Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family –proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.     

Caspase-12 Silencing Attenuates Inhibitory Effects of Cigarette Smoke Extract on NOD1 Signaling and hBDs Expression in Human Oral Mucosal Epithelial Cells

Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human β defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells.     

Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.     

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.     

NOD2 mutation and mice: no Crohn's disease but many lessons to learn

Many patients with ileal Crohn's disease, a chronic intestinal inflammation, carry mutations in the gene encoding NOD2 (CARD15), but the mechanistic details of how this mutation leads to disease are not fully understood. NOD2 is expressed in antigen-presenting cells and Paneth cells, which are secretory epithelial cells of the small intestine. Two complementary studies using genetically engineered murine models help to explain the association of NOD2 malfunction and mucosal disease. One study observes a dysregulation of proinflammatory responses, suggesting that the most common NOD2 mutation in humans results in a gain of function. The other study determined that NOD2-null mutations impair the Paneth-cell antimicrobial response, which is consistent with recent findings in humans. Together, these studies fuel optimism that new therapeutic directions might emerge to better treat this severe mucosal disease.     

NOD1 and NOD2 receptors in mrigal (Cirrhinus mrigala): Inductive expression and downstream signalling in ligand stimulation and bacterial infections

Nucleotide binding and oligomerization domain (NOD)1 and NOD2 are important cytoplasmic pattern recognition receptors (PRRs) and key members of the NOD-like receptor (NLR) family. They sense a wide range of bacteria or their products and play a key role in inducing innate immunity. This report describes the role of NOD1 and NOD2 receptors signalling in innate immunity in the Indian major carp, mrigal (Cirrhinus mrigala). Tissue-specific expression analysis of NOD1 and NOD2 genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs/tissues. In the untreated fish, the highest expression of NOD1 and NOD2 was detected in liver and blood, respectively. Stimulation with NOD1- and NOD2-specific ligands, i.e. iE-DAP and MDP, activated NOD1 and NOD2 receptor signalling in vivo and in vitro resulting in significant (p<0.05) induction of downstream signalling molecule RICK, and the effector molecules IL-1β, IL-8 and IFN-γ in the treated group as compared to their controls. In response to both Gram-positive and Gram-negative bacterial infections, NOD1 and NOD2 receptors signalling were activated and IL-1β, IL-8 and IFN-γ were induced. These findings highlight the important role of NOD receptors in eliciting innate immune response during the pathogenic invasion to the fish.     

Modulation of the NOD-like receptors NOD1 and NOD2: A chemist’s perspective

The innate immune system is the body’s first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist’s perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.     

Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection

Host immune response influences the clinical outcome of Helicobacter pylori infection leading to ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A genetic risk profile for gastric cancer has been identified, but genetic susceptibility to develop MALT lymphoma is still unclear. We investigated the role of NOD1 and NOD2 as intracellular recognition molecules for pathogen-associated molecules in H. pylori infection in vitro and analysed the influence of single nucleotide polymorphisms on susceptibility to ulcer disease and MALT lymphoma. Expression of NOD1 and NOD2 significantly sensitized HEK293 cells to H. pylori-induced NF-kappaB activation in a cag pathogenicity island (cagPAI)-dependent manner. In cells carrying the Crohn-associated NOD2 variant R702W the NF-kappaB response was significantly diminished. NOD1/NOD2 expression levels were induced in the gastric epithelium in H. pylori-positive patients. No mutations were found to be associated with gastritis or gastric ulcer development. However, the R702W mutation in the NOD2/CARD15 gene was significantly associated with gastric lymphoma. Carrier of the rare allele T had a more than doubled risk to develop lymphoma than controls [odds ratio (OR): 2.4, 95% confidence interval (CI): 1.2-4.6; P < 0.044]. H. pylori-induced upregulation of NOD1 and NOD2 in vivo may play a critical role in the recognition of this common pathogen. A missense mutation in the leucine-rich region of CARD15 is associated with gastric lymphoma.     

Insights into the molecular basis of the NOD2 signalling pathway

The cytosolic pattern recognition receptor NOD2 is activated by the peptidoglycan fragment muramyl dipeptide to generate a proinflammatory immune response. Downstream effects include the secretion of cytokines such as interleukin 8, the upregulation of pro-interleukin 1β, the induction of autophagy, the production of antimicrobial peptides and defensins, and contributions to the maintenance of the composition of the intestinal microbiota. Polymorphisms in NOD2 are the cause of the inflammatory disorder Blau syndrome and act as susceptibility factors for the inflammatory bowel condition Crohn''s disease. The complexity of NOD2 signalling is highlighted by the observation that over 30 cellular proteins interact with NOD2 directly and influence or regulate its functional activity. Previously, the majority of reviews on NOD2 function have focused upon the role of NOD2 in inflammatory disease or in its interaction with and response to microbes. However, the functionality of NOD2 is underpinned by its biochemical interactions. Consequently, in this review, we have taken the opportunity to address the more ‘basic’ elements of NOD2 signalling. In particular, we have focused upon the core interactions of NOD2 with protein factors that influence and modulate the signal transduction pathways involved in NOD2 signalling. Further, where information exists, such as in relation to the role of RIP2, we have drawn comparison with the closely related, but functionally discrete, pattern recognition receptor NOD1. Overall, we provide a comprehensive resource targeted at understanding the complexities of NOD2 signalling.     

Crystal Structure of a Complex of NOD1 CARD and Ubiquitin

The Caspase Recruitment Domain (CARD) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance.     

TRIM27 negatively regulates NOD2 by ubiquitination and proteasomal degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.     

Identification of indole scaffold-based dual inhibitors of NOD1 and NOD2

NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target in this respect. To gain additional insight into the structure–activity relationships of NOD1 inhibitors, a series of novel analogs has been designed and synthesized and then screened for their NOD1-inhibitory activity. Selected compounds were also investigated for their NOD2-inhibitory activity. Two compounds 4 and 15, were identified as potent mixed inhibitors of NOD1 and NOD2, displaying a balanced inhibitory activity on both targets in the low micromolar range. The results obtained have enabled a deeper understanding of the structural requirements for NOD1 and NOD2 inhibition.     

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.     

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells

Gram‐negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1‐dependent manner to Gram‐negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram‐negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram‐negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF‐κB and NOD1‐dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1‐dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice‐induced innate and adaptive immune responses via a NOD1‐dependent but TLR‐independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram‐negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.     

Identification of Selective Small Molecule Inhibitors of the Nucleotide-Binding Oligomerization Domain 1 (NOD1) Signaling Pathway

NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.     

The effects of NOD activation on adipocyte differentiation

Objective:

Obesity is associated with chronic inflammation. Toll‐like receptors (TLR) and NOD‐like receptors (NLR) are two families of pattern recognition receptors that play important roles in immune response and inflammation in adipocytes. It has been reported that TLR4 and TLR2 activation induce proinflammatory changes that impair adipocyte differentiation. However, the effects of activation of NOD1 and NOD2, the two prominent members of NLR, on adipocyte differentiation have not been studied.

Design and Methods:

3T3‐L1 and human adipose‐derived stem cells were tested for adipocyte differentiation in the presence or absence of NOD ligand. Adipocyte differentiation was evaluated by the adipocyte markers gene expression and Oil Red O staining for lipid accumulation.

Results:

Activation of NOD1, but not NOD2, by a synthetic ligand dose‐dependently suppressed 3T3‐L1 adipocyte differentiation as revealed by Oil Red O stained cell morphology, lipid accumulation, and attenuated gene expression of adipocyte markers (PPARγ, C/EBPα, SCD, FABP4, Adiponectin). Activation of NOD1, but not NOD2, induced NF‐κB activation, which correlated with their abilities to suppress ligand‐induced PPARγ transaction. Moreover, the suppressive effect by NOD1 activation was reversed by IκB super‐repressor which blocks NF‐κB activation. The suppression by NOD1 ligand C12‐iEDAP on adipocyte differentiation was reversed by small RNA interference targeting NOD1, demonstrating the specificity of NOD1 activation. In contrast, activation of NOD1 and NOD2 both significantly suppressed adipocyte differentiation of human adipose‐derived adult stem cells, demonstrating the species specific effects of NOD activation. In contrast to enhanced leptin mRNA by LPS and TNFα, NOD1 activation suppressed leptin mRNA in adipocytes, suggesting the differential effects of NOD1 activation in adipocytes.

Conclusions:

Overall, our results suggest that NOD1 represents a novel target for adipose inflammation in obesity.     

Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation

Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are intracellular pattern recognition receptors (PRRs) of the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family involved in innate immune responses. Their centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses. Mutations affecting key residues of the ATPase domain of NOD2 are linked to severe auto-inflammatory diseases, such as Blau syndrome and early-onset sarcoidosis. By mutational dissection of the ATPase domain function, we show that the NLR-specific extended Walker B box (DGhDE) can functionally replace the canonical Walker B sequence (DDhWD) found in other ATPases. A requirement for an intact Walker A box and the magnesium-co-ordinating aspartate of the classical Walker B box suggest that an initial ATP hydrolysis step is necessary for activation of both NOD1 and NOD2. In contrast, a Blau-syndrome associated mutation located in the extended Walker B box of NOD2 that results in higher autoactivation and ligand-induced signaling does not affect NOD1 function. Moreover, mutation of a conserved histidine in the NACHT domain also has contrasting effects on NOD1 and NOD2 mediated NF-κB activation. We conclude that these two NLRs employ different modes of activation and propose distinct models for activation of NOD1 and NOD2.