Host Recognition of Bacterial Muramyl Dipeptide Mediated through NOD2

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 (sBLP) (17Aliprantis A.O. Yang R.B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Google Scholar, 18Lee H.K. Tobias P.S. J. Immunol. 2002; 168: 4012-4017Google Scholar). Therefore, we tested the ability of TLR2 and NOD2 to respond to MDP and sBLP. TLR2 mediated a cellular response to sBLP but not to MDP (Fig.2 D). Conversely, MDP, but not sBLP, stimulated NOD2 activity (Fig. 2 D). In addition, we tested whether co-expression of TLR2 with TLR1 and/or TLR6 could mediate recognition of MDP. TLR2 in combination with TLR1, TLR6, or TLR1/TLR6 did not activate NF-κB in response to MDP (Fig. 2 D). Thus, NOD2 and TLR2 recognize different bacterial structures. The NOD2 variants associated with Crohn's disease, R702W, G908R, and L1007fsinsC, occur on the same haplotype background, which includes the common P268S polymorphism (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 mutant NOD2 proteins to induce NF-κB activity in response to MDP, we expressed the NOD2 proteins in cells and evaluated their activity in a functional assay. Both normal and P268S NOD2 induced similar levels of NF-κB activation in response to MDP (Fig.3 A), which is consistent with the observation 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 contrast, the P268S/R702W, P268S/G908R, and P268S/L1007fsinsC variants induced greatly reduced levels of NF-κB activation in response to MDP when compared with normal NOD2 (Fig.3 A). Notably, whereas the P268S/R702W and P268S/R702W point mutants retained some ability to respond to MDP, the truncated P268/L1007fsinsC protein did not respond at all concentrations of NOD2 and MDP tested (Fig. 3 A). Immunoblotting analysis revealed that the normal and NOD2 mutants were appropriately expressed (Fig.3 B), indicating that the observed differences in MDP responses could not be explained by differential expression of the NOD2 constructs. We next determined the role of NOD2 in the recognition of MDP by primary cells from normal and Crohn's disease patients. A panel of healthy and Crohn's disease individuals were genotyped for the disease-associated NOD2 alleles, and two individuals (one without clinical evidence of disease and one individual with Crohn's disease) who were homozygous for the loss-of-function L1007fsinsC mutation were identified. The identification of apparently healthy individuals homozygous for L1007fsinsC is expected in that the penetrance of the L1007fsinsC mutation is not complete (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 known to express NOD2 were isolated, incubated with MDP or LPS, and NF-κB activation was assessed in nuclear extracts by an electrophoretic mobility shift assay (EMSA). Stimulation of PBMNC from individuals carrying normal or heterozygous NOD2 alleles with either LPS or MDP resulted in induction of the NF-κB-DNA complex (Fig. 3 C). In contrast, PBMNC from individuals homozygous for L1007fsinsC did respond to LPS, but not to MDP (Fig. 3 C). The DNA binding complex induced by MDP was shifted by incubation of the nuclear extracts with an antibody specific for the p50 subunit of NF-κB (Fig.3 C), indicating that the detected protein-DNA complex contained NF-κB. To further assess NF-κB activation, the mRNA levels of IL-1β and A1, two NF-κB target genes expressed in PBMNC (19Cogswell J.P. Godlevski M.M. Wisely G.B. Clay W.C. Leesnitzer L.M. Ways J.P. Gray J.G. J. Immunol. 1994; 153: 712-723Google Scholar, 20Zong W.X. Edelstein L.C. Chen C. Bash J. Gelinas C. Genes Dev. 1999; 13: 382-387Google Scholar), were evaluated by quantitative real-time PCR analysis. Both IL-1β and A1 mRNA levels were induced by incubation with LPS and MDP in cells from individuals carrying normal or heterozygous NOD2 alleles (Fig. 3, D and E). In contrast, LPS but not MDP, increased the levels of both IL-1β and A1 mRNA in PBMNC from individuals homozygous for L1007fsinsC (Fig. 3, D andE). Thus, PBMNC require the expression of normal NOD2 for their response to MDP but not to LPS. The lack of response to MDP in apparently normal individuals suggest that the presence of certain bacteria in the intestine and/or additional genetic factors may be required for clinical disease. MDP is the minimal essential structure of bacterial peptidoglycan required for biological effects, including activity in Freund's complete adjuvant (21Takada H. Kotani S. Stewart D.E.S. The Theory and Practical Application of Adjuvants. Wiley, Chichester, UK1995: 171-202Google Scholar). MDP has been shown to signal through TLR2- and TLR4-independent mechanisms (22Yang S. Tamai R. Akashi S. Takeuchi O. Akira S. Sugawara S. Takada H. Infect. Immun. 2001; 69: 2045-2053Google Scholar, 23Wolfert M.A. Murray T.F. Boons G.-J. Moore J.N. J. Biol. Chem. 2002; 277: 39179Google Scholar), but the host recognition system for MDP has not been identified. We present evidence that NOD2 mediates the recognition of MDP in mammalian cells. Macrophages contain intracellular hydrolases that digest bacterial PGN and release PGN fragments including GlcNAc-MurNAc-dipeptide (24Johannsen L. Wecke J. Obal Jr., F. Krueger J.M. Am. J. Physiol. 1991; 260: R126-R133Google Scholar, 25Vermeulen M.W. Gray G.R. Infect. Immun. 1984; 46: 476-483Google Scholar). These muropeptides derived from intracellular and/or phagocytosed bacteria as well as PGN fragments released during bacterial growth would be available for recognition by NOD2. The synthetic MDP and GlcNAc-MurNAc-dipeptides mimicking the natural muropeptides induced NOD2-dependent activation of NF-κB. Thus, NOD2 is likely to be activated by muropeptides derived from bacteria in vivo. Although NOD2 can mediate the recognition of muropeptides, the mechanism involved is unclear and remains to be determined. Because the LRRs are required for recognition, muropeptides could interact directly with NOD2 through its LRRs or via as yet to be identified cellular factor(s). Crohn's disease-associated NOD2 variants and PBMNC from individuals homozygous for L1007fsinsC are defective in their response to muramyl dipeptide. This result is consistent with the observation that homozygocity for L1007fsinsC is needed 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 pathogens, NOD2-mediated susceptibility to disease may be caused by a failure to trigger a protective NF-κB pathway in response to muropeptides. Such a defective response against certain bacterial products may result secondarily in the diffuse activation of NF-κB found in intestinal tissue by NOD2-independent mechanisms. The results presented here suggest that restoring the activity against bacterial muropeptides may be beneficial to Crohn's disease patients harboring NOD2 mutations. We are grateful to T. Kirikae for stimulating discussions, C. Kirschning for HEK293-TLR2 cells, A. Zychlinsky for sBLP, and A. Hajjar for plasmids. We thank P. Lucas for critical review of the manuscript.