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Epithelial Cells Through NOD1 Bacterial Peptidoglycans, Activate Meso-Diaminopimelic Acid and Meso -Lanthionine, Amino Acids Specific to Bacterial Peptidoglycans, Activate Human Epithelial Cells through NOD1 This information is current as of September 26, 2021. Akiko Uehara, Yukari Fujimoto, Akiko Kawasaki, Shoichi Kusumoto, Koichi Fukase and Haruhiko Takada J Immunol 2006; 177:1796-1804; ; doi: 10.4049/jimmunol.177.3.1796 http://www.jimmunol.org/content/177/3/1796 Downloaded from References This article cites 25 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/177/3/1796.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Meso-Diaminopimelic Acid and Meso-Lanthionine, Amino Acids Specific to Bacterial Peptidoglycans, Activate Human Epithelial Cells through NOD11 Akiko Uehara,* Yukari Fujimoto,† Akiko Kawasaki,† Shoichi Kusumoto,† Koichi Fukase,† and Haruhiko Takada2* Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls and exhibit various immunobiological activities. Two types of minimum essential PGN structures for immunobiological activities were chemically synthesized and designated as mu- ramyldipeptide; N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and ␥-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which are common constituents of both Gram-positive and Gram-negative bacteria, as well as most Gram-negative and some Gram- positive bacteria, respectively. Recently, intracellular receptors for MDP and iE-DAP have been demonstrated to be nucleotide- Downloaded from binding oligomerization domain (NOD)1 and NOD2, respectively. In this study, we demonstrated that chemically synthesized meso-DAP itself activated human epithelial cells from various tissues, through NOD1 to generate antibacterial factors, PGN recognition proteins and ␤-defensin 2, and cytokines in specified cases, although the activities of meso-DAP were generally weaker than those of known NOD agonists. However, stereoisomers of meso-DAP, LL-DAP, and DD-DAP were only slightly activated or remained inactive, respectively. Synthetic meso-lanthionine, which is another diamino-type amino acid specific to PGN of the specified Gram-negative bacteria, was also recognized by NOD1. In human monocytic cells, in the presence of cytochalasin D http://www.jimmunol.org/ meso-DAP induced slightly but significantly increased production of cytokines, although the cells did not respond to meso-DAP in the absent of cytochalasin D. Our findings suggest that NOD1 is a special sentinel molecule, especially in the epithelial barrier, allowing the intracellular detection of bacteria through recognizing meso-DAP or comparable moiety of PGN from specified bacteria in cooperation with NOD2, thereby playing a key role in innate immunity. The Journal of Immunology, 2006, 177: 1796–1804. he peptidoglycans (PGNs)3 are ubiquitous constituents of that intracellular protein carrying a nucleotide-binding oligomer- the cell walls of Gram-positive and Gram-negative bac- ization domain (NOD), NOD2, was intracellular receptor for MDP by guest on September 26, 2021 T teria. Numerous studies have reported on the immunobio- (6, 7). Then, the two groups also independently revealed that DMP activities of bacterial PGN, most of which have been reproduced containing DAP was recognized by another NOD protein, NOD1 by a chemically synthesized low-molecular PGN fragment, mu- (8, 9). Recently, it has been reported that iE-DAP is the minimum ramyldipeptide (MDP) N-acetylmuramyl-L-alanyl-D-isoglutamine PGN structure capable of stimulating NOD1 on human embryonic (or MurNAc-L-Ala-D-isoGln) (Fig. 1A) (1, 2). Another type of kidney cell line cells (HEK 293T) transfected with human NOD1 PGN fragment, desmuramylpeptides (DMPs), has also been chem- (6, 10). In these reports, Chamaillard and colleagues (6) and Gi- ically synthesized to mimic PGN containing meso-diaminopimelic rardin et al. (10) described that DAP alone is completely inactive acid (meso-DAP), and the DMPs exerted similar bioactivities to in this respect by using a commercial (Sigma-Aldrich) DAP, a MDP (Fig. 1B) (3, 4). In 1982, the Fujisawa Pharmaceutical group mixture of meso-DAP, LL-DAP and DD-DAP, and meso-DAP pu- ␥ (5) reported that -D-glutamyl-meso-DAP (iE-DAP) is the mini- rified from the commercial DAP, respectively. mum structural unit capable of eliciting bioactivities induced by In various mucosal tissues, epithelial cells interact with bacteria DMPs. Recently, two research groups independently demonstrated in normal flora and have been reported to produce several proin- flammatory cytokines in response to bacterial stimuli. We re- *Department of Microbiology and Immunology, Tohoku University Graduate School vealed, however, that oral epithelial cells did not show enhanced of Dentistry, Sendai, Japan; and †Department of Chemistry, Graduate School of Sci- production of proinflammatory cytokines such as IL-8 in response ence, Osaka University, Osaka, Japan to various bacterial cell surface components, whereas several co- Received for publication December 6, 2005. Accepted for publication April 26, 2006. lonic epithelial cell lines produced inflammatory cytokines upon The costs of publication of this article were defrayed in part by the payment of page stimulation with bacterial components (11). In contrast, we re- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cently demonstrated that chemically synthesized bacterial cell sur- 1 This work was supported in part by Grant-in-Aid 16390519 for Scientific Research face components increase the expression of PGN recognition pro- from the Japan Society for the Promotion of Science. A.U. was supported by Research teins (PGRPs) via TLRs, NOD1 and NOD2 in human oral Fellowship PD6961 from the Japan Society for the Promotion of Science. epithelial cells (12). PGRPs are a novel family of pattern recog- 2 Address correspondence and reprint requests to Dr. Haruhiko Takada, Department nition receptor molecules in innate immunity conserved from in- of Microbiology and Immunology, Tohoku University Gradate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail address: dent- sects to mammals. These findings suggest that epithelial cells in- [email protected] teracting constitutionally with normal flora might respond to 3 Abbreviations used in this paper: PGN, peptidoglycan; MDP, muramyldipeptide; bacterial cell surface components with the enhanced production of DMP, desmuramylpeptide; DAP, diaminopimelic acid; iE-DAP, ␥-D-glutamyl-meso- DAP; NOD, nucleotide-binding oligomerization domain; PGRP, PGN recognition antibacterial molecules, such as PGRPs, to prevent bacterial inva- protein; siRNA, short interfering RNA. sion of host cells without producing proinflammatory cytokines to Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 1797 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 FIGURE 1. Chemically synthesized bacterial PGN fragments. Chemical structure of Lys-type (A) and DAP-type (B) PGN and the respective minimum active moiety reported to sense NOD2 and NOD1 and chemical structures of synthetic DAP isomers and reference synthetic NOD1 agonists (C). NOD2 recognizes MDP (MurNAc-L-Ala-D-isoGln) moiety from Lys-type PGN (A) and probably also MurNAc-L-Ala-D-Glu moiety from DAP-type PGN (B). NOD1 recognizes the DMP moiety containing meso-DAP (B), whose minimum essential structure was reported to be iE-DAP (␥-D-Glu-meso-DAP) until this study. iE-DAP, FK156 (D-lactyl-L-Ala-␥-D-Glu-meso-DAP-Gly), and FK565 (heptanoly-␥-D-Glu-meso-DAP-D-Ala) were used as reference NOD1 agonists. prevent possible tissue destruction through by excessive innate im- Materials and Methods mune responses. Reagents In the course of the study to examine the innate immune system in oral epithelium, we found unexpectedly that a commercial DAP spec- The synthetic MurNAc-L-Ala-D-isoGln (MDP) was purchased from Protein Research Foundation, Peptide Institute. A synthetic Escherichia imen activated human oral epithelial cells. Therefore, to further char- coli-type lipid A (LA-15-PP) was purchased from Daiichi Chemical. acterize the bacterial moiety recognized by NOD1, we have con- ␥-D-glutamyl-meso-DAP (iE-DAP) was synthesized as previously de- ducted an exhaustive study using three chemically synthesized scribed (8). Meso-lanthionine was synthesized by coupling of N-ben- ␤ stereoisomers of DAP, meso-DAP, LL-DAP, and DD-DAP, in human zyloxycarbonyl-D-cysteine with -L-chloroalanine according to the pro- epithelial and monocytic cells in culture. Furthermore, we examined cedure
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