NLRs: Nucleotide-Binding Domain and Leucine-Rich-Repeat-Containing Proteins
LETICIA A. M. CARNEIRO,1 JÖRG H. FRITZ,2 THOMAS A. KUFER,3 LEONARDO H. TRAVASSOS,2 SZILVIA BENKO,1 AND DANA J. PHILPOTT2*
[SECTION EDITORS: FERRIC FANG AND MARTIN KAGNOFF]
Posted December 07, 2009
Departments of Immunology2 and Laboratory Medicine and Pathobiology,1 University of Toronto, Toronto, Ontario M5S 1A8, Canada, and Molecular Innate Immunobiology Group, Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 50931 Cologne, Germany3
*Corresponding author. Mailing address: Department of Immunology, University of Toronto, 1 King's College Circle, Medical Sciences Building, Rm. 4366, Toronto, Ontario M5S 1A8, Canada. E-mail:
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Eukaryotes have evolved strategies to detect microbial intrusion and to instruct immune responses to limit damage from infection. Recognition of microbes and cellular damage relies on the detection of microbe-associated molecular patterns (MAMPs) (also called pathogen-associated molecular patterns, or PAMPS) and so-called danger signals by various families of host pattern recognition molecules (PRMs). Members of the recently identified protein family of nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs), including Nod1, Nod2, NLRP3, and NLRC4, have been shown to detect specific microbial motifs and danger signals for regulating host inflammatory responses. Moreover, with the discovery that polymorphisms in NOD1, NOD2, NLRP1, and NLRP3 are associated with susceptibility to chronic inflammatory disorders, the view has emerged that NLRs not only act as sensors, but also serve as signaling platforms for instructing and balancing host immune responses.
All animals and plants possess systems that recognize microorganisms to mount efficient defenses to maintain commensalism and to inhibit the spread of harmful pathogens. Only in the jawed vertebrates, however, did microbial recognition mechanisms evolve by way of two distinct but functionally overlapping systems described as adaptive (or acquired) and innate immunity. The main distinction between these two systems is the receptors used to recognize pathogens. While adaptive immunity relies on somatic recombination of genes to yield an unlimited repertoire of antigen-specific receptors, e.g., antibodies, innate immune recognition is mediated by germ line-encoded PRMs. Since only a limited repertoire of innate PRMs exists, these molecules possess broad specificities for conserved and largely invariant components of microorganisms. These components are termed MAMPs, and include lipopolysaccharide, microbial nucleic acids, and peptidoglycan, to name a few. Recognition of MAMPs by PRMs triggers a number of responses in the host to limit infection. Indeed, PRM activation can drive direct antimicrobial activities, such as phagocytosis and degranulation, but also regulates the expression of innate response genes including those encoding antimicrobial peptides, costimulatory molecules, and cytokines and chemokines. Induction of these genes is essential not only for first-line defense responses to fight pathogen intrusion, but also for instruction and shaping of adaptive immunity. In mammals, the repertoire of PRMs that can regulate gene expression includes the Toll-like receptor family (TLRs) (4), the retinoic acid-inducible gene (RIG)-like helicases (127), and the NLRs (50). Other PRMs can also bind microbes or derivatives thereof and thereby have an impact on innate defense. These include certain complement receptors (151), scavenger receptors (133), peptidoglycan-binding proteins (23), proteins of the triggering receptors expressed on myeloid cells (TREM) family (90), sialic acid-binding immunoglobulin-like lectins (Siglecs) (29), pentraxins (110), and C-type lectin (CLR; collectins) families (147). This chapter outlines the current knowledge of NLRs and their implications for innate immune sensing and antimicrobial immunity.
The first members of the NLR family were identified in a search for factors homologous to apoptotic protease-activating factor-1 (Apaf-1), which is the human homologue of the nematode apoptosis regulator CED-4 (Caenorhabditis elegans death protein 4) (153). These proteins contain a caspase activation and recruitment domain (CARD) thought to play a role in caspase activation and the subsequent induction of apoptosis. A search for homologues with CARD domains in humans led to the identification of the nucleotide-binding oligomerization domain proteins Nod1 and Nod2 (77, 137). These proteins exhibit the CARD-NBD-LRR domain organization, in which the presence of the nucleotide-binding, leucine-rich repeat (NBD-LRR) is archetypal for the NLR family (see Fig. 1) and strikingly similar to the NB-LRR subfamily of R genes in plants (85). NB-LRR proteins confer species-specific resistance of plants to a number of different microbes, which gave researchers the first clue that the mammalian counterparts might have a similar function in host defense.
Since then, more than 20 members of the NLR family have been identified in the sequenced mammalian genomes (50, 76, 120, 165). Unfortunately, the nomenclature of this protein family is quite diverse: “Nod-like receptors,” “NBD-LRR proteins/receptors,” “NBS-LRR proteins/receptors,” “NACHT-LRR-proteins/receptors,” and “CATERPILLER” are all used to refer to this family (50, 76, 120, 165). Attempts to find a consensus (166) have arrived at the nomenclature “nucleotide-binding domain and leucine-rich repeat-containing gene family,” or NLR, which will be used throughout this chapter.
NLRs are characterized by three distinct domains (see Fig. 1): an N-terminal effector domain, consisting of a pyrin (PYD), a CARD or a baculovirus inhibitor-of-apoptosis repeat (BIR) domain; a central NBD domain that can be either a NACHT domain only or a NACHT domain extended by a helical NACHT-associated domain (NAD); and a C-terminal leucine-rich repeat (LRR) domain. (NACHT stands for: domain present in neuronal apoptosis inhibitor protein [NAIP], CIITA [major histocompatibility complex class II (MHC-II) transactivator], HET-E [plant het gene product involved in vegetative incompatibility], TP-1 [telomerase-associated protein 1].) Both NACHT and NAD domains are key features of a recently defined signal transduction ATPase with numerous domains (STAND) protein family of P-loop NTPases, which are distantly related to AAA+ ATPases (ATPases associated with diverse cellular activities). STAND proteins are thought to work as switches, regulating signal transduction by conformational changes regulated by nucleotide binding (103). The LRR domain is thought to constitute, directly or indirectly, the MAMP-sensing portion of the molecule.
NLRs comprise two large subfamilies: the CARD-containing Nod and NLRC proteins and the pyrin-containing NLRP proteins. The BIR-containing NLRPb protein and NLRPa (CIITA) constitute the remaining NLR members (see Fig. 1).
The functional implications of NLR proteins are very diverse (50, 76, 120, 165). However, as demonstrated for some plant R-proteins (50, 95), recent studies have shown that several mammalian NLRs can detect microbes, microbe-derived molecules, and danger signals to elicit inflammatory responses. The triggering of these potentially hazardous events requires control mechanisms to avoid superfluous activation of NLRs. Recent work has started to elucidate these control mechanisms of the NLR proteins and the specific functions of the individual domains. For an excellent and comprehensive review on sequence- and structure-modeling analysis of the NLR family and its functional implications see reference 143.
The LRR domain.
The LRR domain is thought to act as the elicitor recognition domain in TLRs, NLRs, and R-proteins. Tandem arrays of LRRs form domains that often contain protein-protein interaction motifs and are found in a variety of proteins with diverse functions. Crystal diffraction data from several LRR-containing proteins suggest that the most LRR domains fold in a nonglobular horseshoe-like structure with a curved parallel β-sheet lining the concave surface and helixes at the outer surface (12, 93). However, as with most TLRs and R-proteins, the nature of the interaction between NLR proteins and their agonists remains elusive at present. The recently solved structure of the LRR domain of TLR3 provides some insight into these questions. Posttranslational modifications and oligomerization of the LRRs may play key roles in sensing specificity (10, 11, 26). This might also apply to NLR proteins, but confirmation awaits further experimental evidence.
Studies of the LRR domain of NLRs with regard to function have primarily focused on Nod1 and Nod2. Deletion studies indicate that the LRR domains in both Nod1 and Nod2 are responsible, either directly or indirectly, for peptidoglycan recognition. In fact, the LRR seems to be sufficient for elicitor detection, because the specificity of Nod1 and Nod2 can be partially switched through exchange of their LRR domains (57). Furthermore, certain residues in distinct regions of the Nod1 LRR that are predicted to fold in proximity of each other were shown to be essential for specific agonist sensing (57). These amino acids are thought to form a binding pocket. Switching some of these amino acids in human Nod1 to the murine sequence changes the specificity of the protein, in accordance with the differential sensing of Nod1 in humans and mice (108). Similar studies of Nod2 also identified critical residues for muramyl dipeptide (MDP) sensing in the LRR and identified a negative regulatory role of the amino-terminal region of this domain (162).
Recognition of elicitors by PRMs, including NLRs, does not necessarily rely on a direct interaction with the LRR domain. The involvement of cis-acting cofactors, such as CD14 and the lipopolysaccharide (LPS)-binding protein (LBP), as in the case of LPS recognition by TLR4, or trans-acting elicitor-modifying enzymes such as Spätzle, in the case of Drosophila Toll receptors, is a common theme (125). Indeed, only the plant R-gene products Pi-ta, RRS1, and the L-protein seem to interact in a direct manner with the cognate “avirulence” proteins (Avr) expressed by particular plant pathogens (85). This also appears to be the case for some of the mammalian TLRs, specifically TLR5, TLR9, and perhaps the other nucleotide recognition TLRs. For the most part, however, agonist sensing appears to be indirect. This, together with the fact that a relatively limited number of genome-encoded NLR R-genes confers resistance to a vast array of potential pathogens, led to the proposal of the so called “guard-hypothesis.” This idea posits that sensing of specific elicitors by R-proteins in plants is achieved by monitoring the molecular action of Avr proteins, rather than the elicitors themselves (85). This model predicts that Avr proteins interact either with a cellular complex, including the R-protein, or that binding of an Avr-containing complex activates R-protein-mediated signaling. In the case of the R-proteins RPM1, RPS2, and RPS5 from Arabidopsis thaliana and Prf from Solanum lycoperiscum, it was demonstrated that these proteins sense posttranslational modification and/or degradation of their binding partner RIN4 and Pto, respectively (189). Recent studies suggest that the guard hypothesis might also apply to Drosophila sensing of fungal pathogens (61) and possibly in the recognition of protein allergens in mammals (156). Future studies will clarify whether mammalian NLR proteins might also be activated in a similar manner.
Studies of several NLR members suggest that elicitor sensing and activation follows homo-oligomerization of these molecules, which often adopt sixfold or sevenfold symmetries, leading to downstream signaling events (143). This process is mediated by the NACHT domain whose ATPase and/or GTPase activity is proposed to play an essential role. To date, experimental data on nucleotide binding are only available for NLRP3/Nalp3 and NLRP12, which bind ATP (39, 185) and NLRA/CIITA, which uses GTP (65). The recent resolution of the structures of NLR-related proteins, Apaf-1 and CED-4, has shed light on the regulatory mechanisms of NLR proteins. Accordingly, ATP and ADP, respectively, are coordinated in the active form and proposed to induce structural changes presumed necessary for oligomerization and activation (146). Interestingly, elicitor binding might facilitate nucleotide binding and the subsequent regulation of the activity, as has been observed for cytochrome c binding to Apaf-1; this interaction facilitates ATP binding to the NACHT domain (83).
Point mutations in the nucleotide-binding site abolish signaling on microbial recognition as shown for a number of NLRs, including Nod1 and Nod2, thus underscoring the importance of nucleotide binding for NLR activity (77, 78, 137). As discussed further below, disease-associated mutations in NLR proteins frequently lie within this region. Interestingly, NACHT domains might not only mediate homo-oligomerization, but also heterotypic interactions with other NLR partners (34). The effect of heterotypic interactions could increase the complexity of protein-interaction networks that modulate the immune response. Regarding the site of oligomerization, a distinct motif in the NACHT domain of CIITA, LxxLL, was found to be necessary for homo-oligomerization independent of nucleotide binding (155). This sequence motif is also conserved in other members of the NLR family. However, at present, the biological relevance of the oligomerization process and cross talk between NLR members has not been elucidated in infection models.
Biochemical data from plants suggest that back-folding of the LRR to the NBD domain maintains certain R-proteins in an inactive state (189). In support of this notion, the LRR domains of both plant and mammalian NLR proteins seem to have negative regulatory functions, because truncation of this domain can yield higher activity for some proteins (94).
These observations may be combined into a single coherent model in which NLR activation is initiated by a triggering event consisting of the direct or indirect “sensing” of a microbial product and/or danger signal. A conformational change subsequently takes place wherein the molecule unfolds to allow GTP or ATP binding and subsequent oligomerization. This brings the effector domains into close proximity, thereby providing a platform for the subsequent recruitment of adaptor molecules that transmit the signal to downstream activation pathways (79).
Effector domain usage.
In contrast to the highly conserved structure of the elicitor recognition domain, NLRs use different effector domains as links to distinct downstream signaling pathways. NLRs are subgrouped according to these domains (Fig. 1). Both the CARD domain and the PYD domain present in mammalian NLR proteins belong to the death domain-fold family and are thought to be involved in homophilic protein interactions. However, as seen for plant R-genes, some mammalian NLRs use other effector domains, such as the DNA-binding protein interaction domain of NLRA/CIITA and the BIR domain of NLRPB proteins. In addition, some NLRs have effector domains of unknown function, such as NLRX1, while others have truncated domains or even combinations of effector domains (50).
Recently, the structure of the CARD domain of human Nod1 was solved both by nuclear magnetic resonance and X-ray diffraction. This led to the identification of residues critically important for the interaction of RIP2 and Nod1 (111). Surprisingly, the proposed crystal structure differs from the nuclear magnetic resonance structure in that one helix protrudes out from the globular fold. Furthermore, it was proposed that Nod1 CARD domains might form homodimers (28). Further analysis will show which conformation is the most biologically relevant.
The following sections review members of the NLR family and, when possible, outline the specific trigger and function in innate immune defense for each. Known disease associations for specific NLRs will also be discussed (summarized in Table 1). Nothing to date is known about NLRC5 within the NLRC subfamily or about NLRP2 and NLRP4 to NLRP11 within the NLRP subfamily, and so these proteins will not be discussed further.
TABLE 1.Human genetic diseases associated with polymorphisms in NLR genes| NLR | Disease association | Suspected impact of polymorphism |
| NLRA | Bare lymphocyte syndrome | Lack of MHC-II expression |
| NLRB (murine Naip5) | Susceptibility to Legionella infection | Inability to restrict growth through autophagy? |
| Nod1 | Asthma, eczema, atopic disease | Altered expression of Nod1 isoforms with downregulatory function? |
| Nod2 | Crohn's disease
Blau syndrome
Early-onset sarcoidosis
Graft-versus-host disease | Altered regulation of responses toward gut
flora?
Constitutive activation?
Unknown
Unknown |
| NLRP1 | Vitiligo | Dysregulated inflammasome activity? |
| NLRP3 | Muckle-Wells Syndrome
FCAS
CINCA | Dysregulated inflammasome activity with increased IL-1β production |
| NLRP12 | Periodic fever syndromes | Dysregulated inflammasome activity? |
One of the founding members of the NLR family is the MHC2TA gene, encoding a transcriptional regulator known as CIITA (167), and subsequently renamed NLRA. To date, NLRA is the only NLR with direct impact on transcriptional regulation through its ability to interface with specific transcription factors. NRLA is the “oldest” NLR, having been discovered in the early 1990s as the key factor necessary for MHC-II gene expression.
Unlike other members of the NLR family, no specific trigger for NLRA has been identified so far. Its ability to regulate gene expression might depend solely on induction of its own transcription. Accordingly, stimuli such as interferon gamma (IFN-γ) treatment upregulate NLRA expression, which promotes the expression of NLRA-regulated genes such as MHC-II (66). However, studies in which NLRA is overexpressed suggest that NLRA might exist in the cell cytoplasm in an inactive state, awaiting triggering by microbial and/or danger signals like other NLRs. “Sensing” of these factors by NLRA could, in theory, result in its nuclear translocation and allow it to regulate gene expression.
As is commonly observed with NLRs, in general, intracellular levels of NLRA appear to be insufficient for immunohistochemical staining, and Western blotting requires large numbers of cells for detection. Therefore, structure/function studies to date have been driven mostly by sequence comparison and have focused on the ability of transfected CIITA to activate transcription and subsequent expression from endogenous or engineered class II promoters. Moreover, overexpression studies have allowed insight into the subcellular localization of NLRA, its molecular regulation, and its capacity to interact with other proteins.
As discussed above, GTP regulates the activity and subcellular localization of NLRA. GTP binding of NLRA correlates with nuclear import; mutants defective for GTP binding fail to translocate into the nucleus and also fail to activate class II gene expression (66). Although the GTP-binding motif is not itself a nuclear localization signal, GTP binding most likely causes a conformational change that somehow unmasks multiple nuclear localization signals to promote nuclear translocation.
Although NLRA does not bind DNA, it serves as an interaction interface for DNA-binding transcription factors that recognize the MHC-II promoter. NLRA also coordinates the recruitment of histone-modifying enzymes and contains a histone acetylase domain within its N terminus (182). NLRA is a potent transcriptional activator for essential genes involved in antigen presentation such as MHC-II, HLA-DM, Ii, MHC-I, and plexin-A1 (14). Plexin-A1 has been shown to be a modulator of actin polarization and plays an important role in dendritic cells (DCs) (181). Deficiency of NLRA, as discussed in detail below, results in the near abrogation of MHC-II expression in mice and the near-absence of MHC-II expression in humans (145). NLRA, as a master regulator of MHC-II gene transcription, is targeted by a variety of pathogens, illustrating its central role in host-pathogen counterbalance (2).
Mutations in the MHC2TA gene include splice site, nonsense, and missense mutations that result in an autosomal recessive hereditary immunodeficiency termed “bare lymphocyte syndrome” (BLS) (145). Clinical manifestations include frequent bacterial, fungal, viral, or protozoan infections accompanied by infections of the gastrointestinal tract and respiratory system. Patients with BLS lack constitutive or inducible expression of MHC-II genes but also exhibit deficiencies of MHC-I expression, T-cell activation, and CD4+ T-cell number. Recent studies have shown that, in addition to mutations within the gene, single-nucleotide polymorphisms in the promoter region of MHC2TA are associated with various disorders, including rheumatoid arthritis, multiple sclerosis, and myocardial infarction, demonstrating an important linkage between NLRA and immunological disorders in addition to BLS.
NLRB, or NAIP (neuronal apoptosis inhibitor protein), was first described as an apoptosis inhibitor in neurons implicated in childhood spinal muscular atrophy (SMA), a common autosomal recessive disorder characterized by muscle weakness from degeneration of motor neurons in the spinal cord and brainstem nuclei. However, given its classic domain organization, NLRB was classified as an NLR. NLRB is unique among the NLRs because of its N-terminal BIR domains that appear to mediate its antiapoptotic function in neurons. Few studies have focused on human NLRB in host defense, and most of our understanding of the potential function of NLRB comes from studies of the murine homologue, NAIP5 (also known as Birc1a) (45). While humans appear to have a single NLRB gene, mice have at least eight homologues of NAIP. Therefore, comparisons between murine NAIP5 and human NLRB must be interpreted with caution.
Our understanding of the triggers of Naip5 function has come from studies on the susceptibility of mice to infection with Legionella pneumophila, an intracellular parasite of freshwater protozoa. During opportunistic infection of humans, Legionella can infect human alveolar macrophages and cause an acute form of pneumonia called “Legionnaires’ disease.” A self-limiting and milder infection, called Pontiac fever, can also occur. In most strains of mice, macrophages can restrict the growth of Legionella. However, macrophages derived from A/J mice, like human macrophages, are unable to contain Legionella. Further analysis of A/J mice linked Legionella susceptibility to polymorphisms in the Naip5 gene. Studies have since focused on understanding the nature of the microbial product that triggers Naip5-mediated restriction of bacterial replication. Early studies suggested that, like NLRC4 (below), Naip5 is involved in cytosolic sensing of flagella from bacteria. However, there are studies that both support and challenge this possibility (reviewed in reference 129). Naip5 interacts with NLRC4 and does seem to enhance the responses of cells to flagellated bacteria. However, A/J macrophages still appear to respond to flagellin, suggesting that Naip5 is not absolutely required for flagellin sensing (99). A detailed discussion of flagellin sensing by NLRC4 is provided in the following sections. At present, Naip5 might be viewed as an enhancer of NLRC4-mediated flagellin sensing, with additional triggers of Naip5 remaining to be identified. Whether human NLRB has a function similar to Naip5 is still unknown, although recent data suggest that this may be the case (173).
As mentioned earlier, Naip5 interacts with NLRC4 and also with caspase-1. Therefore, one aspect of Naip5 function appears to involve enhancement of NRC4-mediated inflammasome activation and interleukin 1β (IL-1β) secretion (186). The mechanisms of inflammasome activation will be discussed in more detail in the section specifically discussing NLRC4 (see below).
Functional Naip5 is required for growth restriction of Legionella, but this seems to be independent of inflammasome and caspase-1 activation (99). Naip5 was recently reported to promote the fusion of Legionella-containing phagosomes with lysosomes, thus antagonizing the ability of Legionella to remodel its phagosome into a specialized replicative vacuole (44). This study suggests a direct role of Naip5 in targeting phagosomal cargoes to the lysosome for degradation. Moreover, additional data support the notion that Naip5 may play a role in autophagy, a regulatory pathway of programmed cell death that can be important in the clearance of intracellular infections. Indeed, macrophages from A/J mice appear to have less efficient autophagic responses to Legionella (6). Together, these findings implicate Naip5 in cellular responses that are required to eliminate infecting pathogens from the intracellular compartment. Again, whether human NLRB plays a similar role is still open to speculation.
Recent data have attempted to clarify the role of Naip5 in Legionella infection. Vance and colleagues, concerned that the Naip5 mutation might not be a null mutation in A/J mice, constructed a Naip5-deficient mouse on a C57BL/6 strain background. Their studies showed a requirement for Naip5 in inflammasome activation in response to a C-terminal fragment of flagellin (105). These results have begun to clarify the role of Naip5 in flagellin sensing and the consequences for inflammasome function.
As mentioned, NLRB was originally identified as a susceptibility locus in SMA. However, recent studies have demonstrated that mutations in SMN1 (survival motor neuron-1) are actually responsible for SMA. NLRB appears to act as a modifier that affects the severity of disease. In terms of function, NLRB inhibits caspase-3- and caspase-7-dependent apoptosis through its BIR domains (109). This ability to regulate apoptosis has thus far not been linked to its potential involvement as a pattern recognition receptor in host defense.
Although a Naip5 mutation in A/J mice renders macrophages unable to restrict Legionella growth, human macrophages are also unable to restrict Legionella growth even though they express NLRB. Knocking down NLRB expression in human macrophages and other human cells does enhance the growth of intracellular Legionella (173). NLRB therefore appears to contribute to the restriction of Legionella growth inside human macrophages, but additional unidentified factors are also involved.
The NLRC subfamily includes Nod1, Nod2, NLRC3, NLRC4, and NLRC5 (although nothing to date has been published on NLRC5). This subfamily is distinguished from other NLRs by the presence of an N-terminal CARD domain. As described earlier, this domain appears to be responsible for homotypic interactions with other CARD-containing proteins. Nod1, Nod2, and NLRC4 sense microbial products, while the only information regarding NLRC3 thus far suggests that it is a negative regulator of T-cell function.
Nod1 is a sensor of a peptidoglycan component from the cell walls of bacteria. This NLR is ubiquitously expressed, and its subcellular localization suggests that it is associated in part with the plasma membrane (96). Stimulation of Nod1 leads to proinflammatory signaling. Nod1 has been implicated in combating infection and linking innate and adaptive immune responses. In addition, Nod1 dysregulation has been linked to inflammatory disorders, including asthma and atopy.
Triggers.
Peptidoglycan is a major constituent of the gram-positive bacterial cell wall consisting of glycan chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid cross-linked to each other by short peptides, which allows the formation of a rigid polymer surrounding the bacteria (Fig. 2). In gram-negative bacteria, a thin layer of peptidoglycan is located in the periplasmic space. In contrast, peptidoglycan typically represents the major constituent of the gram-positive bacterial cell wall. Apart from the thickness and the degree of stem peptide cross-linking, an important difference between gram-positive and gram-negative peptidoglycan resides in the nature of the third amino acid of the peptide moiety. In gram-positive bacteria, the canonical stem peptide is the lysine-containing pentapeptide L-Ala-D-Glu-L-Lys-D-Ala-D-Ala (PentaLys), while meso-diaminopimelic acid is found in place of L-lysine in most gram-negative bacteria (51).
As mentioned, Nod1 detects peptidoglycan, with a strict sensing specificity toward diaminopimelic acid (DAP)-type peptidoglycan (21, 55). Indeed, the human form of Nod1 detects a single muropeptide, GM-TriDAP, produced as a peptidoglycan degradation product in gram-negative bacterial metabolism (Fig. 2). Surprisingly, the murine form of Nod1 detects GM-TetraDAP, thus revealing some host specificity in the sensing of muropeptides (108). In Escherichia coli and Salmonella, the outermost D-Ala is lost and the inner D-Ala is linked to the side-chain carboxyl of DAP in cross-linking reactions during peptidoglycan synthesis. It is possible that this could result in the generation of some free GM-TetraDAP and subsequent detection by murine but not human Nod1. Some gram-positive bacteria such as Listeria monocytogenes, Bacillus cereus, and Bacillus anthracis, also contain DAP instead of lysine in their peptidoglycan; in Bacillus subtilis, the DAP amino acid is modified through an amidation reaction, resulting in the loss of peptidoglycan sensing through Nod1 (18).
The list of bacteria detected by a Nod1-dependent pathway is growing. The detection of Shigella flexneri by cultured epithelial cells represented the first demonstration of Nod1-dependent sensing (58). Subsequently, several studies revealed that Nod1 is involved in the recognition of a number of invasive bacteria, such as gram-negative enteroinvasive E. coli, Pseudomonas spp., Campylobacter jejuni, and Chlamydia spp., as well as the gram-positive L. monocytogenes (reviewed in reference 18). In addition, Nod1 has been shown to be activated when cells are stimulated with crude extracts of gram-negative bacteria such as Salmonella enterica or L. pneumophila, as well as by extracts of gram-positive bacterial species including Bacillus spp. (reviewed in reference 18), B. anthracis spores (60), and Mycobacterium tuberculosis (43), demonstrating that, at least in vitro, Nod1 can act as mediator of innate immune responses to a number of different bacteria. Furthermore, Nod1 activation has also been reported to occur in the absence of bacterial invasion. Indeed, Helicobacter pylori (172) and Pseudomonas aeruginosa (168) have been shown to trigger Nod1 signaling in epithelial cells in an invasion-independent manner (see below). Moreover, cooperative interactions among microbes during a polymicrobial infection might facilitate Nod1 activation. A recent report showed that a pore-forming toxin, pneumolysin, from Streptococcus pneumoniae can allow the passage of peptidoglycan fragments of Haemophilus influenzae into the host cell cytosol where they are recognized by Nod1 (144). Strikingly, the activation of Nod1 by H. influenzae helps to clear the pneumococcal infection, providing an interesting example of how one species overcomes competition in a polymicrobial infection by exploiting the host's immune response (106).
These findings lead to questions regarding the mechanism of entry and presentation of peptidoglycan to Nod1 (Nod2 as well; see below) in the cytosol. In an infection with invasive bacteria that gain access to the host cytosol, such as Listeria or Shigella, it seems conceivable that some free peptidoglycan may be released and detected by Nod molecules. However, for bacteria that remain in phagosomes (such as Mycobacteria or Salmonella) or colonize specific cellular organelles without accessing the cytosol (like Legionella), detection by Nod proteins raises the question of how peptidoglycan reaches the cytosol. Similarly, although it has been known for decades that free peptidoglycan or muramyl peptides can stimulate cells, it remains unclear how these molecules, which are hydrophilic in nature, can cross the cell membrane barrier to gain access to the cytosol. Several transport systems for muramyl peptides have been proposed recently, including PepT1, PepT2, and pannexin (86, 161, 171). How these transporters function and might interact with Nod1 is still not known.
Another possible cell entry mechanism for peptidoglycan fragments is inadvertent translocation by bacterial secretory systems. Bacteria that remain extracellular yet possess systems to directly translocate effector proteins into the host cytosol could potentially transfer peptidoglycan fragments as the secretory systems traverse the bacterial cell wall and host plasma membrane. Accordingly, pathogens that possess type 3 or type 4 secretory systems (T3SS or T4SS) may contaminate the host cytoplasm with peptidogylcan fragments that trigger innate immune responses. This is likely the case for P. aeruginosa, and perhaps other pathogens as well. In the case of H. pylori, there is good evidence that a T4SS is responsible for transferring peptidoglycan into the host cell and triggering a Nod1-dependent response (172). As will subsequently be discussed for flagellar sensing by NLRC4, such adventitious transfer of microbial products can be detected by host sensory machinery.
One report has suggested that Nod proteins have molecular triggers other than peptidoglycan by-products. Indeed, a saturated fatty acid, lauric acid, has been shown to trigger inflammatory responses through Nod1 activation. Moreover, unsaturated fatty acids were shown to dampen inflammatory signaling through Nod1 (187). This observation provides an interesting possible link between diet and the modulation of immune function.
It should be noted that, although TLR2 was initially identified as a sensor for peptidoglycan, more recent studies have demonstrated that TLR2-dependent recognition of peptidoglycan is probably attributable to contaminants such as lipoteichoic acid that are commonly found in peptidoglycan preparations, leaving Nod proteins as the only known peptidoglycan sensors (75, 169). These results have important implications for host-pathogen interactions. If Nods are the sole peptidoglycan sensors, and these receptors detect peptidoglycan degradation products, then the detection of peptidoglycan by the host requires prior processing of the peptidoglycan layer either by endogenous bacterial hydrolases or by host enzymes. Consequently, any mechanism that inhibits the release of muropeptides might constitute a mechanism for evasion of innate immunity. One group has recently advanced the hypothesis that TLR2 may detect peptidoglycan from Staphylococcus aureus, if high concentrations are present (40). Further study will be required to establish the biological relevance of this observation.
Function.
The function of Nod1 in innate immune activation has been studied extensively. Most recently, studies have focused on how innate induction by Nod1 can shape the adaptive immune response.
Signal transduction. Signal transduction pathways downstream of Nod1 and Nod2 are quite similar. Therefore, this subsection discusses both of these molecules, and issues unique to Nod2 signaling are addressed in the subsequent section. (See Fig. 3 and Fig. 4 for diagrams of Nod1 and Nod2 signaling.)
Upon activation, Nod1 and Nod2 initiate a proinflammatory response that largely depends on nuclear factor κB (NF-κB) activation (18). Once activated, Nod1 and Nod2 recruit a serine-threonine kinase called Rip2 (also know as RICK or CARDIAK), through homotypic CARD-CARD interactions. Kobayashi and colleagues demonstrated that mouse embryonic fibroblasts from Rip2-deficient mice are unable to activate NF-κB in response to Nod1 and Nod2 agonists, thus revealing the crucial role of Rip2 downstream of these to PRMs (91). Oligomerization of Rip2 following Nod stimulation results in polyubiquitination and subsequent recruitment and activation of transforming growth factor-activated kinase 1 (TAK1) (67). Active TAK1 promotes both NF-κB and mitogen-activated protein kinase (MAPK) activation downstream of Nod signaling (88). Indeed, the Rip2/TAK1 complex likely interacts with the IKK regulatory subunit IKKγ (also called NEMO), promoting its ubiquitination (1) and the subsequent sequence of events leading to NF-κB activation: the phosphorylation and degradation of IkBα, which under steady-state conditions retains NF-κB in the cytosol. Once degraded, NF-κB is released and translocates into the nucleus, where it drives the transcription of target genes. Although Rip2, like TAK1, has been shown to be required for the activation of c-Jun N-terminal kinase (JNK) pathways (140), the molecular details of how this signal transduction pathway is activated remain unclear. The activation of JNK following Nod1 stimulation was first shown in epithelial cells, in which infection with virulent S. flexneri leads to Nod1-dependent activation of JNK (58).
Some regulators of the Nod-dependent NF-κB pathways have been described (Fig. 3). A novel CARD-containing protein, CARD6, was first identified as a modulator of Nod1- and Rip2-dependent activation of NF-κB (38). However, a recent report shows that CARD6-deficient mice have normal responses following Nod protein stimulation (37). Another potential regulator, TRIP-6, which is a LIM-domain-containing protein, can potentiate Nod1 activation, most likely through its interaction with Rip2 (104). SGT1, which is an ubiquitin ligase-associated protein, and heat-shock protein 90 (HSP90), both of which have plant orthologs essential for R-protein responses, appear to potentiate Nod-dependent responses (32, 122). Furthermore, another plant orthologous complex implicated in R-protein responses, designated the COP9 signalosome, is also implicated in Nod1 signaling. Indeed, several components of the COP9 signalosome were shown to interact with Nod1 through the CARD domain (33). The COP9 signalosome performs diverse functions from modulating the cell cycle to regulating some aspects of the immune response. However, it is not yet clear how this complex affects Nod1 signaling, although modulation of apoptosis has been proposed (33). Centaurin β-1 also modulates Nod function (184). This protein, which is a member of the ADP-ribosylation factor family, was shown to interact with both Nod1 and Nod2 in a yeast-two-hybrid screen. Centaurin β-1 appears to downregulate Nod signaling by interfering with the NF-κB cascade (184). Finally, caspase-12, a member of the caspase-1 subfamily, has been implicated in signaling downstream of Nod1 and Nod2. Caspase-12 has been shown to interact with Rip2 to mediate its effects on Nod signaling (100). Interestingly, caspase-12-deficient mice have enhanced production of antimicrobial peptides, cytokines, and chemokines in response to infection with enteric pathogens that are sensed by Nod proteins (100). Although most human populations express a truncated form of caspase-12, 20% of African descendants possess the full-length molecule. Strikingly, expression of the full-length variant is detrimental to host defense since caspase-12 acts as a suppressor of inflammation and innate immunity. Accordingly, individuals with full-length caspase-12 are much more susceptible to septic shock (152). In agreement with these observations in humans, mice deficient in caspase-12 exhibit enhanced bacterial clearance and are resistant to sepsis (152). Taken together, these findings demonstrate that the repressor function of caspase-12 on the Nod signaling cascade at the level of the epithelium may contribute to its deleterious effects during sepsis.
Nod1 has also been shown to play a role in the development of cancer. The lack of Nod1 expression in a breast cancer cell line leads to increased tumor growth in a SCID mouse xenograft model (30). Limited tumor growth in Nod1-expressing cells may be linked to the ability of Nod1 to induce apoptosis. Indeed, activation of Nod1 in the presence of cycloheximide results in caspase-8-dependent apoptosis. However, the biological relevance of these findings is unclear since apoptosis downstream of Nod1 is only observed when protein synthesis is inhibited by cycloheximide (31). There are many examples in the literature in which blockade of NF-κB-dependent synthesis of prosurvival factors leads to cell death. Therefore, the apoptosis observed downstream of Nod1 may represent a nonspecific cell death process resulting when the cell is unable to activate the expression of prosurvival genes. Indeed, in the context of a bacterial infection, Nod1-dependent NF-κB activation is crucial for host cell survival by maintaining the levels of the prosurvival protein Bcl-2 and preventing subsequent mitochondrial dysfunction and necrosis. In MEFs from Nod1-deficient mice, activation of the Rip2/IKKβ/NF-κB axis following Shigella infection is impaired, resulting in massive cell death. Thus, the overall outcome of Nod1-dependent sensing of the bacteria seems to lean toward host cell survival rather than death (19).
Inflammatory mediators and immune response. As mentioned, many downstream activation pathways are common to both Nod1 and Nod2. Therefore, unless indicated, the following discussion applies to both NLRs. Nod2-specific effects are highlighted in the next section.
Because of the relatively recent discovery of Nod1, Nod2, and NLRs in general, many of their functions are not yet known. However, many studies have highlighted the crucial role of these NLRs in the inflammatory response. In vitro studies have shown that, upon ligand recognition, Nod1 and Nod2 activation result in the transcription of a large repertoire of genes, many of which depend on NF-κB activation. In dendritic cells, macrophages, and monocytes, activation of Nod1 and Nod2 leads primarily to the production of proinflammatory cytokines (IL-1β, IL-6, tumor necrosis factor α [TNF-α], CXC chemokine ligand 8 [CXCL8]/IL-8, KC, RANTES, IL-10, IL-18, IL-12p40, IL-12p70), nitric oxide, and expression of costimulatory molecules and adhesion molecules (reviewed in reference 18). It can also be said, however, that the effect of Nod1 and Nod2 ligands appears to be generally weak in promoting DC activation (52), and as we will see below, coactivation of TLRs is often required for full immune responses. In epithelial cell lines, triggering of the Nod pathway induces the production of proinflammatory mediators (TNF-α, IL-6, CXCL8/IL-8, MIP2, CCL2/MCP-1, CXCL5/epithelial neutrophil-activating peptide 78 [ENA-78]) and antimicrobial peptides (β-defensin 2) (reviewed in reference 18). Each of these factors is critical for the recruitment and activation of effector cells and inflammatory processes that result in the establishment of an appropriate immune response.
In the context of an infection, bacteria likely activate the immune response through several PRMs activated simultaneously. Even though some PRMs may play a more relevant role than others, depending on the nature of the pathogen, it is likely that costimulation of multiple PRMs is responsible for a maximal microbe-induced immune response in vivo. To date, the mechanisms by which the signals elicited by distinct PRM activation are integrated into a single global immune response are still not clear. It has been shown that Nod1 and Nod2 agonists can synergize with TLR agonists to induce proinflammatory mediators and cell maturation in dendritic cells and monocytes (reviewed in reference 18). In addition, it has been shown that some proinflammatory mediators can synergize with Nod proteins: (i) IL-32 has been shown to synergize with Nod1 and Nod2 agonists to enhance the secretion of IL-1β and IL-6 and (ii) TNF-α can synergize with the Nod2 agonist MDP to induce increased IL-1β secretion by human monocytes (134).
As mentioned above, simultaneous recognition of bacteria or bacterial products through Nod1, Nod2, and TLRs acts synergistically to induce proinflammatory cytokines and to generate protective immune responses. From a functional standpoint, synergy between TLR and Nod proteins allows the induction of a global and potent response. Another advantage of the existence of two independent detection systems that rely on distinct signaling pathways would be the possibility that one can act as a backup for the other. This becomes obvious in the demonstration that recognition of pathogenic bacteria by intestinal cells lacking TLRs relies on Nod1 (58, 89, 188). More recently, it has been shown that macrophages or mice made insensitive to TLRs by previous exposure to microbial ligands remain responsive to Nod1 and Nod2 stimulation. Further analyses have revealed that innate immune responses induced by bacterial infection rely on Nod1 and Nod2 in macrophages pretreated with TLR ligands but not in naïve macrophages. Finally, bacterial clearance upon systemic infection with Listeria critically depends on Nod1 and Nod2 when mice are previously tolerized with LPS or E. coli (89). These data demonstrate that Nod1 and Nod2 detection of invasive bacteria is critical to protect the host when proinflammatory responses are compromised by TLR-induced tolerization.
In contrast to these studies, additional findings have suggested that some NLRs may act as inhibitors of other NLR pathways or TLR signaling. A cross talk between Nod1 and Nod2 pathways has been demonstrated in patients with Crohn's disease carrying a homozygous Nod2fs (frame-shift) mutation (see more on Nod2 in “Crohn's disease” below). Consequently, in addition to being unresponsive to MDP, peripheral blood mononuclear cells from these patients appear to be hyporesponsive to Nod1 agonists as well (135). Finally, Watanabe et al. have proposed an unexpected role for Nod2 in negatively regulating the TLR2-mediated induction of the Th1-type cytokines IL-12 and IFN-γ in CD11b+ splenocytes (174, 175). Although subsequent studies have challenged these conclusions, it is an interesting hypothesis that, at least for certain stimuli and in certain cell types, Nods and TLRs might antagonize each other. In the study by Watanabe et al., the possibility that Nod2 influences the Th1/Th2 balance, and thereby modulates the adaptive immune response to bacteria, could have a significant impact on the pathogenesis of Crohn's disease, which is associated with mutations in Nod2 (see the next section).
Links to adaptive immunity. Recent studies reveal that Nod1 and Nod2 not only have a role in initiating the innate immune response but also are implicated in driving adaptive immunity (reviewed in reference 52). It has been shown that Nod2 is able to mediate adjuvant activity since coinjection of a protein antigen and MDP leads to the production of IgG1 antibody against the T-cell-dependent antigen (92, 107). Moreover, Nod1 is important for priming antigen-specific T-cell immunity and maximal antibody responses in vivo. Indeed, a crucial role for Nod1 in synergizing with TLRs was demonstrated for priming Th1 as well as Th17 immune pathways in vivo. In contrast, when administered alone, a Nod1 agonist elicits priming of antigen-specific T- and B-cell immunity with a predominant Th2 polarization profile (53). In distinction with the current model that DCs are the key players integrating microbial and antigen signals to instruct adaptive immune responses, this study showed that bone marrow cell transfer from wild-type to Nod1-deficient mice could not compensate for Nod1 deficiency, suggesting that innate immune priming by Nod1 is mediated by cells other than those in the myeloid compartment (53).
An additional role for Nod1 as a key component in the maintenance of intestinal homeostasis and the generation of adaptive lymphoid tissues has been recently proposed, based on the findings that peptidoglycan from gram-negative bacteria is necessary and sufficient to induce the genesis of isolated lymphoid follicles (ILFs) in mice through recognition by Nod1. The maturation of these ILFs into large B-cell clusters requires subsequent detection of bacteria by Toll-like receptors. However, in the absence of ILFs, the composition of the intestinal bacterial flora is profoundly altered. This reciprocal regulation of the intestinal bacterial flora and ILFs was shown to be crucial for intestinal homeostasis, and its disruption can potentially lead to an array of severe illnesses, in particular, inflammatory bowel disease characterized by self-destructive intestinal immunity (15).
Disease association.
Recently, polymorphisms in the human Nod1 gene have been linked to asthma, eczema, and atopy (74, 124, 177). The polymorphisms that have been identified map to an intronic region within the Nod1 gene, and it is speculated that these mutations may affect the relative expression of spliced variants of Nod1. For the moment, however, it is not clear how this might contribute to the development of disease. Recent findings suggest that these isoforms are unable to sense bacterial products and initiate an inflammatory process (57). How this might then contribute to the development of asthma and atopic disease is currently unknown. Indeed, it is not currently known whether asthmatic and atopic patients express greater or lesser quantities of full-length Nod1 or the various isoforms. Potentially, expression of inactive Nod1 isoforms may represent a normal means by which inflammatory signaling is downregulated at the level of the respiratory epithelium, and asthmatic and atopic patients might not be able to mount this protective response. Consequently, such individuals would exhibit enhanced Nod1-dependent Th2-driven responses; indeed, these conditions are characterized by heightened Th2-biased inflammation.
Nod2 is a general sensor for both gram-positive and gram-negative bacteria since biochemical and functional analyses have identified MDP, the minimal motif common to all peptidoglycans, as the essential structure recognized by Nod2 (56, 81) (Fig. 2). Nod2 has gained notoriety since mutations in the gene encoding Nod2 were linked to susceptibility to Crohn's disease, an inflammatory disorder that affects the intestinal tract (73, 136). As discussed above, many of the features of Nod2, including signal transduction pathways, are common with Nod1. An important difference with respect to Nod1, however, is the expression pattern of Nod2. Nod2 is characteristically expressed in lymphocytes and myeloid cells. However, Nod2 expression can be upregulated in other cell types such as epithelial cells by various proinflammatory mediators, including TNF-α and IFN-γ, or by bacterial infection (9, 63, 148). As discussed below, this may have important consequences for the development of disease. As with Nod1, subcellular localization of Nod2 appears to be predominantly at the plasma membrane, and this location is required for sensing MDP and triggering the NF-κB pathway (7, 101, 102).
Triggers.
As discussed above, Nod2 is a general sensor of bacteria through its ability to detect MDP, the common motif in all peptidoglycans. Further analysis of the peptidoglycan structural requirements that allow sensing by Nod2 have shown that, in addition to MDP, Nod2 can detect Muramyl-TriLys but not Muramyl-TriDAP (59) (Fig. 2). Interestingly, the most common mutation in Nod2 associated with Crohn's disease is a frame-shift mutation that truncates the terminal LRR, resulting in a protein product that no longer detects peptidoglycan (56, 81). Although the implications of these findings are still not fully understood, it appears that lack of bacterial sensing may contribute to the pathology of Crohn's disease, at least in some cases.
Function.
Much of Nod2 function parallels that which has been reported for Nod1. Indeed, these two NLRs activate similar pathways leading to the induction of NF-κB and MAPK/JNK. Furthermore, many inflammatory mediators are regulated in common by Nod1 and Nod2. As discussed above, expression of Nod2 under most conditions is generally restricted to the myeloid compartment, which must be kept in mind when considering biological function.
Signal transduction. Nod2 triggering stimulates NF-κB induction through the so-called classical pathway, similar to Nod1. However, in addition, Nod2 triggers the noncanonical or alternative pathway of NF-κB activation, which utilizes the protein NIK (NF-κB-inducing kinase; see Fig. 4). Activation of this pathway leads to the formation of NF-κB comprised by dimers of RelB and p52; consequently, Nod2 may regulate a distinct subclass of NF-κB-dependent genes. Nod2 stimulation has been shown to trigger NIK, and during costimulation with LPS, this alternative pathway regulates a NIK-specific gene called CXCL13 (or BLC; B lymphocyte chemokine), encoding a potent chemoattractant for B cells (138). Collectively, these findings suggest that Nod2 may regulate distinct gene subsets, perhaps depending on cell type, and this may influence the resulting inflammatory response and the induction of adaptive immunity.
With regard to regulation, interacting partners unique for Nod2 have been described. GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts specifically with Nod2 and is required for NF-κB activation following Nod2 recognition of MDP and Nod2-dependent antibacterial actions (8). These observations are somewhat puzzling, since GRIM19 is a mitochondrial protein, and Nod2 does not appear to colocalize with this organelle. More recently, CARD9 has also been shown to associate with both Nod2 and Rip2 and to play a critical role in Nod2-dependent activation of MAPKs (72). In addition, CARD9 has been shown to induce NF-κB signaling through Bcl10 and Malt-1 (176).
Negative regulators of the Nod2-dependent pathway have also been identified. Mitogen-activated protein kinase kinase TAK1 is an essential component of the signaling pathways of many inflammatory cytokines. TAK1 and Nod2 have been shown to interact through the LRR domain of Nod2 and to negatively regulate each other's ability to activate NF-κB in a reciprocal fashion (24). A20, which is a ubiquitin-modifying enzyme, restricts Nod2-dependent activation pathways by turning off NF-κB signaling (70). Erbin, a member of the leucine-rich repeat- and PDZ domain-containing (LAP) family, has also been shown to physically interact with Nod2 and to negatively regulate Nod2-mediated NF-κB activation. During infection with S. flexneri, Nod2 and Erbin were shown to colocalize at the entry foci of the bacteria with maximal affinity between the two proteins observed after 30 to 40 minutes of infection. Furthermore, the knockdown of Erbin expression enhanced NF-κB signaling through Nod2. Taken together, these results suggest an important role for Erbin in modulating Nod2 signaling during bacterial infection (97, 123). More recently, it has been proposed that Rac1 binds to Nod2 (102) and thereby contributes to Nod2-Erbin interactions; knockdown of Rac1 or an associated protein called β-PIX seems to abrogate their binding (41). A short form of Nod2, called Nod2-S, was found to act in a dominant-negative fashion to inhibit Nod2 signaling (150). This molecule is an alternatively spliced form of Nod2 truncated within the second CARD domain. Nod2-S is upregulated by the anti-inflammatory cytokine IL-10, and is thought to bind to full-length Nod2. However, because of its truncated CARD, Nod2-S cannot propagate the signal. Therefore, production of Nod2-S may represent a downregulatory loop by which Nod2 is able to modulate its own activation. Finally, CD147 was shown to interact with the CARD domain of Nod2 in a bacterial two-hybrid screen (164). CD147, a membrane-bound protein involved in the activation of matrix metalloproteases, appears to downregulate signaling downstream of Nod2, perhaps by competing with Rip2 for binding to the CARD domain. Moreover, CD147 appears to enhance the ability of bacteria to invade cells. A detailed picture of the regulators of Nod2 signaling is shown in Fig. 4.
In addition to its role in regulating inflammation through the activation of the NF-κB cascade, Nod2 has also been implicated in the generation of IL-1β through its participation in the assembly of the inflammasome. The inflammasome is discussed in detail in the following sections concerning NLRC4 and the NLRP subfamily. Some observations have implicated the Nod2 ligand MDP as a trigger for inflammasome activation (116, 139). However, whether Nod2 is involved in this process is presently uncertain (115).
Inflammatory mediators and immune response. The common inflammatory response stimulated by Nod1 and Nod2 is discussed in “Nod1,” above.
Links to adaptive immunity. As discussed in “Nod1,” above, Nod2 triggering leads to a predominantly Th2-adaptive immune response (92, 107). Nod2 was also found to be critical for the induction of both Th1- and Th2-type responses following costimulation with TLR agonists (107). However, the Nod2-dependent mediators responsible for this response are still unknown. Interestingly, the synergistic responses to Nod2 and TLR agonists seen in vivo are recapitulated by dendritic cells in vitro, suggesting that these cells likely play a central role in the integration of Nod2- and TLR-dependent signals to drive the adaptive immune response (107).
Nod2 has also been shown to influence the Th17 axis. This distinct T-cell subset appears to play a role in driving inflammation during infection with extracellular bacteria and in autoimmunity. Nod2 has been shown to act in synergy with TLR ligands to drive IL-17 production from memory T cells, which implicates Nod2 in the regulation of the effector function of Th17 cells (170).
Disease association.
In 2001, two independent studies revealed that mutations in the gene Nod2/CARD15 are associated with increased susceptibility to Crohn's disease, a chronic inflammatory disease that can affect the entire gastrointestinal tract (73, 80). In general, Crohn's disease is believed to result from inappropriate hyperresponsiveness to microorganisms constituting the commensal flora of the intestinal tract. The most persuasive support for this hypothesis comes from mouse models of inflammatory bowel disease demonstrating that intestinal inflammation from a variety of causes does not develop if animals are reared under germ-free conditions. Moreover, patients receiving antibiotics or who have intestinal shunts to divert intestinal contents away from inflamed areas experience some clinical benefit. Crohn's disease can thus be viewed as an autoimmune disease; antigens associated with the commensal flora are present for the life of the individual and consequently represent “self-antigens.” In Crohn's disease, patients appear to respond to commensal flora with a Th1-cell-mediated reaction driven by the excessive production of IL-12 and IFN-γ.
The most common Nod2 mutation linked to Crohn's disease, L1007fsC, results in a protein product that can no longer be stimulated by bacterial MDP (56, 81). This is somewhat unexpected and paradoxical since Crohn's disease is characterized by increased intestinal inflammation. This concept has been debated over the years with many hypotheses advanced in an attempt to explain how deficient Nod2-dependent signaling might result in enhanced inflammation. A detailed discussion is beyond the scope of this chapter, and the interested reader is directed to a recent review (25). In brief, it is thought that the loss of Nod2 function may lead to the defective production of defensins and the inability of local intestinal mucosal responses to contain the normal flora, leading to tissue invasion and the elicitation of aberrant inflammation that is characteristic of Crohn's disease (25, 183).
Mutations in Nod2 have also been associated with other disorders including Blau syndrome, increased mortality in graft-versus-host disease, allograft rejection, and early-onset sarcoidosis (68). The molecular mechanisms behind these associations are by and large unknown. For Blau syndrome, which is an inflammatory granulomatous disorder affecting the eyes, skin, and joints, the associated mutation in Nod2 represents what appears to be a gain-of-function mutation that might constitutively activate the NF-κB pathway (22). With relation to infection and cancer, an interesting finding has demonstrated a link between a Nod2 mutation and the development of gastric lymphoma in individuals infected with H. pylori (149). How a specific polymorphism in Nod2 leads to enhanced susceptibility to neoplasia in the context of Helicobacter infection is not yet known.
NLRC3, also known as Nod3, is expressed predominantly in T cells (27). Nod3 expression levels decrease upon T-cell stimulation, which is consistent with a potential negative regulatory role and observed decreases in NF-κB, nuclear factor of activated T cells, and activator protein 1 induction in response to T-cell activation by anti-CD3 and anti-CD28 antibodies or PMA/ionomycin. After T-cell stimulation, Nod3 has been shown to reduce levels of endogenous transcripts for IL-2 and CD25 that play a central role in maintaining T-cell activation and preventing T-cell anergy. Thus, it has been suggested that NLRC3 attenuates T-cell activation via the TCR and costimulatory molecules, while the downregulation of NLRC3 expression during T-cell stimulation allows cellular activation (27). Nothing is currently known about how NLRC3 is triggered, how it functions, or whether there are disease associations linked to NLRC3 gene variants.
Studies have demonstrated that NLRC4 (also known as IPAF and Card12) is an intracellular detector of bacterial flagella (Table 2). The recognition of this bacterial structure results in the activation of a molecular platform called the “inflammasome,” which results in caspase-1 activation and the secretion of active forms of IL-1β, IL-18 and IL-33 (a pathway to be discussed in greater detail in the section on NLRPs). Inflammasome and caspase-1 activation have been strongly implicated in a type of host cell death (see Chapter Host Cell Death).
TABLE 2.Triggers of NLR inflammasomes| NLR | Trigger |
| NLRC4 | Flagellin
Type 3 and type 4 secretion systems? |
| NLRP1 | Anthrax lethal toxin
MDP |
| NLRP3 | A. Products of bacteria or viruses
Bacterial RNA
Microbial DNA
Viral dsRNA
MDP
Bacterial toxins |
B. Endogenous danger signals
ATP
Host DNA
Uric acid
Amyloid β |
C. Exogenous products
Asbestos
Alum
Silica
UVB light |
The Toll-like receptor TLR5 is also a sensor of flagella, indicating that cells have the potential to sense this bacterial product both outside and inside the cell. As discussed below, additional coactivators or processes might be required for NLRC4 activation. Nevertheless, these findings suggest the interesting possibility that cellular responses to microbes may differ qualitatively depending on whether the stimuli are located inside or outside of the plasma membrane.
Triggers.
Flagellin is a bacterial protein that can self-assemble to form the structural backbone of the flagellum, a macromolecular complex that plays a key role in the motility of both gram-negative and gram-positive bacteria. Flagellin monomers from a number of flagellated bacteria are extremely conserved, which has probably influenced the evolution of innate immune mechanisms for bacterial flagellin detection (129). It must be noted that flagellin is one on the very few protein-derived MAMPs characterized thus far. Plants also detect bacterial flagellin; Fls-2, a trans-membrane protein in Arabidopsis, detects a conserved peptide from flagellin to trigger defensive responses to flagellated pathogens (129).
The direct delivery of flagellin monomers into the host cytosol by transfection or other biochemical means can trigger NLRC4 (46, 128). Interestingly, however, in infection with flagellated bacteria, NLRC4-dependent detection of flagellin requires a functional virulence protein secretion system expressed by the invading pathogen, such as the SPI1 (Salmonella pathogenicity island 1) T3SS of Salmonella or the T4SS of Legionella (reviewed in reference 129). These virulence-associated secretion systems are evolutionarily distinct but functionally convergent structures that directly transfer bacterial effector proteins into the cytoplasm of eukaryotic cells. The flagellar apparatus and the virulence-associated secretion systems form similar macromolecular structures. Accordingly, it has been hypothesized that during infection of cells with Salmonella or Legionella, flagellin might “cross over” and be released adventitiously by heterologous bacterial secretion systems, which might in turn be detected by the host as a trigger for the activation of NLRC4 (129). In the studies mentioned above, a role for TLR5 in mediating responses to flagellin was excluded, because bone-marrow-derived macrophages do not express this TLR.
Recent reports from three groups identified NLRC4 as a critical mediator of inflammasome activation by another gram-negative bacterium, Pseudomonas aeruginosa, which also possesses a T3SS (48, 130, 158). Although two of the studies found a crucial role for flagellin in Pseudomonas detection by NLRC4, the report by Sutterwala et al. (158) surprisingly found no role for flagellin in NLRC4-dependent sensing of the bacterium. Rather, the authors speculate that NLRC4 may sense membrane damage triggered by insertion of the T3SS into the host membrane. This idea is in accordance with a recent report showing that the T3SS of Yersinia, likely through its pore-forming activity, results in IL-1β secretion (154). This hypothesis would also offer an explanation for the dependence of Shigella-induced inflammasome activation on NLRC4 (160). Indeed, the inability of Shigella to produce flagella implicates stimuli distinct from cytosolic flagellin in the activation of NLRC4 during Shigella infection. Moreover, flagellin-independent but T3SS/T4SS-dependent activation of IL-1β secretion has also been reported in other systems (128, 131), reinforcing this hypothesis. Further research will be required to establish whether an alternative ligand, the secretion itself, or pore formation is responsible for NLRC4 or other NLR activation.
Function.
NLRC4 triggering can lead to activation of the inflammasome, resulting in proinflammatory cytokine release and cell death (Fig. 5). Moreover, NLRC4 has been also shown to play a role in trafficking of internalized pathogens within an infected cell.
Inflammasome activation. NLRC4 was initially identified as a critical mediator of inflammasome activation in bone marrow-derived macrophages infected with Salmonella (113). The inflammasome is a molecular platform that comprises different proteins whose assembly is required for the activation of caspase-1, the key effector protein of the complex. Tschopp and colleagues were the first to show that caspase-1, which is synthesized as an inactive proenzyme, is processed and activated within this multiprotein complex (117). Upon triggering, the complex assembles from different combinations of molecular components depending on the upstream activator. Flagellin triggers the formation of the “NLRC4-inflammasome,” consisting of oligomerized NLRC4, pro-caspase-1, and variably, the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD; see below). This platform is thought to favor the autocatalytic processing of pro-caspase-1 into its active form, which then cleaves a number of inactive cytokines like pro-IL-1β, pro-IL-18, and pro-IL-33 into their active and secretable forms. Interestingly, activated caspase-1 also plays a role in the “unconventional” secretion of a number of other proteins, although how this relates to host defense is still unknown (87).
The activation of inflammasomes is thought to require a two-step sequential process (reviewed in reference 112). The initiating signal involves TLR (or perhaps NLR) -mediated activation of the targeted cell. In vitro studies usually use LPS as a pretreatment for mouse macrophages prior to triggering with the second signal, consisting of flagellin in the case of the NLRC4 inflammasome or other triggers in the case of NLRP3 (see section on NLRP3). The first signal is thought to upregulate the expression of proforms of caspase-1 targets or other components of the inflammasome that might be necessary for proper assembly, which may include caspase-11 (caspase-4 and -5 in human cells) (112). How this works in vivo is presently unknown.
NLRC4-dependent pyroptosis. Caspase-1-dependent cell death has been termed “pyroptosis” to distinguish this form of cell death from apoptosis mediated by caspases-3, -6, and -7, which is typically not inflammatory. Pyroptosis has features of apoptosis, including DNA fragmentation and nuclear condensation, but cells undergoing pyroptosis ultimately lyse from the loss of membrane integrity. Over the years, a number of bacteria have been shown to induce pyroptosis, and it now is clear that they do so through the activation of NLRC4. NLRC4−/− macrophages infected with different bacteria, including Salmonella, Shigella, Pseudomonas, and Legionella, fail to induce not only caspase-1 cleavage and IL-1β secretion, but also pyroptotic cell death (112). Interestingly, ASC, which is essential for flagellin-induced caspase-1 activation as discussed above, does not seem to be involved in the cell death process mediated by flagellated bacteria (112).
Collectively, these findings implicate NLRC4 in two distinct cellular outcomes, cytokine secretion and cell death, but the activation of one or the other of these processes may be dictated by context. For example, cell death may be favored by the NLRC4 inflammasome only when the cell is overwhelmed by infection. When a high multiplicity of infection is used, pyroptotic cell death predominates. This may stress an important role of ASC in the regulation of cell death. If levels of ASC are limiting, and the activation of NLRC4 inflammasomes is increased because of high cellular infection, the absence of ASC from these inflammasomes might favor the induction of cell death. However, at lower infection rates, ASC-dependent generation of proinflammatory cytokines through the NLRC4 inflammasome might prevail in the absence of cell death. This scenario is presently speculative, and it is not known how the balance between inflammasome-related functions of cell death and cytokine processing are regulated at the molecular level.
Intracellular trafficking and autophagy. NLRC4 and caspase-1 have been shown to be critical for the restriction of intracellular Legionella growth. NLRC4 appears to be required for the fusion of the Legionella-containing vacuole (LCV) with lysosomes and subsequent degradation of the bacteria (5). Indeed, NLRC4−/− macrophages are permissive for Legionella replication, and the LCV matures by acquiring vesicles from the endoplasmic reticulum (5). However, the relationship between NLRC4 and Naip5 (as discussed above) in this context remains unclear. The dispensability of caspase-1 for Naip5-mediated restriction of Legionella growth (99) implies that NLRC4 and Naip5 might trigger independent signaling pathways that lead to the same outcome, i.e., restriction of Legionella growth.
Recently, NLRC4 and caspase-1 were also implicated in the regulation of autophagy following bacterial infection. NLRC4- and caspase-1-deficient macrophages induce much higher levels of autophagy following Shigella infection compared with wild-type cells. Moreover, blockage of the autophagic response appears to promote pyroptotic cell death following Shigella infection (160). Taken together, these findings implicate the NLRC4 inflammasome in regulating autophagy. Moreover, autophagy might account for the slow cell death process that has been reported in caspase-1-deficient cells infected with Shigella.
Before concluding this section, a special note must be added about another gram-negative bacterial pathogen, Francisella tularensis, which is responsible for the zoonotic infection known as tularemia. Recent research has been focused on this pathogen because of its possible application in biological warfare. Francisella was shown to activate caspase-1 via ASC. Surprisingly, however, no evidence for the involvement of NLRC4 or any other NLR in this pathway has been discovered so far. Moreover, Francisella also induces pyroptosis, but in contrast to other host cell-pathogen interactions, ASC is implicated in this process (69). Therefore, this represents an example in which inflammasomes are implicated, but neither the trigger nor the sensor has been discovered. Furthermore, Francisella provides the sole example, thus far, in which ASC has been implicated in the death of bacteria-infected cells.
Disease association.
To date, no specific disease associations have been attributed to NLRC4 mutations or polymorphisms. However, as discussed above in the section on NLRB, polymorphisms in NLRC4 might affect susceptibility to infectious disease.
Phylogenetic analysis of the LRR domain of NLRX1 reveals close homology with Nod1, Nod2, NLRC3, and NLRC5. This NLR surprisingly localizes to the mitochondria where it is thought to associate with mitochondrial antiviral signaling protein (MAVS), a key adaptor protein in the retinoic acid-inducible gene I/melanoma 43 differentiation-associated gene 5 (RIG-I/MDA5) viral-sensing pathway. Here, NLRX1 appears to act as a negative regulator in the viral-sensing pathway by binding to MAVS and inhibiting its interaction with RIG-I/MDA5 (132). In contrast, another study has shown that NLRX1 localizes to the mitochondrial matrix (unpublished data) where it promotes the production of reactive oxygen species (ROS) and amplifies the immune response (163). Nothing thus far is known about the triggers of NLRX1 or the molecular mechanisms underlying its activation.
The NLRP subfamily represents the largest group of NLRs, comprising NLRP1 to NLRP14. Information is presently only available for NLRP1, NLRP3, and NLRP12. Interestingly, the triggers for at least two of the family members, NLRP1 and NLRP3, appear to be “danger” related (see Table 2), indicating that these NLR proteins respond to signals released by dead or dying cells. NLRPs are subdivided from the other NLRs because of the presence of an N-terminal pyrin domain. This domain was first described in a protein called PYRIN, whose gene is mutated in a disease called familial Mediterranean fever (FMF) (20). FMF is an inherited disorder resulting in periodic episodes of fever and inflammation. PYRIN can form an inflammasome and lead to the production of proinflammatory IL-1β and activation of cell death (see below). Mice expressing a hypomorphic Pyrin, similar to that seen in FMF patients, have heightened lethality from endotoxin and impaired apoptosis (20).
NLRP1 is an unusual NLR since it possesses both a pyrin and a CARD domain. This molecule was the first NLR family member characterized with respect to inflammasome assembly and caspase-1 activation (117) (Fig. 5). The cellular roles of human NLRP1 and its murine orthologs are yet to be defined, but at least one of the isoforms of murine Nlrp1 is involved in the cell death induced by anthrax toxin (16). Polymorphisms in NLRP1 have been associated with vitiligo and its associated autoimmune diseases (84).
NLRP1 presents a pattern of expression somewhat distinct from the related NLRP3. Both of these NLRPs are expressed in myeloid and lymphocyte cell populations. However, NLRP1 localizes to glandular epithelial in the stomach, gut, and lung, as well as in testis and neurons. NLRP1 is also expressed in Langerhans cells, which may point to a key role in mediating cutaneous inflammation. Interestingly, the subcellular distribution of NLRP1 appears to be nuclear (98). How this relates to its function is unknown.
Triggers.
The virulence of B. anthracis (the causative agent of anthrax) depends on the secretion of factors that form functional toxins such as edema toxin and lethal toxin (LT) (16). Almost two decades ago, macrophages from C57Bl/6J mice were found to be resistant to LT-induced death, while macrophages from the 129/S1 strain were extremely susceptible (49). Recently, susceptibility to LT-induced cell death in these mice has been associated with polymorphisms in Nlrp1b. The Nlrp1 gene is highly polymorphic in mice with three closely related Nlrp1 paralogues: Nlrp1a, Nlrp1b, and Nlrp1. Transgenic expression of the Nlrp1b allele from 129/S1 mice in LT-resistant macrophages confers LT sensitivity. Conversely, inhibition of Nlrp1b renders LT-sensitive macrophages more resistant. These observations show that Nlrp1b acts as a key determinant of macrophage sensitivity to LT. The process depends on the adaptor protein ASC and involves caspase-1 (16). However, the molecular basis of LT susceptibility and the biochemical link between LT detection by Nlrp1b and macrophage killing remains unknown. Recent data indicate that potassium efflux and proteasome-mediated effects take place upstream of Nlrp1b activation by LT (178). As will be discussed in greater detail with NLRP3, low intracellular potassium levels are required for both NLRP1 and NLRP3 inflammasome assembly (142). An explanation for the ability of only some Nlrp1b variants to confer LT susceptibility will provide important insights into the role of NLRs in host-specific immunity in mammals and its subversion by certain pathogens.
Triggering of the NLRP1 inflammasome has also been shown to take place after MDP treatment, which is normally involved in Nod2 activation. By using recombinant proteins to reconstitute the NLRP1 inflammasome in vitro, MDP was shown to be sufficient to activate caspase-1 (42). Whether or not this activation occurs in vivo has not yet been shown. Moreover, NLRP1 activation by MDP may be species-specific; murine cells do not exhibit the same sensitivity to MDP as human cells with regard to NLRP1 activation.
Function.
The function of NLRP1 is to mediate inflammasome assembly leading to activation of caspase-1 and the cleavage of IL-1β, IL-18, and IL-33 into active and secretable forms. NLRP1 also potentially induces pyroptosis, although this has only been shown for the murine ortholog Nlrp1b after LT treatment.
Using in vitro reconstitution of the NLRP1 inflammasome, Reed and colleagues have demonstrated that activation of this molecular complex is a two-step process (42). MDP induces a conformational change in NLRP1 that allows the protein to bind ribonucleoside triphosphates (NTPs) and oligomerize, thus creating a platform for caspase activation. Similar to what has been proposed for plant R-proteins, it is thought that MDP interacts with the LRR domain of NLRP1 to relieve repression of the NACHT domain, allowing NTP-dependent oligomerization to occur. In vitro studies also show that the role of ASC may be to enhance NLRP1 inflammasomes, as ASC is strictly required (42). The relevance of these observations to in vivo activation of the NLRP1 inflammasome awaits further research.
Recently, Bcl-2 and Bcl-XL were shown to regulate NLRP1 inflammasome activation. Bcl-2 and Bcl-XL are known to act as antiapoptotic proteins but the mechanism by which they regulate caspase activation was previously unclear. In this report, Bcl-2 and Bcl-XL were shown to interact with NLRP1 and suppress its ability to activate caspase-1 (17). Accordingly, mice deficient in Bcl-2 produced more IL-1β after MDP administration than their wild-type counterparts (17). This represents a mechanism by which Bcl-2 family members may blunt pathogen-induced inflammatory responses and perhaps downregulate cell death.
Disease association.
Several polymorphisms in NLRP1 have been associated with vitiligo (84). Patients with this skin disorder often also manifest other autoimmune disorders including autoimmune thyroid disease, rheumatoid arthritis, and psoriasis. Although the molecular implication of NLRP1 in these disorders is so far unknown, one may speculate that dysregulated inflammasome activity might contribute to the development of disease. One can also posit that polymorphisms in the gene encoding NLRP1, in addition to increasing susceptibility to vitiligo, may play modulatory roles in the development of other autoinflammatory disorders.
NLRP3 (also known as Nalp3, cryopyrin, and Cias1) activates the inflammasome in response to a number of microbial factors and danger signals. The gene encoding NLRP3 was first described as Cias1 (for cold-induced autoinflammatory syndrome), and polymorphisms in NLRP3 have been correlated with susceptibility to a number of autoinflammatory diseases. NLRP3 is expressed in myeloid cells and lymphocytes as well as in nonkeratinizing epithelia of the oropharynx, esophagus, and ectocervix (98).
Triggers.
The NLRP3 inflammasome has been shown to be triggered by stimuli belonging to one of the following classes: (a) products of viruses or bacteria, (b) endogenous end- or byproducts of danger or stress stimuli, and (c) xenogenous particles (see Table 2 and Fig. 5) (reviewed in reference 13). Within these classes, triggers include (a) bacterial RNA, dsRNA from viruses, microbial DNA, MDP, and bacterial toxins; (b) host DNA, uric acid, ATP, amyloid-β (64); and (c) chemical irritants, including silica and asbestos crystals, aluminum adjuvants, and UVB light (reviewed in reference 13 unless otherwise indicated). The ability of the NLRP3 inflammasome to be activated by this multitude of factors suggests that this protein acts as an integrator of signaling, responding to a common factor downstream of diverse agonists. One essential factor for NLRP inflammasome activation is the lowering of intracellular potassium (142). However, it is unclear whether this by itself is sufficient to activate the NLRP3 inflammasome (see below).
ATP is one of the best-studied activators of the NLRP3 inflammasome. ATP is released at relatively high concentrations by dead or dying cells and activates the cation-selective purinergic receptor P2X7 (157), leading to potassium efflux. This event is followed by the opening of a large pore formed by pannexin-1 (141). Pannexin-1 forms a nonselective channel that has structural similarity to a nonmammalian, gap junction-forming protein family. One hypothesis is that the opening of pannexin-1 allows the influx of microbial triggers that enable NLRP3 inflammasome activation (86). A remaining conceptual problem with this hypothesis is that the diverse known triggers seem unlikely to be directly responsible for NLRP3 activation.
One possible intermediary has been suggested to be ROS. Indeed, studies demonstrating activation of the NLRP3 inflammasome by chemical irritants have suggested a role for ROS generated by the NADPH oxidase during so-called “frustrated phagocytosis” of large particles (36). Unexpectedly, however, mice that lack superoxide dismutase 1 (SOD1) and thereby produce higher levels of ROS show caspase-1 inhibition and actually produce less caspase-1-dependent cytokines (126). Whether this results from a negative regulatory feedback loop on caspase activation in the presence of chronically high levels of ROS is not known.
Others have suggested that silica crystals and alum as well as amyloid β (64) cause lysosomal membrane damage during phagocytosis, and this damage is somehow sensed by NLRP3 (71). Strikingly, the inhibition of a single lysosomal protease, cathepsin B, during crystal phagocytosis leads to a substantial decrease in activation of the NLRP3 inflammasome (71). In this context, it is noteworthy that nigericin, thought to activate the NLRP3 inflammasome solely by potassium efflux, induces lysosomal leakage and subsequent caspase-1 activation through the function of cathepsin B. It is possible that pannexin-1 opening might affect lysosomal stability. Since other studies have shown a role for lysosomal enzymes in inflammasome activation (180), one can speculate that these enzymes, which are normally sequestered inside phagosomes, might cleave a cytosolic target that in turn triggers the NLRP3 inflammasome. Confirmation of this idea awaits further investigation.
Function.
The NLRP3-inflammsome, like the NLRP1- and NLRC4-inflammasomes, has been implicated in the activation of the cytokines, IL-1β, IL-18 and IL-33. Pyroptosis through NLRP3 signaling has not been examined in detail, although one study has demonstrated a role for NLRP3 in pathogen-induced necrotic cell death (180). Interestingly, a few reports have suggested that NLRP3 mutations associated with disease may favor cell death (see below). The following sections focus on the function of the NLRP3 inflammasome and its impact on inflammation and the induction of adaptive immunity.
Inflammasome activation. As with the NLRC4 and NLRP1 inflammasomes, it is thought that NLRP3-induced activation of the inflammasome involves two consecutive signals: a TLR-mediated induction of proforms of IL-1β, IL-18, IL-33, and possibility other targets (87), followed by a second trigger, such as the ones listed above, to trigger catalytic activation. The NLRP3 inflammasome invariably contains ASC and possibly another CARD-containing protein called CARDINAL, although the latter remains to be confirmed (3).
The role of NLRP3 in endotoxic shock is controversial. Some studies have shown that NLRP3-deficient mice are resistant to LPS-induced sepsis (114) while others have revealed little or no survival difference between NLRP3-deficient and wild-type mice (159). Therefore, it is presently uncertain whether NLRP3 deficiency phenocopies the resistance of caspase-1-deficient mice to LPS-induced shock.
In addition to the direct role of NLRP3 on caspase-1-dependent cytokine processing, one study has identified a potentially unique role of the NLRP3 inflammasome (and NLRC4) in directing transcriptional responses involving lipid metabolic pathways. A pore-forming toxin called aerolysin, which induces NLRP3-dependent activation of caspase-1, was also found to induce activation of the central regulators of membrane biogenesis, the sterol regulatory element-binding proteins. These proteins promote cell survival upon toxin challenge, possibly by facilitating membrane repair (62). This is the first example of NLRP3 influencing a transcriptional response and opens up the possibility that other targets downstream of NLRP3 activation might play a similar role.
NLRP3-dependent induction of adaptive immunity. NALP3-deficient mice demonstrate an impaired contact hypersensitivity response to the hapten trinitrophenylchloride, similar to caspase-1-deficient mice (159). Moreover, transfer of primed T cells to NLRP3-deficient mice restores a normal hypersensitivity response. These findings suggest that NLRP3-mediated induction of caspase-1 is required during the sensitization phase in which skin resident antigen-presenting cells are stimulated by a hapten and prime T-cell responses (159). These findings link the irritant effect of sensitizing chemicals with the activation of NLRP3 inflammasome, which facilitate activation of the adaptive immune system.
Following on this idea, recent studies have sought to find a role for NLRP3 in the ability of alum to act as an adjuvant and stimulate antigen-specific immunity. Surprisingly, there is some controversy as to whether this is the case. Several studies have indicated that NLRP3 function is required for Th2 responses induced by alum. NLRP3-, ASC-, and caspase-1-deficient mice are unable to mount an alum-dependent antigen-specific antibody response (reviewed in reference 35). However, a recent study shows no role of NLRP3 in this effect (47). Further studies will be required to resolve this controversy. If NLRP3 does in fact play a role in the adjuvanticity of alum, the downstream effector of this process remains to be identified.
Disease associations.
In 2001, NLRP3 was identified as the gene responsible for a number of autoinflammatory syndromes, namely, Muckle-Wells syndrome (MWS), familial cold-associated syndrome (FCAS), and chronic infantile neurological cutaneous and articular syndrome (CINCA) (reviewed in reference 118). These diseases are characterized by periodic fever, increase in the serum levels of acute-phase proteins, joint inflammation, skin rash, and, eventually, amyloidosis. CINCA is the most severe of these diseases with the eventual development of blindness and mental retardation. In FCAS patients, as the name suggests, attacks are usually triggered by exposure to cold. The identified mutations in NLRP3 are primarily gain-of-function mutations within the NACHT domain that lead to increased activation of the inflammasome, resulting in aberrantly high production of IL-1β. Monocytes from MWS patients secrete more mature IL-1β than healthy donors, even in the absence of NLRP3 agonists (3). Strikingly, treatment with IL-1 receptor antagonist can reverse symptoms in some of these conditions (118).
Interestingly, disease-associated mutations in NLRP3 have also been associated with necrotic cell death. The transfection of human monocytes with disease-associated mutant NLRP3 proteins leads to lysosomal leakage, mitochondria damage, and cell death, effects that depend on cathepsin B (54). These responses appear to be independent of IL-1β generation. These striking findings are suggestive of a pathway that involves lysosomal leakage as a downstream effect, rather than a trigger, of NLRP3 activation. Moreover, as discussed in the previous section on NLRC4, cell death may result from the overactivation of the inflammasome that bypasses ASC. Whereas engagement of ASC by NLRP3 activates caspase-1, NLRP3 activity in the absence of ASC might favor the induction of cell death. Future studies can determine whether this form of cell death is ASC dependent. Moreover, information is needed regarding the sequence of events in NLRP3 activation and the role of the lysosome in this process.
Gout and pseudogout are inflammatory conditions affecting the joints because of the deposition of crystals: monosodium urate in the case of gout, and calcium pyrophosphate dihydrate in pseudogout. Both types of crystal, which can be released as “danger signals” from dying cells, are potent triggers of the NLRP3-dependent inflammasome (119). The importance of NLRP3-dependent generation of IL-1β in the pathology of gout is highlighted by promising studies in humans using inhibitors of IL-1β to treat patients with gout. These data suggest that targeting IL-1 or the inflammasome is an effective therapeutic alternative in gout (118). These clinical observations, together with the promising trials of anti-IL-1 therapy in MWS patients, provide excellent examples of how an improved basic understanding of inflammatory signaling pathways can be useful in the clinic.
A recent study has shown that a pathogen can subvert NLRP3 activation to evade host immune responses. M. tuberculosis parasitizes macrophages by suppressing the activation of the inflammasome (121). An M. tuberculosis gene product, called zmp1, which encodes a zinc-dependent metalloprotease, was shown to be required for this effect (121). Further research is needed to elucidate the function of Zmp1 and whether other pathogens are similarly able to manipulate the host's innate immune system by bypassing NLRP3 induction.
The NLR family member NLRP12 (also referred to as Nalp12, Monarch-1, or Pypaf7) is expressed in resting myeloid/monocytic cells. NLRP12 expression is reduced by TLR agonists or TNF-α. NLRP12 has been demonstrated to be an antagonist of TLR- or TNF-α-induced proinflammatory mediator production, suggesting that NLRP12 participates in a negative-feedback mechanism (179). Interestingly, a recent study showed that mutations in NLRP12 are associated with patients with periodic fever syndromes (82). These findings point to a possible role of NLRP12 in inflammasome function. However, more insight into the function of NLRP12 awaits further research.
Although the discovery of the NLR family of proteins occurred fairly recently, a surprisingly large number of findings concerning the function of these effectors in the innate immune response has already been amassed. A striking observation is the number of genetic diseases that have been linked to mutations or polymorphisms in NLR-encoding genes. In the coming years, the challenge of ongoing research will be to understand why two distinct innate systems have developed for pathogen detection; indeed, elucidating TLR- versus NLR-specific effects will help us to understand the immune response to infection, as well as how adaptive immunity is primed and might be manipulated for vaccine applications. As demonstrated by the promise of anti-IL-1β therapeutic approaches in the treatment of inflammasome-linked disorders, an increased understanding of NLR function will help to shape treatment strategies for NLR-linked diseases.
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Module8.8.3
