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Key Points Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a newly described family of intracellular sensors of microbial infections and danger signals. NLRs detect microbial motifs such as bacterial peptidoglycan (sensed by NOD1 and NOD2) or bacterial flagellin (sensed by NLR family, CARD-domain-containing 4 (NLRC4; also known as IPAF)) and NLR family, apoptosis inhibitory protein 5 (NAIP5). NLRs also sense danger signals, including uric acid, K + efflux, extracellular ATP, silica, asbestos and β-amyloid peptide through NLR family, pyrin domain-containing 3 (NLRP3). NLRs trigger innate immune responses by inducing signalling pathways, such nuclear factor-κB, mitogen-activated protein kinases, and the caspase 1 inflammasome. This results in the activation of inflammatory cytokines and/or chemokines. NLRs also work in synergy with Toll-like receptors to potentiate signal transduction pathways. Mutations in several NLR genes are associated with autoinflammatory disorders, including NOD2 (associated with Crohn's disease and Blau syndrome), NLRP3 (associated with Muckle–Wells syndrome, chronic infantile neurologic cutaneous and articular syndrome, and familial cold urticaria), NOD1 (associated with asthma, allergy and atopic eczema) and NLRP1 (associated with vitiligo). NLRs have a crucial role in the detection of molecules that were initially known as adjuvants, such as muramyl peptides and complete Freund's adjuvant (sensed by NOD1 and NOD2) and aluminium hydroxide (sensed by NLRP3). On detection of these molecules, NLRs shape the immune response to antigens, highlighting the link between NLRs and adaptive immunity. Because of their importance in innate immunity and adjuvanticity, NLRs and NLR-triggered pathways are promising target candidates for therapeutic strategies against autoinflammatory disorders. The recent development of interleukin 1-specific strategies against gout and Muckle–Wells syndrome illustrates the translation of NLR basic research into clinical practice. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular sensors that have key roles in innate immunity and inflammation. This Review discusses the effect that research on NLRs will have on vaccination, treatment of chronic inflammatory disorders and acute bacterial infections. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular sensors that have key roles in innate immunity and inflammation. Whereas some NLRs — including NOD1, NOD2, NAIP (NLR family, apoptosis inhibitory protein) and NLRC4 — detect conserved bacterial molecular signatures within the host cytosol, other members of this family sense 'danger signals', that is, xenocompounds or molecules that when recognized alert the immune system of hazardous environments, perhaps independently of a microbial trigger. In the past few years, remarkable progress has been made towards deciphering the role and the biology of NLRs, which has shown that these innate immune sensors have pivotal roles in providing immunity to infection, adjuvanticity and inflammation. Furthermore, several inflammatory disorders have been associated with mutations in human NLRgenes. Here, we discuss the effect that research on NLRs will have on vaccination, treatment of chronic inflammatory disorders and acute bacterial infections.

Mutations in NOD2—a bacterial sensor in dendritic cells—and mutations in genes related to autophagosome function have been linked to Crohn's disease. Alison Simmons and her colleagues link these susceptibility genes in a single functional pathway. They show that triggering of NOD2 induces autophagy, resulting in increased bacterial antigen presentation on the surface of the dendritic cell. They also show that this process goes awry in dendritic cells expressing the susceptibility variants from individuals with Crohn's disease. Nucleotide-binding oligomerization domain–containing-2 (NOD2) acts as a bacterial sensor in dendritic cells (DCs), but it is not clear how bacterial recognition links with antigen presentation after NOD2 stimulation. NOD2 variants are associated with Crohn's disease, where breakdown in self-recognition of commensal bacteria leads to gastrointestinal inflammation. Here we show NOD2 triggering by muramyldipeptide induces autophagy in DCs. This effect requires receptor-interacting serine-threonine kinase-2 (RIPK-2), autophagy-related protein-5 (ATG5), ATG7 and ATG16L1 but not NLR family, pyrin domain containing-3 (NALP3).We show that NOD2-mediated autophagy is required for both bacterial handling and generation of major histocompatibility complex (MHC) class II antigen-specific CD4 + T cell responses in DCs. DCs from individuals with Crohn's disease expressing Crohn's disease—associated NOD2 or ATG16L1 risk variants are defective in autophagy induction, bacterial trafficking and antigen presentation. Our findings link two Crohn's disease–associated susceptibility genes in a single functional pathway and reveal defects in this pathway in Crohn's disease DCs that could lead to bacterial persistence via impaired lysosomal destruction and immune mediated clearance.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular sensor for small peptides derived from the bacterial cell wall component, peptidoglycan. Recent studies have uncovered unexpected functions of NOD2 in innate immune responses such as induction of type I interferon and facilitation of autophagy; moreover, they have disclosed extensive cross-talk between NOD2 and Toll-like receptors, which has an indispensable role both in host defense against microbial infection and in the development of autoimmunity. Of particular interest, polymorphisms of CARD15 encoding NOD2 are associated with Crohn's disease and other autoimmune states such as graft vs. host disease. In this review, we summarize recent findings regarding normal functions of NOD2 and discuss the mechanisms by which NOD2 polymorphisms associated with Crohn's disease lead to intestinal inflammation.

Key Points Toll-like receptors (TLRs) and NOD-like receptors (NLRs) sense pathogen-associated molecules and trigger effector pathways to facilitate the destruction and removal of the pathogen. Post-translational modifications, such as ubiquitylation, have important roles in regulating the activities and the levels of signalling molecules in these pathways. The Pellino family (Pellino 1, Pellino 2 and Pellino 3) were initially identified as interleukin-1 receptor-associated kinase (IRAK)-interacting proteins with E3 ubiquitin ligase activity. They are activated by kinases such as IRAKs and TANK-binding kinase 1 (TBK1) and can promote ubiquitylation of their upstream kinases. Pellino 1 regulates TIR domain-containing adaptor protein inducing IFNβ (TRIF)-dependent signalling in the TLR3 and TLR4 pathways by promoting the ubiquitylation of the kinase receptor-interacting protein 1 (RIP1) and the downstream activation of nuclear factor-κB (NF-κB), and by mediating the TRIF-dependent induction of type I interferons (IFNs). In addition, in microglial cells, Pellino 1 can mediate the myeloid differentiation primary-response protein 88 (MYD88)-dependent activation of mitogen-activated protein kinase (MAPK) pathways to trigger neuroinflammation. Pellino 1 also functions as a negative regulator of T cell activation to ensure self tolerance is maintained and to avoid the development of autoimmunity. Pellino 3 negatively regulates the TLR3-induced expression of type I IFNs by ubiquitylating TNF receptor-associated factor 6 (TRAF6) to inhibit the downstream activation of IFN-regulatory factor 7 (IRF7). In addition, Pellino 3 mediates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) signalling and regulates intestinal inflammation by functioning as the E3 ubiquitin ligase for the RIP2 kinase. Pellino 3 is cytoprotective in response to tumour necrosis factor (TNF) challenge, as it targets RIP1 and impairs the formation of the death-inducing signalling complex (DISC). Further investigation of the role of Pellino proteins in immunity will provide a molecular and a functional understanding of their contribution to physiological and pathological conditions. Ubiquitylation is an important regulatory process that controls numerous signalling pathways, including those that drive immune responses. As described in this Review, Pellino proteins are E3 ubiquitin ligases that regulate both innate and adaptive immune signalling pathways. Pellino proteins were initially characterized as a family of E3 ubiquitin ligases that can catalyse the ubiquitylation of interleukin-1 receptor-associated kinase 1 (IRAK1) and regulate innate immune signalling pathways. More recently, physiological and molecular roles for members of the Pellino family have been described in the regulation of innate and adaptive immune responses by ubiquitylation. This Review describes the emerging roles of Pellino proteins in innate and adaptive immunity and discusses the mechanistic basis of these functions.

Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation.

ObjectiveAutophagy has recently been shown to modulate the production of pro-inflammatory cytokine production and to contribute to antigen processing and presentation through the major histocompatibility complex. Genetic variation in the autophagy gene ATG16L1 has been recently implicated in Crohn's disease pathogenesis. The mechanisms underlying this association are not yet known, although experimental models suggest an inhibitory effect of autophagy on interleukin 1β (IL-1β) responses. Here, the effect of ATG16L1 genetic variation on cytokine responses has been assessed in humans.Design and settingPeripheral blood mononuclear cells from healthy individuals and patients with Crohn's disease with different ATG16L1 genotypes were stimulated with ligands for Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerisation domain 2 (NOD2), with or without the autophagy inhibitor 3-methyladenine. Induction of cytokine production and related factors were measured at the mRNA and protein level. Furthermore, protein levels of ATG16L1 were assessed by western blot.ResultsThe present study demonstrates that cells isolated from individuals bearing the ATG16L1 Thr300Ala risk variant, which is shown to affect ATG16L1 protein expression upon NOD2 stimulation, display increased production of the pro-inflammatory cytokines IL-1β and IL-6, specifically after stimulation with NOD2 ligands. In contrast, no differences were found when cells were stimulated with TLR2 or TLR4 agonists. These findings were confirmed in two independent cohorts of volunteers and in a group of patients with Crohn's disease. The increased production could be ascribed to increased mRNA expression, while processing of pro-IL-1β by caspase-1 activation was not affected. The effect of the ATG16L1 polymorphism was abrogated when autophagy was blocked.ConclusionsThe present study is the first to link the ATG16L1 polymorphism with an excessive production of IL-1β and IL-6 in humans, which may explain the effects of this polymorphism on the inflammatory process in Crohn's disease.

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC.

Induction of nucleotide-binding oligomerization domain 2 (NOD2) and downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2) by human cytomegalovirus (HCMV) is known to up-regulate antiviral responses and suppress virus replication. We investigated the role of nucleotide-binding oligomerization domain 1 (NOD1), which also signals through RIPK2, in HCMV control. NOD1 activation by Tri-DAP (NOD1 agonist) suppressed HCMV and induced IFN-β. Mouse CMV was also inhibited through NOD1 activation. NOD1 knockdown (KD) or inhibition of its activity with small molecule ML130 enhanced HCMV replication in vitro. NOD1 mutations displayed differential effects on HCMV replication and antiviral responses. In cells overexpressing the E56K mutation in the caspase activation and recruitment domain, virus replication was enhanced, but in cells overexpressing the E266K mutation in the nucleotide-binding domain or the wild-type NOD1, HCMV was inhibited, changes that correlated with IFN-β expression. The interaction of NOD1 and RIPK2 determined the outcome of virus replication, as evidenced by enhanced virus growth in NOD1 E56K mutant cells (which failed to interact with RIPK2). NOD1 activities were executed through IFN-β, given that IFN-β KD reduced the inhibitory effect of Tri-DAP on HCMV. Signaling through NOD1 resulting in HCMV suppression was IKKα-dependent and correlated with nuclear translocation and phosphorylation of IRF3. Finally, NOD1 polymorphisms were significantly associated with the risk of HCMV infection in women who were infected with HCMV during participation in a glycoprotein B vaccine trial. Collectively, our data indicate a role for NOD1 in HCMV control via RIPK2- IKKα-IRF3 and suggest that its polymorphisms predict the risk of infection.

The intestinal microbiota regulates key host functions. It is unknown whether modulation of the microbiota can affect a genetically determined host phenotype. Polymorphisms in the Nucleotide oligomerization domain (Nod)-like receptor family confer genetic risk for inflammatory bowel disease (IBD). We investigated whether the intestinal microbiota and the probiotic strain Bifidobacterium breve NCC2950 affect intestinal barrier function and responses to intestinal injury in Nod1−/−;Nod2−/− mice.MethodsSpecific pathogen-free (SPF) Nod1−/−;Nod2−/− mice and mice gnotobiotically derived with altered Schaedler flora (ASF) biota were used. SPF Nod1+/−;Nod2+/−littermates (generated by crossing SPF Nod1−/−;Nod2−/− and germ-free C57BL/6 mice) and ASF Nod1+/−;Nod2+/− mice were used as controls. SPF mice were gavaged daily with 109-CFU B. breve for 14 days before colitis induction. Denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR) were used to assess microbiota composition. Intestinal permeability was assessed by in vitro and in vivo techniques. Expressions of epithelial apical junction proteins, mucin, and antimicrobial proteins were assessed by quantitative reverse-transcription PCR (qRT-PCR) and immunofluorescence. Responses to intestinal injury were investigated using an acute experimental model of colitis.ResultsUnder SPF conditions, Nod1−/−;Nod2−/− mice had increased paracellular permeability, decreased E-cadherin, and lower colonic antimicrobial RegIII-γ expression compared to Nod1+/−;Nod2+/− littermate controls. These changes were associated with increased susceptibility to colitis. ASF colonization or B. breve supplementation normalized RegIII-γ expression and decreased susceptibility to dextran sodium sulfate (DSS) colitis in Nod1−/−;Nod2−/− mice.ConclusionsThe intestinal microbiota influences colitis severity in Nod1−/−;Nod2−/− mice. The results suggest that colonization strategies with defined commensals or exogenous specific probiotic therapy may prevent intestinal inflammation in a genetically predisposed host. (Inflamm Bowel Dis 2012)

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3- and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway.