Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2

Nod1 and Nod2 are mammalian proteins implicated in the intracellular detection of pathogen-associated molecular patterns. Recently, naturally occurring peptidoglycan (PG) fragments were identified as the microbial motifs sensed by Nod1 and Nod2. Whereas Nod2 detects GlcNAc-MurNAc dipeptide (GM-Di), Nod1 senses a unique diaminopimelate-containing GlcNAc-MurNAc tripeptide muropeptide (GM-TriDAP) found mostly in Gram-negative bacterial PGs. Because Nod1 and Nod2 detect similar yet distinct muropeptides, we further analyzed the molecular sensing specificity of Nod1 and Nod2 toward PG fragments. Using a wide array of natural or modified muramyl peptides, we show here that Nod1 and Nod2 have evolved divergent strategies to achieve PG sensing. By defining the PG structural requirements for Nod1 and Nod2 sensing, this study reveals how PG processing and modifications, either by host or bacterial enzymes, may affect innate immune responses.     

Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection

Nod2 activates the NF-kappaB pathway following intracellular stimulation by bacterial products. Recently, mutations in Nod2 have been shown to be associated with Crohn's disease, suggesting a role for bacteria-host interactions in the etiology of this disorder. We show here that Nod2 is a general sensor of peptidoglycan through the recognition of muramyl dipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria. Moreover, the 3020insC frameshift mutation, the most frequent Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP. Together, these results impact on the understanding of Crohn's disease development. Additionally, the characterization of Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well known biological activities of this immunomodulatory compound.     

A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling

Nod2 is an intracellular sensor of a specific bacterial cell wall component, muramyl dipeptide, and activation of Nod2 stimulates an inflammatory response. Specific mutations of Nod2 have been associated with two inflammatory diseases, Crohn disease and Blau syndrome, and are thought to contribute to disease susceptibility through altering Nod2 signaling. Association of disease with inappropriate activation of Nod2 highlights the importance of proper regulation of Nod2 activity. However, little is known about specific regulation of the Nod2 pathway. We performed a biochemical screen to discover potential regulators of Nod2 and identified Erbin, a protein involved in cell polarity, receptor localization, and regulation of the mitogen-activated protein kinase pathway, as a novel Nod2-interacting protein. In our studies, we demonstrate specific interaction of Erbin and Nod2 both in vitro and in vivo and characterize the regions required for interaction in both proteins. We found that Nod2-dependent activation of NF-kappaB and cytokine secretion is inhibited by Erbin overexpression, whereas Erbin-/- mouse embryo fibroblasts show an increased sensitivity to muramyl dipeptide. These studies identify Erbin as a regulator of Nod2 signaling and demonstrate a novel role for Erbin in inflammatory responses.     

hPepT1 selectively transports muramyl dipeptide but not Nod1-activating muramyl peptides

Muramyl peptides derived from bacterial peptidoglycan are detected intracellularly by Nod1 and Nod2, 2 members of the newly characterized nod-like receptor (NLR) family of pattern recognition molecules. In the absence of bacterial invasion into the host cytosolic compartment, it remains unclear whether muramyl peptides can cross the plasma membrane and localize into the cytosol. We have recently demonstrated that the plasma membrane transporter, hPepT1, was able to efficiently translocate muramyl dipeptide (MDP), a specific Nod2-activating molecule, into host cells. We aimed to characterize the transport properties of hPepT1 towards a spectrum of muramyl peptides, including Nod1-activating molecules. To do so, we designed an original procedure based on the ectopic expression of hPepT1 in oocytes from Xenopus laevis. Our results demonstrated that hPepT1 transports MDP but no other Nod2-activating molecule. Moreover, we observed that Nod1-stimulating muramyl peptides were not transported by hPepT1. Since hPepT1 expression is strongly associated with intestinal epithelial cells, where Nod1 and Nod2 have been shown to play a key role, these observations suggest a distinct contribution of Nod1 and Nod2 in mucosal homeostasis following the cellular uptake of muramyl peptides by hPepT1.     

pH-dependent Internalization of Muramyl Peptides from Early Endosomes Enables Nod1 and Nod2 Signaling

Nod1 and Nod2 are members of the Nod-like receptor family that detect intracellular bacterial peptidoglycan-derived muramyl peptides. The biological effects of muramyl peptides have been described for over three decades, but the mechanism underlying their internalization to the cytosol remains unclear. Using the human epithelial cell line HEK293T as a model system, we demonstrate here that Nod1-activating ligands entered cells through endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-β-cyclodextrin. In the endocytic pathway, the cytosolic internalization of Nod1 ligands was pH-dependent, occurred prior to the acidification mediated by the vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl peptides likely required processing by endosomal enzymes, prior to transport into the cytosol, suggesting the existence of a sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an oligopeptide transporter expressed in early endosomes, in Nod1-dependent NF-κB signaling. Interestingly, SLC15A4 expression was also up-regulated in colonic biopsies from patients with inflammatory bowel disease, a disorder associated with mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl peptides get access to the host cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as inflammatory bowel disease.Innate immunity relies on the detection of conserved microbial- or danger-associated molecular patterns (MAMPs or DAMPs),² by pattern-recognition molecules. In mammals, several families of pattern-recognition molecules have been recently identified, including the transmembrane Toll-like receptors (TLRs), cytosolic Nod-like receptors (NLRs), and RIG-I-like receptors (1). NLR proteins include Nod1 and Nod2, which trigger pro-inflammatory pathways such as NF-κB and mitogen-activated protein kinases, in response to bacterial peptidoglycan (2), and NLRPs (also known as Nalps), such as NLRP1 and NLRP3, which induce the activation of caspase-1 inflammasomes in response to various MAMPs and DAMPs (3).In the case of TLRs, there is accumulating evidence that the subcellular localization and the function of these pattern-recognition molecules is tightly associated, at multiple levels, with endocytosis and phagocytosis, which represent evolutionary conserved mechanisms for the internalization of small (<0.5 μm) and large (>0.5 μm) particles, respectively. Indeed, whereas some TLRs are expressed at the plasma membrane, others (such as TLR3, -7, and -9) are found predominantly associated with the endoplasmic reticulum and endosomal compartments, where they detect their respective microbial-derived nucleic acid MAMPs (4). In particular, TLR9 has been shown to move from the endoplasmic reticulum to CpG DNA-containing endosomes, concurrent with the accumulation of MyD88, thus showing that endosomes represent the physiological location where TLR9-dependent signaling arises (5). In addition, studies on TLR4 have demonstrated that lipopolysaccharide (LPS) is endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and co-localized with TLR4 on early/sorting endosomes (6). In the case of this TLR, it is believed that endosomal trafficking is associated with termination of the MyD88-dependent pro-inflammatory signal (6). In contrast, TLR4 in early endosomes has been shown recently to engage TRAM and TRIF adaptors, resulting in the ignition of type I interferon signaling in response to LPS (7). Therefore, the nature of the cellular response to LPS is dependent upon the subcellular localization of TLR4, thus reinforcing the importance of the interplay between TLR signaling and endosomal trafficking.A number of studies have also linked TLR signaling with phagosome maturation. Although it remains controversial whether TLR-dependent signaling actually drives phagosomal maturation (8, 9), it is clear that the processing of engulfed microbes within phagosomes regulates the availability of MAMPs within this compartment. Accordingly, Herskovits et al. have recently demonstrated that, in interferon Γ-activated macrophages, the degradation of Listeria monocytogenes in the phagolysosome generates bacterial molecules, which could specifically trigger type I interferon responses through a Nod2-dependent pathway (10). This interesting observation suggests that innate immune signaling and microbial degradation within the phagolysosome are processes that are intimately linked. It also provides support to the concept that Nod-dependent signaling is associated with intracellular vesicular trafficking.Nod1 and Nod2 both detect specific structures from bacterial peptidoglycan (11). Whereas Nod2 detects muramyl dipeptide (MDP) (12, 13), a motif found in almost all bacteria, Nod1 specifically senses diaminopimelic acid (DAP)-containing muramyl peptides (14, 15). In particular, human Nod1 preferentially detects N-acetylmuramyl-l-Ala-d-Glu-mesoDAP (M-Tri-DAP) (16), and the minimal motif for Nod1-dependent sensing is the dipeptide d-Glu-mesoDAP (iE-DAP) (11, 14). Interestingly, long before the identification of Nod1 and Nod2 as sensors of muramyl peptides and bacterial peptidoglycan, the biological activities of these bacterial-derived molecules had been studied extensively (17, 18). It is well documented that these muramyl peptides trigger a multitude of immune responses, such as the induction of cytokines/chemokines, the production of nitric oxide and reactive oxygen species, and the clearance of microbes by phagocytic cells (17, 18). A considerable literature also demonstrated that these muramyl peptides synergize with MAMPs detected by TLRs, such as LPS (19). Although the identification of Nod1 and Nod2 as sensors of muramyl peptides has provided an acceleration in this field of investigation, it also brought the question of how such microbial molecules could get access to the host cytosol, where Nod1 and Nod2 reside. Interestingly, research aiming at improving the biological activities of these muramyl peptides demonstrated early on that the addition of lipophilic groups to these molecules enhanced their activity considerably, suggesting that their internalization was likely a key factor in determining their efficiency (20–23).The mechanisms by which muramyl peptides get access to the host cytosol remain unclear. This question is of fundamental importance for our understanding of Nod-dependent signaling and potentially holds broad therapeutic implications. Indeed, mutations in Nod1 and Nod2 have been associated with inflammatory bowel disease (IBD) in humans (24). In particular, Nod2 has been identified as the first susceptibility gene for Crohn''s disease (25, 26).In this report, we used the HEK293T epithelial cell line to study the mechanism of internalization of Nod1 ligands. We demonstrated that these peptidoglycan-derived molecules enter cells by endocytosis, and that the composition of the Nod1-activating molecules dramatically affected their intrinsic uptake capacity. Our data also suggested that this internalization was mediated by clathrin-dependent endocytosis, because internalization of Nod1 ligands required dynamin and was independent from caveolae. Further, we showed that, within endosomes, the internalization of Nod1 ligands was critically dependent on pH, and was optimal at pH ranging from 5.5 to 6, which are characteristic of early endosomes. Accordingly, internalization of Nod1-activating molecules did not require the action of the vacuolar ATPase (V-ATPase) complex. We also provide evidence that the Nod2 ligand MDP enters cells through a similar endocytic process. Our results also show that the internalization of Nod1 ligands is a process that is sterically gated, and likely requires the action of hydrolytic endosomal enzymes prior to transport into the cytosol, thus suggesting the existence of one or several specific transporters for Nod1 ligands in early endosomes. Using knockdown assays, we identified SLC15A4 as a putative transporter for Nod1 ligands in early endosomes. SLC15A4 expression was found to be significantly up-regulated in tissue biopsies from IBD patients, therefore highlighting a potential role for the modulation of peptidoglycan access to the cytosol in IBD etiology. Together, our results uncover the mechanism by which Nod ligands traffic into cells and get access to the cytosol where they are detected by Nod1 and Nod2. Our observations also highlight the previously unappreciated link between endosomal acidification/maturation and Nod-dependent signaling.     

Nods and 'intracellular' innate immunity

Innate immunity relies on the detection of microbial invaders by two distinct systems. One system comprises a family of membrane-bound receptors, termed the Toll-like receptors, while the other family, termed the nucleotide-binding site/leucine-rich repeat (NBS/LRR) proteins, consists of molecules that are found in the cytoplasmic compartment. These two detection systems recognize conserved molecular components of microbes including such structural motifs as lipopolysaccharide from the Gram-negative bacterial cell wall and peptidoglycan (PGN) found in the cell wall of both Gram-negative and Gram-positive bacteria. This review focuses on two members of the NBS/LRR family of proteins, Nod1 and Nod2. Recently, the microbial motifs sensed by these two molecules have been characterized. Both Nod1 and Nod2 recognize PGN, however, each requires distinct molecular motifs to attain sensing. Nod1 recognizes a naturally occurring muropeptide of PGN that presents a unique amino acid at its terminus called diaminopilemic acid (DAP). This amino acid is found mainly in the PGN of Gram-negative bacteria designating Nodl as a sensor of Gram-negative bacteria. In contrast, Nod2 can detect the minimal bioactive fragment of PGN, called muramyl dipeptide. Thus Nod2 is a general sensor of bacterial PGN. Since mutations in the gene encoding Nod2 were recently shown to be associated with the chronic inflammatory disease, Crohn's disease, these results are discussed in the context of how disrupting the interplay between host detection and bacterial aggression may lead to inflammatory diseases.     

Nucleotide-binding oligomerization domain 2 (Nod2) is dispensable for the innate immune responses of macrophages against Yersinia enterocolitica

Nucleotide-binding oligomerization domain 2 (Nod2) is a cytosolic sensor for muramyl dipeptide, a component of bacterial peptidoglycan. In this study, we have examined whether Nod2 mediates the immune response of macrophages against Yersinia enterocolitica. Bone-marrow-derived macrophages (BMDMs) were isolated from WT and Nod2-deficient mice and were infected with various strains of Y. enterocolitica. ELISA showed that the production of IL-6 and TNF-α in BMDMs infected with Y. enterocolitica was not affected by the Nod2 deficiency. iNOS mRNA expression was induced in both WT and Nod2-deficienct BMDMs in response to Y. enterocolitica, beginning 2 h after infection. Nitric oxide (NO) production by Y. enterocolitica did not differ between WT and Nod2-deficient BMDMs. Western blot analysis revealed that Y. enterocolitica induces activation of NF-κB, p38, and ERK MAPK through a Nod2-independent pathway. Neither LDH release by Y. enterocolitica nor the phagocytic activity of the macrophages was altered by Nod2 deficiency. An in vivo experiment showed that bacterial clearance ability and production of IL-6 and KC in serum were comparable in WT and Nod2-deficient mice infected with Y. enterocolitica. These findings suggest that Nod2 may not be critical for initiating the innate immune response of macrophages against Yersinia infection.     

Nod proteins link bacterial sensing and autophagy

Autophagy is one of the main cellular degradation systems in eukaryotes, being responsible for the elimination of long-lived proteins and damaged organelles. Besides its well-documented role as a housekeeping mechanism, autophagy has recently caught the attention of groups working in the fields of microbiology and immunology, especially those working in innate immunity. In particular, the highly specific segregation and degradation of intracellular bacteria by the autophagic machinery was a matter of great interest. However, it was still unclear how the autophagy machinery could target intracellular bacteria with such specificity. We have recently analyzed the role of the intracellular peptidoglycan (PG) receptors Nod1 and Nod2 as a link between intracellular bacterial sensing and the induction of autophagy. Our results demonstrated that Nod2 recruits the critical autophagy protein ATG16L1 to the plasma membrane during bacterial invasion and that cells expressing mutations in these proteins, two of the most important associated with Crohn disease, autophagy is defective upon infection or stimulation with the bacterial peptidoglycan fragment MDP. Thus, our findings put together two genes previously reported as independent risk factors for the development of Crohn disease and open a venue in the study of new therapies to cure the disease.     

RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs

RICK is a kinase that has been implicated in Nod1 and Nod2 signaling. In addition, RICK has been proposed to mediate TLR signaling in that its absence confers reduced responses to certain bacterial products such as LPS. We show here that macrophages and mice lacking RICK are defective in their responses to Nod1 and Nod2 agonists but exhibit unimpaired responses to synthetic and highly purified TLR agonists. Furthermore, production of chemokines induced by the bacterial dipeptide gamma-d-glutamyl-meso-diaminopimelic acid was intact in MyD88 deficient mice but abolished in RICK-null mice. Stimulation of macrophages with muramyl dipeptide, the Nod2 activator, enhanced immune responses induced by LPS, IFN-gamma, and heat-killed Listeria in wild-type but not in RICK- or Nod2-deficient macrophages. Finally, we show that the absence of RICK or double deficiency of Nod1 and Nod2 was associated with reduced cytokine production in Listeria-infected macrophages. These results demonstrate that RICK functions in innate immunity by mediating Nod1 and Nod2 signaling but not TLR-mediated immune responses.     

Identification of the critical residues involved in peptidoglycan detection by Nod1

Nod1 is an intracellular pattern recognition molecule activated following bacterial infection, which senses a specific muropeptide (l-Ala-d-Glu-meso-DAP (diaminopimelic acid); "Tri(DAP)") from peptidoglycan. Here we investigated the molecular basis of Tri(DAP) sensing by human (h) Nod1. Our results identified the domain responsible for Tri(DAP) detection in the center of the concave surface of hNod1 leucine-rich repeat domain. Amino acid residues critical for sensing define a contiguous surface patch that is largely conserved in Nod1 proteins from different species. Accordingly, the distinct specificities of human versus murine Nod1 toward muropeptide detection were also found to lie in this central cleft. Several splicing variants of Nod1 lacking repeats 7-9 have been characterized recently, the relative balance of which is thought to correlate with the onset of asthma or inflammatory bowel disease. We demonstrated that these isoforms failed to transduce NF-kappaB activation upon muropeptide stimulation. This study provided insights into the molecular mechanisms responsible for the detection of bacterial peptidoglycan by Nod1 and suggested that defects in Nod1-dependent peptidoglycan sensing may contribute to elicit certain inflammatory disorders.     

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

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 or D-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.     

Reciprocal cross-talk between Nod2 and TAK1 signaling pathways

Mutations in the leucine-rich repeat (LRR) domain of Nod2 have been implicated in the pathogenesis of Crohn's disease, yet the function of Nod2 and regulation of the Nod2 pathway remain unclear. In this study, we determined that mitogen-activated protein kinase kinase transforming growth factor (TGF)-beta-activated kinase 1 (TAK1) interacts with Nod2 and is required for Nod2-mediated NF-kappaB activation. The dominant negative form of TAK1 abolished muramyl dipeptide-induced NF-kappaB activation in Nod2-expressing cells. Nod2, acting in a reciprocal manner, inhibited TAK1-induced NF-kappaB activation in RICK-deficient embryonic fibroblasts. Nod2 appears to interact with TAK1 through its LRR region to exert its inhibitory effect on TAK1-induced NF-kappaB activation. Further, wild-type LRR more effectively suppressed NF-kappaB activation induced by TAK1 than LRR with a 3020insC mutation. Considered together, these findings demonstrate a critical role for TAK1 in Nod2-mediated innate immune responses and reveal a novel function for Nod2 in the regulation of the TAK1 signaling pathway.     

Cross-tolerization between Nod1 and Nod2 signaling results in reduced refractoriness to bacterial infection in Nod2-deficient macrophages

Nod2 is an intracellular innate immune receptor that plays a role in host defense and susceptibility to inflammatory disease. We show in this study that macrophages rendered refractory to TLR4 and Nod2 signaling by exposure to LPS and muramyl dipeptide (MDP) exhibit impaired TNF-alpha and IL-6 production in response to pathogenic Listeria monocytogenes and Yersinia pseudotuberculosis as well as commensal bacteria including Escherichia coli and Bacteroides fragilis. Surprisingly, Nod2 deficiency was associated with impaired tolerization in response to pathogenic and commensal bacteria. Mechanistically, reduced tolerization of Nod2-null macrophages was mediated by recognition of bacteria through Nod1 because it was abolished in macrophages deficient in Nod1 and Nod2. Consistently, Nod2-null macrophages tolerant to LPS and MDP showed enhanced production of TNF-alpha and IL-6 as well as increased NF-kappaB and MAPK activation in response to the dipeptide KF1B, the Nod1 agonist. Furthermore, reduced tolerization of Nod2-deficient macrophages in response to bacteria was abolished when mutant macrophages were also rendered tolerant to the Nod1 ligand. Finally, MDP stimulation induced refractoriness not only to MDP, but also to iE-DAP stimulation, providing a mechanism to explain the reduced tolerization of Nod2-deficient macrophages infected with bacteria. These results demonstrate that cross-tolerization between Nod1 and Nod2 leads to increase recognition of both pathogenic and commensal bacteria in Nod2-deficient macrophages pre-exposed to microbial ligands.     

NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2

Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frame-shift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-kappaB inhibitor Helenalin as well as a super-repressor form of the NF-kappaB inhibitor IkappaB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated down-regulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.     

Role of nod2 in the response of macrophages to toll-like receptor agonists

Nod2 (CARD15) is a macrophage-specific protein containing two CARD domains, a large nucleotide binding domain and leucine-rich repeats. Human genetic studies have linked mutations in NOD2/CARD15 with Crohn's disease, although the mechanisms involved are unknown. However, Nod2 has been proposed to directly bind bacterial lipopolysaccharide (LPS) and subsequently act as an activator of NF-kappaB via the association of the CARD domains with Rip2/RICK/CARDIAK. This is hypothesized to constitute a pathogen recognition pathway distinct from Toll-like receptor 4-mediated recognition of LPS. Using targeted mutagenesis, we introduced a mutation to delete the CARD domains of mouse Nod2. Mice lacking Nod2 were indistinguishable from controls and showed no signs of intestinal pathology. Macrophages responded normally to multiple Toll-like receptor agonists in terms of NF-kappaB target activation, mitogen-activated protein kinase activation, and cytokine secretion. However, Nod2(-/-) mice were significantly protected in endotoxin challenge experiments, and Nod2(-/-) macrophages were refractory to muramyl dipeptide stimulation. These results argue that Nod2 does not play an essential, nonredundant role in the response of macrophages to bacterial products but rather plays unexpected roles in regulating systemic responses to pathogens.     

Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components

The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock.     

Distinct roles for Nod2 protein and autocrine interleukin-1beta in muramyl dipeptide-induced mitogen-activated protein kinase activation and cytokine secretion in human macrophages

Elucidating factors regulating Crohn's disease-associated nucleotide-binding oligomerization domain 2 (Nod2) responses is critical to understanding the mechanisms of intestinal immune homeostasis. Stimulation of primary monocyte-derived macrophages by muramyl dipeptide (MDP), a component of bacterial peptidoglycan and specific Nod2 ligand, produces cytokines, including IL-1β. We found that IL-1β blockade profoundly inhibits MDP-induced cytokine production in human monocyte-derived macrophages, demonstrating a key role for IL-1β autocrine secretion in Nod2-mediated responses. Importantly, although MAPK activation has previously been attributed directly to Nod2 signaling, we determined that the IL-1β autocrine loop is responsible for the majority of MDP-induced MAPK activation. Because the critical effects of IL-1β autocrine secretion on MAPK activation are observed as early as 10 min after Nod2 stimulation, we hypothesized that secretion of IL-1β from preexisting intracellular pro-IL-1β stores is necessary for optimal MDP-mediated cytokine induction. Consistently, we detected IL-1β secretion within 10 min of MDP treatment. Moreover, caspase-1 inhibition significantly attenuates MDP-mediated early MAPK activation. Importantly, selective JNK/p38 activation is sufficient to rescue the decreased cytokine secretion during Nod2 stimulation in the absence of autocrine IL-1β. Finally, we found that the IL-1β autocrine loop significantly enhances responses by a broad range of pattern recognition receptors. Taken together, MDP stimulation activates Nod2 to process and release preexisting pro-IL-1β stores in a caspase-1-dependent fashion; this secreted IL-1β, in turn, contributes to the majority of MDP-initiated MAPK activation and leads to subsequent cytokine secretion. Our findings clarify mechanisms of IL-1β contributions to Nod2 responses and elucidate the dominant role of IL-1β in MDP-initiated MAPK and cytokine secretion.     

Innate immune recognition of microbes through Nod1 and Nod2: implications for disease

Nod1 and Nod2 are cytosolic proteins involved in intracellular recognition of microbes and their products. Recently, it was shown that these proteins recognize different moieties of bacterial peptidoglycan (PGN) mediating non-specific pathogen resistance and possibly generating signals for the adaptive immune response. Moreover, mutations in the gene encoding Nod2 are associated with increased susceptibility to chronic inflammatory disorders.     

Nod1 participates in the innate immune response to Pseudomonas aeruginosa

The mammalian innate immune system recognizes pathogen-associated molecular patterns through pathogen recognition receptors. Nod1 has been described recently as a cytosolic receptor that detects specifically diaminopimelate-containing muropeptides from Gram-negative bacteria peptidoglycan. In the present study we investigated the potential role of Nod1 in the innate immune response against the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that Nod1 detects the P. aeruginosa peptidoglycan leading to NF-kappaB activation and that this activity is diminished in epithelial cells expressing a dominant-negative Nod1 construct or in mouse embryonic fibroblasts from Nod1 knock-out mice infected with P. aeruginosa. Finally, we demonstrate that the cytokine secretion kinetics and bacterial killing are altered in Nod1-deficient cells infected with P. aeruginosa in the early stages of infection.