Advances and Perspectives in the Genetics of Inflammatory Bowel Diseases

Advances and Perspectives in the Genetics of Inflammatory Bowel Diseases

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2006;4:143–151 CLINICAL GENOMICS Advances and Perspectives in the Genetics of Inflammatory Bowel Diseases MAT...

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CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2006;4:143–151

CLINICAL GENOMICS Advances and Perspectives in the Genetics of Inflammatory Bowel Diseases MATHIAS CHAMAILLARD,*,‡,§ RAZVAN IACOB,*,‡,§ PIERRE DESREUMAUX,*,‡,§ and JEAN–FREDERIC COLOMBEL*,‡,§ *INSERM 0114, Hôpital Swynghedauw, Lille; ‡University Lille 2, Lille; and §CHRU Lille, Hôpital Huriez, Service des Maladies de l’Appareil Digestif et de la Nutrition, Lille, France

Several clinical and biological phenotypes define complex diseases such as inflammatory bowel disease (IBD). There is a critical role of the caspase recruitment domain protein 15/nucleotide-binding oligomerization protein 2– dependent (CARD15-NOD2) sensing of bacterial cell wall components in health and disease. The current etiologic model for IBD emphasizes an interaction between susceptibility and modifier genes along with environmental factors. Together, these lead to disease progression. However, further work should clarify the pathophysiological mechanisms leading to IBD and how innate immune signaling confers susceptibility to intestinal inflammation. This is a prerequisite for rational clinical management of IBD. Genetic, functional, serologic testing and development of therapeutics in IBD are discussed.

fter the discovery of the first susceptibility gene for Crohn’s disease (CD) on chromosome 16 (the caspase recruitment domain protein 15/nucleotide-binding oligomerization protein 2 [CARD15/NOD2 gene), rapid advances have been made in the field of inflammatory bowel disease (IBD) genetics. Three other CD genes, SLC22A4 and SLC22A5 on chromosome 5 and DLG5 on chromosome 10, have been identified and other candidate genes are under investigation. A complex etiologic model has been proposed for IBD, in which the individual expression of the disease could be influenced by interactions between environmental factors and promoting and modifying genes. Recent insights into the functions of normal and variant CARD15/NOD2 proteins have focused attention on the innate immune system and the pivotal role of host defense against microbes at the mucosal interface in the pathogenesis of CD. In this review, we discuss whether or not these discoveries already can be translated into clinical practice and their implications for future therapeutic strategies.

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Inflammatory Bowel Disease Susceptibility Genes: Where Do We Stand? Numerous susceptibility loci have been implicated in IBD. Replication has been shown for 9 of these loci, termed IBD1–9 (Figure 1). Major CD susceptibility alleles have been identified in genes encoding critical regulators of the intestinal barrier function, referred to the CARD15/NOD2, SLC22A4/A5, and DLG5 genes.1–5 On the other hand, ulcerative colitis (UC) susceptibility alleles or haplotypes (ie, a unique pattern of sequential single-nucleotide polymorphisms from the same chromosome) with a significant population-attributable risk have yet to be identified. CARD15/NOD2 is a cytoplasmic molecule involved in sensing unique microbial cell-wall components. Notably, CARD15/NOD2 confers responsiveness to bacterial peptidoglycan (PGN) through in vitro and in vivo recognition of muramyl dipeptide (MDP).6 –9 Three CARD15/NOD2 mutations (namely R702W, G908R, and 1007fs) are established independent risk factors for CD in Caucasians, but the role in CD development of other rare mutations would require the recruitment of more patients. Interestingly, the CARD15/NOD2dependent population-attributable risk is modest among Northern European populations (Irish, Norwegians, Scots, Fins), as compared with populations residing in Abbreviations used in this paper: CARD, caspase recruitment domain; CD, Crohn’s disease; DLG5, disc large homolog 5; IBD, inflammatory bowel disease; LRR, leucine-rich repeat; NOD-LRR protein, LRR-containing nucleotide-binding oligomerization domain protein; MDP, muramyl dipeptide; MDR1, multidrug resistance 1; NF-␬B, nuclear factor-␬B; NOD2, nucleotide-binding oligomerization protein 2; PRM, pathogen-recognition molecule; PGN, peptidoglycan; SLC22A4/ A5, solute carrier family 22A4/22A5; TLRs, Toll-like receptors; UC, ulcerative colitis. © 2006 by the American Gastroenterological Association Institute 1542-3565/06/$32.00 PII: 10.1053/S1542-3565(05)01096-7

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Figure 1. Overview of the IBD-susceptibility and IBD-modifying genes in human beings. The confirmed and suggested IBD-susceptibility loci are indicated by black and grey rectangles, respectively. The confirmed and suggested IBD-susceptibility genes are indicated by black and grey letters, respectively.

the lower latitudes. Furthermore, CARD15/NOD2 mutations are absent or very rare in Asians, Arabs, Africans, and African Americans.1,2,10 –14 Based on haplotype analysis, CARD15/NOD2 mutations might have arisen recently (Chamaillard et al, unpublished data). Individuals with 1 of the 3 major disease-associated alleles have a 2– 4-fold increased risk for developing CD, whereas homozygous or compound heterozygous carriers have an up to 40-fold increase in genotype-relative risk.2 This strong gene-dosage effect is consistent with a recessive model of a major gene, as suggested initially by epidemiogenetic analysis.15 Despite their strong association with CD, genetic alterations of the CARD15/NOD2 gene are neither sufficient nor necessary for the development of CD because CARD15/NOD2-deficient mice do not develop intestinal inflammation spontaneously,9 0%–5% of the general Caucasian population are homozygous for CD-associated mutations, and 60%–70% of CD patients do not have both CARD15/NOD2 alleles mutated.2 Besides the CARD15/NOD2 gene, particular interest has been focused on other replicated IBD loci such as the IBD5 region on chromosome 5. By using fine mapping, linkage disequilibrium methods, and haplotype analysis the IBD5 region subsequently was replicated and narrowed to an 11-SNP (single nucleotide polymorphism) haplotype of 250 kb. Two novel polymorphisms in the

solute carrier family 22A4/22A5 (SLC22A4/A5) genes within this region were identified in the Canadian population and proposed as 2 CD-associated alleles carried by the same haplotype SLC22A-TC,4,16 but genetic analysis could not make any distinction between such variations. SLC22A4 and SLC22A5 encode the organic cation transporters 1 and 2, respectively. Consistent with previously reported epistatic interactions between the IBD5 and IBD1 loci (encompassing the CARD15/NOD2 gene), the disease risk was enhanced in the presence of both CD-associated SLC22A4-A5 and CARD15/NOD2 mutations.17 However, replicating studies have been inconsistent between populations and it is not yet clear whether or not the IBD5 locus is explained only by the SLC22A4-A5 variants. By using a positional cloning approach, Stoll et al5 identified disease-associated variants responsible for the previously described linkage of CD with chromosome 10q23. The investigators described 3 IBD-associated genetic variations in the disk large homolog 5 (DLG5) gene, a gene encoding for a scaffolding protein involved in the maintenance of epithelial integrity by modulating cell death/proliferation. Gene– gene interactions also were suggested for DLG5 and CARD15/NOD2, but have been replicated only partially16,18 –20 and would benefit from a population-based analysis.

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Figure 2. Host-dependent and environmental-dependent steps from bacterial exposure toward clinical expression of IBD. Gene-gene and gene-environment interactions may affect multiple clinical (ie, disease onset, location of the initial lesions, and severity) and biological (ie, serology to microbial antigen) aspects of the disease.

Given the pivotal role of the host-bacterial interactions in health and disease, etiologic IBD variants are mined among other critical molecules for the innate immune response. Despite the critical protective role of certain Toll-like receptors (TLRs) at the intestinal mucosal interface,21 only minor (namely the Asp299Gly allele at the TLR4 gene) genetic variations affecting the TLR signaling pathway have been identified so far in IBD.22–26 Furthermore, a complex intronic polymorphism of the CARD4/NOD1 gene has been linked recently to CD development.27 CARD4/NOD1 confers cellular responsiveness to the unique PGN motif ␥-Dglutamyl-meso-diaminopimelic acid,28 suggesting a more general involvement of bacterial PGN sensing in this inflammatory disease. Similarly, predisposing and protective roles have been proposed, respectively, to a frameshift mutation of CARD8/TUCAN/CARDINAL, an anti-apoptotic and inhibiting molecule of nuclear factor ␬B (NF-␬B) signaling,29 and a nonsense mutation of TLR5, which recognizes bacterial flagellin (J. Cho, personal communication).30 Multiple other IBD loci and alleles have been proposed throughout the human and mice genome. Notably, IBD3, IBD4, and IBD6 –9 also have shown consistently replicated linkage. Many susceptibility variants can be expected to confer low relative risks, and independent replication of their association will require a population-based analysis. Notably, a CD but not UC locus has been located on the short arm of human chromosome 3,31–37 with a potential role of the gene encoding the peroxisome proliferator activated receptor-␥.37 However, it remains to be established whether a defect in peroxisome proliferator activated receptor-␥ function and/or expression is involved in CD development, as suggested by the increased susceptibility of peroxisome proliferator activated receptor-␥⫹/⫺ mice to experimental colitis.38 Finally, modifying loci on 4q23–25 and on 7q have been unraveled,34,35,39 – 41 with a suggested role of the NF-␬B1 gene expression39 and of the multidrug resistance 1 gene (MDR1), respectively. Consistently NF-

␬B– and MDR1-deficient mice develop bacterial-induced and/or spontaneous colitis.42– 44 Finally, the pericentromeric region of human chromosome 134,45 and the mice genome harbors an IBD-modifier locus at its human homologous region that remains to be identified.46 For more details on replicated and suggested IBD susceptibility loci, we direct the reader to recent excellent reviews on this subject.47,48

Genotype–Phenotype Correlation: Toward a Molecular Classification of Inflammatory Bowel Disease Complex diseases such as IBD are controlled by multiple risk factors that are evolving and interacting together. From environmental exposure to the clinical and biological expression of any IBD-related phenotypes, there are multiple processing steps controlled by the host and the environment (Figure 2). The currently proposed genetic model for IBD phenotypes emphasizes complex interactions between environmental factors, promoting and modifying genetic determinants, resulting in the clinical expression of the disease at the gastrointestinal tract of genetically predisposed individuals. Specific mutations that occur in the disease-promoting genes influence the development of distinct clinical phenotypes, whereas mutations in the modifying genes influence specific features of the disease phenotype such as disease penetrance, progression, complications, or response to treatment. CARD15/NOD2 mutations have been studied extensively in genotype–phenotype correlation studies (reviewed in47). A consistent pattern is that CARD15/ NOD2 variants are associated with younger age at onset, presence of ileal involvement, and a tendency to develop strictures and/or fistulas.49 –52 Furthermore, a significant gene dosage effect has been observed for CD site and complications. For instance, at least 95% of the patients homozygous for CARD15/NOD2 mutations present with ileal lesions. Existing data still are conflicting as to

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whether CARD15/NOD2 carriage is associated with a more severe disease course, suggesting that this feature depends on modifying genes and/or environmental risk factors. Interestingly, pediatric studies have shown that stricturing complications leading to early surgery were found more frequently in patients with the 1007fs mutation compared with those children without mutations.53,54 Children with this mutation had a 6.6-fold increased risk for developing a stricturing phenotype requiring surgery.54 Kugathasan et al54 suggest that genotyping at presentation might identify a subgroup of CD children who are at risk for more rapid development of complications and that these patients may benefit from the early use of more aggressive therapies. The results from genotype–phenotype analysis using other IBD susceptibility genes are still preliminary. In a Canadian study, the SLC22A4-TC haplotype did not influence the age of disease diagnosis. The effect of this haplotype on risk for CD was the same in familial and sporadic cases and stronger in the non-Jewish than in the Ashkenazi Jewish white populations.17 Given the epistasis effect (ie, the interaction of 2 or more genes controlling the expression of a phenotype) between CARD15/ NOD2 and SLC22A4-TC with ileal involvement in CD, genetic effects of this SLC22A4-A5 and CARD15/NOD2 mutation may result from a common physiopathologic mechanism in the ileum. An association between the IBD5 risk haplotype and ileal disease was shown initially in British patients to occur only in the context of coexisting perianal disease.55–57 In addition, a Canadian study showed an association between homozygosity for the SLC22A-TC haplotype and ileal disease without any perianal lesions. Finally, the presence of the SLC22A-TC haplotype was not associated with UC in the presence or absence of CARD15/NOD2 risk alleles.17 Newman et al17 indicate that, in their study population, the SLC22A-TC haplotype constitutes a CD-specific variant that acts together with CARD15/NOD2 risk alleles to predispose to CD and to ileal involvement in patients with CD. The studies conducted to date have indicated no correlation between DLG5 and any particular phenotypes. Finally, there is increasing evidence that the genes in the IBD3 locus located on chromosome 6p and that contain the human leukocyte antigen coding region may have a greater role in modifying the IBD phenotype rather than on initiating or triggering the disease.48 The haplotypic complexity of this locus has challenged many of the other reported genotype–phenotype associations in IBD. In conclusion, the confirmed association of the CARD15/NOD2 variants with ileal locations establishes

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that disease location defines different pathophysiological subsets of CD. Unlike CARD15/NOD2, SLC22A4-A5, and DLG5, the other association data have been inconsistent and/or their low sensitivity and specificity preclude driving in clinical practice. Large population-based studies integrating larger panels of genetic and serologic markers and using updated clinical classifications are needed. In parallel, experimental studies in mice and ex vivo studies in human beings will help to validate the reported genotype–phenotype associations and to identify environmental factors triggering and/or promoting the clinical expression of the disease.

Genetic Diagnostic Tests in Inflammatory Bowel Disease and Their Role in Clinical Management There is considerable optimism that clinical applications will follow the identification of IBD susceptibility genes. These include the use of genetic testing to strengthen the diagnosis in patients presenting with symptoms compatible with IBD, and to offer particular preventive therapy to genetically atrisk individuals. Currently, genetic testing can be considered only for CARD15/NOD2 mutations. PROGenologix (Prometheus Laboratories, San Diego, CA) is the first genetic test for CARD15/NOD2 mutations and is available commercially in the United States for physicians and patients. Interestingly, the CD-associated CARD15/NOD2 mutations predict the outcome of allogeneic bone-marrow transplantation and contribute to the development of graft-versus-host disease, suggesting a major CARD15/NOD2-dependent defect in peripheral blood myeloid cells and the importance of CARD15/NOD2 genotyping before allogeneic stem-cell transplantation.58,59 Surveys suggest that IBD patients and relatives are very interested in undergoing genetic testing, as reported by the Mount Sinai Hospital IBD Center in Toronto, Canada, where more than 85% of individuals affected by IBD were interested in pursuing genetic testing, and a similar proportion was interested in pursuing genetic testing for their young children while they were presymptomatic.60 For patients with established CD, genetic testing may impact therapeutic management. Based on available data, it has been argued that knowledge of CARD15/ NOD2 variant status already could be useful because an increased risk for fibrostenotic disease would require more aggressive care. However, the potential use of CARD15/NOD2 variants in prediction of time to surgery or postoperative recurrence has not yet been evaluated

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prospectively.61 CARD15/NOD2 status does not alter response to infliximab.62 Notably, further work is required to determine the influence of CARD15/NOD2 genotypes on the response to other CD medical therapies targeting host-bacterial interactions, such as antibiotics and probiotics. It has been suggested that before surgery, UC patients carrying one CARD15/NOD2 variant should have careful clinical evaluation of the small bowel to rule out surreptitious CD. Given the weak penetrance of such a mutation, we will need a preliminary prospective analysis of the phenotype of a large set of patients initially diagnosed with UC who are CARD15/NOD2 double mutant carriers.63 Therefore, the combination of several genetic, biological, and/or functional tests might help in predicting specific clinical entities, such as UC. Should CARD15/NOD2 screening be used in people with symptoms suggestive of CD? The effectiveness of any diagnostic test needs to be evaluated using sensitivity, specificity, and likelihood ratios. Data from Oxford showed that the 3 common CD-associated CARD15/ NOD2 variants have a sensitivity of 38.5% and a specificity of 88.6% for CD.50 These equate to positive and negative likelihood ratio of 3.38 and .69, respectively, although it has been suggested that positive likelihood ratios of more than 10 and negative likelihood ratios of less than .1 are necessary for diagnostic purposes. This shows the limitations of CARD15/NOD2 genotyping for the diagnosis of CD. As proposed for UC patients, a combination of serologic analysis and/or genetic and functional analysis of susceptibility alleles as a diagnostic panel might help to reach sufficient likelihood ratios to drive clinical management. Considering that IBD relatives are at a higher risk for developing the disease, family members frequently request a presymptomatic screening or diagnosis. However, the estimated penetrance of CARD15/NOD2 carriers developing CD is approximately 3% for the highest-risk genotype. Even if the sensitivity of genetic testing was sufficiently high, we do not have preventive strategies yet and we do not know which potential environmental covariates trigger the onset of CD in genetically predisposed individuals. With the current limited positive benefits achievable by performing genetic testing in the IBD population, there also may be substantial negative individual outcomes, such as on psychological or economic aspects. Taken together, a better understanding of normal and defective CARD15/ NOD2 function is a prerequisite for the clinical use of diagnostic panel and the development of rational therapeutic molecules.

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From Genes to Function: A Proposed Unifying Pathophysiologic Mechanism for Crohn’s Disease Functions of Normal Nucleotide-Binding Oligomerization Domain 2 The innate immune system is the most ancestral and ubiquitous system of defense against pathogenic infection. Specific host pathogen-recognition molecules (PRMs) sense invading microbes through the detection of pathogen-associated molecular patterns. On the side of the microbe, pathogen-associated molecular patterns are highly conserved structural components of the microbe, such as lipopolysaccharides or other components of the bacterial cell wall. In mammals, TLRs and leucine-rich repeat (LRR)-containing nucleotide-binding oligomerization domain (NOD-LRR) proteins function as host PRMs by regulating antimicrobial processes, proinflammatory responses, and/or cell death.64,65 Notably, activation of membrane-bound TLR molecules and cytosolic NODLRR has been shown to act synergistically for cytokine production by myeloid cells.9,28 NOD2/CARD15, CRYOPYRIN, NOD1/CARD4, and other members of the NOD-LRR family recognize PGN-derived products through their carboxyl-terminal LRRs. NOD-LRR proteins possess a tripartite structure including a C-terminal LRR domain, a central NOD, and at the N-terminus, a protein–protein interaction domain, CARD, pyrin domain, or baculovirus inhibitor of apoptosis protein repeat. PGN is composed of glycan chains containing alternating Nacetylglucosamine and N-acetylmuramic acid sugars cross-linked to each other by short peptides. PGN signaling functions as an antibacterial and leukocytic function because CARD4/NOD1- and CARD15/ NOD2-deficiency in human beings and mice conferred defective antimicrobial peptides and/or chemokine production (Figure 3).9,66 CARD15/NOD2 is expressed constitutively in antigen-presenting cells and in Paneth cells, which are specialized intestinal cells located primarily at the base of the crypts of Liberkühn in the small intestine (Figure 3). They play an important role in the innate immunity of the ileal mucosa by releasing proteinaceous granules into the lumen. These secretory granules are rich in antibiotic peptides, such as ␣-defensins (called cryptdins in the mouse). CARD15/NOD2 expression also is inducible in colonic epithelial cells under inflammatory conditions.67

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Figure 3. Gain- or loss-of-CARD15/NOD2 function in CD. (A) Muramyl dipeptide–induced NF-␬B activation in myeloid and Paneth cells is dependent on CARD15/NOD2. (B) The loss-of-function hypothesis is suggested by human and mice data using peripheral blood monocytes from mutated patients and knock-out mice. Such scenarios might be complemented by activating downstream molecules of CARD15/NOD2 (ie, specific RIP-like interacting CLARP kinase agonist) and/or by stimulating impaired gene expression by the CARD15/NOD2-independent signaling pathway (stimulated by probiotics and/or vaccine adjuvants). (C) CARD15/NOD22939iC/2939iC bone-marrow– derived macrophages showed a hypersensitivity to the CARD15/NOD2 agonist, suggesting the gain-of-function hypothesis. If this situation is confirmed, anti-inflammatory molecules could be proposed.

How Nucleotide-Binding Oligomerization Protein 2 Variants Might Predispose to Crohn’s Disease Human genetic data on CARD15/NOD2 have shown a gene-dosage effect consistent with a recessive model and a predisposition to the development of lesions in the terminal ileum where Paneth cells are prominent. Similarly to what has been observed with CARD15/ NOD2⫺/⫺ myeloid cells, the 3 main CD-associated CARD15/NOD2 variants and 13 additional variants carried by CD patients shown in vitro and ex vivo impaired MDP or PGN-induced NF-␬B activation and cytokine production (Figure 3).6 – 8,68 –70 Mice lacking CARD15/ NOD2 showed a defect in defense against per os infection by Listeria monocytogenes, which was paralleled by a decreased expression of particular Paneth cell– derived cryptdins.9 Consistent with those data, CD patients homozygous for CARD15/NOD2 mutations showed decreased expression of Paneth cell ␣-defensins HD-5 and HD-6.71 Further work should clarify to what extent such immunodeficiencies might affect the microbial flora and promote intestinal infection and inflammation. Ongoing studies using clinically relevant enteropathogens and humanized flora might help to identify the essential

priming and/or triggering environmental risk factor(s) in the development of CD-related phenotype in CARD15/ NOD2 knock-out animals. Recent data from Maeda et al72 have contradicted this loss-of-function hypothesis when introducing the 3020insC variant into a mouse model (namely CARD15/ NOD22939iC/2939iC knock-in mice). Unlike observation of peripheral blood monocytic cells from CD patients homozygous for this mutation, bone-marrow– derived macrophages from CARD15/NOD22939iC/2939iC mice showed increased MDP-induced NF-␬B activation and interleukin 1-␤ signaling compared with macrophages isolated from their wild-type littermates (Figure 3). In addition, unlike CARD4/NOD1-, CARD15/NOD2-, and RIP2/ RIPK2/RICK/CARDIAK-deficient mice9,73 (and our own personal observations), preliminary experiments with these transgenic mice showed more pronounced dextran sodium sulfate–induced mucosal inflammation, ulceration, and intestinal macrophage apoptosis. Therefore, it appears that mice mutated for both alleles show a gain-of-CARD15/NOD2 function. Further experiments using this model of transgenic mice should clarify why this frame-shift mutation shows distinct phenotypes in mice and human beings. It also remains to be deter-

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Figure 4. Implications of decreased TLR and NOD-LRR signaling for future therapies. Specific molecules acting on redundant signaling pathways (probiotics and PRM agonists are shown as (1) and (2), respectively) or downstream of the functional defect (PRM kinase agonist, IkappaB kinase agonist, and nuclear receptor agonists are shown (3), (4), and (5), respectively) could complement TLR and/or NOD-LRR deficiencies (noted in gray), as shown by decreased gene expression. Several therapeutic molecules and their proposed targets of action are indicated. (A) Normal innate immune signaling; (B) decreased innate immune signaling.

mined if the CARD15/NOD22939iC allele is dominant or dominant-negative in vivo and if this hyperresponsiveness might result somehow by activation of inflammatory caspases and processing of interleukin-1␤.73,74 Taken together, the innate immune system has a pivotal role in preventing bacterial-induced intestinal inflammation and repelling pathogenic infection. Given the nature of the CD-susceptibility gene identified to date, it seems reasonable to postulate that impaired bacterial PGN sensing is a major primary event leading to local inflammation, hyporesponsiveness, and/or hyperresponsiveness of the innate immune system and/or abnormal activation of T and B cells. The physiological function of the other susceptibility genes (SLC22A4, SLC22A5, and DLG5) in Paneth cells function, intestinal immunity, and/or cell death remain speculative.

Lessons for Therapy and Future Strategies On recognition of bacterial cell-wall components, subsets of genes are induced in a TLR- and/or NODLRR– dependent manner through the activation of several protein kinases and transcriptional factors, including NF-␬B, activator protein 1, Elk-1, nuclear factor of activated T cells, and interferon regulatory factors. Notably, TLR9 and CARD15/NOD2 activation play a key role in Paneth cell function.9,75 However, further work should clarify the nature and the trafficking of the molecules involved in the TLR- and/or NOD-LRR–signaling pathways. Given the hyporesponsiveness to MDP of peripheral blood monocytes from CD patients homozygous for CARD15/NOD2 mutations, it seems of interest to propose a combination of functional testing (cellular response to bacterial agonists such as MDP) together with genetic testing (ie, screening for CARD15/NOD2

mutations) in the diagnosis and management of CD. Indeed, hyporesponsiveness to MDP could result from rare CARD15/NOD2 mutations not taken into account by the usual tests but also from mutations occurring in other innate immune genes. Furthermore, it is worthwhile to note that CARD4/ NOD1 and CARD15/NOD2 signaling are both dependent on the adaptor protein kinase RIP2/RIPK2/RICK/ CARDIAK, suggesting functional complementation of CARD15/NOD2 deficiency by stimulation with CARD4/NOD1 agonists.28,76 Therefore, in the case of the loss-of-function hypothesis, a preventive complementation of CARD15/NOD2 function could be proposed at different steps from the bacterial exposure to the host response (Figure 4). We could propose stimulation (either by probiotics) and/or priming by other natural and/or synthetic PRM agonists (vaccine adjuvants such as TLR9 agonists); alternatively, we could target specific regulatory and/or effecting molecules such as kinases or nuclear receptors/NF-␬B/MAPK-dependent genes (Figure 4). Taken together, work is needed to clarify the nature of the multiple immune defects before a preventive approach could be proposed in the treatment of IBD.

References 1. Hampe J, Cuthbert A, Croucher PJ, et al. Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 2001;357:1925–1928. 2. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599 – 603. 3. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001;411:603– 606. 4. Peltekova VD, Wintle RF, Rubin LA, et al. Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet 2004;36:471– 475. 5. Stoll M, Corneliussen B, Costello CM, et al. Genetic variation in

150

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

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DLG5 is associated with inflammatory bowel disease. Nat Genet 2004;36:476 – 480. Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 2003;278:8869 – 8872. Chamaillard M, Philpott D, Girardin SE, et al. Gene-environment interaction modulated by allelic heterogeneity in inflammatory diseases. Proc Natl Acad Sci U S A 2003;100:3455–3460. Inohara N, Ogura Y, Fontalba A, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem 2003;278:5509 –5512. Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 2005;307:731–734. Yamazaki K, Takazoe M, Tanaka T, et al. Absence of mutation in the NOD2/CARD15 gene among 483 Japanese patients with Crohn’s disease. J Hum Genet 2002;47:469 – 472. Sugimura M, Kinouchi Y, Takahashi S, et al. CARD15/NOD2 mutational analysis in Japanese patients with Crohn’s disease. Clin Genet 2003;63:160 –162. Croucher PJ, Mascheretti S, Hampe J, et al. Haplotype structure and association to Crohn’s disease of CARD15 mutations in two ethnically divergent populations. Eur J Hum Genet 2003;11:6 – 16. Leong RW, Armuzzi A, Ahmad T, et al. NOD2/CARD15 gene polymorphisms and Crohn’s disease in the Chinese population. Aliment Pharmacol Ther 2003;17:1465–1470. Inoue N, Tamura K, Kinouchi Y, et al. Lack of common NOD2 variants in Japanese patients with Crohn’s disease. Gastroenterology 2002;123:86 –91. Kuster W, Pascoe L, Purrmann J, et al. The genetics of Crohn disease: complex segregation analysis of a family study with 265 patients with Crohn disease and 5,387 relatives. Am J Med Genet 1989;32:105–108. Torok HP, Glas J, Tonenchi L, et al. Polymorphisms in the DLG5 and OCTN cation transporter genes in Crohn’s disease. Gut 2005;129:1854 –1864. Newman B, Gu X, Wintle R, et al. A risk haplotype in the Solute Carrier Family 22A4/22A5 gene cluster influences phenotypic expression of Crohn’s disease. Gastroenterology 2005;128: 260 –269. Daly MJ, Pearce AV, Farwell L, et al. Association of DLG5 R30Q variant with inflammatory bowel disease. Eur J Hum Genet 2005; 13:835– 839. Noble CL, Nimmo ER, Drummond H, et al. DLG5 variants do not influence susceptibility to inflammatory bowel disease in the Scottish population. Gut 2005;54:1421–1427. Yamazaki K, Takazoe M, Tanaka T, et al. Association analysis of SLC22A4, SLC22A5 and DLG5 in Japanese patients with Crohn disease. J Hum Genet 2004;49:664 – 668. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, et al. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004;118:229 –241. Torok HP, Glas J, Tonenchi L, et al. Polymorphisms of the lipopolysaccharide-signaling complex in inflammatory bowel disease: association of a mutation in the Toll-like receptor 4 gene with ulcerative colitis. Clin Immunol 2004;112:85–91. Arnott ID, Nimmo ER, Drummond HE, et al. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn’s disease patients: evidence for genetic heterogeneity within Europe? Genes Immun 2004;5:417– 425. Pierik M, De Hertogh G, Vermeire S, et al. Epithelioid granulomas, pattern recognition receptors, and phenotypes of Crohn’s disease. Gut 2005;54:223–227. Franchimont D, Vermeire S, El Housni H, et al. Deficient hostbacteria interactions in inflammatory bowel disease? The toll-like

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26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42. 43.

receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis. Gut 2004;53:987–992. Ouburg S, Mallant-Hent R, Crusius JB, et al. The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with colonic localisation of Crohn’s disease without a major role for the Saccharomyces cerevisiae mannan-LBP-CD14-TLR4 pathway. Gut 2005;54:439 – 440. McGovern DP, Hysi P, Ahmad T, et al. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease. Hum Mol Genet 2005;14:1245–1250. Chamaillard M, Hashimoto M, Horie Y, et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 2003;4:702–707. Picornell Y, Abreu MT, Ippoliti A, et al. CARD8 variant and the expression of anti-OmpC are synergistically associated with internal penetrating Crohn’s disease (abstr). Gastroenterology 2005;128:A-113. Hawn TR, Verbon A, Lettinga KD, et al. A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to Legionnaires’ disease. J Exp Med 2003;198:1563–1572. Brant SR, Fu Y, Fields CT, et al. American families with Crohn’s disease have strong evidence for linkage to chromosome 16 but not chromosome 12. Gastroenterology 1998;115:1056 –1061. Dechairo B, Dimon C, van Heel D, et al. Replication and extension studies of inflammatory bowel disease susceptibility regions confirm linkage to chromosome 6p (IBD3). Eur J Hum Genet 2001; 9:627– 633. Hampe J, Lynch NJ, Daniels S, et al. Fine mapping of the chromosome 3p susceptibility locus in inflammatory bowel disease. Gut 2001;48:191–197. Vermeire S, Rutgeerts P, Van Steen K, et al. Genome wide scan in a Flemish inflammatory bowel disease population: support for the IBD4 locus, population heterogeneity, and epistasis. Gut 2004;53:980 –986. Rioux JD, Silverberg MS, Daly MJ, et al. Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci. Am J Hum Genet 2000;66:1863–1870. Satsangi J, Parkes M, Louis E, et al. Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12. Nat Genet 1996; 14:199 –202. Sugawara K, Olson TS, Moskaluk CA, et al. Linkage to peroxisome proliferator-activated receptor-gamma in SAMP1/YitFc mice and in human Crohn’s disease. Gastroenterology 2005; 128:351–360. Desreumaux P, Dubuquoy L, Nutten S, et al. Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/ peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies. J Exp Med 2001;193:827– 838. Karban AS, Okazaki T, Panhuysen CI, et al. Functional annotation of a novel NFKB1 promoter polymorphism that increases risk for ulcerative colitis. Hum Mol Genet 2004;13:35– 45. Cho JH, Nicolae DL, Gold LH, et al. Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: evidence for epistasis between 1p and IBD1. Proc Natl Acad Sci U S A 1998;95:7502–7507. Hampe J, Schreiber S, Shaw SH, et al. A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort. Am J Hum Genet 1999;64:808 – 816. Erdman S, Fox JG, Dangler CA, et al. Typhlocolitis in NF-kappa B-deficient mice. J Immunol 2001;166:1443–1447. Panwala CM, Jones JC, Viney JL, et al. A novel model of inflammatory bowel disease: mice deficient for the multiple drug resis-

February 2006

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

tance gene, mdr1a, spontaneously develop colitis. J Immunol 1998;161:5733–5744. Maggio-Price L, Bielefeldt-Ohmann H, Treuting P, et al. Dual infection with Helicobacter bilis and Helicobacter hepaticus in p-glycoprotein-deficient mdr1a⫺/⫺ mice results in colitis that progresses to dysplasia. Am J Pathol 2005;166:1793–1806. Hugot JP, Laurent-Puig P, Gower-Rousseau C, et al. Mapping of a susceptibility locus for Crohn’s disease on chromosome 16. Nature 1996;379:821– 823. Farmer MA, Sundberg JP, Bristol IJ, et al. A major quantitative trait locus on chromosome 3 controls colitis severity in IL-10deficient mice. Proc Natl Acad Sci U S A 2001;98:13820 – 13825. Schreiber S, Rosenstiel P, Albrecht M, et al. Genetics of Crohn disease, an archetypal inflammatory barrier disease. Nat Rev Genet 2005;6:376 –388. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 2003;3:521– 533. Cuthbert AP, Fisher SA, Mirza MM, et al. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. Gastroenterology 2002;122:867– 874. Ahmad T, Armuzzi A, Bunce M, et al. The molecular classification of the clinical manifestations of Crohn’s disease. Gastroenterology 2002;122:854 – 866. Lesage S, Zouali H, Cezard JP, et al. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002;70:845– 857. Hampe J, Grebe J, Nikolaus S, et al. Association of NOD2 (CARD 15) genotype with clinical course of Crohn’s disease: a cohort study. Lancet 2002;359:1661–1665. Sun L, Roesler J, Rosen-Wolff A, et al. CARD15 genotype and phenotype analysis in 55 pediatric patients with Crohn disease from Saxony, Germany. J Pediatr Gastroenterol Nutr 2003;37: 492– 497. Kugathasan S, Collins N, Maresso K, et al. CARD15 gene mutations and risk for early surgery in pediatric-onset Crohn’s disease. Clin Gastroenterol Hepatol 2004;2:1003–1009. Negoro K, McGovern DP, Kinouchi Y, et al. Analysis of the IBD5 locus and potential gene-gene interactions in Crohn’s disease. Gut 2003;52:541–546. Armuzzi A, Ahmad T, Ling KL, et al. Genotype-phenotype analysis of the Crohn’s disease susceptibility haplotype on chromosome 5q31. Gut 2003;52:1133–1139. McGovern DP, Van Heel DA, Negoro K, et al. Further evidence of IBD5/CARD15 (NOD2) epistasis in the susceptibility to ulcerative colitis. Am J Hum Genet 2003;73:1465–1466. Sonwalkar SA, James RM, Ahmad T, et al. Fulminant Crohn’s colitis after allogeneic stem cell transplantation. Gut 2003;52: 1518 –1521. Holler E, Rogler G, Herfarth H, et al. Both donor and recipient NOD2/CARD15 mutations associate with transplant-related mortality and GvHD following allogeneic stem cell transplantation. Blood 2004;104:889 – 894.

IBD GENES AND INTESTINAL IMMUNITY

151

60. Appelton JI, Mascarenhas J, Esplen MJ, et al. Demand for genetic testing for inflammatory bowel disease. Gastroenterology 2004; 128:A352–A353. 61. Rubin DT. To test or “NOD-2” test: what are the questions? The balanced viewpoint. Inflamm Bowel Dis 2005;11:510 –512. 62. Vermeire S, Louis E, Rutgeerts P, et al. NOD2/CARD15 does not influence response to infliximab in Crohn’s disease. Gastroenterology 2002;123:106 –111. 63. Brant SR, McGovern DP. NOD2, not yet: con. Inflamm Bowel Dis 2005;11:507–509. 64. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499 –511. 65. Inohara N, Chamaillard M, McDonald C, et al. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 2005;74:355–383. 66. Viala J, Chaput C, Boneca IG, et al. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 2004;5:1166 –1174. 67. Berrebi D, Maudinas R, Hugot JP, et al. Card15 gene overexpression in mononuclear and epithelial cells of the inflamed Crohn’s disease colon. Gut 2003;52:840 – 846. 68. van Heel DA, Ghosh S, Butler M, et al. Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn’s disease. Lancet 2005;365:1794 –1796. 69. Li J, Moran T, Swanson E, et al. Regulation of IL-8 and IL-1beta expression in Crohn’s disease associated NOD2/CARD15 mutations. Hum Mol Genet 2004;13:1715–1725. 70. Gutierrez O, Pipaon C, Inohara N, et al. Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factorkappa B activation. J Biol Chem 2002;277:41701– 41705. 71. Wehkamp J, Harder J, Weichenthal M, et al. NOD2 (CARD15) mutations in Crohn’s disease are associated with diminished mucosal alpha-defensin expression. Gut 2004;53:1658 –1664. 72. Maeda S, Hsu LC, Liu H, et al. Nod2 mutation in Crohn’s disease potentiates NF-kappaB activity and IL-1beta processing. Science 2005;307:734 –738. 73. Mariathasan S, Newton K, Monack DM, et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 2004;430:213–218. 74. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 2002;10:417– 426. 75. Rumio C, Besusso D, Palazzo M, et al. Degranulation of Paneth cells via toll-like receptor 9. Am J Pathol 2004;165:373–381. 76. Girardin SE, Boneca IG, Carneiro LA, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003;300:1584 –1587.

Address requests for reprints to: Mathias Chamaillard, PhD, INSERM E114, Pathogenesis of Inflammatory Digestive Diseases, Hôpital Swynghedauw, Rue A. Verhaeghe, F-59037, Lille, France. e-mail: [email protected]; fax: (33) 3-20-446-717.