GRIM-19 interacts with nucleotide oligomerization domain 2 and serves as downstream effector of anti-bacterial function in intestinal epithelial cells

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-kappaB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.     

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.     

Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition

Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.     

L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.     

ASC-mediated NF-kappaB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP

ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several Apaf-1-like molecules, including CARD12/Ipaf, cryopyrin/PYPAF1, PYPAF5, PYPAF7, and NALP1. To characterize the signaling pathway mediated by ASC, we established cell lines in which muramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited cell autonomous NF-kappaB activation. This response required caspase-8, and was suppressed by CLARP/FLIP, an inhibitor of caspase-8. The catalytic activity of caspase-8 was required for the ASC-mediated NF-kappaB activation when caspase-8 was expressed at an endogenous level, although it was not essential when caspase-8 was overexpressed. In contrast, FADD, the adaptor protein linking Fas and caspase-8, was not required for this response. Consistently, ASC recruited caspase-8 and CLARP but not FADD and Nod2 to its speck-like aggregates in cells. Finally, muramyl dipeptide induced interleukin-8 production in MAIL8 cells. These results are the first to indicate that caspase-8 plays an important role in the ASC-mediated NF-kappaB activation, and that the ASC-mediated NF-kappaB activation actually induces physiologically relevant gene expression.     

Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2

The nucleotide-binding domain and leucine-rich repeat containing protein NOD2 serves as a cytoplasmic pattern recognition molecule sensing bacterial muramyl dipeptide (MDP), whereas TLR2 mediates cell surface recognition of bacterial lipopeptides. In this study, we show that NOD2 stimulation activated Rac1 in human THP-1 cells and primary human monocytes. Rac1 inhibition or knock-down, or actin cytoskeleton disruption increased MDP-stimulated IL-8 secretion and NF-kappaB activation, whereas TLR2-dependent cell activation was suppressed by Rac1 inhibition. p21-activated kinase [Pak]-interacting exchange factor (beta-PIX) plays a role in this negative regulation, because knock-down of beta-PIX also led to increased NOD2-mediated but not TLR2-mediated IL-8 secretion, and coimmunoprecipitation experiments demonstrated that NOD2 interacted with beta-PIX as well as Rac1 upon MDP stimulation. Moreover, knock-down of beta-PIX or Rac1 abrogated membrane recruitment of NOD2, and interaction of NOD2 with its negative regulator Erbin. Overall, our data indicate that beta-PIX and Rac1 mediate trafficking and negative regulation of NOD2-dependent signaling which is different from Rac1's positive regulatory role in TLR2 signaling.     

The c-Jun N-terminal Kinase (JNK)-binding Protein (JNKBP1) Acts as a Negative Regulator of NOD2 Protein Signaling by Inhibiting Its Oligomerization Process

NOD2 is one of the best characterized members of the cytosolic NOD-like receptor family. NOD2 is able to sense muramyl dipeptide, a specific bacterial cell wall component, and to subsequently induce various signaling pathways leading to NF-κB activation and autophagy, both events contributing to an efficient innate and adaptive immune response. Interestingly, loss-of-function NOD2 variants were associated with a higher susceptibility for Crohn disease, which highlights the physiological importance of proper regulation of NOD2 activity. We performed a biochemical screen to search for new NOD2 regulators. We identified a new NOD2 partner, c-Jun N-terminal kinase-binding protein 1 (JNKBP1), a scaffold protein characterized by an N-terminal WD-40 domain. JNKBP1, through its WD-40 domain, binds to NOD2 following muramyl dipeptide activation. This interaction attenuates NOD2-mediated NF-κB activation and IL-8 secretion as well as NOD2 antibacterial activity. JNKBP1 exerts its repressor effect by disturbing NOD2 oligomerization and RIP2 tyrosine phosphorylation, both steps required for downstream NOD2 signaling. We furthermore showed that JNKBP1 and NOD2 are co-expressed in the human intestinal epithelium and in immune cells recruited in the lamina propria, which suggests that JNKBP1 contributes to maintain NOD2-mediated intestinal immune homeostasis.     

Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn's disease.     

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.     

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.     

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.     

Interaction between NOD2 and CARD9 involves the NOD2 NACHT and the linker region between the NOD2 CARDs and NACHT domain

NOD2 activation by muramyl dipeptide causes a proinflammatory immune response in which the adaptor protein CARD9 works synergistically with NOD2 to drive p38 and c-Jun N-terminal kinase (JNK) signalling. To date the nature of the interaction between NOD2 and CARD9 remains undetermined. Here we show that this interaction is not mediated by the CARDs of NOD2 and CARD9 as previously suggested, but that NOD2 possesses two interaction sites for CARD9; one in the CARD–NACHT linker and one in the NACHT itself.     

The NOD2-RICK complex signals from the plasma membrane

NOD2 plays an important role in the innate immunity of the intestinal tract. By sensing the muramyl dipeptide (MDP), a bacterial wall component, NOD2 triggers the NF-kappaB signaling pathway and promotes the release of proinflammatory cytokines such as interleukin-8. Mutations in Nod2 (1007FS, R702W, G908R) impinge on NOD2 functions and are associated with the pathogenesis of Crohn disease, a chronic inflammatory bowel disease. Although NOD2 is usually described as a cytosolic receptor for MDP, the protein is also localized at the plasma membrane, and the 1007FS mutation delocalizes NOD2 to the cytoplasm (Barnich, N., Aguirre, J. E., Reinecker, H. C., Xavier, R., and Podolsky, D. K. (2005) J. Cell Biol. 170, 21-26; McDonald, C., Chen, F. F., Ollendorff, V., Ogura, Y., Marchetto, S., Lecine, P., Borg, J. P., and Nunez, G. (2005) J. Biol. Chem. 280, 40301-40309). In this study, we demonstrate that membrane-bound versions of NOD2 and Crohn disease-associated mutants R702W and G908R are capable of responding to MDP and activating the NF-kappaB pathway from this location. In contrast, the 1007FS mutant remains unable to respond to MDP from the plasma membrane. We also show that NOD2 promotes the membrane recruitment of RICK, a serine-threonine kinase involved in NF-kappaB activation downstream of NOD2. Furthermore, the artificial attachment of RICK at the plasma membrane provokes a constitutive and strong activation of the NF-kappaB pathway and secretion of interleukin-8 showing that optimal RICK activity depends upon its subcellular localization. Finally, we show that endogenous RICK localizes at the plasma membrane in the THP1 cell line. Thus, our data suggest that NOD2 is responsible for the membrane recruitment of RICK to induce a regulated NF-kappaB signaling and production of proinflammatory cytokines.     

NOD2 activation induces oxidative stress contributing to mitochondrial dysfunction and insulin resistance in skeletal muscle cells

Nucleotide-binding oligomerization domain protein-2 (NOD2) activation in skeletal muscle cells has been associated with insulin resistance, but the underlying mechanisms are not yet clear. Here we demonstrate the implication of oxidative stress in the development of mitochondrial dysfunction and insulin resistance in response to NOD2 activation in skeletal muscle cells. Treatment with the selective NOD2 ligand muramyl dipeptide (MDP) increased mitochondrial reactive oxygen species (ROS) generation in L6 myotubes. MDP-induced ROS production was associated with increased levels of protein carbonyls and reduction in citrate synthase activity, cellular ATP level, and mitochondrial membrane potential, as well as altered expression of genes involved in mitochondrial function and metabolism. Antioxidant treatment attenuated MDP-induced ROS production and restored mitochondrial functions. In addition, the presence of antioxidant prevented NOD2-mediated activation of MAPK kinases and the inflammatory response. This was associated with reduced serine phosphorylation of insulin receptor substrate-1 (IRS-1) and improved insulin-stimulated tyrosine phosphorylation of IRS-1 and downstream activation of Akt phosphorylation. These data indicate that oxidative stress plays a role in NOD2 activation-induced inflammatory response and that MDP-induced oxidative stress correlates with impairment of mitochondrial functions and induction of insulin resistance in skeletal muscle cells.     

Interleukin-1beta induces macrophage inflammatory protein-1beta expression in human hepatocytes

The investigation of factors that regulate expression of CC-chemokines, the important mediators in immune responses and inflammation processes, has an important significance in understanding the immunopathogenesis of liver diseases. We examined the role of interleukin-1beta (IL-1beta), a multifunctional cytokine, in regulating the expression of macrophage inflammatory protein (MIP)-1beta in human hepatocytes (Huh7 and HepG2). IL-1beta significantly enhanced MIP-1beta expression in these cells at both the mRNA and protein levels. Cytokine-enriched supernatants from monocyte-derived macrophage (MDM) cultures also induced MIP-1beta expression. IL-1beta is responsible for MDM supernatant-mediated up-regulation of MIP-1beta since the antibody to IL-1beta abolished MDM supernatant action. Investigation of the mechanism involved in MIP-1beta induction by IL-1beta showed that IL-1beta activated the nuclear factor kappa B (NF-kappaB) promoter in Huh7 cells. In addition, caffeic acid phenethyl ester (CAPE), a specific inhibitor of the activation of NF-kappaB, not only abolished IL-1beta-mediated NF-kappaB promoter activation, but also blocked IL-1beta-induced MIP-1beta expression. These observations suggest that IL-1beta-mediated up-regulation of MIP-1beta production in the hepatic cells may contribute a critical mechanism for continuous recruitment of inflammatory cell to liver and maintenance of inflammation.     

Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.     

PAN1/NALP2/PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages

Genes encoding proteins with PYRIN/PAAD/DAPIN domains, a nucleotide binding fold (NACHT), and leucine rich repeats have recently been recognized as important mediators in autoimmune inflammatory disorders. Here we characterize the expression and function of a member of the PYRIN and NACHT domain (PAN) family, PAN1 (also known as NALP2 and PYPAF2). PAN1 protein expression is regulated by lipopolysaccharide (LPS) and interferons (IFNbeta and IFNgamma) in THP-1 macrophage cells. In gene transfection studies PAN1 manifests an inhibitory influence on NF-kappaB activation induced by various pro-inflammatory stimuli, including tumor necrosis factor TNFalpha and interleukin-1beta (IL-1beta). Gene transfer-mediated elevations in PAN1 protein also suppressed activation of IkappaB kinases induced by inflammatory cytokines. Conversely, reducing endogenous levels of PAN1 using small interfering RNA enhanced LPS-induced production of ICAM-1 (intercellular adhesion molecule 1), an NF-kappaB-dependent gene. We also show here that PAN1 binds via its PYRIN domain to ASC, an adapter protein involved in caspase-1 activation. This binding is disrupted by mutation of the alpha1 helix of ASC. In gene transfer experiments PAN1 enhances caspase-1 activation and IL-1beta secretion in collaboration with ASC. Conversely, reducing endogenous levels of PAN1 using small interfering RNA significantly reduced LPS-induced secretion of IL-1beta in monocytes. We propose that PAN1 functions as a modulator of the activation of NF-kappaB and pro-caspase-1 in macrophages.