NOD2-associated diseases: Bridging innate immunity and autoinflammation

Clinical Immunology (2010) 134, 251–261

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

Clinical Immunology w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

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FOCIS Centers of Excellence Review

NOD2-associated diseases: Bridging innate immunity and autoinflammation Arturo Borzutzky ⁎, Ari Fried, Janet Chou, Francisco A. Bonilla, Susan Kim, Fatma Dedeoglu Division of Immunology, Children's Hospital Boston, 300 Longwood Ave, Boston, MA 02115, USA Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA Received 5 May 2009; accepted with revision 6 May 2009 Available online 24 May 2009 KEYWORDS NOD2; Blau syndrome; Early onset sarcoidosis; Crohn's disease

Abstract NOD2 is an intracellular microbial sensor of the innate immune system that can act as a potent activator and regulator of inflammation. Mutations in the gene encoding NOD2 in humans have been associated with Crohn's disease (CD), Blau syndrome (BS), and early onset sarcoidosis (EOS). These diseases have in common features of dysregulated inflammation, but have very distinct phenotypes, which have been hypothesized to result from either loss-offunction (CD) or gain-of-function (BS/EOS) mutations. Here we describe an infant with early onset sarcoidosis who presented with systemic inflammation and disseminated granulomatous disease, including the triad of granulomatous arthritis, uveitis and dermatitis, as well as unusual gastrointestinal tract granulomas. The patient had a susceptibility polymorphism of NOD2 previously described in CD, but not in BS or EOS. We discuss the complex role of NOD2 in innate immunity to microbes and the clinical consequences of disturbances in this system. © 2009 Elsevier Inc. All rights reserved.

⁎ Corresponding author. Fax: +1 617 730 0249. E-mail address: [email protected] (A. Borzutzky). 1521-6616/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2009.05.005

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Case presentation A nine-month-old Caucasian boy was transferred to our institution for further evaluation of fever, irritability, and failure to thrive. He was healthy until about 2 weeks of age, when he developed intermittent fevers that became more constant by 3 months. By 4 months of age he developed feeding intolerance, failure to thrive, a migratory rash, and non-tender subcutaneous nodules. His illness progressed and one month prior to transfer to our institution, he was increasingly irritable, not wanting to be touched or held, with nonspecific swelling of his extremities, worsening rash, and daily fevers as high as 40 °C. He had an uneventful perinatal course: birth weight was 3.4 kg at 38 weeks' gestation, and he was discharged without complications. During the course of his many febrile episodes, he was diagnosed with various infections including streptococcal pharyngitis, otitis media on two occasions, recurrent nonspecific viral illnesses including rotavirus, and recurrent oral candidiasis. Due to his recurrent illnesses and fevers, he was only immunized for hepatitis B at 2 weeks of age.

A. Borzutzky et al. His family history revealed no consanguinity or miscarriages, no history of early death from infections or unknown causes, and no immunodeficiencies or autoimmune conditions. His mother, father and two older sisters were healthy. His examination on admission was notable for irritability, pallor, and toxic appearance with a weight of 7.8 kg (3rd percentile) and a length of 69.5 cm (14th percentile). He could not sit unassisted. He had scaly erythematous patches over his cheeks and diffuse red-brown, non-blanching papules on his chest, abdomen, and neck (Fig. 1A). There were numerous palpable subcutaneous nodules most prominent on his back, and diffuse non-pitting edema. He had a normal hair and nail pattern. He had clear conjunctiva with reactive pupils, no oral lesions, and no teeth. He had diffuse nontender shotty cervical and inguinal lymph nodes. He had arthritis with associated calor and effusions most pronounced in hands, feet, knees and ankles. He also had a 2/6 systolic heart murmur, and hepatomegaly, with his liver edge palpable 3 cm below the right costal margin, but no splenomegaly. Laboratory studies were notable for an elevated leukocyte count (24,300 cells/mm3) with neutrophil predominance

Figure 1 Photograph of the patient's skin showing characteristic rash of granulomatous dermatitis (A). Plain X-ray films of the patient's lower legs showing prominent periosteal reaction of bilateral tibial diaphyses (B). Light microscopy of hematoxylin and eosin stained tissue section of affected skin showing discrete non-necrotizing granulomas throughout the reticular dermis (C), and of esophageal wall biopsy with a non-necrotizing granuloma in the lamina propria (D). Arrows indicate granulomas. (Histopathology images are courtesy of William C. Klingensmith IV, M.D., Pathology Department, Children's Hospital Boston).

NOD2-associated diseases: Bridging innate immunity and autoinflammation (83%), normal lymphocyte count (3400 cells/mm3), and no eosinophilia. He had anemia (hematocrit 25%) with a normal platelet count. C-reactive protein was elevated (26.1 mg/dL). Serum calcium was elevated (13 mg/dL), with normal phosphorus, low albumin (2.6 mg/dL), and low 25-OH vitamin D (15 ng/mL). His skeletal survey showed bilateral symmetric diaphyseal changes with prominent periosteal reactions (Fig. 1B). Urinalysis demonstrated eosinophiluria, suggestive of interstitial nephritis. Renal and hepatic function studies, and cerebrospinal fluid analysis were normal. He was treated empirically with broad spectrum antibiotics, but his work-up was negative for infections, including routine bacterial and fungal cultures of stool, blood, urine, and cerebrospinal fluid, stains for acid fast bacilli, and specific testing for HIV, tuberculosis, syphilis, Lyme, Toxoplasma, CMV, and RSV. Autoantibodies, including ANA, double stranded DNA, and antineutrophil cytoplasmic antibodies, were negative. Angiotensin-converting enzyme level was elevated at 206 U/L (normal range 18–90 U/L). Immunological studies, including serum immunoglobulin levels, complement, lymphocyte subpopulations and dihydrorhodamine test, were normal. He had a normal karyotype. A skin biopsy showed discrete tuberculoid granulomas throughout the reticular dermis and hypodermis (Fig. 1C). An ophthalmologic evaluation revealed granulomatous panuveitis. A bone marrow biopsy demonstrated a normocellular marrow and maturing trilineage hematopoiesis without lymphohistiocytic aggregates or granulomata. An esophageal biopsy revealed squamous mucosa with regenerative epithelial changes and non-necrotizing granulomata in the lamina propria (Fig. 1D). The remainder of his endoscopic and colonoscopic exams were normal. The patient was diagnosed with early onset sarcoidosis, and treated initially with 2 mg/kg/day of systemic corticosteroids, weekly oral methotrexate at a dose of 1 mg/kg, and monthly immunomodulatory therapy with the TNF antagonist infliximab at a dose of 6 mg/kg every 4 weeks. Within one month, he improved considerably, with resolution of his arthritis and uveitis, as well as normalization of his laboratory parameters, including inflammatory markers, anemia, hypercalcemia, and ACE levels. Twelve months following the initiation of therapy he has remained generally healthy and has reached age-appropriate developmental milestones with improved growth parameters (55th percentile for weight and 35th percentile for height), but continues to have intermittent low-grade fevers and rashes. He has not had any Table 1

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flares of arthritis or uveitis and he is tolerating a tapering dose of corticosteroids. Nucleotide sequence analysis of the gene encoding the nucleotide-binding oligomerization domain containing 2 protein (NOD2; also known as CARD15) yielded a heterozygous Crohn's disease susceptibility mutation at IVS8+158CNT, not previously described in patients with Blau syndrome or early onset sarcoidosis.

Discussion The innate immune system functions as a first line of host defense against microbes, constantly surveying the environment for invading pathogens. It senses microbial elements through pattern-recognition receptors (PRRs), which are structurally related proteins that detect conserved microbial components, termed pathogen-associated molecular patterns (PAMPs). Our knowledge about the different families of PRRs has grown rapidly over the past decade, led by early studies focused on the Toll-like receptors (TLRs) [1]. TLRs are membrane-associated; therefore, they recognize pathogens at the cell surface and within endosomes. In contrast, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are localized in the cytosol, and mediate recognition of intracellular pathogen structures [2]. NOD2 is a member of the NLR family that acts as an intracellular sensor of bacterial cell wall components and, thus, is involved in the innate immune response to infection. Over the last decade, mutations or polymorphisms of this gene have been identified in human diseases, most notably Crohn's disease, Blau syndrome and early onset sarcoidosis. In the following sections of this review, we will discuss the current understanding about the structure, function and signaling pathways of NOD2. Subsequently, we will focus on the human diseases associated with NOD2 mutations and polymorphisms, with the aim of providing a global view of this protein from the bench to the bedside.

Structure and mutations of NOD2 The NLR family includes key regulators of apoptosis and several proteins involved in immunity against pathogens. In humans, this protein family contains a total of 22 members in 6 subfamilies (Table 1), which are classified based on structural characteristics (Fig. 2A). Each member of this group of proteins contains functional domains or subunits

Members and subfamilies of the NOD-like receptor (NLR) family of proteins.

Name

Family

Gene

Chromosome

MHC class II (C-II) transactivator Neuronal apoptosis inhibitory protein Nucleotide-binding oligomerization domain (NOD) 1 and 2 NOD-like receptor caspase recruitment domain containing proteins 3–5 NACHT, LRR, and PRD containing proteins 1–14

NLR NLRB NLRC NLRC

CIITA NAIP NOD1,2 NLRC3–5

16p13 5q13 7p15, 16q12 16p13, 2p22, 16q13

NLRP

NOD-like receptor with “unknown” domain

NLRX

NLRP1 NLRP2, 4, 5, 7–9, 11–13 NLRP3 NLRP6, 10, 14 NLRX1

17p13 19q13 1q44 11p15 11q23

Data from GeneCards®, http://www.genecards.org/index.shtml.

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Figure 2 (A) Schematic representations of the structures of some NLRs. See text for abbreviations. Note that LRRs may sometimes overlap with or be contained in other domains such as a CARD or NACHT. NOD1 is similar to NOD2 except that it contains only 1 CARD and 9 LRRs. NLRC4 contains a total of 14 LRRs. In spite of their names, neither NLRC3 (17 LRRs) nor NLRC5 (27 LRRs) contains a CARD. NLRP1 is the only member of the NLRP family shown; it is the only member of this family that contains a CARD. All other NLRPs each have 1 PYRIN, 1 NACHT, and 0–13 LRRs. NLRP10 is the only member of this family that does not contain LRRs. (B) The structure of NOD2 is shown to scale in more detail. The numbers over the diagram are the first and last amino acids of each structural domain or motif. The diamonds represent the positions of mutations in the sequence that have been associated with various diseases. Only the most clearly disease-related mutations are labeled (black diamonds); the white diamonds represent mutations associated with Crohn's disease that have been reported, but are less clearly disease-causing. There are 42 missense, 1 nonsense, and 1 insertion/duplication related to Crohn's disease shown. One splice mutation (intron 1) and one small deletion (6 bp interrupting codon 557) are not shown. Also shown are mutations associated with Blau syndrome/early onset sarcoidosis. Data from GeneCards®, http://www.genecards.org/ index.shtml.

that are shared among many families of proteins engaging in an wide variety of signaling pathways involving one or more of the following: the binding/recognition of PAMPs or dangerassociated molecular patterns (DAMPs), recruitment and activation or inhibition of caspases, and homo- or heteromultimerization. Distinct structural domains within these proteins mediate each of these functions. Most NLRs have a tripartite configuration with a C-terminal region constituted by a series of leucine-rich repeat (LRR) sequences, a central NACHT domain also known as nucleotide-binding domain (NBD), and an N-terminal effector domain. LRRs are found in a wide variety of protein families (including TLRs) and mediate protein–protein interactions and pattern-recognition of various conserved or invariant pathogen or host

molecules that recruit, activate, and/or otherwise initiate immune or inflammatory signaling cascades [3]. However, the exact mechanism of how LRRs sense PAMPs or DAMPs is still unclear. The NACHT domain is a nucleotide triphosphatase (NTPase) domain whose name is a meta-acronym standing for the acronyms designating other proteins or protein families in which it is found: NAIP (the NLR neuronal apoptosis inhibitory protein), CIITA (the NLR class II transactivator), HET-E (vegetative incompatibility protein from the fungus Podospora anserina), and TP1 (telomerase protein 1) [4]. The NACHT domain is believed to be responsible for the oligomerization of NLRs, forming active high molecular weight complexes, commonly referred to as inflammasomes or NOD signalosomes depending on the

NOD2-associated diseases: Bridging innate immunity and autoinflammation structural protein that oligomerizes. The N-terminal region of NLRs varies among different family members, with caspase recruitment domains (CARDs) and pyrin being the most common domains. This region is responsible for mediating signal transduction to downstream targets, triggering activation of inflammasomes and/or NFκB. CARDs mediate associations with other CARD-containing proteins, including caspases 1, 2, 9, 11, and 12 [5]. NOD2 is a member of the NLRC subgroup of the NLRs; it contains 6 LRRs, 1 NACHT and 2 CARD domains (Fig. 2A). The related protein NOD1 is similar to NOD2 but has one CARD domain and 9 LRRs. The genes encoding these variably homologous NLR molecules are rather widely scattered in the genome, although some clustering occurs (Table 1). The NOD2 gene is on chromosome 16q12.1 [6]. It has 12 exons and spans 36 kilobases. As is the case with all of the NLRs, several alternative mRNAs may be formed by alternative initiation of transcription, or splicing. Ten different mRNAs for NOD2 have been identified. They all appear to be capable of coding for protein and use anywhere from 2 to all 12 exons. The coding portions range in size from 270 to 3120 nucleotides (90–1040 amino acids). Little is yet known regarding the relative expression and function of all of the isoforms. Two hundred twelve single nucleotide polymorphisms have been identified in the NOD2 gene. At least 58 diseaseassociated or disease-causing mutations in NOD2 have been reported (Fig. 2B). The majority of these (46, or 79%) are associated with Crohn's disease [7–10], while other NOD2 mutations have been associated with Blau syndrome and early onset sarcoidosis [11–14], as we will discuss in later sections of this review. Allele frequencies (where known) range from b 0.01 to 0.49.

Role of NOD1 and NOD2 as intracellular pathogen sensors In the cytoplasm, NOD1 and NOD2 detect distinct muropeptide structures derived from peptidoglycan (PGN), a major component of bacterial cell walls. The recognized fragments are PGN degradation products released by growing bacteria. NOD1 detects meso-diaminopimelic acid (meso-DAP), which is predominantly found in Gram-negative bacteria. The minimal epitope required for NOD1 activation is γ-D-glutamyl-meso-diaminopimelic (iE-DAP). NOD2 senses muramyl dipeptide (MDP), which is contained in the PGN of nearly all Gram-positive and Gram-negative bacteria [15,16]. NOD1 and NOD2 are mainly expressed by two cell types that are in direct contact with microbial organisms: antigen-presenting cells (APCs) and epithelial cells. APCs, including dendritic cells (DCs) and macrophages express both NOD1 and NOD2 [17]. Most epithelial cells constitutively express NOD1, whereas NOD2 is restricted to Paneth cells — secretory epithelial cells located at the base of intestinal crypts [18]. Specific inflammatory stimuli upregulate NOD expression. NOD1 can be upregulated by IFN-γ, NOD2 by both IFN-γ and TNF-α, suggesting augmentation of host defense in inflammatory conditions [19,20]. The mechanisms by which the PGN fragments reach the cytosol to interact with NOD proteins are not completely understood, but have been found to vary in different cell

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types and for different pathogens. In the case of APCs the peptide ligands are thought to be released into the cytoplasm after phagocytosis and digestion of bacteria [21]. In epithelial cells, invasive bacterial pathogens such as Shigella flexneri and Listeria monocytogenes enter the cell directly and deliver cell wall fragments to the cytosol [22]. Further, NOD1 agonists can enter epithelial cells through pore-forming toxins produced by bacteria [23]. As an example of a noninvasive pathogen, Helicobacter pylori was found to ‘inject’ PGN into the cytoplasm of gastric epithelial cells through a Type IV secretion apparatus, resulting in NOD1-dependent induction of pro-inflammatory responses [24]. The human peptide transporter 1 (PepT1), whose main function is absorption of dietary di- and tripeptides in the intestinal mucosa, has been shown to selectively transport the NOD2 agonist MDP into the cytosol. The opposite is true for the human peptide transporter PepT2, which is abundantly expressed in respiratory epithelium and selectively internalizes iE-DAP, suggesting that transport mechanisms play a role in tissue-specific responses to NOD1 vs. NOD2 ligands [25].

NOD2 signaling pathways The signaling pathways activated by the NLRs are diverse and can be differentiated by the receptor's effector domain [26]. NOD1 and NOD2 have a CARD effector domain and cause inflammation via activation of the NF-κB and MAP kinase pathways (Fig. 3). Other members of the NLR family, such as NLRP1, NLRP3, and NLRC4, activate the caspase-1 pathway via an adaptor protein called PYCARD (also known as ASC or CARD5) [27]. The NLRs remain inactive through autoinhibition of the NACHT region by the LRRs until ligand binding results in conformational changes [6,28]. Ligand recognition results in oligomerization via the NACHT domain, exposure of the CARD-containing effector domain, and recruitment of the serine-threonine kinase RICK (also known as RIP2) through a CARD–CARD interaction [18,29]. RICK facilitates ubiquitination of the IκB kinase (IKK) regulatory subunit NEMO, thereby activating the IKK complex [30]. The IKK complex is also activated by TGF-β activated kinase 1 (TAK1), which is downstream of TLR activation. Activated IKK subsequently relieves inhibition of NF-κB by phosphorylating IκB, thus releasing NF-κB for translocation to the nucleus and gene transcription. Alternatively, RICK and TAK1 can activate the MAP kinases p38, ERK, and JNK [30–32]. The activation of downstream kinases depends in part on the pathogen presented: the gram-negative bacteria S. flexneri activates JNK through NOD1 [33], while gram-positive L. monocytogenes causes p38 and ERK activation through NOD2 [34]. Activation of NOD proteins also results in production of inhibitory proteins such as the LRR-containing protein, Erbin, and the GTPase activating protein Centaurin-β1. While Erbin inhibits NOD2-mediated MAP kinase signaling, Centaurin-β1 suppresses NF-κB activation stimulated by NOD1 and NOD2 [35–37]. The NF-κB and MAP kinase pathways culminate in an inflammatory response mediated by IL-6, TNF-α, IL-12, IL8, CXCL1, CXCL2, and CCL5 [34,38,39]. The release of chemokines such as CCL5 also augments the innate immune response by promoting neutrophil influx [40]. Furthermore,

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Figure 3 NOD1 recognizes diaminopimelic acid-containing peptides (DAP) found in gram-negative bacteria and NOD2 recognizes muramydipeptide (MDP) found in both gram-negative and gram-positive bacteria. Ligand recognition results in NOD receptor oligomerization, exposure of the effector domain, and RICK recruitment. RICK and TAK1 can lead to activation of either (a) the NF-kB pathway or (b) the MAP kinase pathway, culminating in an inflammatory response.

NOD1 and NOD2 activation stimulates production of antibacterial peptides such as α- and β-defensins [18,41,42].

NOD1 and NOD2 in host defense While a sensing mechanism involving NOD1 and NOD2 has been well established for multiple bacteria, an essential role of NOD-mediated responses in host defense against these pathogens has been confirmed only in a few in vivo models. As an example for dependence on an intact NOD pathway, NOD1-deficient mice were found to be more susceptible to oral infection with a subtype of H. pylori [24]. Similarly, it was demonstrated that NOD2-deficient mice infected orally with L. monocytogenes had a significantly greater bacterial burden in liver and spleen than wild-type mice. However, this was not observed when they were infected through intravenous or intraperitoneal routes [34]. In the same model, it was found that the expression of the antimicrobial peptide cryptdin (an αdefensin protein), which is produced by intestinal Paneth cells, was significantly decreased in NOD2-deficient mice after infection with L. monocytogenes compared to wildtype mice [34]. This is consistent with other studies that have demonstrated that NOD stimulation can induce the production of antimicrobial peptides, including α- and βdefensins in epithelial cells [41,43]. Another NOD-responsive mediator of innate host defense is the microbicidal compound nitric oxide (NO), which can be induced in

murine mesothelial cells in response to NOD1 agonists coupled with IFN-γ [44]. The interaction between the NOD proteins and another key aspect of innate immunity, the TLRs, is nuanced and remains somewhat controversial. Considering that NOD proteins and TLRs can be activated by the same microbial organisms and converge in the same signaling pathways that lead to production of pro-inflammatory cytokines and antimicrobial peptides, a redundant role of these systems has been suggested [45]. Several studies have proposed that the NOD receptors cooperate with TLRs since the addition of NOD agonists augments the inflammatory response (specifically, IL-6, IL-8, IL-10, and TNF-α) produced by human and murine macrophages or monocytes exposed to TLR ligands [32,46,47]. In contrast, a role of NOD as a negative regulator of TLR responses has also been demonstrated, as NOD2 stimulation by MDP leads to downregulation of TLR2-mediated secretion of the Th1promoting cytokine IL-12 [48]. As this finding is specific to PGN stimulation of TLR2 and IL-12 production, these differences in the cellular response to TLR and NOD receptor costimulation suggest that activation of NOD receptors can have either a positive or negative regulatory role on TLR responses, depending on the ligand and inflammatory response studied. Recently, NOD1 and NOD2 have been postulated to play a critical role in host defense by priming of immune responses in cells that have acquired insensitivity to TLR ligands [45]. This hypothesis is based on the observation that activation of TLR pathways in mice can result in a state of tolerance to subsequent TLR stimulation using the same intracellular

NOD2-associated diseases: Bridging innate immunity and autoinflammation pathway [49]. Murine monocytes made insensitive to TLRs by previous TLR activation remained responsive to NOD1 and NOD2 stimulation. This provides the host with a potential mechanism for detection of invasive pathogens by cells that do not express TLRs or are rendered TLR tolerant through continuous exposure to commensal bacteria [45,50]. NOD1 and NOD2 contribute not only to the innate immune response, but to adaptive immunity as well. NOD1 agonists stimulate a Th2 response in antigen-specific T and B cells that is abrogated in NOD1-deficient mice [51]. NOD1 also cooperates with other aspects of the immune system to augment adaptive immunity. Th1, Th2, and Th17 responses to adjuvants such as complete Freud's adjuvant, are significantly decreased in NOD1-null mice [51]. In addition, PGN recognition through NOD1 significantly increases TLR-mediated IL-12, IL-6, and IL-23 production by DCs. Similarly, NOD2 deficient murine DCs have markedly decreased production of IL-6 and IL-12 in response to stimulation with MDP and TLR [34]. Therefore, the diverse roles of NOD1 and NOD2 in immune responses highlight the importance of these receptors in linking innate and adaptive immunity.

Clinical manifestations of NOD2 mutations NOD2 primarily targets the activation pathway of NF-κB, therefore, diseases associated with mutations and polymorphisms in this gene have been termed NF-κB activation disorders [52]. At least 58 disease-associated or disease-causing mutations in NOD2 have been reported (Fig. 2B). Different mutations of NOD2 can lead to decreased or increased NF-κB activity, causing two distinct phenotypes. While gain-of-function mutations have been described in Blau syndrome (BS) and early onset sarcoidosis (EOS), other variants considered to be loss-offunction mutations have been associated with Crohn's disease (CD) [8,11,53]. Interestingly, though these conditions are characterized as granulomatous disorders, their pathogenesis and clinical features appear to be quite different (Table 2). Table 2

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Blau syndrome and early onset sarcoidosis Blau syndrome was first described in 1985 in 11 members of a family that had the triad of granulomatous arthritis, uveitis, and dermatitis. Although the disease resembled some aspects of sarcoidosis, the authors considered it to be a new syndrome because of the autosomal dominant inheritance pattern and early age of onset [54]. Sporadic cases of children with EOS with granulomatous involvement of different organs, primarily affecting joints, eyes and skin, had been described earlier in the literature [55,56]. Although these cases were not familial and did not have dominant inheritance, it was suspected that BS and EOS were the same disease, since patients' characteristics were nearly identical [57]. In 2001, three missense spontaneous mutations in the nucleotide-binding domain of NOD2 were described in four different families with BS. Subsequently, NOD2 mutations were described by two independent groups in patients with EOS, confirming earlier suspicions that EOS and BS had a common genetic etiology and, therefore, were the same disease [58,59]. Due to the increased activity of NF-κB signaling, BS/EOS is generally considered an autoinflammatory disorder. Individuals with BS and EOS typically present during early childhood, but clinical manifestations at presentation may vary. Over 90% of affected patients in a recent Japanese study had arthritis, uveitis and skin rash [60], although other studies have shown a smaller incidence of ocular involvement [14]. In the Japanese study, the median age at onset of rash, arthritis, and uveitis was 24 months, 33 months and 4.5 years, respectively [60]. The rash is usually described as tan-colored or erythematous scaly plaques, sometimes with multiple lichenoid papules. Some patients also develop erythema nodosum. Joint disease is polyarticular in the majority of cases, but some patients have oligoarticular involvement. A report from the International Registry of Pediatric Granulomatous Arthritis revealed that 76% of affected patients that developed arthritis had boggy inflammatory synovitis, while 24% had a dry arthritis. Camptodactyly due to hypertrophic tenosynovitis and synovial cysts is frequently described as a late consequence in

Comparison of typical genetic and clinical features of BS/EOS and CD.

Genetic features Inheritance Location of NOD2 mutations Functional consequences Clinical features Age of onset Arthritis Skin manifestations Uveitis Gastrointestinal tract inflammation Location of granulomas Treatment

Blau syndrome/early onset sarcoidosis

Crohn's disease

Monogenic disorder (NOD2): dominant (BS) and recessive (EOS) NACHT domain Increased NF-k B activity (gain-of-function)

Complex polygenic disorder: NOD2 is a susceptibility gene LRR domain Decreased NF-k B activity (loss-of-function)

b 4 years N 90% N 90% N 90% Rare

Late childhood or young adulthood 10–35% 6–15% 4–6% 100%

Multisystemic Anti-inflammatory and immunosuppressive drugs

Gastrointestinal Anti-inflammatory and immunosuppressive drugs

258 these patients. Uveitis is generally the last of the triad's symptoms to develop, but nonetheless, can lead to serious sequelae. Ocular disease manifests as varying degrees of panuveitis, but about one third of the patients have isolated anterior uveitis. Over 50% of the patients develop chronic ocular damage such as cataracts, glaucoma or visual impairment [60,61]. Besides the usual triad of granulomatous arthritis, uveitis and dermatitis, patients can have pathologic changes in many different organs including liver, kidney, bone marrow, lymph nodes, parotid glands, brain and bone [55,61]. Persistent or intermittent fever is reported in over 50% of patients in one report, and appears simultaneously with other clinical manifestations at a young age [60]. Other less common manifestations described include panniculitis, cranial neuropathy, hypertension and large vessel vasculitis [14,54,61,62]. Patients with BS/EOS typically lack the pulmonary involvement seen in patients with adult onset sarcoidosis, although interstitial pneumonitis and bronchial granulomas have been described [56]. In the patient reported in the clinical vignette, we report esophageal involvement, which was confirmed by histopathology. To our knowledge, this is the first report of gastrointestinal tract granulomas in EOS, a manifestation that is more typical of CD. Another unusual feature not previously reported is the diffuse periosteal reaction that our patient developed, which may be due to granulomatous invasion of the periosteum or linked to the disturbance of bone metabolism that can lead to hypercalcemia in these patients. Most patients with EOS/BS have mutations affecting the NBD/NACHT domain of NOD2. These mutations have been associated with increased basal and MDP-induced NF-κB activity [63]. Some patients with BS/EOS, particularly those with atypical presentations, have not been found to have mutations in the NOD2 gene [61]. Thus, it is plausible to hypothesize that there may be other genes involved in the pathogenesis of this disease. Histopathological analyses in BS/EOS reveal non-necrotizing and noncaseating granulomas in the affected tissues. Laboratory markers of systemic inflammation can range between normal and severely increased inflammatory parameters, such as white blood cells, platelets, erythrocyte sedimentation rate and C-reactive protein. One study showed increased pro-inflammatory cytokines in plasma, including IL-6, TNF-α and to a lesser degree IL-1β [14]. However, a recent report showed that peripheral blood mononuclear cells from BS patients with NOD2 mutations did not produce IL-1β, TNF-α and IL-12/23 p40 in excess after stimulation with MDP and a TLR2 agonist [64]. Serum angiotensin-converting enzyme levels can be elevated and patients with a large number of granulomas can also have hypercalcemia, as evidenced in our patient. Treatment of this condition is usually directed at blocking inflammation by using potent anti-inflammatory medicines such as corticosteroids. Some cases, including the one presented here, have shown positive responses to biologics against TNF-α and IL-1β.

Crohn's disease Crohn's disease is a multifactorial inflammatory bowel disease (IBD) characterized by transmural granulomatous

A. Borzutzky et al. inflammation of the gastrointestinal tract. Most patients have involvement of the small intestine, but the large intestine, perianal area, stomach, esophagus and mouth may also be affected. Inflammation can lead to complications of varying severity including bleeding, obstruction, fibrosis, perforation, fistulization and malignancy. CD is generally limited to the gastrointestinal tract, but extraintestinal manifestations, including arthritis and uveitis may occur. This relative organ selectivity is in contrast with BS/EOS, which typically has diffuse, multisystemic granulomatous inflammation. The immunological derangements that lead to IBD affect innate and adaptive immunity, having characteristics of both autoinflammation and autoimmunity. In the current model of pathogenesis, CD is thought to be due to a disturbance in mucosal immunologic tolerance to components of the normal intestinal microflora [65]. In susceptible hosts, continuous enteric bacterial antigenic stimulation leads to activation of the adaptive immune system that responds with a Th1-cell mediated inflammation that damages the gastrointestinal tissues. Genetic susceptibility to IBD can be linked to mucosal barrier dysfunction, deficient innate bacterial killing or immune dysregulation [66]. In 2001, susceptibility polymorphisms in the NOD2 gene were identified in patients with CD using linkage analysis and linkage disequilibrium mapping [8,53]. Three major polymorphisms of NOD2 are most prevalent in patients with CD (Arg702Trp, Gly908Arg and Leu1007fsinsC) [10]. These are in or near the LRR region of NOD2, thus interfering with ligand binding. Therefore, cells with these NOD2 mutations have a decreased NFκB response to MDP [67]. Several other less common variants have been shown to be more frequent in CD patients compared to the normal population. Carrying one mutated NOD2 allele confers 2–4-fold higher odds of developing CD, whereas double carriers (homozygous or compound heterozygous), a 17.1-fold risk for CD [68]. Over 50% of CD patients have at least a heterozygous mutation of NOD2 and about 15% of CD patients bear homozygous or compound heterozygous mutations [8]. There have been great advances in understanding how NOD2 mutations confer susceptibility to IBD, although different hypotheses are still being considered. Normally, the intestinal mucosa is tolerant of the bacterial microflora that is present throughout an individual's life. Evidence exists that CD-associated NOD2 mutations render the protein hyporesponsive to its ligand, MDP, impairing the NF-κB response and production of α-defensins that normally keep the commensal bacterial population downregulated [69,70]. This hypothetically leads to overgrowth of the intestinal microflora and increased susceptibility to enteric pathogens, triggering an adaptive immunity hyper-responsiveness that causes and perpetuates inflammation. As discussed earlier, some studies suggest that NOD2 signaling can participate in the suppression of responses to TLR2 ligands, resulting in a homeostatic mechanism of inflammation control [48]. Although other studies have contradicted these findings [71,72], it has been hypothesized that NOD2 mutations cause disruption of this homeostasis with an exaggerated Th1-mediated pro-inflammatory response to the continuous stimuli intestinal microflora constitute for the intestinal immune system. A third theory for NOD2-associated susceptibility to CD has been generated

NOD2-associated diseases: Bridging innate immunity and autoinflammation through studies done in knock-in mice expressing NOD2 with a common CD-associated mutation (Leu1007fsinsC) [73]. In this model, macrophages had increased IL-1β production after stimulation with MDP, and the mice had increased susceptibility to experimental colitis. However, these results are contradictory to studies done in peripheral blood mononuclear cells of patients with this mutation, that have shown decreased IL-1β production after stimulation with MDP [74].

Other diseases Multiple studies have looked at possible associations between NOD2 polymorphisms and different diseases, including rheumatologic conditions, allergic diseases, infections, malignancies and complications of stem cell transplantation. A comprehensive review of these studies was published recently by Henckaerts and Vermeire [75]. Although promising findings have revealed a link between NOD2 and graft versus host disease and some malignancies, a clear association of mutations in NOD2 with most of these conditions remains to be proven. Studies in patients with ulcerative colitis, the other major form of inflammatory bowel disease, have not revealed a relevant role of NOD2 mutations in the pathogenesis of this disease [8]. However, it has been suggested that these variants may have some epistatic effect with the IBD5 risk haplotype that confers genetic susceptibility to IBD [76].

Conclusions NOD2 is an excellent example of the crosstalk between innate immunity and control of inflammation. Physiologically, NOD2 is involved in intracellular sensing of bacterial components, but when dysregulated, it leads to inflammatory diseases. In addition, there are distinct genotype–phenotype correlations, as in the cases of CD and BS/EOS. The patient with EOS presented in the clinical vignette raises interesting questions. Although he had classic features of EOS, some features were quite unique, including the granulomas of the gastrointestinal tract, more characteristic of CD than BS/EOS, and the prominent periosteal reactions of long bones. Sequencing of his NOD2 gene revealed a polymorphism previously found to predispose to CD, but not described in patients with BS/ EOS. It is possible that sections of the gene including promoter regions that were not sequenced carried additional mutations that led to his severe phenotype. However, epigenetic phenomena and possible variants in related genes in the NOD signaling pathway may also be considered. Many issues regarding NOD2 remain to be resolved by future research including the mechanisms of ligand detection, the interaction with TLR signaling and other proinflammatory pathways, as well as the pathogenic mechanisms by which NOD2 mutations and polymorphisms lead to human diseases.

References [1] R. Medzhitov, C.A. Janeway Jr., Decoding the patterns of self and nonself by the innate immune system, Science 296 (2002) 298–300.

259

[2] N. Inohara, G. Nunez, NODs: intracellular proteins involved in inflammation and apoptosis, Nat. Rev., Immunol. 3 (2003) 371–382. [3] B. Kobe, A.V. Kajava, The leucine-rich repeat as a protein recognition motif, Curr. Opin. Struct. Biol. 11 (2001) 725–732. [4] E.V. Koonin, L. Aravind, The NACHT family — a new group of predicted NTPases implicated in apoptosis and MHC transcription activation, Trends Biochem. Sci. 25 (2000) 223–224. [5] K. Hofmann, P. Bucher, J. Tschopp, The CARD domain: a new apoptotic signalling motif, Trends Biochem. Sci. 22 (1997) 155–156. [6] Y. Ogura, N. Inohara, A. Benito, F.F. Chen, S. Yamaoka, G. Nunez, Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB, J. Biol. Chem. 276 (2001) 4812–4818. [7] K. King, M.F. Sheikh, A.P. Cuthbert, S.A. Fisher, C.M. Onnie, M.M. Mirza, et al., Mutation, selection, and evolution of the Crohn disease susceptibility gene CARD15, Hum. Mutat. 27 (2006) 44–54. [8] J.P. Hugot, M. Chamaillard, H. Zouali, S. Lesage, J.P. Cezard, J. Belaiche, et al., Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease, Nature 411 (2001) 599–603. [9] M. Sugimura, Y. Kinouchi, S. Takahashi, H. Aihara, S. Takagi, K. Negoro, et al., CARD15/NOD2 mutational analysis in Japanese patients with Crohn's disease, Clin. Genet. 63 (2003) 160–162. [10] S. Lesage, H. Zouali, J.P. Cezard, J.F. Colombel, J. Belaiche, S. Almer, et al., CARD15/NOD2 mutational analysis and genotype– phenotype correlation in 612 patients with inflammatory bowel disease, Am. J. Hum. Genet. 70 (2002) 845–857. [11] C. Miceli-Richard, S. Lesage, M. Rybojad, A.M. Prieur, S. Manouvrier-Hanu, R. Hafner, et al., CARD15 mutations in Blau syndrome, Nat. Genet. 29 (2001) 19–20. [12] M.M. van Duist, M. Albrecht, M. Podswiadek, D. Giachino, T. Lengauer, L. Punzi, et al., A new CARD15 mutation in Blau syndrome, Eur. J. Hum. Genet. 13 (2005) 742–747. [13] N. Kanazawa, I. Okafuji, N. Kambe, R. Nishikomori, M. NakataHizume, S. Nagai, et al., Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome, Blood 105 (2005) 1195–1197. [14] J.I. Arostegui, C. Arnal, R. Merino, C. Modesto, M. Antonia Carballo, P. Moreno, et al., NOD2 gene-associated pediatric granulomatous arthritis: clinical diversity, novel and recurrent mutations, and evidence of clinical improvement with interleukin-1 blockade in a Spanish cohort, Arthritis Rheum. 56 (2007) 3805–3813. [15] C. McDonald, N. Inohara, G. Nunez, Peptidoglycan signaling in innate immunity and inflammatory disease, J. Biol. Chem. 280 (2005) 20177–20180. [16] S.E. Girardin, D.J. Philpott, Mini-review: the role of peptidoglycan recognition in innate immunity, Eur. J. Immunol. 34 (2004) 1777–1782. [17] O. Gutierrez, C. Pipaon, N. Inohara, A. Fontalba, Y. Ogura, F. Prosper, et al., Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B activation, J. Biol. Chem. 277 (2002) 41701–41705. [18] Y. Ogura, S. Lala, W. Xin, E. Smith, T.A. Dowds, F.F. Chen, et al., Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis, Gut 52 (2003) 1591–1597. [19] P. Rosenstiel, M. Fantini, K. Brautigam, T. Kuhbacher, G.H. Waetzig, D. Seegert, et al., TNF-alpha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells, Gastroenterology 124 (2003) 1001–1009. [20] T. Hisamatsu, M. Suzuki, D.K. Podolsky, Interferon-gamma augments CARD4/NOD1 gene and protein expression through interferon regulatory factor-1 in intestinal epithelial cells, J. Biol. Chem. 278 (2003) 32962–32968.

260 [21] W. Strober, P.J. Murray, A. Kitani, T. Watanabe, Signalling pathways and molecular interactions of NOD1 and NOD2, Nat. Rev., Immunol. 6 (2006) 9–20. [22] L. Le Bourhis, S. Benko, S.E. Girardin, Nod1 and Nod2 in innate immunity and human inflammatory disorders, Biochem. Soc. Trans. 35 (2007) 1479–1484. [23] A.J. Ratner, J.L. Aguilar, M. Shchepetov, E.S. Lysenko, J.N. Weiser, Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria, Cell. Microbiol. 9 (2007) 1343–1351. [24] J. Viala, C. Chaput, I.G. Boneca, A. Cardona, S.E. Girardin, A.P. Moran, et al., Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island, Nat. Immunol. 5 (2004) 1166–1174. [25] P.W. Swaan, T. Bensman, P.M. Bahadduri, M.W. Hall, A. Sarkar, S. Bao, et al., Bacterial peptide recognition and immune activation facilitated by human peptide transporter PEPT2, Am. J. Respir. Cell Mol. Biol. 39 (2008) 536–542. [26] T.A. Kufer, Signal transduction pathways used by NLR-type innate immune receptors, Mol. Biosyst. 4 (2008) 380–386. [27] J.M. Wilmanski, T. Petnicki-Ocwieja, K.S. Kobayashi, NLR proteins: integral members of innate immunity and mediators of inflammatory diseases, J. Leukoc. Biol. 83 (2008) 13–30. [28] M. Albrecht, F.S. Domingues, S. Schreiber, T. Lengauer, Identification of mammalian orthologs associates PYPAF5 with distinct functional roles, FEBS Lett. 538 (2003) 173–177. [29] N. Inohara, T. Koseki, L. del Peso, Y. Hu, C. Yee, S. Chen, et al., Nod1, an Apaf-1-like activator of caspase-9 and nuclear factorkappaB, J. Biol. Chem. 274 (1999) 14560–14567. [30] M. Windheim, C. Lang, M. Peggie, L.A. Plater, P. Cohen, Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide, Biochem. J. 404 (2007) 179–190. [31] J. da Silva Correia, Y. Miranda, N. Leonard, J. Hsu, R.J. Ulevitch, Regulation of Nod1-mediated signaling pathways, Cell Death Differ. 14 (2007) 830–839. [32] J.H. Park, Y.G. Kim, C. McDonald, T.D. Kanneganti, M. Hasegawa, M. Body-Malapel, et al., RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs, J. Immunol. 178 (2007) 2380–2386. [33] S.E. Girardin, R. Tournebize, M. Mavris, A.L. Page, X. Li, G.R. Stark, et al., CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri, EMBO Rep. 2 (2001) 736–742. [34] K.S. Kobayashi, M. Chamaillard, Y. Ogura, O. Henegariu, N. Inohara, G. Nunez, et al., Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract, Science 307 (2005) 731–734. [35] C. McDonald, F.F. Chen, V. Ollendorff, Y. Ogura, S. Marchetto, P. Lecine, et al., A role for Erbin in the regulation of Nod2dependent NF-kappaB signaling, J. Biol. Chem. 280 (2005) 40301–40309. [36] D. Weichart, J. Gobom, S. Klopfleisch, R. Hasler, N. Gustavsson, S. Billmann, et al., Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells, J. Biol. Chem. 281 (2006) 2380–2389. [37] J.K. Yamamoto-Furusho, N. Barnich, R. Xavier, T. Hisamatsu, D.K. Podolsky, Centaurin beta1 down-regulates nucleotide-binding oligomerization domains 1- and 2-dependent NF-kappaB activation, J. Biol. Chem. 281 (2006) 36060–36070. [38] K. Kobayashi, N. Inohara, L.D. Hernandez, J.E. Galan, G. Nunez, C.A. Janeway, et al., RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems, Nature 416 (2002) 194–199. [39] J. Li, T. Moran, E. Swanson, C. Julian, J. Harris, D.K. Bonen, et al., Regulation of IL-8 and IL-1beta expression in Crohn's disease associated NOD2/CARD15 mutations, Hum. Mol. Genet. 13 (2004) 1715–1725.

A. Borzutzky et al. [40] C. Werts, L. le Bourhis, J. Liu, J.G. Magalhaes, L.A. Carneiro, J.H. Fritz, et al., Nod1 and Nod2 induce CCL5/RANTES through the NFkappaB pathway, Eur. J. Immunol. 37 (2007) 2499–2508. [41] P.K. Boughan, R.H. Argent, M. Body-Malapel, J.H. Park, K.E. Ewings, A.G. Bowie, et al., Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor: critical regulators of beta-defensins during Helicobacter pylori infection, J. Biol. Chem. 281 (2006) 11637–11648. [42] S. Lala, Y. Ogura, C. Osborne, S.Y. Hor, A. Bromfield, S. Davies, et al., Crohn's disease and the NOD2 gene: a role for paneth cells, Gastroenterology 125 (2003) 47–57. [43] L. Eckmann, Defence molecules in intestinal innate immunity against bacterial infections, Curr. Opin. Gastroenterol. 21 (2005) 147–151. [44] J.H. Park, Y.G. Kim, M. Shaw, T.D. Kanneganti, Y. Fujimoto, K. Fukase, et al., Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells, J. Immunol. 179 (2007) 514–521. [45] L. Franchi, N. Warner, K. Viani, G. Nunez, Function of Nod-like receptors in microbial recognition and host defense, Immunol. Rev. 227 (2009) 106–128. [46] M. Chamaillard, M. Hashimoto, Y. Horie, J. Masumoto, S. Qiu, L. Saab, et al., An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid, Nat. Immunol. 4 (2003) 702–707. [47] J.H. Fritz, S.E. Girardin, C. Fitting, C. Werts, D. MenginLecreulx, M. Caroff, et al., Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1and NOD2-activating agonists, Eur. J. Immunol. 35 (2005) 2459–2470. [48] T. Watanabe, A. Kitani, P.J. Murray, W. Strober, NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses, Nat. Immunol. 5 (2004) 800–808. [49] A. Bagchi, E.A. Herrup, H.S. Warren, J. Trigilio, H.S. Shin, C. Valentine, et al., MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists, J. Immunol. 178 (2007) 1164–1171. [50] Y.G. Kim, J.H. Park, M.H. Shaw, L. Franchi, N. Inohara, G. Nunez, The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Tolllike receptor ligands, Immunity 28 (2008) 246–257. [51] J.H. Fritz, L. Le Bourhis, G. Sellge, J.G. Magalhaes, H. Fsihi, T.A. Kufer, et al., Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity, Immunity 26 (2007) 445–459. [52] S.L. Masters, A. Simon, I. Aksentijevich, D.L. Kastner, Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease (⁎), Annu. Rev. Immunol. 27 (2009) 621–668. [53] Y. Ogura, D.K. Bonen, N. Inohara, D.L. Nicolae, F.F. Chen, R. Ramos, et al., A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease, Nature 411 (2001) 603–606. [54] E.B. Blau, Familial granulomatous arthritis, iritis, and rash, J. Pediatr. 107 (1985) 689–693. [55] A.F. North Jr., C.W. Fink, W.M. Gibson, J.E. Levinson, S.L. Schuchter, W.K. Howard, et al., Sarcoid arthritis in children, Am. J. Med. 48 (1970) 449–455. [56] S. Hetherington, Sarcoidosis in young children, Am. J. Dis. Child 136 (1982) 13–15. [57] J.J. Miller 3rd, Early-onset “sarcoidosis” and “familial granulomatous arthritis (arteritis)”: the same disease, J. Pediatr. 109 (1986) 387–388. [58] N. Kanazawa, S. Matsushima, N. Kambe, T. Tachibana, S. Nagai, Y. Miyachi, Presence of a sporadic case of systemic granulomatosis syndrome with a CARD15 mutation, J. Invest. Dermatol. 122 (2004) 851–852. [59] C.D. Rose, T.M. Doyle, G. McIlvain-Simpson, J.E. Coffman, J.T. Rosenbaum, M.P. Davey, et al., Blau syndrome mutation of

NOD2-associated diseases: Bridging innate immunity and autoinflammation

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

CARD15/NOD2 in sporadic early onset granulomatous arthritis, J. Rheumatol. 32 (2005) 373–375. I. Okafuji, R. Nishikomori, N. Kanazawa, N. Kambe, A. Fujisawa, S. Yamazaki, et al., Role of the NOD2 genotype in the clinical phenotype of Blau syndrome and early-onset sarcoidosis, Arthritis Rheum. 60 (2009) 242–250. C.D. Rose, C.H. Wouters, S. Meiorin, T.M. Doyle, M.P. Davey, J.T. Rosenbaum, et al., Pediatric granulomatous arthritis: an international registry, Arthritis Rheum. 54 (2006) 3337–3344. D. Rotenstein, D.L. Gibbas, B. Majmudar, E.A. Chastain, Familial granulomatous arteritis with polyarthritis of juvenile onset, N. Engl. J. Med. 306 (1982) 86–90. T. Tanabe, M. Chamaillard, Y. Ogura, L. Zhu, S. Qiu, J. Masumoto, et al., Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition, EMBO J. 23 (2004) 1587–1597. T.M. Martin, Z. Zhang, P. Kurz, C.D. Rose, H. Chen, H. Lu, et al., The NOD2 defect in Blau syndrome does not result in excess interleukin-1 activity, Arthritis Rheum. 60 (2009) 611–618. L. Quaglietta, A. te Velde, A. Staiano, R. Troncone, D.W. Hommes, Functional consequences of NOD2/CARD15 mutations in Crohn disease, J. Pediatr. Gastroenterol. Nutr. 44 (2007) 529–539. C.D. Packey, R.B. Sartor, Commensal bacteria, traditional and opportunistic pathogens, dysbiosis and bacterial killing in inflammatory bowel diseases, Curr. Opin. Infect. Dis. (2009). N. Inohara, Y. Ogura, A. Fontalba, O. Gutierrez, F. Pons, J. Crespo, et al., Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease, J. Biol. Chem. 278 (2003) 5509–5512. M. Economou, T.A. Trikalinos, K.T. Loizou, E.V. Tsianos, J.P. Ioannidis, Differential effects of NOD2 variants on Crohn's disease risk and phenotype in diverse populations: a metaanalysis, Am. J. Gastroenterol. 99 (2004) 2393–2404.

261

[69] S.E. Girardin, I.G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas, et al., Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection, J. Biol. Chem. 278 (2003) 8869–8872. [70] J. Wehkamp, J. Harder, M. Weichenthal, M. Schwab, E. Schaffeler, M. Schlee, et al., NOD2 (CARD15) mutations in Crohn's disease are associated with diminished mucosal alphadefensin expression, Gut 53 (2004) 1658–1664. [71] M.A. Wolfert, T.F. Murray, G.J. Boons, J.N. Moore, The origin of the synergistic effect of muramyl dipeptide with endotoxin and peptidoglycan, J. Biol. Chem. 277 (2002) 39179–39186. [72] A. Uehara, S. Yang, Y. Fujimoto, K. Fukase, S. Kusumoto, K. Shibata, et al., Muramyldipeptide and diaminopimelic acidcontaining desmuramylpeptides in combination with chemically synthesized Toll-like receptor agonists synergistically induced production of interleukin-8 in a NOD2- and NOD1dependent manner, respectively, in human monocytic cells in culture, Cell. Microbiol. 7 (2005) 53–61. [73] S. Maeda, L.C. Hsu, H. Liu, L.A. Bankston, M. Iimura, M.F. Kagnoff, et al., Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing, Science 307 (2005) 734–738. [74] D.A. van Heel, S. Ghosh, M. Butler, K.A. Hunt, A.M. Lundberg, T. Ahmad, et al., Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn's disease, Lancet 365 (2005) 1794–1796. [75] L. Henckaerts, S. Vermeire, NOD2/CARD15 disease associations other than Crohn's disease, Inflamm. Bowel Dis. 13 (2007) 235–241. [76] D.P. McGovern, D.A. Van Heel, K. Negoro, T. Ahmad, D.P. Jewell, Further evidence of IBD5/CARD15 (NOD2) epistasis in the susceptibility to ulcerative colitis, Am. J. Hum. Genet. 73 (2003) 1465–1466.