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The Role of Osteopontin (OPN/SPP1) Haplotypes in the Susceptibility to Crohn's Disease

  • Jürgen Glas ,

    Contributed equally to this work with: Jürgen Glas, Julia Seiderer

    Affiliations Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany, Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany, Department of Human Genetics, Rheinisch-Westfälische Technische Hochschule (RWTH), Aachen, Germany

  • Julia Seiderer ,

    Contributed equally to this work with: Jürgen Glas, Julia Seiderer

    Affiliation Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany

  • Corinna Bayrle,

    Affiliation Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany

  • Martin Wetzke,

    Affiliation Department of Pediatrics, Hannover Medical School, Hannover, Germany

  • Christoph Fries,

    Affiliations Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany, Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany

  • Cornelia Tillack,

    Affiliation Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany

  • Torsten Olszak,

    Affiliations Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany, Division of Gastroenterology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America

  • Florian Beigel,

    Affiliation Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany

  • Christian Steib,

    Affiliation Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany

  • Matthias Friedrich,

    Affiliations Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany, Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany

  • Julia Diegelmann,

    Affiliations Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany, Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany

  • Darina Czamara,

    Affiliation Max-Planck-Institute of Psychiatry, Munich, Germany

  • Stephan Brand

    Stephan.Brand@med.uni-muenchen.de

    Affiliation Department of Medicine II - Grosshadern, Ludwig-Maximilians-University, Munich, Germany

The Role of Osteopontin (OPN/SPP1) Haplotypes in the Susceptibility to Crohn's Disease

  • Jürgen Glas, 
  • Julia Seiderer, 
  • Corinna Bayrle, 
  • Martin Wetzke, 
  • Christoph Fries, 
  • Cornelia Tillack, 
  • Torsten Olszak, 
  • Florian Beigel, 
  • Christian Steib, 
  • Matthias Friedrich
PLOS
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Abstract

Background

Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients.

Methodology/Principal Findings

Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn's disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10−8). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10−5). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10−2) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10−2) but none of these associations remained significant after Bonferroni correction.

Conclusions/Significance

Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.

Introduction

The pathogenesis of inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) is only partially understood. Currently, these diseases are assumed to be triggered by an exaggerated immune response to intestinal bacteria in a genetically susceptible host. In addition to the nucleotide-binding oligomerization domain 2/caspase recruitment domain-containing protein 15 (NOD2/CARD15) [1], [2], various novel susceptibility loci such as the interleukin-23 receptor (IL23R) [3], [4], the ATG16L1 (autophagy-related 16-like 1) gene [5], [6] and variants in the 5p13.1 region [7] have been identified as susceptibility variants in CD patients. Based on new insights in the genetic background of CD, there is raising evidence for a key role of innate immunity and CD-related inflammatory pathways such as IL-23/IL-17 mediated T cell responses [8]. Recently, osteopontin (OPN, also known as Eta-1), an extracellular matrix glycosylated phosphoprotein produced by immune cells, epithelial cells and osteoblasts has been identified as an important molecule involved in tissue repair, inflammation and autoimmunity as well as tumour growth [9], [10], [11], [12]. So far, two forms of osteopontin have been identified - secreted osteopontin (sOPN) seems to be involved in the production of pathogenetic Th1 and Th17 cells, while an intracellular form of osteopontin (iOPN) is a key regulator for Toll like receptor-9 (TLR9) and/or TLR7-dependent interferon-α (IFN-α) expression by plasmacytoid dendritic cells (DCs) and Th17 development [13]. There is evidence for a key role of osteopontin in Th1- and Th17-mediated diseases [10], [14], [15] such as rheumatoid arthritis [16], [17], [18], psoriasis [19] and multiple sclerosis [20], [21], [22], [23]. In addition, osteopontin has also shown to be involved in granuloma formation [10], cell migration [24], [25], [26], and IL-12 production [27], [28], [29].

Osteopontin is expressed in the terminal ileum of CD patients [30] and seems to be closely involved in the Th1 immune response associated with CD [31], [32], [33], [34]. Moreover, it has also been reported to play an important role in the pathogenesis of UC [35], [36], [37], [38]. Analyzing the exact role of osteopontin in a murine model of acute colitis, a recent study demonstrated that Opn−/− mice showed increased serum levels of TNF-α but also reduced mRNA expression of IL-1β and matrix metalloproteinases as well as decreased blood levels of IL-22 [39]. In contrast, in a chronic DSS model, Opn−/− mice were protected from mucosal inflammation showing lower serum IL-12 levels compared to wildtype mice and neutralization of OPN in wildtype mice abrogated colitis [39]. These findings implicate a dual function of osteopontin in intestinal inflammation characterized by activation of innate immunity and Th17 cytokines such as IL-22 initiating mucosal repair in acute inflammation; while under conditions of chronic intestinal inflammation it may promote the Th1 response and thereby enhancing inflammation [39]. Further investigations by daSilva et al. in a DSS model demonstrated that osteopontin administration reduced the disease activity index, improved red blood cell counts, and reduced gut neutrophil activity compared with the DSS-treated wildtype mice [37]. Interestingly, the study by Heilmann et al. demonstrated a significant correlation of osteopontin serum levels with disease activity in human CD [39].

In this study, we aimed to analyze the role of OPN gene variants on IBD disease susceptibility and phenotype. We also investigated for potential epistasis with IBD-associated IL23R gene variants. In total, we genotyped nine common single nucleotide polymorphisms (SNPs) in the OPN gene, which were previously shown to be associated with other immune-mediated diseases [40], [41], [42], [43]. Last, based on the important role demonstrated for IL-22 in colitis experiments in Opn−/− mice [39], we analyzed the effect of OPN gene variants on IL-22 serum levels.

Methods

Ethics statement

Written, informed consent was obtained from all patients prior to inclusion into the study. In the case of minors, the consent was provided by the parents. This study was approved by the Ethics committee of the Medical Faculty of Ludwig-Maximilians-University Munich. The study protocol adhered to the ethical principles for medical research involving human subjects of the Helsinki Declaration (as described in detail under: http://www.wma.net/en/30publications/10policies/b3/index.html).

Study population

Our study population comprised 2819 individuals of Caucasian origin including n = 841 patients with CD, n = 473 patients with UC and n = 1505 healthy unrelated controls. All phenotypic data were collected blind to the results of genotyping and included detailed demographic and clinical parameters (disease behaviour, anatomic manifestation of IBD, complications, surgical or immunosuppressive therapy). The diagnosis of CD and UC was based on established guidelines according to endoscopic, radiological, and histopathological parameters. For classification of CD patients, the Montreal classification [44] based on age at diagnosis (A), location (L), and behaviour (B) of disease was used. In patients with UC, anatomic location was also based on the Montreal classification, based on the criteria ulcerative proctitis (E1), left-sided UC (distal UC; E2), and extensive UC (pancolitis; E3). Patients with indeterminate colitis were excluded from the study. The clinical characteristics of the IBD study population are shown in Table 1.

DNA extraction

From all study participants, blood samples were taken and genomic DNA was isolated from peripheral blood leukocytes using the DNA blood mini kit from Qiagen (Hilden, Germany) according to the manufacturer's guidelines.

Genotyping of OPN gene variants

Nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138) were genotyped by PCR and melting curve analysis using a pair of fluorescence resonance energy transfer (FRET) probes in a LightCycler® 480 Instrument (Roche Diagnostics, Mannheim, Germany) as previously described in detail [45], [46], [47], [48]. The selection of these SNPs was based on previous studies in which associations for several of these OPN variants with autoimmune and Th1- and Th17-mediated diseases have been shown [40], [41], [42], [43], [49], [50], [51], [52], [53]. The donor fluorescent molecule (fluorescein) at the 3′-end of the sensor probe (or the anchor probe in the case of rs2853744 and rs11730582) is excited at its specific fluorescence excitation wavelength (533 nm) and the energy is transferred to the acceptor fluorescent molecule at the 5′-end (LightCycler Red 610, 640 or 670) of the anchor probe (or the sensor probe in the case of rs2853744 and rs11730582). The specific fluorescence signal emitted by the acceptor molecule is detected by the optical unit of the LightCycler. The sensor probe is exactly matching to one allele of each SNP, preferentially to the rarer allele, whereas in the case of the other allele, there is a mismatch resulting in a lower melting temperature. The total volume of the PCR was 5 µl containing 25 ng of genomic DNA, 1× Light Cycler 480 Genotyping Master (Roche Diagnostics), 2.5 pmol of each primer and 0.75 pmol of each FRET probe (TIB MOLBIOL, Berlin, Germany). In the case of rs11739060, the concentration of the forward primer, and in the case of rs1126772, the concentration of the reverse primer was reduced to 0.5 pmol. The PCR comprised an initial denaturation step (95°C for 10 min) and 45 cycles (95°C for 10 sec, primer annealing temperature as given in the Supplementary data (Table S1) for 10 sec, 72°C for 15 sec). The melting curve analysis comprised an initial denaturation step (95°C for 1 min), a step rapidly lowering the temperature to 40°C and holding for 2 min, and a heating step slowly (1 acquisition/°C) increasing the temperature up to 95°C and continuously measuring the fluorescence intensity. The results of the melting curve analysis have been confirmed by analyzing two patient samples for each possible genotype using sequence analysis. For sequencing, the total volume of the PCR was 100 µl containing 250 ng of genomic DNA, 1× PCR buffer (Qiagen, Hilden, Germany), a final MgCl2 concentration of 2 mM, 0.5 mM of a dNTP mix (Sigma, Steinheim, Germany), 2.5 units of HotStar Plus Taq™ DNA polymerase (Qiagen) and 10 pmol of each primer (TIB MOLBIOL). The PCR comprised an initial denaturation step (95°C for 5 min), 35 cycles (denaturation at 94°C for 30 sec, primer annealing at 60°C for 30 sec, extension at 72°C for 30 sec) and a final extension step (72°C for 10 min). The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and sequenced by a commercial sequencing company (Sequiserve, Vaterstetten, Germany). All sequences of primers and FRET probes and primer annealing temperatures used for genotyping and for sequence analysis are given in Tables S1 and S2.

Genotyping of IL23R gene variants

Genotypes of 10 IBD-associated IL23R gene variants (rs1004819, rs7517847, rs10489629, rs2201841, rs11465804, rs11209026 = p.Arg381Gln, rs1343151, rs10889677, rs11209032, rs1495965) were available for all study patients and controls from previous studies [4], [54].

Analysis of IL-22 serum levels in CD patients

In order to investigate a potential correlation between IL-22 serum expression and OPN/SPP1 genotype, IL-22 serum levels were determined in a subcohort of CD patients, in which serum samples and genomic DNA was available. IL-22 serum levels for the majority of these patients were available from a previous study [55]. For the ELISA analysis, the human IL-22 Quantikine Elisa Kit (R&D Systems, Minneapolis, MN) was used following the manufacturer's guidelines. The following steps were performed: First, all reagents, working standards, and samples were prepared as outlined in the manufacturer's guidelines. Next, 100 µL of assay diluent RD1-88 were added to each well. After this step, 100 µL of standard, control, or sample were added per well and incubated for two hours at room temperature. Then, each well was aspirated and washed four times. 200 µL of a mouse monoclonal antibody against IL-22 conjugated to horseradish peroxidase were added and the plates were incubated for two hours at room temperature. After this, wells were aspirated and washed four times. Next, 200 µL of substrate solution were added to each well. The plates were incubated for 30 minutes at room temperature to allow colour development while being protected from light. Next, 50 µL of stop solution were added to each well and the optical density of each well was determined within 30 minutes, using a microplate reader set to 450 nm. IL-22 serum levels (pg/ml) were calculated from a standard curve of known IL-22 concentrations.

Statistical analyses

Each genetic marker was tested for Hardy-Weinberg equilibrium in the control population. Single-marker allelic tests were performed with Fisher's exact test. All tests were two-tailed, considering p-values<0.05 as significant. Odds ratios were calculated for the minor allele at each SNP. For multiple comparisons, Bonferroni correction was applied where indicated. rs4754 deviated from the Hardy-Weinberg equilibrium in the control population (p = 0.0005) and was therefore excluded from the haplotype analysis. Haplotype analysis was conducted with PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/) and the –hap-logistic option using a sliding-window approach with 2 up to 8 included SNPs. Interaction between different polymorphisms were also tested with PLINK and the –epistasis command. For analyzing potential differences of IL-22 serum levels between the carriers of the different OPN gene variants, the mean IL-22 serum level of carriers of the wildtype allele of each SNP was compared with the mean IL-22 serum level of carriers of the minor allele ( = combined group of heterozygous and homozygous carriers) using Student's t-test.

Results

Frequency distribution of OPN gene variants and their role in IBD susceptibility

For all three subgroups (CD, UC, and controls), the minor allele frequencies of the nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138) are summarized in Table 2. With the exception of rs4754, no significant differences in the allele frequencies were observed comparing CD and UC patients to healthy controls (Table 2). Our analysis revealed a weak association of SNP rs4754 (p.Asp80Asp) with CD susceptibility (p = 1.28×10−2; OR (95% CI) 0.85 [0.74–0.96]). Similar to CD, rs4754 (p.Asp80Asp) decreased susceptibility to UC, although this association did not reach significance in univariate analysis (p = 5.25×10−2; OR (95% CI) 0.85 [0.70–1.00]) (Table 2). Moreover, both associations of rs4754 (regarding CD and UC susceptibility) were not statistically significant after Bonferroni correction, suggesting that these OPN variants are not major contributors to IBD susceptibility on their own. In addition, rs4754 deviated from the Hardy-Weinberg equilibrium in the control population (p = 0.0005) and was therefore excluded from the haplotype analysis. However, several OPN haplotypes were associated with CD susceptibility. As shown in table 3, the strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 with an omnibus p-value of 2.07×10−8 (Table 3); if rs4754 would be included into this haplotype block, the omnibus p-value would increase further to p = 3.67×10−12. In contrast, there were no associations of certain OPN haplotypes with UC susceptibility (Table 4).

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Table 2. Associations of OPN/SPP1 gene markers in CD and UC case-control association studies.

https://doi.org/10.1371/journal.pone.0029309.t002

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Table 3. Haplotypes of OPN SNPs in Crohn's disease (CD) case-control sample (846 cases and 1510 controls) and omnibus p-values for association with CD susceptibility.

https://doi.org/10.1371/journal.pone.0029309.t003

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Table 4. Haplotypes of OPN SNPs in ulcerative colitis (UC) case-control sample (501 cases and 1510 controls) and omnibus p-values for association with UC susceptibility.

https://doi.org/10.1371/journal.pone.0029309.t004

Analysis for gender-specific differences in OPN variants

Previous studies demonstrated significant gender-specific effects of OPN variants in systemic lupus erythematosus (SLE) and type 1-diabetes, particularly in male patients [43], [50]. Considering the deviation of rs4754 from the Hardy-Weinberg equilibrium, we therefore investigated potential gender-specific effects in IBD susceptibility. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. Interestingly, for these SNPs, there was an opposite direction of the association results for males and females (rs4754: p = 0.0004, OR = 0.69 [95% CI: 0.56–0.85] (males), p = 0.7693, OR = 1.03 (females); rs1126616: p = 0.1187, OR = 0.85 (males), p = 0.2676, OR = 1.12 (females); rs1126772: p = 0.1679, OR = 0.85 (males), p = 0.0893, OR = 1.21 (females); rs9138: p = 0.1256, OR = 0.85 (males), p = 0.0864, OR = 1.19 (females)). Given that the most pronounced difference between male and female CD patients was found for rs4754, which deviated from the Hardy-Weinberg equilibrium in the control population, we next investigated if the deviation from Hardy-Weinberg equilibrium is based on a gender-specific effect. This analysis revealed that there was significant deviation from Hardy-Weinberg equilibrium in male controls (n = 917; p = 0.0018), but not in female controls (n = 547; p = 0.1347), confirming the gender-specific effect of this OPN SNP found in CD patients.

Analysis for epistasis between OPN variants and IL23R variants

To investigate if OPN variants modify IBD susceptibility by epistastic interaction with other Th17-related IBD susceptibility genes, we next analyzed for potential epistasis of OPN variants with main IBD-associated IL23R variants. We found evidence of weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10−2) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10−2) but none of these associations remained significant after Bonferroni correction (Table 5).

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Table 5. Analysis for epistatic interactions between OPN SNPs and IL23R SNPs regarding CD susceptibility (based on 1510 controls and 704 cases).

https://doi.org/10.1371/journal.pone.0029309.t005

Correlation between OPN variants and IL-22 serum levels in CD patients

Based on the recent data of Heilmann et al. [39] demonstrating decreased blood levels of IL-22 in acute colitis in Opn−/− mice, we next investigated a potential association of OPN variants and IL-22 serum levels in a subcohort of CD patients. No correlation was found between OPN SNPs and IL-22 serum levels (Table 6). However, overall the IL-22 serum levels tended to be lower in the carriers of OPN minor alleles, which was statistically significant when the mean IL-22 expression level of carriers of the 9 investigated OPN SNPs minor alleles (homo- and heterozygous carriers) were compared to the homozygous carriers of the wildtype allele (p = 3.6×10−5). Interestingly, for 7 out of 8 OPN SNPs forming the haplotype rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138, which was strongly associated with CD susceptibility (omnibus p-value 2.07×10−8), the IL-22 serum levels were nominally lower in CD carriers of the minor allele than in wildtype carriers, although these differences were for each SNP only small and statistically not significant (Table 6).

Discussion

The presented study represents the first detailed analysis of OPN gene variants in IBD patients. In this study, there were no significant associations of single OPN SNPs with CD or UC susceptibility after Bonferroni correction for multiple testing; however, several OPN haplotypes were associated with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs (rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138; omnibus p-value 2.07×10−8). However, considering the strength of the association signals found for a number of other recently identified IBD susceptibility genes [56], [57], this argues against a major role for OPN in the genetic susceptibility for IBD. Given the strong association of osteopontin with Th1- and Th17-mediated diseases, the finding of an association of OPN haplotypes with CD, a Th1- and Th17-mediated disease, but not UC susceptibility is not surprising. In contrast, UC has been associated with a predominantly modified Th2 response but partially also with a Th17 immune response. The results of our haplotype analysis suggest that certain rare haplotypes significantly contribute to the genetic risk of CD. This is in agreement with recent results of the International IBD Genetics Consortium which identified a total of 71 CD susceptibility loci [56]. These 71 susceptibility loci explain only slightly more than 20% of CD heritability. Therefore, it is assumed that a number of rare SNPs and haplotypes contribute to the overall CD risk such as recently shown by us for PXR gene variants [58]. In addition, most likely a high number of common CD risk genes with small effect size are still unidentified but for their identification very large cohorts would be required.

So far, genetic variants in the OPN gene have shown to be involved in susceptibility to other immune-mediated diseases such as SLE [59], [60], oligoarticular juvenile idiopathic arthritis [61] and sarcoidosis [51]. Despite promising functional data, previous genotype analyses could not confirm OPN as significant disease-modifying gene in classical Th17-mediated diseases such as multiple sclerosis [62], [63] and rheumatoid arthritis [64]. Investigating the role of OPN as a susceptibility gene in SLE, a recent study demonstrated a significant association in male patients [50] – a phenomenon also seen in a study investigating OPN variants in type-1 diabetes, implicating a potential gender-specific mechanism acting in the autoimmune process [43]. Similarly, our analysis demonstrated gender-specific effects for four OPN SNPs, particularly for rs4754 which deviated from the Hardy-Weinberg equilibrium in male controls. Moreover, there was a significant association of this SNP with CD in male but not in female patients.

While osteopontin is closely involved in the Th1- and Th17-mediated immune response associated with CD [31], [32], [33], [34], its role in murine colitis models is controversially discussed. In one study, osteopontin deficiency protected mice from DSS-induced colitis [38], while in another study, osteopontin administration in Opn−/− mice reduced the disease activity index, improved red blood cell counts, and reduced gut neutrophil activity compared with the DSS-treated wildtype mice [37]. Interestingly, a recent study demonstrated that Opn−/− mice showed decreased blood levels of IL-22 [39]. Since we recently demonstrated that IL-22 serum levels are increased in CD and correlate with disease activity and the IL23R genotype [55], we next analyzed a potential association between OPN genotypes and IL-22 serum levels in CD patients. Overall, we observed lower IL-22 serum levels in the carriers of OPN minor alleles (homo- and heterozygous carriers), which was statistically significant when the mean IL-22 expression level of carriers of the 9 investigated OPN SNPs minor alleles was compared to the mean IL-22 serum level of the carriers of the homozygous wildtype alleles (p = 3.6×10−5). In 7 out of 8 OPN/SPP1 SNPs forming the haplotype rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138, which was strongly associated with CD susceptibility, the IL-22 serum levels were nominally lower in CD carriers of the minor allele than in homozygous wildtype carriers, although these differences were for each SNP only small and statistically not significant. Similarly, there were no significant associations with CD or UC susceptibility with single OPN SNPs after Bonferroni correction, suggesting that only the combined effect of certain OPN SNPs and haplotypes leads to decreased basal IL-22 levels and increased CD susceptibility. We therefore hypothesize that certain OPN variants may increase the CD risk via decreased basal expression of IL-22, for which we and others demonstrated strong epithelial-protective properties [65], [66], [67], [68]. However, given the multitude of functions mediated by osteopontin, other disease-modulating properties of OPN haplotypes are likely and need further functional investigation.

In addition to increased wound healing, IL-22 mediates also early host defense against attaching and effacing bacterial pathogens [69], [70]. In line with the data of Heilmann et al. [39] demonstrating a dual role of osteopontin in intestinal inflammation, one might therefore hypothesize that carriers of OPN minor alleles with lower IL-22 serum levels are at high risk of developing intestinal inflammation due to the lack of IL-22-induced mucosal protection. Interestingly, Opn−/− mice demonstrated altered wound healing [71], which may be also related to decreased expression of IL-22, which is a strong enhancer of intestinal wound healing [65].

Recent studies in mice showed that osteopontin is involved in Th17 cell differentiation [72] and Opn-expressing DCs induce IL-17 production in T cells [21]. On the other hand, osteopontin expression in DCs is repressed by IFN-α and IFN-γ [73], [74]. This decreased osteopontin expression is associated with high production of IL-27, a Th17 cell-inhibiting cytokine that favors regulatory T cell development [75]. We recently demonstrated that IL-27 is also a protective factor for the intestinal epithelial barrier [76]. IL-27 induces anti-inflammatory and antibacterial responses in intestinal epithelial cells and increases cell restitution after wounding [76]. In mice with Opn-deficient DCs, substantially elevated levels of IL-27 are produced and Opn−/− mice develop delayed experimental autoimmune encephalitis with a Th1 rather than Th17-dominated response [73]. Opn−/− mice display a stronger Th1-mediated proinflammatory response during chronic inflammation while a reduced Th17 response during acute colitis protects them from mucosal inflammation [39], further strengthening the dual role of osteopontin in intestinal inflammation.

In summary, our study identified certain OPN haplotypes to be associated with CD susceptibility. OPN variants may modulate IL-22 secretion which is consistent with data in Opn−/− mice, in which low levels of the epithelial-protective cytokine IL-22 predispose to intestinal inflammation. However, the rather weak association signals found in this study argue against a significant role for OPN as major IBD susceptibility gene which is consistent with the recent IBD meta-analyses [56], [57]. Further functional analysis of large cohorts and detailed fine mapping is required to clarify the role of OPN variants in the genetic susceptibility to IBD.

Supporting Information

Table S1.

Primer sequences, FRET probe sequences, and primer annealing temperatures used for genotyping OPN variants.

https://doi.org/10.1371/journal.pone.0029309.s001

(DOC)

Table S2.

Primer sequences used for the sequence analysis of OPN variants.

https://doi.org/10.1371/journal.pone.0029309.s002

(DOC)

Author Contributions

Conceived and designed the experiments: JG SB. Performed the experiments: JG CB MW CF JG. Analyzed the data: DC JG SB. Contributed reagents/materials/analysis tools: CS JD TO DC MF CS JG SB. Wrote the paper: JS JG SB. Collected phenotype data and DNA samples: JS JG CT TO JD FB CS SB.

References

  1. 1. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, et al. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 411: 599–603.JP HugotM. ChamaillardH. ZoualiS. LesageJP Cezard2001Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.Nature411599603
  2. 2. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, et al. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 411: 603–606.Y. OguraDK BonenN. InoharaDL NicolaeFF Chen2001A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.Nature411603606
  3. 3. Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, et al. (2006) A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 314: 1461–1463.RH DuerrKD TaylorSR BrantJD RiouxMS Silverberg2006A genome-wide association study identifies IL23R as an inflammatory bowel disease gene.Science31414611463
  4. 4. Glas J, Seiderer J, Wetzke M, Konrad A, Torok HP, et al. (2007) rs1004819 is the main disease-associated IL23R variant in German Crohn's disease patients: combined analysis of IL23R, CARD15, and OCTN1/2 variants. PLoS ONE 2: e819.J. GlasJ. SeidererM. WetzkeA. KonradHP Torok2007rs1004819 is the main disease-associated IL23R variant in German Crohn's disease patients: combined analysis of IL23R, CARD15, and OCTN1/2 variants.PLoS ONE2e819
  5. 5. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, et al. (2007) A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 39: 207–211.J. HampeA. FrankeP. RosenstielA. TillM. Teuber2007A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.Nat Genet39207211
  6. 6. Glas J, Konrad A, Schmechel S, Dambacher J, Seiderer J, et al. (2008) The ATG16L1 gene variants rs2241879 and rs2241880 (T300A) are strongly associated with susceptibility to Crohn's disease in the German population. Am J Gastroenterol 103: 682–691.J. GlasA. KonradS. SchmechelJ. DambacherJ. Seiderer2008The ATG16L1 gene variants rs2241879 and rs2241880 (T300A) are strongly associated with susceptibility to Crohn's disease in the German population.Am J Gastroenterol103682691
  7. 7. Libioulle C, Louis E, Hansoul S, Sandor C, Farnir F, et al. (2007) Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4. PLoS Genet 3: e58.C. LibioulleE. LouisS. HansoulC. SandorF. Farnir2007Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4.PLoS Genet3e58
  8. 8. Brand S (2009) Crohn's disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn's disease. Gut 58: 1152–1167.S. Brand2009Crohn's disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn's disease.Gut5811521167
  9. 9. Kawamura K, Iyonaga K, Ichiyasu H, Nagano J, Suga M, et al. (2005) Differentiation, maturation, and survival of dendritic cells by osteopontin regulation. Clin Diagn Lab Immunol 12: 206–212.K. KawamuraK. IyonagaH. IchiyasuJ. NaganoM. Suga2005Differentiation, maturation, and survival of dendritic cells by osteopontin regulation.Clin Diagn Lab Immunol12206212
  10. 10. O'Regan A, Berman JS (2000) Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation. Int J Exp Pathol 81: 373–390.A. O'ReganJS Berman2000Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation.Int J Exp Pathol81373390
  11. 11. Giachelli CM, Steitz S (2000) Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biol 19: 615–622.CM GiachelliS. Steitz2000Osteopontin: a versatile regulator of inflammation and biomineralization.Matrix Biol19615622
  12. 12. Mori R, Shaw TJ, Martin P (2008) Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring. J Exp Med 205: 43–51.R. MoriTJ ShawP. Martin2008Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring.J Exp Med2054351
  13. 13. Uede T (2011) Osteopontin, intrinsic tissue regulator of intractable inflammatory diseases. Pathol Int 61: 265–280.T. Uede2011Osteopontin, intrinsic tissue regulator of intractable inflammatory diseases.Pathol Int61265280
  14. 14. Lund SA, Giachelli CM, Scatena M (2009) The role of osteopontin in inflammatory processes. J Cell Commun Signal 3: 311–322.SA LundCM GiachelliM. Scatena2009The role of osteopontin in inflammatory processes.J Cell Commun Signal3311322
  15. 15. Renkl AC, Wussler J, Ahrens T, Thoma K, Kon S, et al. (2005) Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype. Blood 106: 946–955.AC RenklJ. WusslerT. AhrensK. ThomaS. Kon2005Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype.Blood106946955
  16. 16. Ohshima S, Yamaguchi N, Nishioka K, Mima T, Ishii T, et al. (2002) Enhanced local production of osteopontin in rheumatoid joints. J Rheumatol 29: 2061–2067.S. OhshimaN. YamaguchiK. NishiokaT. MimaT. Ishii2002Enhanced local production of osteopontin in rheumatoid joints.J Rheumatol2920612067
  17. 17. Bazzichi L, Ghiadoni L, Rossi A, Bernardini M, Lanza M, et al. (2009) Osteopontin is associated with increased arterial stiffness in rheumatoid arthritis. Mol Med 15: 402–406.L. BazzichiL. GhiadoniA. RossiM. BernardiniM. Lanza2009Osteopontin is associated with increased arterial stiffness in rheumatoid arthritis.Mol Med15402406
  18. 18. Sennels H, Sorensen S, Ostergaard M, Knudsen L, Hansen M, et al. (2008) Circulating levels of osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor-kappa B ligand, and high-sensitivity C-reactive protein in patients with active rheumatoid arthritis randomized to etanercept alone or in combination with methotrexate. Scand J Rheumatol 37: 241–247.H. SennelsS. SorensenM. OstergaardL. KnudsenM. Hansen2008Circulating levels of osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor-kappa B ligand, and high-sensitivity C-reactive protein in patients with active rheumatoid arthritis randomized to etanercept alone or in combination with methotrexate.Scand J Rheumatol37241247
  19. 19. Buommino E, Tufano MA, Balato N, Canozo N, Donnarumma M, et al. (2009) Osteopontin: a new emerging role in psoriasis. Arch Dermatol Res 301: 397–404.E. BuomminoMA TufanoN. BalatoN. CanozoM. Donnarumma2009Osteopontin: a new emerging role in psoriasis.Arch Dermatol Res301397404
  20. 20. Vogt MH, Ten Kate J, Drent RJ, Polman CH, Hupperts R (2010) Increased osteopontin plasma levels in multiple sclerosis patients correlate with bone-specific markers. Mult Scler 16: 443–449.MH VogtJ. Ten KateRJ DrentCH PolmanR. Hupperts2010Increased osteopontin plasma levels in multiple sclerosis patients correlate with bone-specific markers.Mult Scler16443449
  21. 21. Murugaiyan G, Mittal A, Weiner HL (2008) Increased osteopontin expression in dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune encephalomyelitis and in multiple sclerosis. J Immunol 181: 7480–7488.G. MurugaiyanA. MittalHL Weiner2008Increased osteopontin expression in dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune encephalomyelitis and in multiple sclerosis.J Immunol18174807488
  22. 22. Braitch M, Nunan R, Niepel G, Edwards LJ, Constantinescu CS (2008) Increased osteopontin levels in the cerebrospinal fluid of patients with multiple sclerosis. Arch Neurol 65: 633–635.M. BraitchR. NunanG. NiepelLJ EdwardsCS Constantinescu2008Increased osteopontin levels in the cerebrospinal fluid of patients with multiple sclerosis.Arch Neurol65633635
  23. 23. Niino M, Kikuchi S, Fukazawa T, Yabe I, Tashiro K (2003) Genetic polymorphisms of osteopontin in association with multiple sclerosis in Japanese patients. J Neuroimmunol 136: 125–129.M. NiinoS. KikuchiT. FukazawaI. YabeK. Tashiro2003Genetic polymorphisms of osteopontin in association with multiple sclerosis in Japanese patients.J Neuroimmunol136125129
  24. 24. Standal T, Borset M, Sundan A (2004) Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp Oncol 26: 179–184.T. StandalM. BorsetA. Sundan2004Role of osteopontin in adhesion, migration, cell survival and bone remodeling.Exp Oncol26179184
  25. 25. Zohar R, Cheifetz S, McCulloch CA, Sodek J (1998) Analysis of intracellular osteopontin as a marker of osteoblastic cell differentiation and mesenchymal cell migration. Eur J Oral Sci 106: Suppl 1401–407.R. ZoharS. CheifetzCA McCullochJ. Sodek1998Analysis of intracellular osteopontin as a marker of osteoblastic cell differentiation and mesenchymal cell migration.Eur J Oral Sci106Suppl 1401407
  26. 26. Begum MD, Umemura M, Kon S, Yahagi A, Hamada S, et al. (2007) Suppression of the bacterial antigen-specific T cell response and the dendritic cell migration to the lymph nodes by osteopontin. Microbiol Immunol 51: 135–147.MD BegumM. UmemuraS. KonA. YahagiS. Hamada2007Suppression of the bacterial antigen-specific T cell response and the dendritic cell migration to the lymph nodes by osteopontin.Microbiol Immunol51135147
  27. 27. O'Regan AW, Hayden JM, Berman JS (2000) Osteopontin augments CD3-mediated interferon-gamma and CD40 ligand expression by T cells, which results in IL-12 production from peripheral blood mononuclear cells. J Leukoc Biol 68: 495–502.AW O'ReganJM HaydenJS Berman2000Osteopontin augments CD3-mediated interferon-gamma and CD40 ligand expression by T cells, which results in IL-12 production from peripheral blood mononuclear cells.J Leukoc Biol68495502
  28. 28. Koguchi Y, Kawakami K, Kon S, Segawa T, Maeda M, et al. (2002) Penicillium marneffei causes osteopontin-mediated production of interleukin-12 by peripheral blood mononuclear cells. Infect Immun 70: 1042–1048.Y. KoguchiK. KawakamiS. KonT. SegawaM. Maeda2002Penicillium marneffei causes osteopontin-mediated production of interleukin-12 by peripheral blood mononuclear cells.Infect Immun7010421048
  29. 29. Koguchi Y, Kawakami K, Uezu K, Fukushima K, Kon S, et al. (2003) High plasma osteopontin level and its relationship with interleukin-12-mediated type 1 T helper cell response in tuberculosis. Am J Respir Crit Care Med 167: 1355–1359.Y. KoguchiK. KawakamiK. UezuK. FukushimaS. Kon2003High plasma osteopontin level and its relationship with interleukin-12-mediated type 1 T helper cell response in tuberculosis.Am J Respir Crit Care Med16713551359
  30. 30. Gassler N, Autschbach F, Gauer S, Bohn J, Sido B, et al. (2002) Expression of osteopontin (Eta-1) in Crohn disease of the terminal ileum. Scand J Gastroenterol 37: 1286–1295.N. GasslerF. AutschbachS. GauerJ. BohnB. Sido2002Expression of osteopontin (Eta-1) in Crohn disease of the terminal ileum.Scand J Gastroenterol3712861295
  31. 31. Agnholt J, Kelsen J, Schack L, Hvas CL, Dahlerup JF, et al. (2007) Osteopontin, a protein with cytokine-like properties, is associated with inflammation in Crohn's disease. Scand J Immunol 65: 453–460.J. AgnholtJ. KelsenL. SchackCL HvasJF Dahlerup2007Osteopontin, a protein with cytokine-like properties, is associated with inflammation in Crohn's disease.Scand J Immunol65453460
  32. 32. Gordon JN, MacDonald TT (2005) Osteopontin: a new addition to the constellation of cytokines which drive T helper cell type 1 responses in Crohn's disease. Gut 54: 1213–1215.JN GordonTT MacDonald2005Osteopontin: a new addition to the constellation of cytokines which drive T helper cell type 1 responses in Crohn's disease.Gut5412131215
  33. 33. Sato T, Nakai T, Tamura N, Okamoto S, Matsuoka K, et al. (2005) Osteopontin/Eta-1 upregulated in Crohn's disease regulates the Th1 immune response. Gut 54: 1254–1262.T. SatoT. NakaiN. TamuraS. OkamotoK. Matsuoka2005Osteopontin/Eta-1 upregulated in Crohn's disease regulates the Th1 immune response.Gut5412541262
  34. 34. Mishima R, Takeshima F, Sawai T, Ohba K, Ohnita K, et al. (2007) High plasma osteopontin levels in patients with inflammatory bowel disease. J Clin Gastroenterol 41: 167–172.R. MishimaF. TakeshimaT. SawaiK. OhbaK. Ohnita2007High plasma osteopontin levels in patients with inflammatory bowel disease.J Clin Gastroenterol41167172
  35. 35. Masuda H, Takahashi Y, Asai S, Hemmi A, Takayama T (2005) Osteopontin expression in ulcerative colitis is distinctly different from that in Crohn's disease and diverticulitis. J Gastroenterol 40: 409–413.H. MasudaY. TakahashiS. AsaiA. HemmiT. Takayama2005Osteopontin expression in ulcerative colitis is distinctly different from that in Crohn's disease and diverticulitis.J Gastroenterol40409413
  36. 36. Masuda H, Takahashi Y, Asai S, Takayama T (2003) Distinct gene expression of osteopontin in patients with ulcerative colitis. J Surg Res 111: 85–90.H. MasudaY. TakahashiS. AsaiT. Takayama2003Distinct gene expression of osteopontin in patients with ulcerative colitis.J Surg Res1118590
  37. 37. da Silva AP, Ellen RP, Sorensen ES, Goldberg HA, Zohar R, et al. (2009) Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice. Lab Invest 89: 1169–1181.AP da SilvaRP EllenES SorensenHA GoldbergR. Zohar2009Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice.Lab Invest8911691181
  38. 38. Zhong J, Eckhardt ER, Oz HS, Bruemmer D, de Villiers WJ (2006) Osteopontin deficiency protects mice from Dextran sodium sulfate-induced colitis. Inflamm Bowel Dis 12: 790–796.J. ZhongER EckhardtHS OzD. BruemmerWJ de Villiers2006Osteopontin deficiency protects mice from Dextran sodium sulfate-induced colitis.Inflamm Bowel Dis12790796
  39. 39. Heilmann K, Hoffmann U, Witte E, Loddenkemper C, Sina C, et al. (2008) Osteopontin as two-sided mediator of intestinal inflammation. J Cell Mol Med 13: 1162–1174.K. HeilmannU. HoffmannE. WitteC. LoddenkemperC. Sina2008Osteopontin as two-sided mediator of intestinal inflammation.J Cell Mol Med1311621174
  40. 40. Barizzone N, Marchini M, Cappiello F, Chiocchetti A, Orilieri E, et al. (2011) Association of osteopontin regulatory polymorphisms with systemic sclerosis. Hum Immunol 72: 930–934.N. BarizzoneM. MarchiniF. CappielloA. ChiocchettiE. Orilieri2011Association of osteopontin regulatory polymorphisms with systemic sclerosis.Hum Immunol72930934
  41. 41. Chiocchetti A, Orilieri E, Cappellano G, Barizzone N, S DA, et al. (2010) The osteopontin gene +1239A/C single nucleotide polymorphism is associated with type 1 diabetes mellitus in the Italian population. Int J Immunopathol Pharmacol 23: 263–269.A. ChiocchettiE. OrilieriG. CappellanoN. BarizzoneDA S2010The osteopontin gene +1239A/C single nucleotide polymorphism is associated with type 1 diabetes mellitus in the Italian population.Int J Immunopathol Pharmacol23263269
  42. 42. Schmidt-Petersen K, Brand E, Telgmann R, Nicaud V, Hagedorn C, et al. (2009) Osteopontin gene variation and cardio/cerebrovascular disease phenotypes. Atherosclerosis 206: 209–215.K. Schmidt-PetersenE. BrandR. TelgmannV. NicaudC. Hagedorn2009Osteopontin gene variation and cardio/cerebrovascular disease phenotypes.Atherosclerosis206209215
  43. 43. Marciano R, D'Annunzio G, Minuto N, Pasquali L, Santamaria A, et al. (2009) Association of alleles at polymorphic sites in the Osteopontin encoding gene in young type 1 diabetic patients. Clin Immunol 131: 84–91.R. MarcianoG. D'AnnunzioN. MinutoL. PasqualiA. Santamaria2009Association of alleles at polymorphic sites in the Osteopontin encoding gene in young type 1 diabetic patients.Clin Immunol1318491
  44. 44. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, et al. (2005) Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 19: Suppl A5–36.MS SilverbergJ. SatsangiT. AhmadID ArnottCN Bernstein2005Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Report of a Working Party of the 2005 Montreal World Congress of Gastroenterology.Can J Gastroenterol19Suppl A536
  45. 45. Glas J, Seiderer J, Nagy M, Fries C, Beigel F, et al. (2010) Evidence for STAT4 as a common autoimmune gene: rs7574865 is associated with colonic Crohn's disease and early disease onset. PLoS ONE 5: e10373.J. GlasJ. SeidererM. NagyC. FriesF. Beigel2010Evidence for STAT4 as a common autoimmune gene: rs7574865 is associated with colonic Crohn's disease and early disease onset.PLoS ONE5e10373
  46. 46. Glas J, Seiderer J, Fries C, Tillack C, Pfennig S, et al. (2011) CEACAM6 gene variants in inflammatory bowel disease. PLoS ONE 6: e19319.J. GlasJ. SeidererC. FriesC. TillackS. Pfennig2011CEACAM6 gene variants in inflammatory bowel disease.PLoS ONE6e19319
  47. 47. Glas J, Seiderer J, Tillack C, Pfennig S, Beigel F, et al. (2010) The NOD2 single nucleotide polymorphisms rs2066843 and rs2076756 are novel and common Crohn's disease susceptibility gene variants. PLoS ONE 5: e14466.J. GlasJ. SeidererC. TillackS. PfennigF. Beigel2010The NOD2 single nucleotide polymorphisms rs2066843 and rs2076756 are novel and common Crohn's disease susceptibility gene variants.PLoS ONE5e14466
  48. 48. Glas J, Stallhofer J, Ripke S, Wetzke M, Pfennig S, et al. (2009) Novel genetic risk markers for ulcerative colitis in the IL2/IL21 region are in epistasis with IL23R and suggest a common genetic background for ulcerative colitis and celiac disease. Am J Gastroenterol 104: 1737–1744.J. GlasJ. StallhoferS. RipkeM. WetzkeS. Pfennig2009Novel genetic risk markers for ulcerative colitis in the IL2/IL21 region are in epistasis with IL23R and suggest a common genetic background for ulcerative colitis and celiac disease.Am J Gastroenterol10417371744
  49. 49. Chiocchetti A, Comi C, Indelicato M, Castelli L, Mesturini R, et al. (2005) Osteopontin gene haplotypes correlate with multiple sclerosis development and progression. J Neuroimmunol 163: 172–178.A. ChiocchettiC. ComiM. IndelicatoL. CastelliR. Mesturini2005Osteopontin gene haplotypes correlate with multiple sclerosis development and progression.J Neuroimmunol163172178
  50. 50. Han S, Guthridge JM, Harley IT, Sestak AL, Kim-Howard X, et al. (2008) Osteopontin and systemic lupus erythematosus association: a probable gene-gender interaction. PLoS One 3: e0001757.S. HanJM GuthridgeIT HarleyAL SestakX. Kim-Howard2008Osteopontin and systemic lupus erythematosus association: a probable gene-gender interaction.PLoS One3e0001757
  51. 51. Maver A, Medica I, Salobir B, Tercelj M, Peterlin B (2009) Genetic variation in osteopontin gene is associated with susceptibility to sarcoidosis in Slovenian population. Dis Markers 27: 295–302.A. MaverI. MedicaB. SalobirM. TerceljB. Peterlin2009Genetic variation in osteopontin gene is associated with susceptibility to sarcoidosis in Slovenian population.Dis Markers27295302
  52. 52. Kariuki SN, Moore JG, Kirou KA, Crow MK, Utset TO, et al. (2009) Age- and gender-specific modulation of serum osteopontin and interferon-alpha by osteopontin genotype in systemic lupus erythematosus. Genes Immun 10: 487–494.SN KariukiJG MooreKA KirouMK CrowTO Utset2009Age- and gender-specific modulation of serum osteopontin and interferon-alpha by osteopontin genotype in systemic lupus erythematosus.Genes Immun10487494
  53. 53. Forton AC, Petri MA, Goldman D, Sullivan KE (2002) An osteopontin (SPP1) polymorphism is associated with systemic lupus erythematosus. Hum Mutat 19: 459.AC FortonMA PetriD. GoldmanKE Sullivan2002An osteopontin (SPP1) polymorphism is associated with systemic lupus erythematosus.Hum Mutat19459
  54. 54. Seiderer J, Elben I, Diegelmann J, Glas J, Stallhofer J, et al. (2007) Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): Upregulated colonic IL-17F expression in active crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD. Inflamm Bowel Dis 14: 437–445.J. SeidererI. ElbenJ. DiegelmannJ. GlasJ. Stallhofer2007Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): Upregulated colonic IL-17F expression in active crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD.Inflamm Bowel Dis14437445
  55. 55. Schmechel S, Konrad A, Diegelmann J, Glas J, Wetzke M, et al. (2008) Linking genetic susceptibility to Crohn's disease with Th17 cell function: IL-22 serum levels are increased in Crohn's disease and correlate with disease activity and IL23R genotype status. Inflamm Bowel Dis 14: 204–212.S. SchmechelA. KonradJ. DiegelmannJ. GlasM. Wetzke2008Linking genetic susceptibility to Crohn's disease with Th17 cell function: IL-22 serum levels are increased in Crohn's disease and correlate with disease activity and IL23R genotype status.Inflamm Bowel Dis14204212
  56. 56. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, et al. (2010) Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nat Genet 42: 1118–1125.A. FrankeDP McGovernJC BarrettK. WangGL Radford-Smith2010Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci.Nat Genet4211181125
  57. 57. Anderson CA, Boucher G, Lees CW, Franke A, D'Amato M, 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.CA AndersonG. BoucherCW LeesA. FrankeM. D'Amato2011Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47.Nat Genet43246252
  58. 58. Glas J, Seiderer J, Fischer D, Tengler B, Pfennig S, et al. (2011) Pregnane X receptor (PXR/NR1I2) gene haplotypes modulate susceptibility to inflammatory bowel disease. Inflamm Bowel Dis 17: 1917–1924.J. GlasJ. SeidererD. FischerB. TenglerS. Pfennig2011Pregnane X receptor (PXR/NR1I2) gene haplotypes modulate susceptibility to inflammatory bowel disease.Inflamm Bowel Dis1719171924
  59. 59. D'Alfonso S, Barizzone N, Giordano M, Chiocchetti A, Magnani C, et al. (2005) Two single-nucleotide polymorphisms in the 5′ and 3′ ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus. Arthritis Rheum 52: 539–547.S. D'AlfonsoN. BarizzoneM. GiordanoA. ChiocchettiC. Magnani2005Two single-nucleotide polymorphisms in the 5′ and 3′ ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus.Arthritis Rheum52539547
  60. 60. Xu AP, Bai J, Lu J, Liang YY, Li JG, et al. (2007) Osteopontin gene polymorphism in association with systemic lupus erythematosus in Chinese patients. Chin Med J (Engl) 120: 2124–2128.AP XuJ. BaiJ. LuYY LiangJG Li2007Osteopontin gene polymorphism in association with systemic lupus erythematosus in Chinese patients.Chin Med J (Engl)12021242128
  61. 61. Marciano R, Giacopelli F, Divizia MT, Gattorno M, Felici E, et al. (2006) A polymorphic variant inside the osteopontin gene shows association with disease course in oligoarticular juvenile idiopathic arthritis. Ann Rheum Dis 65: 662–665.R. MarcianoF. GiacopelliMT DiviziaM. GattornoE. Felici2006A polymorphic variant inside the osteopontin gene shows association with disease course in oligoarticular juvenile idiopathic arthritis.Ann Rheum Dis65662665
  62. 62. Hensiek AE, Roxburgh R, Meranian M, Seaman S, Yeo T, et al. (2003) Osteopontin gene and clinical severity of multiple sclerosis. J Neurol 250: 943–947.AE HensiekR. RoxburghM. MeranianS. SeamanT. Yeo2003Osteopontin gene and clinical severity of multiple sclerosis.J Neurol250943947
  63. 63. Mas A, Martinez A, de las Heras V, Bartolome M, de la Concha EG, et al. (2007) The 795CT polymorphism in osteopontin gene is not associated with multiple sclerosis in a Spanish population. Mult Scler 13: 250–252.A. MasA. MartinezV. de las HerasM. BartolomeEG de la Concha2007The 795CT polymorphism in osteopontin gene is not associated with multiple sclerosis in a Spanish population.Mult Scler13250252
  64. 64. Urcelay E, Martinez A, Mas-Fontao A, Peris-Pertusa A, Pascual-Salcedo D, et al. (2005) Osteopontin gene polymorphisms in Spanish patients with rheumatoid arthritis. J Rheumatol 32: 405–409.E. UrcelayA. MartinezA. Mas-FontaoA. Peris-PertusaD. Pascual-Salcedo2005Osteopontin gene polymorphisms in Spanish patients with rheumatoid arthritis.J Rheumatol32405409
  65. 65. Brand S, Beigel F, Olszak T, Zitzmann K, Eichhorst ST, et al. (2006) IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration. Am J Physiol Gastrointest Liver Physiol 290: G827–838.S. BrandF. BeigelT. OlszakK. ZitzmannST Eichhorst2006IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.Am J Physiol Gastrointest Liver Physiol290G827838
  66. 66. Sugimoto K, Ogawa A, Mizoguchi E, Shimomura Y, Andoh A, et al. (2008) IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J Clin Invest 118: 534–544.K. SugimotoA. OgawaE. MizoguchiY. ShimomuraA. Andoh2008IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis.J Clin Invest118534544
  67. 67. Zenewicz LA, Yancopoulos GD, Valenzuela DM, Murphy AJ, Stevens S, et al. (2008) Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease. Immunity 29: 947–957.LA ZenewiczGD YancopoulosDM ValenzuelaAJ MurphyS. Stevens2008Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease.Immunity29947957
  68. 68. Brand S, Dambacher J, Beigel F, Zitzmann K, Heeg MH, et al. (2007) IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro. Am J Physiol Gastrointest Liver Physiol 292: G1019–1028.S. BrandJ. DambacherF. BeigelK. ZitzmannMH Heeg2007IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro.Am J Physiol Gastrointest Liver Physiol292G10191028
  69. 69. Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, et al. (2008) IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat Med 14: 275–281.SJ AujlaYR ChanM. ZhengM. FeiDJ Askew2008IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia.Nat Med14275281
  70. 70. Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, et al. (2008) Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 14: 282–289.Y. ZhengPA ValdezDM DanilenkoY. HuSM Sa2008Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens.Nat Med14282289
  71. 71. Liaw L, Birk DE, Ballas CB, Whitsitt JS, Davidson JM, et al. (1998) Altered wound healing in mice lacking a functional osteopontin gene (spp1). J Clin Invest 101: 1468–1478.L. LiawDE BirkCB BallasJS WhitsittJM Davidson1998Altered wound healing in mice lacking a functional osteopontin gene (spp1).J Clin Invest10114681478
  72. 72. Chen G, Zhang X, Li R, Fang L, Niu X, et al. (2010) Role of osteopontin in synovial Th17 differentiation in rheumatoid arthritis. Arthritis Rheum 62: 2900–2908.G. ChenX. ZhangR. LiL. FangX. Niu2010Role of osteopontin in synovial Th17 differentiation in rheumatoid arthritis.Arthritis Rheum6229002908
  73. 73. Shinohara ML, Kim JH, Garcia VA, Cantor H (2008) Engagement of the type I interferon receptor on dendritic cells inhibits T helper 17 cell development: role of intracellular osteopontin. Immunity 29: 68–78.ML ShinoharaJH KimVA GarciaH. Cantor2008Engagement of the type I interferon receptor on dendritic cells inhibits T helper 17 cell development: role of intracellular osteopontin.Immunity296878
  74. 74. Murugaiyan G, Mittal A, Weiner HL (2010) Identification of an IL-27/osteopontin axis in dendritic cells and its modulation by IFN-gamma limits IL-17-mediated autoimmune inflammation. Proc Natl Acad Sci U S A 107: 11495–11500.G. MurugaiyanA. MittalHL Weiner2010Identification of an IL-27/osteopontin axis in dendritic cells and its modulation by IFN-gamma limits IL-17-mediated autoimmune inflammation.Proc Natl Acad Sci U S A1071149511500
  75. 75. Murugaiyan G, Mittal A, Lopez-Diego R, Maier LM, Anderson DE, et al. (2009) IL-27 is a key regulator of IL-10 and IL–17 production by human CD4+ T cells. J Immunol 183: 2435–2443.G. MurugaiyanA. MittalR. Lopez-DiegoLM MaierDE Anderson2009IL-27 is a key regulator of IL-10 and IL–17 production by human CD4+ T cells.J Immunol18324352443
  76. 76. Diegelmann J, Olszak T, Göke B, Blumberg RS, Brand S (2011) A novel role for IL-27 as mediator of intestinal epithelial barrier protection mediated via differential STAT signaling and induction of antibacterial and anti-inflammatory proteins. J Biol Chem. J. DiegelmannT. OlszakB. GökeRS BlumbergS. Brand2011A novel role for IL-27 as mediator of intestinal epithelial barrier protection mediated via differential STAT signaling and induction of antibacterial and anti-inflammatory proteins.J Biol ChemNov 8. [Epub ahead of print]. Nov 8. [Epub ahead of print].