A Role of Lipophilic Peptidoglycan-related Molecules in Induction of Nod1-mediated Immune Responses

Abstract

Nod1 is an intracellular protein that is involved in recognition of bacterial molecules and whose genetic variation has been linked to several inflammatory diseases. Previous studies suggested that the recognition core of Nod1 stimulatory molecules is ␥-D-glutamyl-meso-diaminopimelic acid (iE-DAP), but the identity of the major Nod1 stimulatory molecule produced by bacteria remains unknown. Here we show that bacteria produce lipophilic molecules capable of stimulating Nod1. Analysis of synthetic compounds revealed stereoselectivity of the DAP residue and that conjugation of lipophilic acyl residues specifically enhances the Nod1 stimulatory activity of the core iE-DAP. Furthermore, we demonstrate that lipophilic molecules induce and/or enhance the secretion of innate immune mediators from primary mouse mesothelial cells and human monocytic MonoMac6 cells, and this effect is mediated through Nod1. These results provide insight into the mechanism of immune recognition via Nod1, which might be useful in the design and testing of novel immunoregulators.The innate immune receptors recognize specific molecules that are commonly found in microbes and induce host defense responses to eliminate invading pathogens (1). These pathogen-recognizing receptors include membrane-bound Toll-like receptors (TLRs) 2 as well as cytosolic Nod-like receptors and RIG-I family proteins. These three classes of pathogen-recognizing receptors regulate innate and acquired immune responses by inducing intracellular signaling pathways that lead to the activation of p38, c-Jun NH 2 -terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), caspase-1, and transcription factors including NF-B and/or IRFs (1).Nod1 is the founding member of the Nod-like receptor protein family that contains nucleotide oligomerization domain (NOD) and ligand-recognizing leucine-rich repeats. This family is comprised of more than 20 members including Nod2, cryopyrin, Ipaf, NAIP, and NALP1 (1, 2). Genetic studies have revealed that variations of the NOD1 gene are associated with susceptibility to several human disorders including allergic diseases (3-5), Crohn disease (6), and sarcoidosis (7), although the mechanism underlying these disease associations remains poorly understood. Although Nod2 recognizes the muramyl dipeptide (MDP) structure in bacterial peptidoglycan (PGN)-related molecules, Nod1 senses the essential iE-DAP dipeptide, which is uniquely found in PGN of all Gram-negative and certain Gram-positive bacteria (8 -12). Previous studies suggested the involvement of Nod1 in the recognition of numerous bacteria including Helicobacter pylori (13,14), Listeria monocytogenes (15, 16), Shigella flexneri (17), several Bacillus species (18), and Propionibacterium acnes (7). Although the iE-DAP structure is found in the insoluble fraction of intact PGN, intermediates of PGN synthesis and cleaved PGN products produced during bacterial growth and PGN recycling (11,19), the identity of the major Nod1 stimulatory molecule (s) remains unknown (18). Previous s...

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