Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan

GASTROENTEROLOGY 2003;124:140 –146

Crohn’s Disease-Associated NOD2 Variants Share a Signaling Defect in Response to Lipopolysaccharide and Peptidoglycan DENISE K. BONEN,* YASUNORI OGURA,‡ DAN L. NICOLAE,§ NAOHIRO INOHARA,‡ LISA SAAB,‡ TSUYOSHI TANABE,‡,㛳 FELICIA F. CHEN,‡ SIMON J. FOSTER,¶ RICHARD H. DUERR,# ˜ EZ‡ STEVEN R. BRANT,** JUDY H. CHO,* and GABRIEL NUN *Martin Boyer Laboratories, Gastroenterology Section, Department of Medicine, University of Chicago Hospitals, Chicago, Illinois; ‡Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan; §Department of Statistics, University of Chicago, Chicago, Illinois; 㛳Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan; ¶Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom; #Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, School of Medicine; and Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania; **Harvey M. and Lyn P. Meyerhoff Inflammatory Bowel Disease Center, and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

Background & Aims: The NOD2 variants R702W, G908R, and L1007fsinsC are strongly associated with Crohn’s disease (CD) in both European and American populations, but whether this susceptibility extends to all ethnic groups remains unknown. Except for the L1007fsinsC mutation, which produces a truncated NOD2 protein, the functional activity of the major CD-associated variants G908R and R702W is unknown. Methods: Individuals were genotyped for R702W, G908R, and L1007fsinsC. The ability of G908R, R702W, and L1007fsinsC variants in the presence and absence of P268S to confer responsiveness to lipopolysaccharide (LPS) and peptidoglycan (PGN) was determined in HEK293T kidney cells. Results: G908R and L1007fsinsC, but not R702W, were associated with disease susceptibility in Ashkenazi Jews. Ashkenazi Jews with CD had significantly higher allele frequency carriage of G908R and lower carriage of R702W compared with non-Jewish whites with CD. Functional studies revealed that the G908R, R702W, and L1007fsinsC variants in the presence and absence of P268S are defective in their ability to respond to bacterial LPS and PGN, whereas P268S alone exhibited wild-type activity. Conclusions: R702W is not associated with susceptibility to CD in Ashkenazi Jews. The G908R, R702W, and L1007fsinsC variants share a common signaling defect in response to bacterial components, providing evidence for a unifying molecular mechanism whereby NOD2 mutations contribute to disease susceptibility.

rohn’s disease (CD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract most commonly affecting the terminal ileum and colon with a prevalence of approximately 100 –200 of 100,000 people in Western countries.1,2 Despite numerous studies performed over several decades, the cause of CD remains

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poorly understood. Several lines of evidence have suggested that CD is a multifactorial disease in which both genetic and environmental factors appear to play a role in disease development.3 A role for intraluminal bacteria in disease pathogenesis is suggested by the absence of inflammation in various genetic models of colitis when animals are raised in germ-free conditions.3 Furthermore, antibiotic therapies are effective and extensively used in the treatment of human CD.4 Familial clustering of disease and genetic analyses in monozygotic twins strongly suggest that CD is a genetic disorder.5 Genomewide searches for inflammatory bowel disease (IBD) susceptibility genes have resulted in the identification of several loci at chromosomes 1, 3–7, 12, 14, 16, and 19 harboring potential predisposing genes for CD.6 –12 Of these, linkage to a gene at 16q12 (IBD1 locus) in CD has been replicated by independent studies.13–18 Recent studies have shown that single nucleotide polymorphisms in the coding region of NOD2 (MIM 605956), a gene that overlaps with the IBD1 locus on chromosome 16q12, are significantly associated with susceptibility to CD.19 –22 The NOD2 product is a protein composed of 2 NH2-terminal caspase recruitment domains (CARDs), a centrally located nucleotide-binding domain (NBD), and multiple COOH-terminal leucinerich repeats (LRRs). Three major polymorphisms within the coding region of NOD2, L1007fsinsC, G908R, and R702W have been genetically associated with CD in European and American populations.19 –23 In addition, a Abbreviations used in this paper: CD, Crohn’s disease; IBD, inflammatory bowel disease; LPS, lipopolysaccharide; LRRs, leucine-rich repeats; NF-␬B, nuclear factor ␬B; PGN, peptidoglycan. © 2003 by the American Gastroenterological Association 0016-5085/03/$35.00 doi:10.1053/gast.2003.50019

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number of rare amino acid polymorphisms within the LRR region of NOD2 have been described uniquely in CD patients, suggesting that alterations in the LRR domain may be critical to CD pathogenesis.20,24 NOD2 is expressed in monocytes and activates nuclear factor ␬B (NF-␬B) via the serine-threonine kinase, RICK.25 Functional studies have shown that NOD2 confers responsiveness to bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN).26 Furthermore, the LRRs of NOD2 are required for recognition of LPS by NOD2.26 These studies have suggested that NOD2 functions as an intracellular receptor for LPS, PGN, and/or another component contained within the bacterial preparations. Notably, the frameshift variant L1007fsinsC truncates most of the terminal LRR of NOD2, resulting in loss of NF-␬B activation in response to LPS.19 However, the functional activity of the major variants G908R and R702W associated with CD susceptibility remains unknown. Furthermore, all disease variants are found in the background of a common P268S polymorphism,19 but the functional role of P268S remains to be defined. In this study, we performed genetic and case-control studies of the 3 major NOD2 variants in familial cases of CD in non-Jewish and Jewish populations and studied the functional activity of the NOD2 variants to define better the role of NOD2 in susceptibility for CD.

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Expression Lasmids and Immunoblotting Expression plasmids producing N-terminal HAtagged NOD2 variants P268S, R702W, G908R, P268S/ R702W, and P268S/G908R were generated by the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). L1007fsinsC and P268S/L1007fsinsC were generated by a PCR method using wild-type and P268S DNA, respectively, as templates. The generated PCR products were cloned into the pMX-puro expression plasmid.28 The authenticity of the constructs was confirmed by DNA sequencing. The amount of LPS in 100 ␮g/mL of plasmid DNA was below detection using the Limulus Amebocyte Lysate assay (BioWhittaker, Walkersville, MD). Expression of NOD2 proteins in transfected cells was determined by immunoblotting using monoclonal anti-HA antibody (Babco, La Jolla, CA) as described.25

NF-␬B Activation Assay NF-␬B activation assays were performed as described.24,25 Briefly, HEK293T cells were cotransfected with 12 ng of the reporter construct pBVI-Luc, plus indicated amounts of each expression plasmid and 120 ng of pEF-BOS␤-gal in triplicate. LPS and PGN were added to the cultures in the presence of calcium phosphate to allow their entry into the cells as reported.26 LPS from Campylobacter jejuni was a gift of A. Moran, National University of Galway, Ireland. PGN was purified from Bacillus subtilis and digested with Cellosyl as reported.29 Twenty-four-hours posttransfection, cell extracts were prepared, and its relative luciferase activity was measured as described.26 Results were normalized for transfection efficiency with values obtained with pEF-BOS-␤-gal.

Materials and Methods Patient Studies A total of 475 independent CD families were recruited through the IBD clinical practices at the University of Chicago, the Johns Hopkins Hospital, and the University of Pittsburgh. In all cases, informed consent for a molecular genetic study was obtained, and the study protocol was approved by the individual institutional review boards. Diagnoses for CD or ulcerative colitis (UC) were obtained from primary review of endoscopic, radiologic, and pathologic data.27 Patients were classified as being Ashkenazi Jewish if at least 2 grandparents were self-defined as Jewish, and their families emigrated from countries in Central or Eastern Europe. Initial trends for ethnic differences were identified in 113 independent Jewish CD patients and confirmed in an independent cohort of 59 Jewish CD patients, with no differences observed between the 2 Jewish cohorts (data not shown). The test for allele frequency differences was based on Pearson ␹2 test for the 2 ⫻ 2 table. When the cell count was equal to or below 5, the P value was calculated by Monte Carlo simulation with 10,000 replicates. Families were analyzed for association of NOD2 variants with disease using the transmission disequilibrium test as previously reported.19

Results For the frameshift variant L1007fsinsC, comparable allele frequencies (P ⫽ 0.62) were observed in Jewish (7.3%) and non-Jewish (8.4%) CD patients (Table 1). Evidence for disease association with L1007fsinsC in Jewish (P ⫽ 0.0082) and non-Jewish patients (P ⫽ 0.0016) was observed by case-control analysis (Table 1). In contrast, the allele frequency of G908R was significantly higher among Jewish (8.7%) patients compared with non-Jewish (4.3%) CD patients (P ⫽ 0.0082). Furthermore, positive evidence for disease association of G908R was observed for both Jewish (P ⫽ 0.024) and non-Jewish (P ⫽ 0.0095) CD patients (Table 1). The presence of higher allele frequencies observed for G908R in Jewish patients (8.7%) and controls (3.2%) compared with non-Jewish patients (4.3%) and controls (1.6%), respectively, is not unexpected for risk alleles, given the higher CD prevalence among Ashkenazi Jews.30 However, for R702W, non-Jewish CD patients (10.7%) have a significantly higher allele frequency compared with Jewish CD patients (2.6%, P ⫽ 1.3 ⫻ 10⫺5). Unlike in

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Table 1. Case-Control Analysis of NOD2 Variants Stratified by Ethnicity

R702W G908R L1007fsinsC

WNJ CD (n ⫽ 303)

WNJ control (n ⫽ 288)

P valueb

Jewish CD (n ⫽ 172)

Jewish control (n ⫽ 62)

P valuec

P value ethnicd

10.7a 4.3 8.4

4.0 1.6 3.8

1.7 ⫻ 10⫺5 0.0095 0.0016

2.6 8.7 7.3

4.8 3.2 1.6

0.24 0.024 0.0082

1.3 ⫻ 10⫺5 0.0082 0.62

NOTE. Genotyping of the L1007fsins mutation was carried out using allele-specific PCR as described previously.19 For the R702W mutation, primers framing a 512-base-pair region surrounding the mutation were used (sense: 5⬘-GAATTCCTTCACATCACTTTCCAGT-3⬘; antisense: 5⬘GTCAACTTGAGGTGCCCAACATT-3⬘). In addition, each PCR contained 2 additional primers designed to detect the R702W allele (sense: 5⬘-GCGCATCTGAGAAGGCCCTGTTCT-3⬘; antisense: 5⬘-CGCCCAGCGGGCACAGGCCTGGCACCG-3⬘). For detection of the G908R mutation, 4 primers, (2 sense: 5⬘-GAAAAAGTGTCACAACTGTAAATTACTC-3⬘; 5⬘-CGGCTTTTGGCCTTTTCAGATTCAGGG-3⬘, and 2 antisense: 5⬘-CCTAACATTGTGGGGTAGAAATAAA-3⬘; 5⬘-GCCGCCCCCTCGTCACCCACTCTGTAGCG-3⬘), were used. WNJ, white non-Jewish; CD, Crohn’s disease. aPercent allele frequency. b-dP values refer to allele frequency differences between bWNJ CD vs. WNJ controls, cJewish CD vs. Jewish controls, and dWNJ CD vs. Jewish CD (ethnic).

the non-Jewish population, we found no significant difference in R702W allele frequency between Jewish CD patients and controls (P ⫽ 0.24) (Table 1). The lack of association of R702W among Jewish CD patients by case-control analysis does not merely reflect a risk allele simply not present in the Jewish cohort; comparable allele frequencies for R702W were observed in Jewish (4.8%) and non-Jewish (4.0%) case-controls (Table 1). Allelic transmission distortion for R702W, G908R, and L1007fsinsC haplotypes was determined in families from both cohorts of patients. Significant evidence of preferential transmission was observed for R702W, G908R, and L1007fsinsC in non-Jewish patients (Table 2), which is in agreement with results reported in European populations.20 –23 Similarly, we found evidence of disease association for G908R and L1007fsinsC in Jewish families by transmission disequilibrium testing (Table 2). However, we could not assess the transmission of R702W in the Jewish cohort because this variant was very rare in Jewish patients (Table 2). All 3 of the white patients CD variants, R702W, G908R, and L1007fsinsC, occur on the same haplotype background,19,20 which includes the common P268S variant in both Jewish and non-Jewish patients (Table 2). NOD2 has been shown to activate NF-␬B and to confer responsiveness to bacterial LPS and PGN.26 To assess the function of the NOD2 variants and to test whether

P268S affects NOD2 activity, we constructed plasmids to express G908R, R702W, and L1007fsinsC proteins in the presence and absence of P268S. We first examined the ability of wild-type and NOD2 variants to activate NF-␬B in the absence of bacterial components. Transient transfection of wild-type and P268S NOD2 plasmids into HEK293T cells induced comparable levels of NF-␬B (Figure 1). Significantly, expression of the R702W, G908R, P268S/R702W, P268S/G908R, L1007fsinsC, and P268S/L1007fsinsC variants yielded reduced levels of NF-␬B activation when compared with wild-type NOD2 (Figure 1). The difference in basal NF-␬B activity between wild-type and NOD2 variants was more pronounced at lower concentrations of plasmid (5- to 14-fold vs. 3- to 6-fold with 100 and 300 ng of plasmid, respectively) (Figure 1). To assess the expression of the NOD2 proteins, we performed immunoblotting analysis of the same cell extracts used in the functional studies. The analysis shown in Figure 1 revealed that the observed differences in functional activity could not be explained by differential expression of the transfected NOD2 constructs. Next, we compared the ability of wild-type and mutant NOD2 proteins to induce NF-␬B activity in response to LPS and PGN. Because overexpression of NOD2 induces potent NF-␬B activation (Figure 1), we transfected the cells with low amounts of plasmids, and, subsequently, LPS or PGN was added to

Table 2. Haplotype Transmission-Disequilibrium Analysis Stratified by Ethnicity Haplotype

P268 P268S P268S P268S P268S

R702 R702 R702W R702 R702

G908 G908 G908 G908R G908

WNJ CD

L1007 L1007 L1007 L1007 L1007fsinsC

Transmission

No transmission

55 28 33 15 25

77 51 11 6 12

WNJ, white non-Jewish; CD, Crohn’s disease; ND, not determined.

Jewish CD P value

Transmission

No transmission

P value

0.00091 0.05 0.033

15 7 0 12 8

22 17 1 0 0

ND 0.00053 0.047

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Figure 1. NF-␬B activation by wild-type and NOD2 variants. HEK293T cells were cotransfected with the indicated amounts of plasmid expressing wild-type NOD2, the indicated NOD2 variants, or empty plasmid and pEF-BOS-␤gal and pBVI-luc reporter plasmids in triplicate. Cell extracts were prepared at 24 hours posttransfection; one tenth of the lysate was used for luciferase assay and the remaining for immunoblotting analysis. Values represent means ⫾ SD of triplicate cultures. The results are representative of at least 3 independent experiments. The difference in values between wild-type and all mutant NOD2 except P268S were significant (P ⬍ 0.0005). Expression of wild-type and mutant NOD2 protein in equal amounts of cell extracts is shown on top.

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nism for the major CD-associated NOD2 variants. These studies need to be verified using monocytes from CD patients harboring wild-type and mutant NOD2 proteins to make sure that the results are unrelated to protein overexpression or another possible artifact of the culture system. However, such experiments are not currently feasible in that LPS and PGN are complex molecules that trigger a potent NF-␬B response in monocytes through surface Toll-like receptors. HEK 293 cells lack expression of Toll-like receptors, which allows us to compare the function of wild-type and mutant NOD2 in response to LPS and PGN. The identification of the structure(s) within LPS and/or PGN, which is recognized by NOD2, would facilitate the analysis of NOD2 function in monocytes. The P268S variant is found in association with the 3 major variants in a common disease-associated haplotype.

the cultures under conditions that allow the internalization of the bacterial components into the cells.26 Expression of both wild-type and P268S NOD2 induced similar levels of NF-␬B activation in response to LPS or PGN (Figure 2A and B). In contrast, the R702W, G908R, P268S/R702W, P268S/G908R, L1007fsinsC, and P268S/L1007fsinsC variants induced greatly reduced levels of NF-␬B activation in response to LPS or PGN when compared with wild-type NOD2 at all plasmid concentrations tested (Figure 2A and B). Notably, whereas the R702W, G908R, P268S/R702W, and P268S/R702W variants retained their ability to respond to LPS or PGN, the L1007fsinsC and P268/L1007fsinsC proteins did not respond at all (Figure 2A and B).

Discussion In this study, we characterize the functional activity of the 3 major CD-associated NOD2 variants R702W, G908R, and L1007fsinsC. The most marked functional differences were observed with the L1007fsinsC variant, which showed a lack of responsiveness to LPS and PGN-induced NF-␬B activation alone and in combination with P268S. In contrast, the R702W and G908R variants responded to LPS and PGN, but the response was significantly reduced because of diminished ability to activate NF-␬B. Thus, the variants G908R, R702W, and L1007fsinsC exhibit a deficit in NF-␬B activation in response to bacterial components, providing a unifying mecha-

Figure 2. Responsiveness of wild-type and NOD2 variants to LPS and PGN. HEK293T cells were cotransfected with the indicated amounts of plasmid expressing wild-type NOD2, the indicated NOD2 variants, or empty plasmid and pEF-BOS-␤gal and pBVI-luc reporter plasmids in triplicate. Eight hours posttransfection, cells were treated with 5 ␮g/mL of LPS from Campylobacter jejuni (A) or 5 ␮g/mL of PGN from Bacillus subtilis (B) under conditions that allow the internalization of the bacterial components into the cells.26 Values represent means ⫾ SD. The difference in the response to LPS or PGN between wild-type and all mutant NOD2 except P268S was significant (P ⬍ 0.005 and P ⬍ 0.01, respectively).

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We observed that P268S does not affect NF-␬B activation in response to LPS or PGN, a finding that is consistent with genetic evidence, indicating that this common variant is not directly associated with disease.19,20,24 We speculate that the basis for the 3 major CD risk alleles’ independent occurrence on a common haplotype is that P268S alone or together with other possible variants present in an extended haplotype exerted positive selection at some point in evolutionary history. This selection pressure may account, at least in part, for the relatively high allele frequencies (2%– 4%) among white patient case-controls for the 3 major CD risk alleles. The presence of disease association with NOD2 variants in multiple ethnic groups provides support for disease causality, as is the case for the L1007fsinsC and G908R variants. The absence of disease association with R702W in Ashkenazi Jews could be interpreted as not being directly causative, but in linkage disequilibrium with the causative variant, reflecting differences in human populations. However, the functional studies showed that R702W is also associated with reduced ability to activate NF-␬B in response to LPS or PGN. Further studies in independent Jewish cohorts will be important to verify these findings. The present findings of decreased NF-␬B activation for all 3 major risk alleles in response to LPS and PGN preparations provide support for the concept that a deficit of NF-␬B activation in the NOD2 signaling pathway is involved in susceptibility to CD. There is mounting evidence that activation of NF-␬B signaling pathways in response to bacterial components mediates protection of the host against invading pathogens. For example, mutant mice deficient in TNF-␣ or Toll-like receptor signaling, which is associated with decreased NF-␬B activation, exhibit increased susceptibility to pathogen invasion.31–33 NF-␬B deficient mice (p50⫺/⫺, p65⫺/⫹) develop spontaneous enterocolitis because of Helicobacter hepaticus infection,34 suggesting that NF-␬B deficiency in certain cell populations could increase susceptibility to intestinal inflammation. Finally, mice deficient in RICK, a factor required for NOD2-mediated NF-␬B activation, exhibit increased susceptibility to Listeria monocytogenes infection.35,36 Together, these results suggest that a deficit in NOD2 activity may lead to an impaired ability of the host to respond normally to a component of enteric bacteria. The observation that CD-associated NOD2 variants exhibit an altered response to PGN might be significant in that intramucosal injection of PGN is known to chronic granulomatous colitis in rats.37 It is possible that altered cellular apoptosis might be an ad-

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ditional mechanism of NOD2-associated disease pathogenesis. However, we have found no evidence that NOD2 variants associated with CD exhibit an altered ability to enhance caspase-mediated apoptosis when compared with wild-type NOD2 (data not shown). However, given the role of NF-␬B in preventing TNF-␣-induced cell death, it is formally possible that NOD2 variants may, through their effects on NF-␬B activation, alter cellular apoptosis as a mechanism of increasing susceptibility to intestinal inflammation. We suggest that the defect in innate immunity involving NOD2 may secondarily trigger an aberrant T-cell inflammatory response leading to abnormal cytokine production, deregulated NF-␬B activation, and tissue damage.38 This model is consistent with the observed gene-dose effect of the 3 major NOD2 mutations19,20 and the recessive model of inheritance proposed for CD.39,40

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Received August 30, 2002. Accepted October 15, 2002. Address requests for reprints to: Gabriel Nun ˜ez, M.D., University of Michigan Medical School, 1500 E. Medical Center Drive, 4219 CCGC, Ann Arbor, Michigan 48109. e-mail: [email protected]; fax: (734) 647-9654. Supported by grants from the National Institutes of Health (to G.N., J.H.C., N.I., and S.R.B.), the Crohn’s and Colitis Foundation of America (to J.H.C. and S.R.B.), the Reva and David Logan Foundation (to J.H.C.),

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the Scaife Family Foundation (to R.H.D.), and the Gastrointestinal Research Foundation (to J.H.C). Y. Ogura was supported by funds from Tokushima University, Japan, and a fellowship from the Crohn’s and Colitis Foundation of America. D.K.B. and Y.O. contributed equally to the work. J.H.C. and G.N. share senior authorship. The authors thank the patients and their families for participating in this study and A. Moran for LPS preparation.