Nod-like proteins in immunity, inflammation and diseaseJörg H Fritz1, Richard L Ferrero2, Dana J Philpott1 & Stephen E Girardin3

The intracellular Nod-like proteins or receptors are a family of sensors of intracellularly encountered microbial motifs and ‘danger signals’ that have emerged as being critical components of the innate immune responses and of inflammation in mammals. Several Nod-like receptors, including Nod1, Nod2, NALP3, Ipaf and Naip, are strongly associated with host responses to intracellular invasion by bacteria or the intracellular presence of specific bacterial products. An additional key function of Nod-like receptors is in inflammatory conditions, which has been emphasized by the identification of several different mutations in the genes encoding Nod1, Nod2 and NALP3 that are associated with susceptibility to inflammatory disorders. Those and other issues related to the Nod-like receptor family are discussed here.

The innate immune system provides a rapid response to pathogens and, in contrast to the adaptive immune system, is immediately activated; it does not require ‘priming’ or the existence of cells with ‘memory’ of earlier encounters with pathogens. Such immediate activation of innate immunity relies on the detection by the host of conserved microbial motifs known as pathogen-associated molecular patterns (PAMPs), a diverse family of molecules including lipopolysaccharide, peptidoglycan, bacterial lipoproteins, flagellin and nucleic acid structures from bacteria and viruses1. After sensing the presence of a PAMP, host innate immune cells initiate a broad spectrum of defense mechanisms that result in the development of inflammation and host resistance to infection: the recruitment of professional phagocytes; the release of antimicrobial peptides; and the establishment of a network of cytokines, chemokines and prostanoids1. In many cases, control of infection does not require adaptive immune responses; nevertheless, when necessary, the innate immune system is also very efficient in ‘instructing’ the cellular media-tors of adaptive immunity to lead a powerful additional ‘strike force’ against an invading pathogen.

In the past decade, research in the field of innate immunity has accelerated enormously with the discovery of many microbial sen-sors called ‘pattern-recognition molecules’ (PRMs). The first family of PRMs receiving considerable interest was the Toll-like receptor (TLR) family. The TLR family of membrane-anchored proteins have exposed leucine-rich repeat (LRR) domains in an extracellular or luminal com-

partment, whereas their Toll–interleukin 1 receptor domains ‘project’ into the cytoplasm of the cell on which the TLR is expressed to interact with signaling modules required for the activation of defense responses against an invading organism2. Although TLRs sense the presence of PAMPs, in most cases, conclusive evidence that TLRs interact directly with their respective PAMPs is lacking.

Although the key function of TLRs in innate immunity is apparent and is supported by a rich and dense literature, certain observations have indicated the possibility that all features of the host response to pathogens cannot be accounted for by TLRs alone. The initial evidence for the likely existence of an intracellular mode of bacterial detection has come with the observation that only an invasive form of the enteric bacterium Shigella flexneri triggers the activation of the transcription factor NF-κB pathway in cultured epithelial cells3; subsequent studies have demonstrated that the protein Nod1 (nucleotide-binding oligo-merization domain 1’) is responsible for the NF-κB–dependent response of epithelial cells to shigella in vitro4. Other members of the Nod-like receptor (NLR) family of PRMs have been linked not only to the detec-tion of different bacterial components and toxins but also to the sens-ing so-called ‘danger signals’ released by dying or injured cells. Those findings have indicated the conclusion that NLRs are the cytoplasmic counterparts of TLRs, which in combination with TLRs constitute a ‘tour de force’ of cellular defense both at the plasma membrane and from within the cell.

The NLR familyThe mammalian NLR family is composed of more than 20 members whose defining feature is a modular domain organization of a C-terminal leucine-rich repeat (LRR) domain, a central nucleotide-binding NACHT domain and an N-terminal protein-protein–interaction domain com-posed of a CARD (caspase activation and recruitment domain), pyrin domain or Bir (baculovirus ‘inhibitor of apoptosis’ repeat) domain5 (Fig. 1). A similar domain organization is found in a large family of proteins in plants, in which the so-called ‘NBS-LRR’ (nucleotide-binding

1Department of Immunology, Medical Sciences Building, University of Toronto,

Toronto, Ontario M5S 1A8, Canada. 2Department of Microbiology, Monash

University, Clayton, 3800 Victoria, Australia. 3Department of Laboratory

Medicine and Pathobiology, Medical Sciences Building, University of Toronto,

Toronto, Ontario M5S 1A8, Canada. Correspondence should be addressed to

D.J.P. (dana.philpott@utoronto.ca).

Received 1 September; accepted 6 October; published online 16 November

2006; doi:10.1038/ni1412

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site–LRR) proteins are involved in the defense against plant pathogens6,7. NLR homologs have been predicted in silico in higher vertebrates (mam-mals, amphibians, birds, reptiles and fish) but seem to be absent from the protochordate ciona and other nonvertebrates animals, including Caenorhabditis elegans and Drosophila melanogaster. However, notably, a large family of NLRs (over 200 members) has been identified in the sea urchin, although their functions remain unknown8.

Based on the absence of reported or predicted signal peptides and transmembrane domains in their amino acid sequences, NLRs are thought to be localized to the cytosol. In most cases, antibodies recog-nizing the endogenous proteins have not been reported, and analysis of the subcellular localization of NLRs has been hampered by the fact that overexpression may result in their aggregation (as noted for Nod1; S.E.G and D.J.P, unpublished results), which can easily lead to artifacts. Nevertheless, overexpression systems have suggested that Nod2 localizes to the plasma membrane9. Notably, a truncated form of Nod2 found in some patients with Crohn disease (discussed below) remains cytosolic, suggesting that plasma membrane targeting is somehow associated with the proper function of the protein. That might be explained by the find-ing that plasma membrane localization of Nod2 is mediated through its

interaction with erbin, a member of the LRR- and PDZ(PSD-95/Discs-large/ZO-1)-containing family of proteins that localize to the basolateral side of polarized epithelial cells10,11.

NLRs as bacterial sensorsStudies have shown that several NLRs can detect bacterial molecules through mechanisms that remain poorly understood. Nevertheless, studies have indicated that many NLRs are necessary sensors of specific PAMPs. However, as mentioned above for TLRs, it is unclear if NLRs directly bind to the PAMPs they detect.

The first NLRs reported to have a direct function as intracellular PRMs were Nod1 (Card4) and Nod2 (Card15); both proteins detect distinct substructures from bacterial peptidoglycan (Fig. 2 and Table 1). Nod2 detects muramyl dipeptide, the largest molecular motif common to Gram-negative and Gram-positive bacteria12,13. In contrast, Nod1 senses peptidoglycan containing meso-diaminopimelic acid (meso-DAP), which is more commonly found in Gram-negative bacteria14,15. The optimal peptidoglycan motifs detected by human and mouse Nod1 are structures containing L-Ala-γ-D-Glu-meso-DAP and L-Ala-D-glu-mesoDAP-D-Ala, respectively, and the minimal activating structure is

Figure 1 NLRs, homologs and adaptors. The NLRs are characterized by three distinct domains: an N-terminal effector domain, which can be a pyrin domain (PYD), a CARD or a Bir domain; a central NACHT domain (NACHT stands for domain present in Naip, CIITA, HET-E (plant het product involved in vegetative incompatibility) and TP-1 (telomerase-associated protein 1)) and a C-terminal LRR domain thought to constitute the microbe-sensing portion. The NACHT domain and NAD (NACHT-associated domain) are homologous to domains of proapoptotic regulators such as Apaf-1 in mammals and CED-4 in C. elegans and to disease resistance-genes encoding R proteins in plants, such as I-2 (tomato) and RPS4 (Arabidopsis thaliana), which confer effector-triggered immunity (also called ‘host-specific immunity’) by recognizing specific elicitors7. The CARD and pyrin domain counterparts in plants are the coiled-coil (CC) and Toll–interleukin 1 receptor (TIR) domains. Bottom, adaptors involved in NLR signaling, including RIP2, ASC and CARDINAL. FIIND, ‘function-to-find’ domain; X, undefined domain; AD, activation domain; WD40,a domain consisting of repeating units of approximately 40 amino acids in length with conserved tryptophan (W) and aspartic acid (D) residues at their C-terminal regions. Naip, neuronal apoptosis inhibitor protein; CIITA, major histocompatibility complex (MHC) class II transactivator; Ipaf, ICE protease-activating factor; Apaf, apoptotic protease-activating factor; CED, Caenorhabditis elegans death protein.

Bir Bir Bir

NLR fam ily

Homologs

Ada ptors

NALP1

NALP2 –NA LP14

Nod1

Nod2

Nod3

Nod4

Nod5

Ipaf

Nai p

CII TA

Apa f-1

CED-4

I-2

RPS4

Proapopto tic proteins

R prot eins

RIP2

ASC

CARDINAL

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the dipeptide γ-D-Glu-meso-DAP14,16,17. However, specific activation of Nod1 by meso-DAP itself has been demonstrated18; the reason for the discrepancy between that observation and previously published reports remains unclear.

Distinct from the Nod proteins, NALP3 ('NACHT-LRR-PYD-con-taining protein 3'; also called cryopyrin and PYPAF1 ('pyrin-containing Apaf-1-like protein'); encoded by Cias1 (‘cold-induced autoinflamma-tory syndrome 1’)) is a pyrin domain–containing NLR that activates the caspase-1 ‘inflammasome’, leading to interleukin 1β (IL-1β) and IL-18 processing19. Studies of NALP3-deficient mice suggest that it is involved in sensing both microbial components and cellular danger signals; two of those studies report that ATP drives the activation of the inflamma-some and the release of IL-1β and IL-18 after TLR stimulation through a NALP3-dependent pathway20,21. Indeed, those results are consistent with published reports demonstrating that IL-1β release from macro-phages requires two stimuli: first, an inflammatory signal through TLR activation to induce expression of pro-IL-1β; and second, a stimulus such as ATP, to induce the inflammasome. The combined effect of these stimuli is the activation of the autocatalytic processing of procaspase 1 and, finally, active caspase-1 cleavage of pro-IL-1β to active and secreted forms22. Reports of NALP3-deficient mice have shown that these mice fail to produce IL-1β in TLR agonist–primed cells additionally stimu-lated with ATP. In particular, one report has further demonstrated that NALP3 does not directly sense ATP or its activated receptor P2X7 but instead senses the occurrence of a secondary event, probably the deple-tion of intracellular potassium, resulting from the ATP-dependent sig-naling cascade20. It has also been speculated20 that toxins from bacterial pathogens (such as listeriolysin O toxin from Listeria monocytogenes) that ‘insert’ themselves into host membranes can directly alter intra-cellular potassium concentrations and activate NALP3. However, one problem with the interpretation of those results is that the study used a listeriolysin O–negative listeria strain (which does not escape the phago-cytosis vacuole) rather than purified listeriolysin O toxin. Nevertheless, a separate study has supported their conclusion by demonstrating that aerolysin, a toxin purified from Aeromonas hydrophila, can trigger cas-pase-1 activation through a pathway dependent, in part, on the NALP3 inflammasome23. In contrast, other studies have concluded that NALP3 is not required for the TLR-induced release of IL-1β24 but instead is an intracellular sensor of bacterial RNA. If true, that conclusion would sug-gest that TLRs and NALP3 control the activation and release of IL-1β and IL-18 through distinct intracellular pathways.

In addition to Nod proteins and NALP3, Ipaf (Card12) and Naip (Birc1a; known as Naip5 in mice) are intracellular sensors that detect intracellular flagellin, leading to inflammasome activation through a TLR5-independent pathway25–28. Notably, those findings provide a mechanistic rationale for the earlier studies demonstrating a require-ment for Naip and Ipaf in the resistance to legionella and salmonella infection, two flagellated Gram-negative bacteria29–32 (discussed below). The relative contributions of Naip and Ipaf to flagellin-mediated activa-tion of caspase-1, however, remain unclear, though it is apparent that both lead to the production of proinflammatory cytokines after stimu-lation by flagellin.

Finally, Nalp1 is highly polymorphic in mice, and five alleles have been identified in a total of eighteen mice strains. Mice carrying an allele of Nalp1 encoding the protein NALP1b are susceptible to the effects of lethal toxin of Bacillus anthracis33. It has been suggested that NALP1β-mediated caspase-1 activation might be linked to macrophage susceptibility to lethal toxin33; however, the molecular basis for the sus-ceptibility and the biochemical link between incorporation of lethal toxin into host membranes and possible NALP1b-mediated signaling remains uncharacterized.

NLRs and danger signalsAlthough NALP3 has been linked to physiologically important immu-nological responses to bacteria-induced triggers (an exogenous danger signal), a separate study has found that NALP3 is also responsible for activating caspase-1 via an inflammasome in response to uric acid, a host molecule released into the extracellular milieu by necrotic cells34. Deposition of uric acid crystals in joints is associated with the inflam-matory condition known as gout35. Such findings extend the functions of NALP3 to that of detecting endogenous danger signals. In some ways, that observation is consistent with the proposed function of NALP3 as a sensor of intracellular potassium depletion (discussed above). Indeed, it is hypothesized that ATP released from necrotic cells could induce, through the activation of P2X7, the opening of potassium channels, resulting in potassium efflux in neighboring cells (although this last point has not been formally demonstrated)20. Therefore, it is possible that NALP3 is able to integrate both host-derived and microbe-derived stimuli, an idea that supports the hypothesis that NALP3, rather than being a direct sensor of a pathogen, signals ‘downstream’ of an initial sensing event mediated by a separate receptor.

The considerations presented above are reminiscent of the functions of Apaf-1 (Apaf1), a central mediator of mitochondrial-dependent apoptosis that does not, strictly speaking, belong to the NLR family but shares similarities with the NLRs in its modular domain organization. Apaf-1 has a central NACHT domain and an N-terminal CARD, yet in contrast to the NLRs, its C terminus contains a WD domain36, which is composed of repeating motifs containing conserved tryptophan (W) and aspartic acid (D) residues at their C-terminal regions. After vari-ous forms of cellular stress (endogenous or microbial), cytochrome c is released from mitochondria and is detected by Apaf-1. Cytochrome c can therefore be considered an ‘intracellular danger signal’ and therefore is conceptually related to the sensing of potassium efflux by NALP3.

NLR signalingUntil now, the well documented signaling pathways downstream of NLRs have included the NF-κB pathway, for Nod1 and Nod2, and acti-vation of the caspase-1 inflammasome, for NALPs, Ipaf and Naip. We will now review those pathways and how they contribute to immune responses and inflammation. Many studies have delineated important signaling ‘crosstalk’ between NLRs and TLRs. Yet despite its physiologi-cal importance, here we will not discuss the crosstalk between the two pathways, which has been reviewed5.

The present model of Nod-mediated signaling holds that after detec-tion of peptidoglycan, Nod1 and Nod2 rapidly form oligomers and then transiently recruit receptor-interacting protein 2 (RIP2) through CARD-CARD interactions37. The complex of Nod1-RIP2 (or Nod2-RIP2) then recruits the inhibitor of NF-κB kinase complex, which leads to activation of NF-κB through phosphorylation and ubiquitin-pro-teasome–dependent degradation of the inhibitory cytoplasmic NF-κB chaperone IκBα. The signaling protein TRIP6 and the NF-κB-modula-tor CARD6 positively modulate Nod1- and Nod2-dependent signaling to NF-κB38,39. Nod2 activation is also modulated by cell death regulatory protein GRIM19, the kinase TAK1 and the basolateral membrane pro-tein erbin10,11,40,41. Because signaling in response to tumor necrosis fac-tor (TNF), IL-1β and TLRs also leads to NF-κB activation, it is unclear how activation of Nod proteins results in the induction of specific NF-κB-mediated innate immune programs. Some studies, however, have provided insight into that issue. First, it has been shown that Nod2-dependent recruitment of the NF-κB modulator NEMO (also called IKKγ) results in the specific ubiquitination of a lysine residue (K285) on the protein different from the residue targeted by TNF signaling (K399)42. Furthermore, a microarray-based study has analyzed the

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transcriptional profile of epithelial cells stimulated with a Nod1 agonist or with TNF43; complete analysis of the data should provide insights into the specific targets of Nod1 signaling.

Several NLRs share the common feature of being able to form the high-molecular-weight structures called ‘inflammasomes’ that result in the recruitment and activation of caspase-1, pro-IL-1β and pro-IL-18 (ref. 44). Several studies have shown that Ipaf, Naip and several NALP family members all can participate in the formation of inflam-masomes, including three distinct inflammasomes (NALP1 inflamma-somes, NALP3 inflammasomes and Ipaf inflammasomes) that form in response to the detection of specific molecular motifs (discussed above). It has been speculated that the formation of large protein complexes in a given inflammasome is sufficient to trigger caspase-1 activation (and further activation of IL-18 and IL-1β), a speculation that is consistent with the so-called ‘close proximity’ model of activation44. In most if not all cases, the recruitment of caspase-1 is accompanied by recruitment of the adaptor protein ASC that (very) likely physically links the inflam-masomes to caspase-1 via modular domain organizations composed of an N-terminal pyrin domain and a C-terminal CARD.

NLRs in innate immune responses to bacteriaAlthough there is little doubt that NLRs function as cytosolic sensors of bacterial products (Fig. 2 and Table 1), whether the NLRs directly interact with PAMPs is less apparent. The detection of invasive S. flexneri in cul-tured epithelial cells by Nod1 (ref. 4) represented the first demonstration that an NLR was required for sensing a bacterial PAMP. Subsequent stud-ies have demonstrated the importance of Nod1 in the cytosolic recogni-

tion of invasive Gram-negative bacteria such as enteroinvasive Escherichia coli45 and Chlamydia pneumonia46,47. Conversely, the related CARD-containing protein Nod2 is linked to innate immune responses to intracellular infection by the Gram-positive pathogens Streptococcus pneumoniae48 and Mycobacterium tuberculosis49. Those studies have thus established a function for NLRs, at least in vitro, as essential cytoplas-mic mediators of innate immune responses to invasive bacterial infection.

Cytosolic NLR signaling to bacterial patho-gens has also been reported to occur in the absence of direct epithelial cell invasion. The pathogens Helicobacter pylori and Pseudomonas aeruginosa, for example, trigger Nod1 signaling in epithelial cells in an invasion-independent way50,51 in addition to TLR activation in cer-tain other cell types52,53. For H. pylori, induc-tion of the Nod1-dependent signaling pathway requires the bacterial type IV secretion sys-tem53. Although the mechanism has yet to be fully elucidated, it seems that the H. pylori type IV secretion system facilitates the intracellular delivery (‘injection’) of a specific Nod1 agonist, in this case, the known human Nod1 agonist meso-DAP-containing peptidoglycan. Other microbial pathogens have since similarly been shown to use their bacterial secretion systems to initiate NLR signaling in nonepithelial host cells (Fig. 2).

NLRs have been also identified being impor-tant in macrophage-mediated detection and control of bacterial infection in vitro. As men-

tioned above, Ipaf and ASC are required for caspase-1 activation and IL-1β secretion in macrophages exposed to the Gram-negative patho-gen Salmonella typhimurium22,26. Francisella-mediated activation of the caspase-1 inflammasome, in contrast, requires ASC but not Ipaf, NALP3 or NOD2 (ref. 54), and inflammasome signaling in response to Gram-positive Staphylococcus aureus and L. monocytogenes has been shown to be mediated by NALP3 and ASC20. Notably, IPAF and NALP3 have been reported to have differing roles in caspase-1 signaling in mac-rophages: IPAF favors cell death in Salmonella infection19,54, whereas NALP3 specifically mediates proinflammatory cytokine processing in response to stimulation with TLR agonists and ATP20, 21 (see above). As mentioned above, a key initiator of caspase-1 signaling in macrophages is bacterial flagellin25–28. Cytosolic recognition of S. typhimurium and Legionella pneumophila flagellins by Ipaf (and possibly Naip5) results in the induction of macrophage cell death and IL-1β secretion25–28,32 independently of signaling by TLR5, the known extracellular sensor of bacterial flagellin.

NLRs such as Nod1 and Nod2 have been detected in diverse cell types, including astrocytes56, microglia57, osteoblasts58, myofibroblasts59 and endothelial cells60. The function of NLRs in those various cell and tis-sue sites, however, is generally not well defined, and their function in host defense against bacterial infection are even less well understood. Exposure of endothelial cells to the intracellular pathogen L. monocyto-genes triggers Nod1-dependent NF-κB activation and IL-8 secretion60. Involvement of host cell invasion in that process has been demonstrated in experiments with L. monocytogenes internalin B and listeriolysin O mutants that are less able to invade host cells or escape into the

Figure 2 NLR activation by microbes, PAMPs and danger signals in mammalian cells. Microbes and their molecular constituents (blue oval), as well as exogenous and endogenous danger-associated components (red oval), are sensed by members of the NLR family. Microbial constituents, such as the PAMPs, peptidoglycan (PGN), flagellin and bacterial RNA (green oval), are presented to the cytosol directly through release from intracellularly localized bacteria or are actively transported into host cells by type III or type IV secretion systems from microbes residing extracellularly. After detection of PAMPs by NLRs or of danger signals such as potassium efflux and uric acid by NALP3 (gray oval), the NLRs and NALP3 induce the activation of ‘downstream’ signaling that includes the NF-κB pathway (via RIP2), leading to an inflammatory response, and the caspase-1 inflammasome (yellow oval), leading to the production of IL-1β and IL-18 and/or cell death. MDP, muramyl dipeptide.

Microbial constituents Danger

Cytokine processingCell deathInflammatory

response

Flagellin

Nod1 Nod2 NALP1b NAIP5 IPAF other NALPs?Pyrin?

NALP3

?

PGNDAP MDP

BacterialRNA

RIP2

NF-κB Caspase-1

Pro-IL1βPro-IL-18

IL-1β, IL-18

P2X7

K+ efflux?

ATP NAD+

Uric acidcrystals

Membranepermeabilization

Bacillus anthracis(lethal toxin)

Salmonella(Type III secretion) Staphyllococcus

Listeria

FrancisellaViruses

Legionella(Type IV secretion)

ASC-CARDINAL

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cytoplasm, respectively. Host cell invasion is also required for induction by S. aureus or S. typhimurium of the genes expressing Nod1 and Nod2 in osteo-blasts58. Similar findings for Nod1 and Nod2 gene expression and protein synthesis have been reported for astrocytes stimulated with two Gram-negative pathogens of the central nervous system, Neisseria meningitidis and Borrelia burgdorferi56. However, the stimulation of astrocytes and osteoblasts with muramyl dipeptide, the specific agonist of Nod2, induces only weak proinflammatory responses56,58. It has been suggested that alterations in the amount of Nod protein in those tissues may be secondary to sig-naling by other pathogen-recognition molecules56,58. A similar synergy has been reported for TLR2 and Nod2 in monocyte responses to M. tuberculosis49. Although it is possible that Nod proteins may act as mediators of inflammation in bacterial infections of connective and nervous tissues, that suggestion remains speculative.

Recognition of live bacteria by NLRs upregulates host cell synthesis of proinflammatory factors such as chemokines (IL-8, MIP-2 and KC) and cytokines (IL-1β, IL-6 and IL-18)3,21,26,27,32,45,47,52–54. Those factors are thought to mediate the recruitment of inflammatory cell populations needed for bacte-rial clearance. Direct evidence for NLR-mediated host defense has come mostly from an in vivo study in which Nod1-deficient mice were reported to be more susceptible to infection by H. pylori strains with functional type IV secretion systems53. That finding was consistent with subsequent in vitro studies dem-onstrating Nod1-dependent regulation of antimi-crobial peptide (β-defensin) production in epithelial cells stimulated with H. pylori strains expressing type IV secretion systems61 (R.L.F. and D.J.P., unpublished observations). Additionally, Nod2 is reported to regu-late antimicrobial peptide synthesis as part of host defense strategies against L. monocytogenes infection in vivo62. Consistent with that function, Nod2-defi-cient mice are more sensitive than wild-type mice to oral challenge with listeria62.

In other locations and tissues, the functions of the NLRs are equivocal. Nod1-deficient mice and wild-type mice that had been inoculated intravaginally with Chlamydia muridarum, for example, show the same cytokine and chemokine secretion and bacterial clearance47. It has been suggested that redundancy between cytosolic NLRs and TLRs in vivo may have contributed to the differences noted in Nod1 depen-dency in the in vitro and in vivo models47. Similarly, despite the demonstrated importance of Ipaf in sal-monella-induced macrophage death in vitro22, Ipaf-deficient mice are no more resistant to the bacteria than are wild-type mice in vivo after oral challenge55. That same study also reported that not only Ipaf but also the caspase-1-inflammasome has no substan-tially involvement in the resistance of mice to infec-tion with salmonella by the oral route55. In contrast, however, a separate study showed that caspase-1- or ASC-deficient mice infected subcutaneously with

Table 1 Sensing of microbes, PAMPs, bacterial toxins and ‘danger signals’ by NLRsNLR Elicitor Refs.

hNod1 Microbial motifs

meso-lanthionine, meso-DAP 18

γ-D-Glu-meso-DAP (iE-DAP) 14,16

L-Ala-γ-D-Glu-meso-DAP (TriDAP) 16

D-lactyl-L-Ala-γ-Glu-meso-DAP-Gly (FK156) 17,79

Heptanoly-γ-Glu-meso-DAP-D-Ala (FK565) 79

Bacterial extracts

Bacillus speciesa 80

B. anthracis spores 81

L. pneumophila 80

S. typhimurium 80

M. tuberculosis 49

Live bacteria

S. flexneri (G– Intra Ep) 4

H. pylori (G– Extra Ep) 53

Enteroinvasive E. coli (G– Intra Ep) 45

Pseudomonas speciesb (G– Intra Ep, F) 52,82

Chlamydia speciesc (G– Intra Ep, En, F) 46,47

L. monocytogenes (G+ Intra Ep, En) 60

mNod1 Microbial motifs

GlcNAc-(anhydro)MurNAc-L-Ala-γ-D-Glu-meso-DAP-D-Ala (TCT) 17

D-lactyl-L-Ala-γ-Glu-meso-DAP-Gly (FK156) 17

Nod2 Microbial motifsMurNAc-L-Ala-D-isoGln (muramyldipeptide) 12,13

MurNAc-L-Ala-γ-D-Glu-L-Lys (M-TRILys) 16

Bacterial extracts

Bacillus speciesa 80

B. anthracis spores 81

Lactobacillus speciesd 80

Corynebacterium xerosis 80

E. coli 80

Pseudomonas speciese 80

L. pneumophila 80

M. tuberculosis 49

Live bacteria

L. monocytogenes (G+ Intra Ep) 62

S. pneumoniae (G+ Intra Ep, M) 48

S. typhimurium (G– Intra Ep) 83

S. flexneri (G– Intra Ep) 10

NALP1b Lethal factor from B. anthracis 33

NALP3 Microbial motifs

MurNAc-L-Ala-D-isoGln (muramyldipeptide) 84

Bacterial RNA 24

Imidazoquinoline compounds (R837, R848) 24

Live bacteria

S. aureus (G+ Intra M) 20

L. monocytogenes (G+ Intra M) 20

Microbial toxins

Aerolysin (A. hydrophila) 23

Maitotoxin (Marine dinoflagellates) 20

Nigericin (Streptomyces hygroscopicus) 20

Danger-associated host components

ATP, NAD+ (P2X7 receptors) 20,21

Uric acid crystals 34

Ipaf Microbial motifs

Cytosolic flagellin 25,26

Live bacteria

S. typhimurium (G– Intra M) 22

Naip5 Microbial motifs

Cytosolic flagellin? 27,28

Live bacteria

L. pneumophila (G– Intra Ep M) 29–32

NLRs are reported to be involved in sensing various microbes and molecular constituents thereof as well as exogenous and endogenous danger-associated components. NLRs detect the defined microbial motifs, bacterial extracts, live bacteria, toxins and danger-associated host components listed here. NLRs are activated by intracellular (Intra) or extracellular (Extra) live bacteria (Gram-positive (G+) or Gram-negative (G–)) in different cell-types, such as epithelial cells (Ep), endothelial cells (En), macrophages-monocytes (M) or fibroblasts (F)). aBacillus species: cereus, simplex, subtilis, megaterium, pumilus. bPseudomonas species: aeruginosa, putida. cChlamydia species: pnuemoniae, trachomatis, muridarum. dLactobacillus species: plantarum, pentosus. ePseudomonas species: aeruginosa, putida.

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francisella are more susceptible to the bacteria than are wild-type mice, whereas Ipaf-deficient mice are no different from wild-type mice54. Yet some functional data for an NLR are consistent both in vitro and in vivo. In the legionella model, for example, detection of bacterial fla-gellin by Naip5 correlates directly with the survival of L. pneumophila bacteria both in macrophages in vitro and in the lungs of experimentally infected mice27,32. Thus, it seems that the detection and control of bacte-rial pathogens may depend on multiple signaling molecules. It may also be true that the route of infection and the types of host cells infected influence the array of PRMs involved in host defense.

NLRs in human diseasesUnderstanding of the in vivo function of NLRs has deepened with the finding that some NLR family members are genetically linked to human immunological disorders. The first disease-based gene discoveries of NLRs were made with the family ‘founding member’, MHC2TA, which encodes the ‘master transcription regulator’ CIITA; this protein regulates the expression of genes important for the function of antigen-presenting cells, including major histocompatibility complex class II (refs. 63,64). Of particular relevance are mutations in MHC2TA, including splice-site, nonsense and missense mutations, associated with the autosomal reces-sive hereditary immunodeficiency called ‘bare lymphocyte syndrome’65. Clinical manifestations of this syndrome include frequent bacterial, fungal, viral or protozoan infections accompanied by infections of the gastrointestinal and respiratory tracts. Patients with bare lymphocyte syndrome lack not only constitutive and inducible expression of major histocompatibility complex class II genes but also major histocompat-ibility complex class I expression; they also have severely hampered T cell activation and much lower numbers of CD4+ T cells. Studies have shown that in addition to mutations in MHC2TA, single-nucleotide polymorphisms in the promoter region of MHC2TA are associated with disorders such as rheumatoid arthritis, multiple sclerosis and myocardial infarction, demonstrating important linkage of CIITA to immunological disorders other than bare lymphocyte syndrome.

Several genetic disorders have also been associated with mutations in Cias1 (which encodes NALP3)19. All Cias1-linked diseases are autoso-mal dominant autoinflammatory disorders characterized by recurrent episodes of systemic inflammatory attacks in the absence of infection. Approximately 40 mutations have been linked to three Cias1-associ-ated diseases: familial cold autoinflammatory syndrome (or familial cold urticaria), Muckle-Wells syndrome, and neonatal onset multisys-tem inflammatory disease (or chronic infantile neurological, cutane-ous and articular syndrome). Almost all of those mutations are located in the region encoding the NACHT domain of NALP3. The systemic inflammation characteristic of those disorders is attributed to increased serum concentrations of C-reactive protein, serum amyloid A, IL-6 and TNF, as well as to peripheral blood leukocyte mRNA expression of Il1b (IL-1β), Il3 (IL-3) and Il5 (IL-5). However, the most consistent finding is increased release of IL-1β from peripheral blood leukocytes and/or monocytes, a finding that is consistent with what is known about the central involvement of NALP3 in activation of the inflammasome and regulation of IL-1β processing. Some mutations in Cias1, however, have been identified in people with no inflammatory diseases and in patients with undefined or other inflammatory disorders, such as rheumatoid arthritis or systemic lupus erythematosus.

Mutations in both Card4 (Nod1) and Card15 (Nod2) are linked to inflammatory mucosal barrier diseases. Mutations in Card4 are also associated with increased susceptibility to asthma66 and inflammatory bowel disease67. In both cases, the mutation has been found to be an insertion-deletion polymorphism in an intron of Card4. Notably, the allele linked to asthma has been shown to be associated with increased

immunoglobulin E independently of the presence or absence of aller-gen-specific immunoglobulin E. Moreover, splice variants of the LRR domain of Nod1 have been found in healthy people as well as in asthma patients66. Future research should address if and to what extent the prod-ucts of splice variants and genetic variations of Card4 have the capacity to sense peptidoglycan and how that contributes to the onset of asthma and inflammatory bowel disease.

In contrast to mutations in Card4, mutations in Card15 in the region encoding the NACHT domain and the LRR are associated with a variety of inflammatory pathologies characterized by granulomatous inflam-mation, such as Crohn disease68,69, Blau syndrome70 and early-onset sarcoidosis71. Blau syndrome and early-onset sarcoidosis are autosomal dominant diseases linked to missense point mutations in the region of Card15 encoding the NACHT domain, resulting in increased basal muramyl dipeptide–independent NF-κB activity, suggesting a gain-of-function phenotype. Those pathologies share characteristic clinical features of juvenile-onset systemic granulomatosis syndrome, which mainly affects the skin, joints and eyes. Notably, some of those muta-tions in the region encoding the NACHT domain of Nod2 correspond to those of NALP3, indicating the important functional analogy of the NACHT domain in various NLR proteins.

The best studied Nod2 variants are those linked to Crohn disease68,69, a chronic relapsing autoinflammation of the digestive tract. Although disease-associated mutations linked to Blau syndrome and early-onset sarcoidosis are found in the region encoding the NACHT domain, vari-ants linked to Crohn disease are mapped to the LRR region of Nod2. Crohn disease is associated with high concentrations of IL-1β, IL-6, IL-12 and TNF. Antibodies to TNF, IL-6 and IL-12, as well as direct inhi-bition of NF-κB, are effective therapies. Because antibiotics have been proven to be an effective treatment for Crohn disease, its cause is prob-ably related to a disruption in host tolerance to the resident microflora. Three potential hypotheses (defective epithelial responses, dysregulation of IL-12 and enhanced IL-1β processing) have been proposed to explain the mechanisms by which NOD2 variants might promote intestinal inflammation. These three hypotheses are elaborated below.

First, in Nod2-deficient mice, lower antimicrobial peptide expres-sion correlates with higher susceptibility to oral infection with L. monocytogenes62. Moreover, Nod2 is expressed in Paneth cells (a cell population present in the intestinal crypts that produces large quantities of antimicrobial peptides) and triggers in vitro the secretion of human β-defensin-2. Along with observations demonstrating that Nod2-deficient mice have a general lack of responsiveness to muramyl dipeptide62,72,73, those observations support a ‘loss-of-function’ scenario, suggesting that normal Nod2 expressed in Paneth cells or enterocytes contributes to epithelial host defense against bacterial insults through the induction of antimicrobial peptides. Nod2 variants might fail to activate those defense mechanisms, resulting in increased bacterial burden followed by mucosal inflammation.

Second, although several reports have demonstrated that antigen-pre-senting cells of Nod2-deficient mice respond normally to various TLR agonists, it has been reported that wild-type Nod2 limits the TLR2-medi-ated macrophage response, suggesting that Card15 variants fail to inhibit that process (‘loss-of-function’ phenotype), causing enhanced activation of NF-κB and IL-12 expression. Moreover, another report has indicated that Nod2 deficiency leads to dysregulated TLR2 signaling and induction of antigen-specific colitis74. That colitis is dependent on (unregulated) TLR2 function, as Nod2-TLR2 double-deficient mice show no signs of disease74. Thus, alterations in Nod2 might change its regulatory func-tion of peptidoglycan sensing, leading to increased IL-12 release, which stimulates the growth and differentiation of T helper type 1 cells, thereby promoting interferon-γ-driven intestinal inflammation.

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Third, in contrast to the mechanisms suggested above, a ‘gain-of-function’ scenario for variant but not normal Nod2 has been reported by the generation of a ‘knockout-knockin’ Card15 mouse through the introduction of a mutation homologous to the main mutation in human Crohn disease75. Stimulation of macrophages with muramyl dipeptide or peptidoglycan resulted in a more effective activation of RIP2 and NF-κB and enhanced caspase-1-mediated IL-1β processing and release. Thus, enhanced IL-1β release by macrophages might con-tribute to intestinal inflammation through the activation of a broad range of proinflammatory genes in an autocrine or paracrine way. If true, such a ‘gain of function’ would provide an explanation for the phenotype of Crohn disease, yet those results are in contrast to reports demonstrating blunted responses to muramyl dipeptide or peptidogly-can in human cells expressing the Card15 mutations associated with Crohn disease13,76–78. Thus, further research on the species-specific and cell type–specific functions of NLR proteins is needed to clarify such discrepancies.

Open issues and concluding remarksAn important lesson from the discoveries of NLRs is that compart-mentalization, extracellular or luminal versus intracellular, of micro-bial innate immune detection may be an important strategy of host defense in vertebrates. Consequently, two types of PRMs, both TLRs and NLRs, would be needed to provide complementary surveillance of the two compartments. Such complementarity is further seen in the well described signaling synergy and crosstalk between the two PRM families.

There are considerable challenges in the coming years for understand-ing how NLRs function in host defense, inflammation and disease. The many areas of yet to be exhaustively addressed include the following. The subcellular localization, trafficking and expression of NLRs in dis-tinct cell populations remain poorly defined. It is unclear if NLRs serve as direct receptors of PAMPs or instead only sense PAMPs that bind to adaptor proteins. It is also mostly unknown if NLRs contribute substan-tially to the development of adaptive immune responses. It is notable that until now, bacterial pathogens, not viruses or parasites, have been identified as the main activators of NLRs. The characterization of the RIG-I–Mda5 family of intracellular viral sensors that act independently of NLRs and TLRs identifies a further sensing strategy for host cells. Finally, even though the identification of a genetic link between NLR gene mutations and inflammatory disorders has helped to advance the field, the road is still long and tortuous before such findings will, if indeed they will, ‘translate’ into new therapeutics for disease.

ACKNOWLEDGMENTSSupported by the National Health and Medical Research Council of Australia (R.L.F.), the ANZ Charitable Trust (R.L.F.), the Canadian Institutes for Health Research (D.J.P. and S.E.G.) and the Austrian Science Fund (Erwin Schrödinger Research Fellowship J2630 to J.H.F.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureimmunology/Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

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