CD39 deletion exacerbates experimental murine colitis and human polymorphisms increase susceptibility to inflammatory bowel disease

David J. Friedmana,1, Beat M. Ku¨ nzlib,c,1, Yousif I. A-Rahimd, Jean Sevignyb, Pascal O. Berberatc, Keiichi Enjyojib, Eva Csizmadiab, Helmut Friessc, and Simon C. Robsonb,2

aRenal Division and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard University, 330 Brookline Avenue, Boston,MA 02215; bTransplantation Center and Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard University, 330 Brookline Avenue, Boston, MA 02215; cDepartment of Surgery, Technische Universita¨t Mu¨ nchen, Ismaningerstrasse 22, D-81675 Munich, Germany; and dDepartment of Gastroenterology, University of Hawaii, 651 Ilalo Street, Honolulu, HI 96822

Edited by Ralph M. Steinman, The Rockefeller University, New York, NY, and approved August 24, 2009 (received for review March 18, 2009) CD39/ENTPD1 hydrolyzes proinflammatory nucleotides to gener- diators such as IFN gamma, IL-1ß, IL-6, and TNF (8). These ate . As purinergic mediators have been implicated in mutant mice are also prone to increased injury in a wide range intestinal inflammation, we hypothesized that CD39 might protect of acute, subacute, and chronic vascular inflammatory models against inflammatory bowel disease. We studied these possibilities including transplant rejection (9), ischemia-reperfusion (10), in a mouse model of colitis using mice with global CD39 deletion. and diabetic microvascular disease (11). We then tested whether human genetic polymorphisms in the Given the prominent role of CD39 in the response to inflam- CD39 gene might influence susceptibility to Crohn’s disease. We mation and adaptive immunity, we asked whether CD39 might induced colitis in mice using Dextran Sodium Sulfate (DSS). Read- participate in regulating the complex immune balance that outs included disease activity scores, histological evidence of governs susceptibility to inflammatory bowel disease (IBD). We injury, and markers of inflammatory activity. We used HapMap cell tested this hypothesis in mice null for CD39 by administering lines to find SNPs that tag for CD39 expression, and then compared Dextran Sodium Sulfate (DSS), an experimental form of IBD, the frequency of subjects with high vs. low CD39-expression and characterized these mice clinically, histologically, and for genotypes in a case-control cohort for Crohn’s disease. Mice null markers of immune activation. for CD39 were highly susceptible to DSS injury, with heterozygote Recent advances in genetics and genomics, such as the Hap- mice showing an intermediate phenotype compared to wild type Map and the advent of genome wide association studies, have (WT). We identified a common SNP that tags CD39 mRNA expres- generated a wealth of human genetic data. Using data from gene sion levels in man. The SNP tagging low levels of CD39 expression expression profiling studies and Crohn’s case-control studies, we was associated with increased susceptibility to Crohn’s disease in are able to identify human SNPs strongly associated with CD39 a case-control cohort comprised of 1,748 Crohn’s patients and 2,936 mRNA expression and have determined the effect of these SNPs .Our data indicate that CD39 deficiency on susceptibility to Crohn’s disease .(0.0006–0.005 ؍ controls (P exacerbates murine colitis and suggest that CD39 polymorphisms are associated with inflammatory bowel disease in humans. Results Initial Conditions. We studied adult wild-type (WT), heterozygous Crohn’s disease ͉ ENTPD1 (hz) for CD39, and CD39-null (KO) C57BL6 mice at 16–22 weeks of age. Mice in the treatment groups for DSS (WT 37.9 Ϯ Ϯ Ϯ Ͼ ϭ nflammatory bowel disease (IBD) results when the complex 1.0 g vs. hz 34.9 3.0 g vs. CD39-null 35.4 1.2 g; P 0.05, n Iimmune balance in the bowel environment is disrupted. Nor- 5 for each group) had comparable body weights at initiation of mally, the immune system suppresses in the pres- the study. ence of resident gut bacteria or food antigens but can respond rapidly to potentially pathogenic bacteria or viruses with vigor- Clinical Course of DSS Colitis. DSS treatment of C57BL6 mice ous inflammatory responses (1). In IBD, gut immunity is epi- resulted in acute colitis in all mice. We evaluated the clinical sodically activated in the absence of a clear ‘‘danger signal,’’ severity of the colitis using a disease activity index (DAI) (Fig. leading to a constellation of symptoms including abdominal pain, 1A), which incorporates weight loss, stool consistency, and GI diarrhea, gastrointestinal bleeding, venous thrombotic compli- bleeding (Fig. 1B). CD39-null mice had significantly worse colitis cations, and often malnutrition (1). Strong genetic components than WT mice starting on day 2 and continuing through day 7. contribute to an individual’s susceptibility to IBD, particularly Heterozygote mice were intermediate between WT and CD39- Crohn’s disease (2). null at all time points, but did not differ significantly from either CD39, also known as ENTPD1, is a vascular and immune cell group at any time point (Fig. 1A). that converts extracellular ATP and ADP to We measured the hematocrit in each group as another indi- AMP. Since ATP is typically proinflammatory and AMP is cator of disease severity. There is no difference at baseline in rapidly converted by CD73 into the largely anti-inflammatory metabolite adenosine, CD39 tends to promote an anti- Author contributions: D.J.F., B.M.K., and S.C.R. designed research; D.J.F., B.M.K., Y.I.A.-R., inflammatory and immune suppressive milieu. Within the im- J.S., and E.C. performed research; J.S. contributed new reagents/analytic tools; D.J.F., mune system, CD39 is expressed on cells of both the innate and B.M.K., Y.I.A.-R., P.O.B., K.E., H.F., and S.C.R. analyzed data; and D.J.F., B.M.K., and S.C.R. adaptive immune systems, including T cells (particularly T- wrote the paper. regulatory cells) (3), B-cells (4), NK cells (5), NKT cells (6), The authors declare no conflict of interest. dendritic cells (4), monocytes (5), and macrophages (7). This article is a PNAS Direct Submission. Studies in our laboratory indicate a particularly important role 1D.J.F. and B.M.K. contributed equally. for CD39 and CD73 in the tandem generation of adenosine with 2To whom correspondence should be addressed. E-mail: [email protected]. subsequent activation of adenosine A2a receptors (3). Mice null This article contains supporting information online at www.pnas.org/cgi/content/full/ for CD39 have heightened expression of proinflammatory me- 0902869106/DCSupplemental.

16788–16793 ͉ PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902869106 Downloaded by guest on September 27, 2021 Fig. 2. Acute DSS colitis pathology and myeloperoxidase (MPO) activity. (A) WT mice showed thickening of the colon wall with discrete inflammatory infiltrates, but the architecture and polarity of tissue layers were maintained. (B) DSS caused moderate inflammation in heterozygous mice and (C) severe inflammation in CD39-null mice, affecting the epithelial and subserosal layer in heterozygous and severe inflammation in CD39-null mice, with mononu- Fig. 1. Experimental assessment of acute DSS colitis. (A) Disease activity clear cell infiltrates into the muscularis. (D) Leukocyte recruitment into the index (DAI) correlated with severity of disease and CD39-null mice displayed colonic wall during DSS colitis was determined by a standard MPO activity worse DAI compared to WT DSS colitis. (B) Definition of disease activity index. assay. DSS treatment was associated with a significant (P Ͻ 0.01) increase in (C) Hematocrit (Hct) values correlated with the pathological findings. A drop MPO activity in CD39-null mice. in the Hct occurred in all mice, but WT mice maintained a significant higher Hct than heterozygous or CD39-null mice. levels in humans. Several SNPs in strong linkage dysequilibrium appeared to correlate with CD39 expression, and we chose hematocrit (approximately 50) between WT and CD39-null mice rs10748643 as the tag SNP because of its proximity to the CD39 (12). However, colitis resulted in decreased hematocrit for both ϩ CD39-null (27.8 Ϯ 0.8%) and heterozygous (28.0 Ϯ 0.4%) mice promoter ( 850 base pairs into intron 1, a region often associated Ϯ P Ͻ with transcriptional regulation). We directed tested cell lines from compared to WT mice (33.8 1.0%; 0.012 for both ϭ comparisons (Fig. 1C)). HapMap subjects of European ancestry who were either AA (n 6) or GG (n ϭ 6) at rs10748643 for CD39 mRNA expression. GG Ͻ Pathology. Next we examined bowel from mice in each group. homozygotes had mRNA levels 43% higher (P 0.0004), with no Histologically, CD39-null mice displayed severe transmural in- overlap between groups (Fig. 3). flammation with dense wall thickening, profound leukocytic infiltration and ulceration of colonic mucosa (Fig. 2C), when High Vs. Low Expressors Across Four Population Groups. After pub- compared to heterozygous (Fig. 2B) and WT mice (Fig. 2A). We lication of complete HapMap gene expression profiling sets, we also examined myeloperoxidase activity in the colons of the mice as a quantitative index of inflammation. CD39 null mice showed significantly increased MPO activity (P Ͻ 0.01) when compared to WT mice, with heterozygous mice demonstrating an inter- mediate level of inflammation (Fig. 2D).

Apyrase Rescue. We pretreated CD39-null mice with parenteral apyrase, a soluble factor with enzymatic activity essentially identical to CD39, before chronic exposure with DSS and used weight as a proxy for colitis severity. Mice receiving apyrase before DSS lost no weight compared to mice receiving only placebo (38.0 Ϯ 1.0 g vs. 37.7 Ϯ 2.3 g), while mice receiving only DSS experienced significant weight loss (32.8 Ϯ 0.8g; P Ͻ 0.05 compared to both groups) (Fig. S1, n ϭ 6–10 per group). Fig. 3. CD39 mRNA expression by genotype at rs10748643. CD39 mRNA Real-Time PCR of CD39 mRNA by Genotype: High Vs. Low ENTPD1 expression levels (by real time PCR) were normalized for ribosomal 18S subunit Expressors. We analyzed available whole-genome expression pro- expression level in HapMap lymphocytes. CD39 expression from cell lines from filing data in public repositories to generate hypotheses about SNPs subjects with GG alleles at rs10748643 was higher than expression from MEDICAL SCIENCES in the ENTPD1 gene associated with CD39 mRNA expression subjects with AA alleles. There was no overlap between groups.

Friedman et al. PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 ͉ 16789 Downloaded by guest on September 27, 2021 Table 2. CD39 expression in HapMap cell lines Relative expression

CEU YRI JPT CHB

AA 1.00 1.00 1.00 1.00 AG 1.34 1.13 1.21 1.20 GG 1.77 1.46 1.61 1.72

Relative CD39 expression for each population based on genotype at rs10748643.

Discussion IBD may represent a tipping of the immune balance toward excessive inflammation in genetically susceptible individuals. Crohn’s disease in particular has been associated with a skewing toward a Th1/IFN gamma-driven inflammatory response (1). Interestingly, CD39-null mice also exhibit a general tendency toward exaggerated Th1/IFN gamma responses (15). In the selected mouse model (acute DSS exposure) that recapitulates important cardinal features of colitis in IBD such Fig. 4. CD39 expression in 210 subjects across four populations. (A) CD39 as diarrhea, weight loss, and gastrointestinal bleeding, we ob- mRNA expression in cell lines from subjects with Caucasian/European (CEU), served that CD39-null mice suffer more severe injury than WT Yoruba (YRI), Chinese (CHB), or Japanese (JPT) ancestry with each rs10748643 mice. Although heterozygous mice did not differ from either genotype. For Chinese subjects, AG and GG were combined because there group in our overall index (DAI) of IBD-activity, these mice did were only two GG subjects. show a potential CD39 dose effect in limiting inflammation. DSS-colitis has traditionally been associated with features of examined CD39 mRNA expression in 210 HapMap subjects from both Crohn’s disease and ulcerative colitis in rodents, with a bias four diverse populations to confirm our in vitro findings in a larger toward the former. set of European subjects and to determine if this expression In CD39-null mice, injury spanned the bowel wall, a typical phenotype could be generalized to people of other ancestries. In and critically important feature of Crohn’s disease that is not Europeans, Africans, Chinese, and Japanese subjects, rs10748643 seen in classic ulcerative colitis (16). Examination of large bowel genotype correlates strongly with CD39 expression. GG is associ- in the treated mouse groups revealed substantially more severe ated with much higher levels than AA (46–77% increase), with histological lesions in the null mice, with loss of crypts and a far carriers of the AG genotype displaying intermediate expression more vigorous inflammatory response. Increased MPO activity levels (Fig. 4 and Tables 1 and 2). In Chinese subjects, the GG in CD39-null mice provided further evidence of this heightened inflammatory response, emphasizing the role of infiltrating genotype was uncommon and AG and GG genotypes were pooled monocyte-macrophages in this disease process. Treatment of for analysis. For all populations combined, the G allele was asso- CD39-null mice with soluble apyrase could reverse weight loss ciated with higher expression levels with a P value on the order of Ϫ from chronic DSS treatment, implicating the nucleotide hydro- 10 22 using an additive, linear model (13). These results were lyzing capacity of CD39 as the functional protective mechanism. confirmed in a second genome wide study, with similarly strong Immune system dysregulation is generally considered the association (10Ϫ28) (14). major cause of IBD, but there is a growing appreciation for the role of the vasculature in modifying this response (17–19). CD39 Association of a SNP Tagging ENTPD1 Expression with Crohn’s Disease. is both the dominant immune cell and vascular ectonucleotidase, Next, we asked whether this SNP strongly correlated to CD39 so its protective effect against DSS colitis in mice may be due to expression might influence human susceptibility to colitis, as we either of these two roles, or perhaps both. CD39 is important for saw in our mouse models. We obtained data from the Wellcome Treg cell function through generation of adenosine (3), and we Trust Case Control Consortium study in Crohn’s disease to test would propose this function as the most likely factor in our this hypothesis. At rs10748643, there was significant enrichment model. However, recently others have shown that CD39 may of the low CD39-expressing AA genotype in Crohn’s disease specifically modify vascular regulation in murine colitis (20). We cases, while controls were enriched for the higher CD39- expect tissue specific deletion of CD39 in immune cells or expressing GG allele (Table 3). Using the Wellcome Trust Case vascular cells will answer this question definitively in the future. Control Consortium (WTCCC) expanded control panel (all In mice, several experimental models have been published without IBD), we noted an increase in statistical significance in showing involvement of purinergic signaling in IBD and toxin- additive, genotypic, allelic, and homozygote vs. homozygote induced colitis. The ultimate products of are models (Table 3). The odds ratio for the A allele was 1.13 nucleosides/adenosine that activate type 1 A2A adenosine re- (1.04–1.23) or 1.14 (1.06–1.22) using the standard control or ceptors on immune and vascular cells, markedly attenuating expanded control panel, respectively. intestinal inflammation in some animal models of IBD (21, 22). CD39 is not expressed on epithelial surfaces but rather has similar tissue distribution to the A2A receptor, and the similar Table 1. HapMap cell lines from 4 ancestral populations response to colitis in CD39- and A2A-null mice suggests a Genotype CD39 CEU YRI JPT CHB functional interaction. A2A activation protects against colitis in these studies, associated with decreased production of inflam- AA 9 10 20 26 matory cytokines such as TNF-␣ and IFN gamma. Notably, these AG 30 35 19 17 cytokines are upregulated in CD39-null mice (23). In contrast, GG 21 15 6 2 conflicting recent reports show paradoxically that activation of Number of cell lines per genotype in each ancestral population. another adenosine receptor, A2B, may be either pathogenic or

16790 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902869106 Friedman et al. Downloaded by guest on September 27, 2021 Table 3. Association of rs10748643 with Crohn’s disease in a case-control cohort AA AG GG Additive: P Allelic: P, O.R. Genotypic: P AA vs GG: P, O.R.

Crohn’s cases 391 (22.4%) 885 (50.6%) 472 (27%) Controls 577 (19.7%) 1472 (50.1%) 887 (30.2%) 0.005 0.005, 1.13 (1.04–1.23) 0.0197 0.005, 1.27 (1.07–1.51) Expanded controls 2108 (19.9%) 5236 (49.4%) 3,256 (30.7%) 0.0006 0.0007, 1.13 (1.05–1.22) 0.0026 0.0008, 1.28 (1.11–1.48)

Frequency of AA, AG, and GG genotypes at rs10748643 in control subjects and patients with Crohn’s disease from the Wellcome Trust Case Control Consortium. Expanded controls include additional subjects without Crohn’s disease from the WTCCC (see Methods). O.R.ϭOdds Ratio, with confidence intervals. The additive model is a 1-degree of freedom ␹2 test for trend. The allelic and homozygote models are 1-degree of freedom ␹2 tests. The genotypic model is a 2-degree of freedom ␹2 test.

of benefit in mouse colitis models (24–26). A2B is a lower equal prior probability of significance, we first identified a gene affinity adenosine receptor that is also expressed on the epithe- with functional importance in the specific disease process in an lial surface and has differing effects to the A2A receptor. animal model. Secondly, we found a SNP that strongly correlates Quite recently, investigators have shown that luminal ATP with expression of that functional gene. Given these first two produced by gut bacteria plays a critical role in Th17 T-cell pieces of evidence, the prior probability of this SNP’s effect on immune deviation in the lamina propria (27). Administration of Crohn’s disease is markedly increased compared to a randomly ATP worsened and luminal administration of apyrase amelio- chosen SNP. In this Bayesian context, a P value of 0.005 (or rated colitis in mouse models (27). These findings suggest that 0.0007 using the expanded controls) provides strong evidence of more efficient ATP scavenging in the gut environment by the an effect of this SNP in human Crohn’s disease. host organism might protect against colitis. The integral role of While many studies identify genetically modified mice sus- the ATP receptor P2ϫ7 in the inflammasome complex (28), a ceptible to an injury model, and many others find SNPs associ- critical coordinator of innate immunity, also suggests a potential ated with human disease, it is uncommon when both functional candidate pathway that may mediate this effect. Other studies data in mice and studies demonstrating relevance in humans are have suggested roles for various P2 receptors in colitis, partic- in alignment. We believe that evidence supporting both of these ularly P2ϫ3, but little consensus has emerged (29–32). criteria makes our study particularly interesting, especially since We sought further evidence for relevance of the purinergic the SNP we identified definitively alters gene expression levels. system to human IBD by identifying SNPs associated with high Our data are particularly intriguing given the links between or low CD39 expression. We took advantage of the fact that inflammation and thrombosis in IBD (34) and the observation CD39 was initially described as a B-cell activation marker (4), that CD39 was first characterized as a vascular thromboregula- and that B-lymphoblast cell lines are available for all HapMap tory factor (35). Our future aim is to further define the patho- subjects, along with genotyping at several million SNPs. Initially genetic mechanisms underlying the role of CD39 in IBD, con- we identified rs10748643 due to its proximity to the promoter, clusively identify the specific functional SNP and molecular gene but because of high LD between this SNP and several dozen regulation mechanisms, and analyze more patients to rigorously others, we are not able to determine if rs10748643 is the establish the genetic association of this CD39 polymorphism functional SNP or a SNP that tags the functional SNP. In either with Crohn’s disease in humans. case, it does appear to distinguish high versus low CD39 expres- sors. We suspect the GG genotype leads to an increase in Materials and Methods expression in Caucasians of closer to the 77% calculated from Animals. Pathogen-free C57BL6 CD39-null and matched wild-type (WT) mice our bioinformatic studies than the 43% observed in our in vitro were studied in accordance with standard institutional animal welfare guide- work, due to chance inclusion of the highest CD39-expressing lines. The derivation and characterization of CD39-null mice have been de- AA cell line in the CEU HapMap collection in our abbreviated scribed elsewhere (12). Genotyping of the CD39 alleles was performed as previously described (12). Animal care and experiments were carried out panel of six AA cell lines. Nonetheless, all six AA cell lines had under the guidelines and protocols approved by the Animal Care and Use lower CD39 mRNA expression than all six GG cell lines tested. Committee, Beth Israel Deaconess Medical Center, Harvard University, Boston. The correlation with this set of SNPs tagged by rs10748643 and CD39 expression was consistent in cell lines across four popu- Induction and Clinical Assessment of Experimental (Acute) Colitis. Dextran lation groups, although the frequency of the A vs. G alleles Sodium Sulfate (DSS) colitis. CD39-null (n ϭ 8), heterozygous for CD39 (n ϭ 11), varied widely between ancestral groups. Previously, we have and WT (n ϭ 12) mice were equally treated with 3% DSS in standard drinking shown that CD39 expression levels in B-lymphoblasts correlate water. There was no restriction regarding the dose of DSS solution that was well with ATPase and ADPase functional activity (33). provided ad libitum for 7 consecutive days. Control mice received standard We examined the effect of rs10748643 in data obtained from drinking water. Body weights, hemoccult, gross blood, and stool consistency a carefully collected and phenotyped case-control study of were analyzed on a daily basis. Disease activity index (DAI) was calculated by scoring percent weight loss, intestinal bleeding [no blood, occult blood Crohn’s disease in people of European ancestry. We found that (hemoccult ϩ), or gross blood], and stool consistency (normal stool, loose control subjects were more likely to have the high CD39- stool, or diarrhea), as previously described (36). The clinical features were expressing GG genotype, and Crohn’s patients were more likely scored separately and then correlated with a histological score: DAI ϭ (body to have the low-expressing AA genotype. Although the effect weight loss) ϩ (diarrhea score) ϩ (rectal bleeding score) (Fig. 1B). size was modest in terms of odds ratio (about 1.27 when comparing homozygote groups), the high frequency of the risk Chronic DSS Colitis. Sixteen- to eighteen-week-old CD39 null mice were (A) allele, about 40% in Caucasians, means that the effect of this further studied with a longer course of DSS, with or without apyrase to variant on populations as a whole (population attributable risk) reconstitute CD39 function. CD39-null mice were treated with four 1-week may be more important than variants in other genes with higher intervals of DSS in standard drinking water followed by 1 week off DSS relative risks but very low frequencies. Conversely, there may be treatment. The total duration of the treatment was 7 weeks. We treated with parenteral apyrase and compared three different conditions: CD39-null mice rare CD39 variants with greater effects on expression level and with DSS (n ϭ 10), CD39-null mice with DSS and apyrase (n ϭ 10), and CD39-null more impact on relative risk. mice receiving only apyrase (n ϭ 6). We used grade-VII apyrase (derived from Although the P value of our variant is also modest, we think S tuberosum) purchased from Sigma at a dose of 2.5 U/g twice a day by i.p. this result is best appreciated from the perspective of Bayesian injection. This apyrase has comparable (approximately1:1) ATPase and MEDICAL SCIENCES probability. Rather than ask 500,000 simultaneous tests with ADPase activity, paralleling the nucleotide preference of CD39.

Friedman et al. PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 ͉ 16791 Downloaded by guest on September 27, 2021 Histological Assessment of Colitis. Specimens were embedded and snap-frozen Bio-Informatic Assessment of ENTPD1 Expression. Expression data for 210 as previously described (37) and stored at Ϫ80 °C. Four- to five-micrometer HapMap cell lines was downloaded from the Wellcome Trust Sanger Institute tissue sections were stained with hematoxylin and eosin. Stained sections (http://www.sanger.ac.uk/humgen/genevar/). The methods for both genotyp- were examined for evidence of colitis as previously published, (38) using as ing and expression profiling of these cell lines have been described in detail criteria the presence of lymphocytic infiltration, macrophages or polymor- (13). ENTPD1 expression level for each genotype was calculated. We used phonuclear cells, elongation and/or distortion of crypts, crypt abscesses, re- log2-transformed data for tests of statistical significance. duction in goblet cell number, ulceration, and edema formation of the colon wall. Crohn’s Disease Genetics. Genotypes at rs10748643 for both control and Crohn’s patients in the Wellcome Trust Case Control Consortium (WTCCC) Colonic Myeloperoxidase (MPO) Activity. Colon samples were obtained from were downloaded from http://www.wtccc.org.uk/. Data were available for control and DSS treated animals, and prepared as previously decribed (39). The 1,748 Crohn’s cases and 2,936 apparently healthy controls. We conducted a samples were thawed for MPO activity assay determination according to the second analysis of the data using an expanded control panel (10,600 subjects) o-dianisidine method as previously described (39). In brief, tissue samples were that included the healthy controls plus additional subjects from the Wellcome thawed, weighed, suspended in 50 mM potassium phosphate buffer (Kpi), pH Trust case cohorts. We included hypertension, coronary artery disease, bipolar 6.0, containing 0.5% hexadecyltrimethylammonium bromide buffer (0.1 g/20 disorder, and type 2 diabetes cohorts that are not expected to have disease mL Kpi), and homogenized. After sonication the sample was microcentri- SNPs in common with Crohn’s disease, but excluded subjects from autoim- fuged. The reaction was started by mixing and incubating the supernatant in mune disease cohorts (type 1 diabetes and rheumatoid arthritis) that are likely 50 mM Kpi, 20 mg/mL o-dianisidine dihydrochloride, and 20 mM hydrogen to have disease SNPs that overlap with Crohn’s disease (40). peroxide. The reaction was stopped by adding 2% sodium azide. The change of the absorbance was monitored at 460 nm for 10 min. MPO activity was Statistical Analysis. Statistical analysis was performed with Microsoft Excel or expressed as the amount of enzyme necessary to produce a change in absor- Graphpad Prism 4 software. For mouse data, we used ANOVA with Newman- bance of 1.0 per min/g wet weight of colonic tissues. Keuls posthoc test for parametric analyses between three or more groups and the Kruskal-Wallis test with Dunn’s posthoc test for nonparametric analyses. Cell Culture. Lymphoblasts from HapMap participants of European (CEU) For cell line data, we used unpaired Student t-test for comparison between ancestry were purchased from Coriell Institute (www.coriell.org). Genotypes groups. For Crohn’s genetic data, we analyzed the data under a genotypic for each cell line were obtained from www.hapmap.org. Cells were grown in model (two-degree of freedom ␹2 test), an allelic model (one-degree of RPMI 1640 media with 2 mM L-glutamine, 15% FBS, penicillin-streptomycin freedom ␹2 test), a homozygote vs. homozygote model (1- degree of freedom mix, and 10 mM HEPES buffer. Cells were seeded at 200,000 per mL and ␹2 test), and an additive model (one-degree of freedom ␹2 test for linear harvested 24–48 h later in log-phase growth. trend).

Real-Time PCR. mRNA was isolated using RNeasy (Qiagen) according to the ACKNOWLEDGMENTS. We thank Mark Daly for advice and suggestions; the manufacturer’s instructions. mRNA (0.5 ␮g) was reversed transcribed to cDNA Wellcome Trust and the authors of the Wellcome Trust Case Control Consor- using the Taqman Reverse Transcription Kit (Applied Biosystems). Probe- tium study (40) for making their data publicly available; Stranger et al. (13) for making their gene expression data publicly available; and the National Insti- primer sets for ENTPD1 and the 18S ribosomal subunit (loading control) were tute of General Medical Sciences and the Coriell Cell Repository for their obtained from Applied Biosystems. Real-time PCR was performed with an service in providing human cell lines for use by the research community. This Applied Biosystems 7900 system. ENTPD1 mRNA expression was normalized work was supported by National Institutes of Health Grants K08 DK076868, using 18S expression. mRNA was quantified twice from each cell line and HL57307, HL63972, and HL076540 (to D.J.F. and S.C.R.) and German Research averaged. Foundation Grants DFG KU 1957/1-1 and DFG KU 1957/3-1 (to B.M.K.).

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