CD, 1.5 for UC and 1.4 for IBD.

[Figure omitted. See PDF.]

Table 1. Description of the study participants.

https://doi.org/10.1371/journal.pone.0145302.t001

Statistical analysis

Logistic regression was used to compare genotype distributions among patients with CD, UC and IBD versus healthy controls. Odds ratio adjusted for age and gender were assessed (Table 2). Odds ratio unadjusted (crude) and adjusted for age, gender and smoking status were included as supplementary Tables (S2 and S3 Tables). A chi-square test was used to test for deviation from Hardy-Weinberg equilibrium among the healthy controls.

[Figure omitted. See PDF.]

Table 2. Odds ratios (OR) (adjusted for age and sex) for genotypes studied among healthy controls and patients with Crohns disease (CD), ulcerative colitis (UC) and combined inflammatory bowel disease (IBD).

https://doi.org/10.1371/journal.pone.0145302.t002

Statistical analyses were performed using STATA version 11 (STATA Corp., Texas, USA).

Ethics Statement

The study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committees of Central (M20100153) and Southern (S-20120113) Denmark and the Danish Data Protection Agency of Central (RM: J. 2010-41-4719) and Southern (RSD: 2008-58-035) Denmark. The Ethics Committees gave suspension for obtaining written informed consent.

Results

Study population

Characteristics of the Danish patients with CD, UC and healthy controls are shown in Table 1. The genotype distributions among the healthy controls deviated from Hardy-Weinberg equilibrium for TLR1 743 T>C (rs4833095) (p = 0.03). After correction for multiple testing, all SNPs studied were in Hardy-Weinberg equilibrium.

Polymorphisms associated with risk of CD

The homozygous variant genotype of TLR1 743 T>C (rs4833095) (OR: 3.15, 95% confidence interval (CI): 1.59–6.26, p = 0.001) and TLR5 936 T>C (rs5744174) (OR: 1.54, 95% CI: 1.04–2.28, p = 0.03) and the combined homozygous and the heterozygous variant genotypes of IL12B G>C (rs6887695) (OR: 1.49, 95% CI: 1.13–1.96, p = 0.004) were associated with increased risk of CD. The homozygous variant genotype of IL18–137 G>C (rs187238) (OR: 0.50, 95% CI: 0.28–0.88, p = 0.02) was associated with reduced risk of CD (Table 2).

After Bonferroni correction for multiple testing, the homozygous variant genotype of TLR1 743 T>C (rs4833095) (OR: 3.15, 95% CI: 1.59–6.26, p = 0.02) was associated with increased risk of CD.

Polymorphisms associated with risk of UC

The homozygous variant genotype of TLR1 743 T>C (rs4833095) (OR: 2.92, 95% CI: 1.42–6.00, p = 0.004) was associated with increased risk of UC. The combined homozygous and the heterozygous variant genotypes of IL18–137 G>C (rs187238) (OR: 0.72, 95% CI: 0.54–0.97, p = 0.03) were associated with reduced risk of UC (Table 2).

Polymorphisms associated with risk of IBD

In order to increase statistical power the analyses for CD and UC were combined (IBD). The studied polymorphisms generally showed the same direction of effect for CD and UC (Table 2).

The homozygous variant genotype of TLR1 743 T>C (rs4833095) (OR: 2.96, 95% CI: 1.64–5.32, p = 0.0003) and the combined homozygous and the heterozygous variant genotypes of IL12B G>C (rs6887695) (OR: 1.29, 95% CI: 1.03–1.62, p = 0.03) were associated with increased risk of IBD. The homozygous variant genotype of IL18–137 G>C (rs187238) (OR: 0.61, 95% CI: 0.39–0.95, p = 0.03) was associated with reduced risk of IBD (Table 2).

After Bonferroni correction for multiple testing, the homozygous variant genotype of TLR1 743 T>C (rs4833095) (OR: 2.96, 95% CI: 1.64–5.32, p = 0.005) was associated with increased risk of IBD.

The biologic effects of the studied SNPs and OR for polymorphisms which have been shown to be associated with risk of CD, UC or IBD in other studies and in this study were summarized in S1 Table.

Linkage disequilibrium

Rs6887695 (IL12B) included in this study was in linkage disequilibrium with rs6556412 (IL12B) associated with CD in another study with r² = 0.96 and D' = 1.00. No linkage disequilibrium was found for the SNPs in TLR5 (rs5744174 and rs2072493), NLRP1 (rs878329 and rs2670660), IL12B (rs3212217 and rs6887695), IL18 (rs1946518 and rs187238) or IFNGR2 (rs8134145, rs8126756 and rs17882748) (r² < 0.8).

Discussion

In this Danish cohort of severely ill patients, 4 functional polymorphisms in 4 genes involved in the Toll-like receptor (TLR1 and TLR5) and the IL-23/IL-17 (IL12B and IL18) pathways were found to be associated with risk of CD or UC (Fig 1).

[Figure omitted. See PDF.]

Fig 1. Eighteen functional single nucleotide polymorphisms (SNPs) in 14 genes were successfully genotyped and 4 SNPs in 4 genes were found to be associated with susceptability of severe Crohn’s disease (CD), ulcerative colitis (UC) or combined CD and UC.

The genes are involved in the Toll-like receptor (TLR1 and TLR5) and the IL-23/IL-17 (IL12B and IL18) pathways. Other studies have also found the IL12B G>C (rs6887695) polymorphism to be associated with risk of CD and our results confirm it as a risk locus. The TLR1 743 T>C (rs4833095)) polymorphism is a novel susceptibility loci.

https://doi.org/10.1371/journal.pone.0145302.g001

In agreement with our study, the variant allele of the IL12B G>C (rs6887695) polymorphism has been associated with increased risk of CD, in the Welcome Trust Case Control Consortium and in a Japanese cohort [25,26]. Our study thereby confirms IL12B G>C (rs6887695) as a susceptibility locus for CD. IL12B encode the IL-12p40 subunit which is part of both the IL12 (IL-12p35/IL-12p40) and IL23 (IL-23p19/IL-12p40) heterodimers. All three subunits are up-regulated in patients with CD [27–30]. Furthermore, IL12B G>C (rs6887695) is in linkage disequilibrium with IL12B (rs6556412) which in another study has been associated with increased risk of CD [5] in agreement with the results in our study for rs6887695. In addition, the variant allele of IL12B G>C (rs6887695) has also been associated with increased risk of UC in a meta-analysis (OR: 1.11), and although the direction of association was the same in our study we were unable to confirm the result probably because of lack of power [31].

In contrast to our study, other case-control studies found opposite direction of association in other ethnic groups of patients with UC for IL18–137 G>C (rs187238) [32] or opposite direction of association in children with CD for TLR5 936 T>C (rs5744174) [33]. There is no consensus of how the stop codon polymorphism in CARD8 A>T (rs2043211) influence risk of CD [34,35].

To the best of our knowledge the polymorphism in TLR1 743 T>C (rs4833095) found to be associated with both CD and UC in our study, has not previously been described as a susceptibility polymorphism.

The biological interpretation of our results indicates that genetically determined high activity of TLR1 (rs4833095) was associated with increased risk of both CD and UC and genetically determined high activity of TLR5 (rs5744174) was associated with increased risk of CD. Both TLR1 and TLR5 can activate NFκB, a central regulator of inflammation regulating the expression of more than 150 genes including IL1B, IL12A (p35), IL12B (p40), IL17A, IL23A (p19) and IFNG [36]. The associations found in TLR1 and TLR5 further supports that the host microbial composition or environmental factors in the gut are involved in risk of IBD in agreement with other studies [2,3].

The pro-protein IL-18 can be activated by the inflammasome and IL-18 can downstream induce the production of the pro-inflammatory cytokine IL-17 [10]. In our study, the associations found for the polymorphism -137 G>C (rs187238) indicates that genetically determined low activity of IL-18 was associated with reduced risk of CD and UC. This suggests that lower activity of the IL-23/IL-17 pathway was associated with reduced risk of CD and UC.

The activity of IL-17 can also be up-regulated through the IL-23 pathway and down-regulated through the IL-12 pathway. Many proteins involved in the IL-12 pathway are also involved in the IL-23 pathway, including IL-12p40 and Jak2. Our study indicates that genetically determined low IL-12p40 level (a subunit of both IL-12 and IL-23) was associated with increased risk of CD (rs6887695), in accordance with other studies [25,26]. It could be speculated, that a lower level of the IL-12p40 subunit results in a lower activity of the IL-12 pathway, lower inactivation of the pro-inflammatory cytokine IL-17 and thus increased inflammatory response, which might explain the increased risk of CD.

Overall, the polymorphism in TLR5 (rs5744174) and IL12B (rs6887695) associated with CD, the polymorphism TLR1 (rs4833095) and IL18 (rs187238) associated with both CD and UC support that genetically determined high inflammatory response is associated with increased risk of both CD and UC. In addition, the polymorphism in IL12B (rs6887695) associated with CD and the polymorphism in IL18 (rs187238) associated with both CD and UC suggest that genetically determined high activity of the IL-23/IL-17 pathway was associated with increased risk of CD and UC. It could be speculated that IL-17 plays a key role as IL-17 can be inhibited by the IL-12 pathway and induced by the IL-23 pathway.

Studies have shown that mice deficient in the IL-23 pathway specific subunit (IL-23p19) or subunits shared with IL12 (IL-12p40 and IL12R-β1) were resistant to inflammation, whereas mice deficient in IL-12 pathway specific subunits (IL-12p35 and IL12R-β2) were more susceptible to inflammation [14]. Antibodies targeting the shared subunit IL-12p40 (Ustekinumab) have been shown to be more effective than placebo in treating patients with severe CD, especially those who failed to respond to anti-TNF therapy [37]. Based on our results it could be speculated, that a better result might be obtained by using antibodies targeting the IL-23 specific subunit IL-23p19, as our study indicates a protective effect of IL-12p40 through the IL-12 pathway.

The results in this study should be interpreted with care. TLR1 743 T>C (rs4833095) was not in Hardy-Weinberg equilibrium among the healthy controls, which is probable due to chance. When corrected adequately for multiple testing no deviation from Hardy-Weinberg equilibrium was found. In the light of the obtained P-values and the number of statistical tests performed we cannot exclude, that some of our positive findings may be due to chance. If the results were corrected for multiple testing the homozygous variant genotype of TLR1 743 T>C (rs4833095) was associated with increased risk of CD (p = 0.02) and marginally with UC (p = 0.07) and the combined homozygous and the heterozygous variant genotypes of IL12B G>C (rs6887695) were marginally associated with increased risk of CD (p = 0.08). We successfully assessed 18 polymorphisms and assuming a 5% acceptance level, one polymorphism would be expected to be associated with susceptibility by pure chance. In this study 5 polymorphisms were found to be associated with susceptibility and most of the found associations were biologically plausible. We cannot exclude that associations were not identified due to insufficient statistical power. The results should therefore be replicated in independent cohorts. A major strength was that this clinically homogeneous and well-characterised cohort was rather large including 1035 patients with IBD and 795 healthy controls. All the patients were considered for anti-TNF treatment and were therefore considered to have a severe disease course. Genetic determinants may be expected to be strong among severely ill cases [38].

In conclusion, 4 functional SNPs in 4 genes involved in regulation of inflammation were found to be associated with susceptibility of severe CD or UC. The SNP in TLR1 743 (rs4833095) has not previously been reported as susceptibility polymorphisms of both CD and UC (Fig 1). The IL12B G>C (rs6887695) polymorphism associated with risk of CD in our study has also been associated with risk of CD in other studies and should therefore be considered a confirmed risk locus. Our results suggest that genetically determined high activity of TLR1 and TLR5 was associated with increased risk of CD and UC. This supports that the host microbial composition or environmental factors in the gut are involved in risk of IBD. Furthermore, genetically determined high activity of the IL-23/IL-17 pathway was associated with increased risk of CD and UC. Overall, our results support that genetically determined high inflammatory response was associated with increased risk of both CD and UC.

Supporting Information

[Figure omitted. See PDF.]

S1 Table. The biologic effect of the studied single nucleotide polymorphism (SNP) and odds ratios (OR) for polymorphisms which have been shown to be associated with risk of Crohn's disease (CD), ulcerative colitis (UC) or inflammatory bowel disease (IBD) in other studies and in this study.

https://doi.org/10.1371/journal.pone.0145302.s001

(DOC)

S2 Table. Odds ratios (OR) (unadjusted) for genotypes studied among healthy controls and patients with Crohns disease (CD), ulcerative colitis (UC) and combined inflammatory bowel disease (IBD).

https://doi.org/10.1371/journal.pone.0145302.s002

(DOC)

S3 Table. Odds ratios (OR) (adjusted for age, sex and smoking status) for genotypes studied among healthy controls and patients with Crohns disease (CD), ulcerative colitis (UC) and combined inflammatory bowel disease (IBD).

https://doi.org/10.1371/journal.pone.0145302.s003

(DOC)

Acknowledgments

The work was funded by Health Research Fund of Central Denmark Region, Colitis-Crohn Foreningen and University of Aarhus (PhD grant).

Author Contributions

Conceived and designed the experiments: SB PSA BAN JS UV VA. Performed the experiments: SB JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH. Analyzed the data: SB PSA BAN JS UV VA. Contributed reagents/materials/analysis tools: SB JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH. Wrote the paper: SB PSA UV VA. Drafting the article or revising it critically: SB PSA JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH BAN JS UV VA.

Citation: Bank S, Andersen PS, Burisch J, Pedersen N, Roug S, Galsgaard J, et al. (2015) Polymorphisms in the Toll-Like Receptor and the IL-23/IL-17 Pathways Were Associated with Susceptibility to Inflammatory Bowel Disease in a Danish Cohort. PLoS ONE 10(12): e0145302. https://doi.org/10.1371/journal.pone.0145302

References

1. Podolsky DK (2002) Inflammatory bowel disease. N Engl J Med 347: 417–429. pmid:12167685

2. Cario E (2010) Toll-like receptors in inflammatory bowel diseases: a decade later. Inflamm Bowel Dis 16: 1583–1597. pmid:20803699

3. Bank S, Skytt AP, Burisch J, Pedersen N, Roug S, Galsgaard J, et al. (2014) Polymorphisms in the Inflammatory Pathway Genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG Are Associated with Susceptibility of Inflammatory Bowel Disease in a Danish Cohort. PLoS One 9: e98815. pmid:24971461

4. Anderson CA, Boucher G, Lees CW, Franke A, D'Amato M, Taylor KD, et al. (2011) Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet 43: 246–252. pmid:21297633

5. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T, et al. (2010) Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nat Genet 42: 1118–1125. pmid:21102463

6. Thompson AI, Lees CW (2011) Genetics of ulcerative colitis. Inflamm Bowel Dis 17: 831–848. pmid:21319274

7. Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R (2008) TLR-4, IL-1R and TNF-R signaling to NF-kappaB: variations on a common theme. Cell Mol Life Sci 65: 2964–2978. pmid:18535784

8. Paramel GV, Folkersen L, Strawbridge RJ, Elmabsout AA, Sarndahl E, Lundman P, et al. (2013) CARD8 gene encoding a protein of innate immunity is expressed in human atherosclerosis and associated with markers of inflammation. Clin Sci (Lond) 125: 401–407.

9. Aguilera M, Darby T, Melgar S (2014) The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases—Lessons learned from experimental models. Cytokine Growth Factor Rev: 10.

10. Dinarello CA, Novick D, Kim S, Kaplanski G (2012) Interleukin-18 and IL-18 Binding Protein. Front Immunol: 289.

11. D'Osualdo A, Reed JC (2012) NLRP1, a regulator of innate immunity associated with vitiligo. Pigment Cell Melanoma Res: 5–8.

12. Sagulenko V, Thygesen SJ, Sester DP, Idris A, Cridland JA, Vajjhala PR, et al. (2013) AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ: 1149–1160. pmid:23645208

13. Hong GS, Jung YK (2002) Caspase recruitment domain (CARD) as a bi-functional switch of caspase regulation and NF-kappaB signals. J Biochem Mol Biol: 19–23. pmid:16248965

14. Hoeve MA, Savage ND, de BT, Langenberg DM, de Waal MR, Ottenhoff TH, et al. (2006) Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells. Eur J Immunol: 661–670. pmid:16482511

15. Chang HD, Radbruch A (2007) The pro- and anti-inflammatory potential of interleukin-12. Ann N Y Acad Sci: 40–46.

16. Schroder K, Hertzog PJ, Ravasi T, Hume DA (2004) Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 75: 163–189. pmid:14525967

17. Li J, Li J, You Y, Chen S (2012) The role of upstream stimulatory factor 1 in the transcriptional regulation of the human TBX21 promoter mediated by the T-1514C polymorphism associated with systemic lupus erythematosus. Immunogenetics 64: 361–370. pmid:22258560

18. Bank S, Andersen PS, Burisch J, Pedersen N, Roug S, Galsgaard J, et al. (2014) Associations between functional polymorphisms in the NFkappaB signaling pathway and response to anti-TNF treatment in Danish patients with inflammatory bowel disease. Pharmacogenomics J: 526–534. pmid:24776844

19. Sode J, Vogel U, Bank S, Andersen PS, Thomsen MK, Hetland ML, et al. (2014) Anti-TNF treatment response in rheumatoid arthritis patients is associated with genetic variation in the NLRP3-inflammasome. PLoS One 9: e100361. pmid:24967817

20. Ernst A, Jacobsen B, Ostergaard M, Okkels H, Andersen V, Dagiliene E, et al. (2007) Mutations in CARD15 and smoking confer susceptibility to Crohn's disease in the Danish population. Scand J Gastroenterol: 1445–1451. pmid:17852840

21. Bank S, Nexo BA, Andersen V, Vogel U, Andersen PS (2013) High-Quality and -Quantity DNA Extraction from Frozen Archival Blood Clots for Genotyping of Single-Nucleotide Polymorphisms. Genet Test Mol Biomarkers 17: 501–503. pmid:23574531

22. https://www.broadinstitute.org/mpg/snap/ldsearchpw.php#

23. http://pngu.mgh.harvard.edu/~purcell/gpc/cc2.html

24. Kappelman MD, Moore KR, Allen JK, Cook SF (2013) Recent trends in the prevalence of Crohn's disease and ulcerative colitis in a commercially insured US population. Dig Dis Sci 58: 519–525. pmid:22926499

25. Yamazaki K, Takahashi A, Takazoe M, Kubo M, Onouchi Y, Fujino A, et al. (2009) Positive association of genetic variants in the upstream region of NKX2-3 with Crohn's disease in Japanese patients. Gut: 228–232. pmid:18936107

26. Parkes M, Barrett JC, Prescott NJ, Tremelling M, Anderson CA, Fisher SA, et al. (2007) Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility. Nat Genet: 830–832. pmid:17554261

27. Liu Z, Yadav PK, Xu X, Su J, Chen C, Tang M, et al. (2011) The increased expression of IL-23 in inflammatory bowel disease promotes intraepithelial and lamina propria lymphocyte inflammatory responses and cytotoxicity. J Leukoc Biol 89: 597–606. pmid:21227898

28. Pugazhendhi S, Jayakanthan K, Pulimood AB, Ramakrishna BS (2013) Cytokine gene expression in intestinal tuberculosis and Crohn's disease. Int J Tuberc Lung Dis 17: 662–668. pmid:23575333

29. Gologan S, Iacob R, Iancu D, Iacob S, Cotruta B, Vadan R, et al. (2013) Inflammatory gene expression profiles in Crohn's disease and ulcerative colitis: a comparative analysis using a reverse transcriptase multiplex ligation-dependent probe amplification protocol. J Crohns Colitis 7: 622–630. pmid:23014361

30. Ng SC, Benjamin JL, McCarthy NE, Hedin CR, Koutsoumpas A, Plamondon S, et al. (2011) Relationship between human intestinal dendritic cells, gut microbiota, and disease activity in Crohn's disease. Inflamm Bowel Dis 17: 2027–2037. pmid:21910165

31. http://www.ibdgenetics.org/

32. Wang Y, Tong J, Chang B, Wang BF, Zhang D, Wang BY (2014) Genetic Polymorphisms in the IL-18 Gene and Ulcerative Colitis Risk: A Meta-Analysis. DNA Cell Biol: 33(7): 438–47. pmid:24621393

33. Sheridan J, Mack DR, Amre DK, Israel DM, Cherkasov A, Li H, et al. (2013) A non-synonymous coding variant (L616F) in the TLR5 gene is potentially associated with Crohn's disease and influences responses to bacterial flagellin. PLoS One 8: e61326. pmid:23593463

34. Franke A, Rosenstiel P, Balschun T, Von KO, Schreiber S, Sina C, et al. (2007) No association between the TUCAN (CARD8) Cys10Stop mutation and inflammatory bowel disease in a large retrospective German and a clinically well-characterized Norwegian sample. Gastroenterology: 2080–2081. pmid:17484912

35. Buning C, Schmidt HH, Molnar T, Drenth JP, Fiedler T, Gentz E, et al. (2008) No association of the CARD8 (TUCAN) c.30T>A (p.C10X) variant with Crohn's disease: a study in 3 independent European cohorts. Inflamm Bowel Dis: 332–337. pmid:18092344

36. http://www.bu.edu/nf-kb/gene-resources/target-genes/. http://www.bu.edu/nf-kb/gene-resources/target-genes/

37. Sandborn WJ, Gasink C, Gao LL, Blank MA, Johanns J, Guzzo C, et al. (2012) Ustekinumab induction and maintenance therapy in refractory Crohn's disease. N Engl J Med: 1519–1528. pmid:23075178

38. Fowler EV, Doecke J, Simms LA, Zhao ZZ, Webb PM, Hayward NK, et al. (2008) ATG16L1 T300A shows strong associations with disease subgroups in a large Australian IBD population: further support for significant disease heterogeneity. Am J Gastroenterol: 2519–2526. pmid:18671817