Naohiro Inohara

The gene encoding the Nod2 protein is frequently mutated in Crohn's disease (CD) patients, although the physiological function of Nod2 in the intestine remains elusive. Here we show that protective immunity mediated by Nod2 recognition of bacterial muramyl dipeptide is abolished in Nod2-deficient mice. These animals are susceptible to bacterial infection via the oral route but not through intravenous or peritoneal delivery. Nod2 is required for the expression of a subgroup of intestinal anti-microbial peptides, known as cryptdins. The Nod2 protein is thus a critical regulator of bacterial immunity within the intestine, providing a possible mechanism for Nod2 mutations in CD.

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-l-Ala-d-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of l-Ala for d-Ala ord-isoGln for l-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development. NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-l-Ala-d-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of l-Ala for d-Ala ord-isoGln for l-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development. Innate immunity recognizes invading microbes and triggers a defense response in the host aimed at clearing the invading pathogen. Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; LPS, lipopolysaccharide; MDP, muramyl dipeptide MurNAc-l-Ala-d-isoGln; NF-κB, nuclear factor-κB, PBMNC, peripheral blood mononuclear cells; PGN, peptidoglycan; sBLP, synthetic bacterial lipoprotein; HEK, human embryonic kidney; LRR, leucine-rich repeat; HA, hemagglutinin; IL, interleukin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; Mur, muramic acid expressed on the surface of myelomonocytic cells play an important role in the recognition of microbial components and activation of innate immunity (1Takeda K. Akira S. Genes Cells. 2001; 6: 733-742Google Scholar). Each membrane-associated TLR recognizes pathogen-associated molecular patterns that are expressed on infectious agents (1Takeda K. Akira S. Genes Cells. 2001; 6: 733-742Google Scholar). Nods, including NOD1 and NOD2, are members of another family of proteins that have been recently implicated in intracellular recognition of bacterial components (2Inohara N. Ogura T. Nuñez G. Curr. Opin. Microbiol. 2002; 5: 76-80Google Scholar, 3Girardin S.E. Sansonetti P.J. Philpott D.J. Trends Microbiol. 2002; 10: 193-199Google Scholar). NOD2 is composed of two NH2-terminal caspase-recruitment domains, a centrally located nucleotide-binding domain and multiple COOH-terminal leucine-rich repeats (LRRs), and is expressed in myelomonocytic and dendritic cells (4Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nuñez G. J. Biol. Chem. 2001; 276: 4812-4818Google Scholar, 5Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nuñez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-47705Google Scholar). Three genetic variants within the coding region of NOD2, L1007fsinsC, G908R, and R702W, have been genetically associated with susceptibility to Crohn's disease in European and American populations (6Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O'Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Nature. 2001; 411: 599-603Google Scholar, 7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar, 8Hampe J. Cuthbert A. Croucher P.J. Mirza M.M. Mascheretti S. Fisher S. Frenzel H. King K. Hasselmeyer A. MacPherson A.J. Bridger S. van Deventer S. Forbes A. Nikolaus S. Lennard-Jones J.E. Foelsch U.R. Krawczak M. Lewis C. Schreiber S. Mathew C.G. Lancet. 2001; 357: 1925-1928Google Scholar, 9Ahmad T. Armuzzi A. Bunce M. Mulcahy-Hawes K. Marshall S.E. Orchard T.R. Crawshaw J. Large O. de Silva A. Cook J.T. Barnardo M. Cullen S. Welsh K.I. Jewell D.P. Gastroenterology. 2002; 122: 854-866Google Scholar). NOD2 has been shown to recognize preparations of lipopolysaccharides (LPS) and peptidoglycan (PGN) through its COOH-terminal LRRs (10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar), and this activity is deficient in the disease-associated variants (7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar). However, the precise bacterial structure recognized by NOD2 remains unknown. In this report we identified muramyl dipeptide (MDP) derived from peptidoglycan as the structure in bacteria recognized by NOD2. LPS from Escherichia coli O55:B5 prepared by the phenol extraction method, further purified by gel-filtration chromatography, detoxified LPS, and purified lipid A were obtained from Sigma. LPS from Salmonella typhimurium was obtained from Sigma. PGN from Staphylococcus aureus was from Fluka-Chemie (Buchs, Germany). PGN fromBacillus subtilis was purified as reported (11Hajjar A.M. O'Mahony D.S. Ozinsky A. Underhill D.M. Aderem A. Klebanoff S.J. Wilson C.B. J. Immunol. 2002; 166: 15-19Google Scholar). Pam3CysSerLys4, a synthetic bacterial lipoprotein analog (sBLP), was a gift of A. Zychlinsky (Max Planck Institute for Infection Biology, Berlin, Germany). MDP and their analogs MurNAc-l-alanyl-l-isoglutamine and MurNAc-d-alanyl-d-isoglutamine were obtained from Bachem (Torrance, CA). Molecules with two and four copies of GlcNAc-MurNAc attached to l-Ala-d-isoGln, and their counterparts lacking dipeptide were synthesized as reported (12Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol. 1996; 24: 324-329Google Scholar). Briefly, the disaccharide glucosaminyl-β (1Takeda K. Akira S. Genes Cells. 2001; 6: 733-742Google Scholar, 2Inohara N. Ogura T. Nuñez G. Curr. Opin. Microbiol. 2002; 5: 76-80Google Scholar, 3Girardin S.E. Sansonetti P.J. Philpott D.J. Trends Microbiol. 2002; 10: 193-199Google Scholar, 4Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nuñez G. J. Biol. Chem. 2001; 276: 4812-4818Google Scholar)-muramic acid was prepared by stereoselective glycosylation of an N-Troc muramic acid acceptor with N-Troc-glucosaminyl trichloroacetimidate. The disaccharide was converted to both disaccharide acceptor and donor, which was then coupled together by the same glycosylation method to give a tetrasaccharide. Octasaccharide was obtained in a good yield in a similar manner. Introduction of the dipeptide moiety ofl-alanyl-d-isoglutamine to 3-O-lactyl groups was performed by deprotection afforded by the peptidoglycan tetrasaccharide and octasaccharide fragments. The plasmids pcDNA3- NOD2, pcDNA3-TLR4, and pDNA3-MD2 have been described (10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar). The plasmids expressing TLR1 and TLR6 have been reported (13Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Google Scholar) and were generously provided by Dr. A. Hajjar (University of Washington, Seattle). Expression plasmids producing NH2-terminal HA-tagged NOD2 variants P268S, P268S/R702W, and P268S/G908R were generated by the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). P268S/L1007fsinsC was generated by a PCR method using P268S DNA as a template. The generated PCR products were cloned into the pMX-puro expression plasmid (14Atrih A. Bacher G. Allmaier G. Williamson M.P. Foster S.J. J. Bacteriol. 1999; 181: 3956-3962Google Scholar). The authenticity of the constructs were confirmed by DNA sequencing. Expression of NOD2 proteins in transfected cells was determined by immunoblotting using monoclonal anti-HA antibody (Babco, La Jolla, CA) as described (5Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nuñez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-47705Google Scholar). NF-κB activation assays were performed as described (10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar). LPS, PGN, and MDP derivaties were added to the cultures in the presence of calcium phosphate to allow their entry into the cells as reported (10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar). Results were normalized for transfection efficiency with values obtained with pEF-BOS-βgal. Expression of NOD2 proteins in transfected cells was determined by immunoblotting using monoclonal anti-HA antibody (Babco, La Jolla, CA) as described (4Ogura Y. Inohara N. Benito A. Chen F.F. Yamaoka S. Nuñez G. J. Biol. Chem. 2001; 276: 4812-4818Google Scholar). PGN from B. subtilus (0.4 mg) was digested with mutanolysin (Sigma) for 24 h. After centrifugation at 100,000 × g for 10 min and filtration with a 0.22-μm nitrocellulose filter, digested PGN was fractionated by Superose 12 gel-filtration column chromatography with 10 mm HEPES, 100 mm NaCl, pH 7.4. Purified bovine serum albumine, chicken egg lysozyme, and bovine cytochromec were used as molecular size standards. Peripheral blood was obtained from normal donors and Crohn's disease patients after informed consent according to Guidelines from the Committee for the Protection of Human Subjects at the Hospital Universitario Marques de Valdecilla. DNA was tested for the 3020insC NOD2 mutation using the SNaPshot method (Applied Biosystems, Foster City, CA) based on the dideoxy single-base extension of an unlabeled oligonucleotide primer (5′-GCCCTCCTGCAGGCCC-3′). PBMNC were cultured for 1 h with 1 μg/ml LPS from S. typhimuriumor 10 ng/ml MDP. Then cells were lysed and nuclear extracts were analyzed for the presence of NF-κB binding activity as described previously (5Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nuñez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-47705Google Scholar). Total RNA was prepared using TRIZOL reagent (Invitrogen). To assess mRNA expression, a quantitative PCR method was used as described previously (5Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nuñez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-47705Google Scholar). The generated cDNA was amplified by using primers for human IL-1β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nuñez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-47705Google Scholar), and A1 (5′-CGGATGTGGATACCTATAAGG-3′ and 5′-GTCATCCAGCCAGATTTAGG-3′). Quantitative real-time PCR was performed in a 7000 sequence detection system (Applied Biosystems). The ratio of the abundance of IL-1β and A1 transcripts to that of GAPDH transcripts was calculated as described (5Gutierrez O. Pipaon C. Inohara N. Fontalba A. Ogura Y. Prosper F. Nuñez G. Fernandez-Luna J.L. J. Biol. Chem. 2002; 277: 41701-47705Google Scholar). Specificity of the PCR products was determined by melting curve analysis. NOD2 was shown to mediate responsiveness to preparations of LPS and PGN (10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar). To further characterize the bacterial moiety recognized by NOD2, we used human embryonic kidney (HEK293T) cells and a NF-κB-dependent luciferase reporter to compare the ability of NOD2 and TLR4 to recognize LPS and PGN. Because NOD2 is an intracellular protein, we assessed NOD2 activity under culture conditions that allow the internalization of the bacterial components into the cells (10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar). Expression of NOD2 and TLR4 together with its co-factor MD2 conferred responsiveness to purified LPS prepared by phenol extraction as reported (7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar, 10Inohara N. Ogura Y. Chen F.F. Muto A. Nuñez G. J. Biol. Chem. 2001; 276: 2551-2554Google Scholar), whereas only NOD2 induced the response to PGN (Fig. 1 A). The lipid A moiety of LPS mediates TLR4/MD2 activation (15Glauner B. Höltje J.-V. Schwartz U. J. Biol. Chem. 1988; 263: 10088-10095Google Scholar). Consistent with the latter, NOD2, but not TLR4, did respond to lipid A-depleted detoxified LPS prepared by alkaline treatment (Fig. 1 A), whereas TLR4, but not NOD2, responded to purified lipid A (Fig.1 A). Notably, TLR4, but not NOD2, was stimulated by highly purified LPS prepared by gel-filtration chromatography (Fig.1 A). These results indicate that TLR4 and NOD2 recognize different bacterial components and suggest that PGN present in LPS preparations may contain the moiety recognized by NOD2. To further characterize the bacterial structure recognized by NOD2, we digested purified PGN with the muramidases mutanolysin or Cellosyl, which degrade the glycan chains and result in the generation of muropeptides which are composed of N-acetylglucosamine (GlcNAc) andN-acetylmuramic acid (MurNAc) linked to short peptides (16Inamura S. Fukase K. Kusumoto S. Tetrahedron Lett. 2001; 42: 7613-7619Google Scholar). Digested PGN was fractionated by gel-filtration chromatography. Analysis of each fraction revealed a major peak of NOD2-stimulating activity induced by mutanolysin digestion with a relative molecular mass of less than 12 kDa, consistent with that expected for muropeptides (Fig. 1 B). Similar results were observed when PGN was digested with Cellosyl, which revealed a single peak of NOD2 stimulatory activity of less than 12 kDa. 2N. Inohara, Y. Ogura, and G. Nuñez, unpublished results. PGN-derived muropeptides derived from most bacterial species are composed of GlcNAc-MurNAc linked to short peptides, which include the conserved dipeptide l-Ala and d-isoGln ord-Glu (Fig. 2 A). To determine more directly if muropeptides are recognized by NOD2, we used a panel of synthetic molecules with the structure of GlcNAc-MurNAc linked to l-Ala-d-isoGln, as well as muramyl dipeptide MurNAc-l-Ala-d-isoGln (MDP), which lacks GlcNAc (Fig. 2 A). GlcNAc-MurNAc-l-Ala-d-isoGln stimulated NF-κB in a NOD2-dependent manner, whereas disaccharide GlcNAc-MurNAc in dimeric or tetrameric forms did not (Fig.2 B), indicating that amino acid residues are required for stimulation of NOD2. However, we cannot formally rule out that the lack of NOD2 response to GlcNAc-MurNAc in dimeric or tetrameric forms is due to poor internalization of the synthetic molecules into the cells. MDP induced potent stimulation of NOD2 (Fig. 2 B), indicating that GlcNAc is not essential for stimulatory activity. To determine further the specificity of the recognition of MDP by NOD2, we tested the MDP analogs MurNAc-l-Ala-l-isoGln and MurNAc-d-Ala-d-isoGln. Notably, replacement ofl-Ala for d-Ala or d-isoGln forl-isoGln eliminated the ability of MDP to stimulate NOD2, indicating stereoselective recognition (Fig. 2 C). Thus, the core structure required for recognition of NOD2 is MurNAc attached tol-Ala and d-isoGln. TLR2 has been proposed to act as a surface receptor for PGN and certain bacterial lipoproteins including synthetic bacterial lipopeptide S. B. P. Zychlinsky A. 1999; Scholar, J. Immunol. 2002; Scholar). we tested the ability of TLR2 and NOD2 to respond to MDP and TLR2 a response to but not to MDP (Fig.2 MDP, but not sBLP, stimulated NOD2 activity (Fig. 2 In we tested co-expression of TLR2 with TLR1 TLR6 mediate recognition of MDP. TLR2 in with or did not NF-κB in response to MDP (Fig. 2 Thus, NOD2 and TLR2 recognize different bacterial The NOD2 variants associated with Crohn's disease, R702W, G908R, and L1007fsinsC, on the same which the P268S (6Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O'Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Nature. 2001; 411: 599-603Google Scholar, 7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar). To compare the ability of normal and NOD2 proteins to NF-κB activity in response to MDP, we expressed the NOD2 proteins in cells and their activity in a functional normal and P268S NOD2 induced similar of NF-κB activation in response to MDP A), which is consistent with the that P268S is not genetically associated with disease (6Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O'Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Nature. 2001; 411: 599-603Google Scholar, 7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar). In the P268S/R702W, and P268S/L1007fsinsC variants induced of NF-κB activation in response to MDP when with normal NOD2 A). Notably, whereas the and mutants ability to respond to MDP, the protein did not respond at of NOD2 and MDP tested (Fig. A). revealed that the normal and NOD2 mutants were expressed B), indicating that the observed in MDP not by expression of the NOD2 determined the role of NOD2 in the recognition of MDP by cells from normal and Crohn's disease A panel of and Crohn's disease individuals were for the disease-associated NOD2 and two individuals of disease and with Crohn's were homozygous for the L1007fsinsC mutation were The of individuals homozygous for L1007fsinsC is expected in that the of the L1007fsinsC mutation is not (6Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O'Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Nature. 2001; 411: 599-603Google Scholar, 7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar, 8Hampe J. Cuthbert A. Croucher P.J. Mirza M.M. Mascheretti S. Fisher S. Frenzel H. King K. Hasselmeyer A. MacPherson A.J. Bridger S. van Deventer S. Forbes A. Nikolaus S. Lennard-Jones J.E. Foelsch U.R. Krawczak M. Lewis C. Schreiber S. Mathew C.G. Lancet. 2001; 357: 1925-1928Google Scholar, 9Ahmad T. Armuzzi A. Bunce M. Mulcahy-Hawes K. Marshall S.E. Orchard T.R. Crawshaw J. Large O. de Silva A. Cook J.T. Barnardo M. Cullen S. Welsh K.I. Jewell D.P. Gastroenterology. 2002; 122: 854-866Google Scholar). PBMNC that are to NOD2 were with MDP or LPS, and NF-κB activation was assessed in nuclear extracts by an electrophoretic mobility shift of PBMNC from individuals normal or NOD2 with LPS or MDP in of the (Fig. C). In PBMNC from individuals homozygous for L1007fsinsC did respond to LPS, but not to MDP (Fig. C). The DNA binding induced by MDP was by of the nuclear extracts with an antibody for the of NF-κB indicating that the To further assess NF-κB the mRNA of IL-1β and two NF-κB expressed in PBMNC J.P. M.M. J.P. J. Immunol. Scholar, Chen C. J. C. Genes 1999; Scholar), were by quantitative real-time PCR analysis. IL-1β and A1 mRNA were induced by with LPS and MDP in cells from individuals normal or NOD2 (Fig. and In LPS but not MDP, the of both IL-1β and A1 mRNA in PBMNC from individuals homozygous for L1007fsinsC (Fig. Thus, PBMNC the expression of normal NOD2 for their response to MDP but not to The lack of response to MDP in normal individuals suggest that the presence of certain bacteria in the genetic may required for disease. MDP is the essential structure of bacterial peptidoglycan required for including activity in adjuvant H. S. The and of Scholar). MDP has been shown to through and S. R. S. O. Akira S. S. Takada H. 2001; Scholar, J. Biol. Chem. 2002; 277: Scholar), but the host recognition system for MDP has not been present that NOD2 mediates the recognition of MDP in cells. contain intracellular that bacterial PGN and PGN including J. F. J. Scholar, Scholar). These muropeptides derived from intracellular bacteria as well as PGN bacterial for recognition by NOD2. The synthetic MDP and the muropeptides induced NOD2-dependent activation of Thus, NOD2 is to by muropeptides derived from bacteria in NOD2 mediate the recognition of the is and remains to Because the LRRs are required for muropeptides directly with NOD2 through its LRRs or as to identified Crohn's disease-associated NOD2 variants and PBMNC from individuals homozygous for L1007fsinsC are in their response to muramyl dipeptide. result is consistent with the that for L1007fsinsC is for susceptibility to Crohn's disease (6Hugot J.P. Chamaillard M. Zouali H. Lesage S. Cezard J.P. Belaiche J. Almer S. Tysk C. O'Morain C.A. Gassull M. Binder V. Finkel Y. Cortot A. Modigliani R. Laurent-Puig P. Gower-Rousseau C. Macry J. Colombel J.F. Sahbatou M. Thomas G. Nature. 2001; 411: 599-603Google Scholar, 7Ogura Y. Bonen D.K. Inohara N. Nicolae D.L. Chen F.F. Ramos R. Britton H. Moran T. Karaliuskas R. Duerr R.H. Achkar J.P. Brant S.R. Bayless T.M. Kirschner B.S. Hanauer S.B. Nuñez G. Cho J.H. Nature. 2001; 411: 603-606Google Scholar). Because activation of NF-κB in response to bacterial components mediates protection of the host against susceptibility to disease may by a to a NF-κB in response to a response against certain bacterial products may result in the activation of NF-κB in by The results suggest that the activity against bacterial muropeptides may to Crohn's disease patients NOD2 are to T. for C. for A. Zychlinsky for sBLP, and A. Hajjar for P. for of the

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-kappaB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor kappaB (NF-kappaB) activation, which required IKKgamma and was inhibited by dominant negative mutants of IkappaBalpha, IKKalpha, IKKbeta, and IKKgamma. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-kappaB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-kappaB signaling pathway.