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NOD-Like Receptors: Role in Innate Immunity and Inammatory Disease

Grace Chen, Michael H. Shaw, Yun-Gi Kim, and Gabriel Nunez
Departments of Pathology and Internal Medicine and the Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109; email: gchenry@umich.edu; mhshaw@umich.edu; yungikim@umich.edu; bclx@umich.edu
Annu. Rev. Pathol. Mech. Dis. 2009. 4:36598 First published online as a Review in Advance on October 17, 2008 The Annual Review of Pathology: Mechanisms of Disease is online at pathmechdis.annualreviews.org This articles doi: 10.1146/annurev.pathol.4.110807.092239 Copyright c 2009 by Annual Reviews. All rights reserved 1553-4006/09/0228-0365$20.00
Key Words
caspase-1, Crohns disease, IL-1, NOD2, NLRP3
Abstract
The NOD-like receptors (NLRs) are a specialized group of intracellular receptors that represent a key component of the host innate immune system. Since the discovery of the rst NLR almost 10 years ago, the study of this special class of microbial sensors has burgeoned; consequently, a better understanding of the mechanism by which these receptors recognize microbes and other danger signals and of how they activate inammatory signaling pathways has emerged. Moreover, in addition to their primary role in host defense against invading pathogens, their ability to regulate nuclear factorkappa B (NF-B) signaling, interleukin-1-beta (IL-1) production, and cell death indicates that they are crucial to the pathogenesis of a variety of inammatory human diseases.
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INTRODUCTION
PAMPs: pathogenassociated molecular patterns PRRs: pathogen recognition receptors LPS: lipopolysaccharide PGN: peptidoglycan TLRs: Toll-like receptors
Essential to human survival is the ability to eradicate pathogenic microorganisms. To ensure the efcient detection and removal of harmful microbes, two effector mechanisms have evolved: the innate and adaptive immune systems. The adaptive immune response is characterized by a delayed response involving gene rearrangements for the clonal selection and expansion of T cell and B cell lymphocytes with antigen-specic receptors. This process results in the generation of a diverse, yet specic repertoire of immune effectors that also contribute to immunologic memory. The innate immune system, however, is rapidly activated and does not require the somatic gene rearrangements that form the cornerstone of adaptive immunity. It represents one of the rst lines of defense against microorganisms, whereby conserved microbial structures known as pathogen-associated molecular patterns (PAMPs) are recognized by germlineencoded innate immune receptors, also known as pathogen recognition receptors (PRRs). Examples of PAMPs include lipopolysaccharides (LPS), peptidoglycan (PGN), agellin, and microbial nucleic acids. Recognition of these PAMPs by PRRs results in the activation of signaling pathways, which promotes an inammatory, antimicrobial response. Emerging data have also demonstrated a link between the innate PRRs and the activation of the adaptive immune response that can act in concert in defense against invasive organisms (1). Moreover, it has also become apparent that these receptors are involved in sensing not only invading pathogens, but also endogenous nonmicrobial danger or stress signals, both of which result in the activation of inammatory signaling pathways such as nuclear factorkappa B (NF-B) and mitogen-activated protein kinases (MAPKs). This upstream link to complex effector pathways highlights the importance of these immune receptors in both microbial defense and the pathogenesis of noninfectious, inammatory diseases when signaling becomes dysregulated.
Three major classes of PRRs have been identied: (a) the Toll-like receptors (TLRs), which are transmembrane proteins with an extramembranous domain involved in ligand recognition on either the extracellular surface or within endosomes and a cytoplasmic domain involved in signal transduction; (b) the NOD-like receptors (NLRs), which are intracellular, cytoplasmic sensors; and (c) the retinoid acidinducible gene1 (RIG-1)-like receptors (RLRs), which are cytosolic helicases that primarily sense viruses. Historically, the TLRs were rst recognized for their role in host defense, but the importance of NLRs in complementing the functions of the TLRs has become increasingly clear. The high evolutionary conservation of the NLRs attests to their efcacy and vitality in host defense. Homologs of the NLRs (e.g., R genes) have been discovered throughout the plant and animal kingdoms, including phylogenetically primitive organisms such as the zebrash (2) and the sea urchin, which has at least 203 identied putative NLRs (3).
DEFINING FEATURES OF THE NOD-LIKE RECEPTOR FAMILY

In humans, the NLR family is composed of 22 proteins (Table 1), and there are at least 33 NLR genes in mice. Although primarily expressed in immune cells, including both lymphocytes and antigen-presenting cells (APCs) such as macrophages and dendritic cells, NLRs can also be expressed in nonimmune cells, including epithelial and mesothelial cells. This family of proteins is dened by a tripartite structure consisting of (a) a variable Nterminal protein-protein interaction domain, dened by the caspase recruitment domain (CARD), pyrin domain (PYD), acidic transactivating domain, or baculovirus inhibitor repeat (BIR); (b) a central nucleotide-binding oligomerization (NOD) domain, which mediates self-oligomerization that occurs during activation (4); and (c) a C-terminal leucine-rich repeat (LRR) that detects PAMPs.
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Table 1
NOD-like receptor (NLR) family members Other names and aliases NLRA; MHC2TA; C2TA Nlra; MHC2TA; C2TA NLRB1; BIRC1; CLR5.1 Birc1a Birc1b Birc1c Birc1d Birc1e Birc1f Birc1g NLRC1; CARD4; CLR7.1 Nod1 Nlrc1; Card4 NLRC2; CARD15; CD; BLAU; IBD1; PSORAS1; CLR16.3 Nlrc2; Card15 NOD3; CLR16.2 CLR16.2 CARD12; CLAN; CLR2.1; IPAF Card12; CLAN; Ipaf NOD27; CLR16.1 NALP1; DEFCAP; NAC; CARD7; CLR17.1 NALP1 NLRP NALP2; PYPAF2; NBS1; PAN1; CLR19.9 Nlrp2 Pypaf2; Nbs1; Pan1 CIAS1; PYPAF1; Cryopyrin; NALP3; CLR1.1 Cias1; Pypaf1; Cryopyrin; Nalp3; Mmig1 NALP4; PYPAF4; PAN2; RNH2; CLR19.5 Nalp4a; Nalp-eta; Nalp9D Nalp4b; Nalp-gamma; Nalp9E Nalp4c; Nalp-alpha; Rnh2 Nalp4d; Nalp-beta Nalp4e; Nalp-epsilon Nalp4f; Nalp-kappa; Nalp9F Nalp4g NLRP NLRP Domain organization NLR family NLRA
HGNC-approved symbol Human CIITA Mouse Cllta
NAIP Naip1 Naip2 Naip3 Naip4 Naip5 Naip6 Naip7 NOD1 NOD2
NLRB
NLRC NLRC
Nod2 NLRC3 Nlrc3 NLRC4 Nlrc4 NLRC5 NIrc5 NLRP1 Nlrp1a Nlrp1b Nlrp1c NLRP2
NLRC NLRC
NLRC
NLRP3
NLRP
Nlrp3 NLRP4 Nlrp4a Nlrp4b Nlrp4c Nlrp4d Nlrp4e Nlrp4f Nlrp4g
(Continued )
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Table 1
(Continued ) Other names and aliases NALP5; PYPAF8; MATER; PAN11; CLR19.8 Mater; Op1 NALP6; PYPAF5; PANS; CLR11.4 NALP7; PYPAF3; NOD12; PAN7; CLR19.4 NALP8; PAN4; NOD16; CLR19.2 NALP9; NOD6; PAN12; CLR19.1 Nalp9a; Nalp-theta Nalp9b; Nalp-delta Nalp9c; Nalp-zeta NALP10; PAN5; NOD8; PYNOD; CLR11.1 Nalp10; Pynod NALP11; PYPAF6; NOD17; PAN10; CLR19.6 NALP12; PYPAF7; Monarch1; RNO2; PAN6; CLR19.3 Nalp12 NALP13; NOD14; PAN13; CLR19.7 NALP14; NOD5; PAN8; CLR11.2 Nalp14; Nalp-iota; GC-LRR NOD9; CLR11.3 NLRX Domain organization NLR family NLRP
HGNC-approved symbol Human NLRP5 Nlrp5 NLRP6 Nlrp6 NLRP7 NLRP8 Mouse
NLRP NLRP NLRP
NLRP9 Nlrp9a Nlrp9b Nlrp9c NLRP10 Nlrp10 NLRP11 NLRP12 Nlrp12 NLRP13 NLRP14 Nlrp14 NLRX1
NLRP
NLRP
NLRP NLRP
NLRP NLRP
The N-terminal domain of the NLRs is critical for downstream signaling. CARD domains were originally associated with proteins involved in apoptosis and inammation such as many of the caspases, including caspase1; however, CARDs have also been shown to mediate caspase-independent interactions.
The structure of PYD is homologous to that of CARD and promotes homophilic interactions with other PYD-containing proteins that are important for downstream signaling events. Both CARD and PYD are members of the death domainfold superfamily, members of which are involved in both apoptosis and
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inammation. Finally, the BIR-containing proteins can be classied into two major groups, inhibitor of apoptosis proteins (IAPs) and neuronal apoptosis inhibitor proteins (NAIPs), both of which are NLR family members. Because of the variety in domain structures for each NLR, the nomenclature of the different NLRs has been inconsistent and confusing. Recently, in order to promote uniformity in the nomenclature of the various NLRs, a new system approved by the Human Genome Organization Gene Nomenclature Committee was established (5). This new annotation system recognizes the evolutionarily conserved NOD and LRR domains that are the central dening features of these PRRs, but it further subdivides the NLR family into four subfamilies (indicated by letters A through C and P) based on the type of N-terminal effector domain (Table 1). An additional subfamily, NLRX, has no strong homology to the N-terminal domain of any of the other four subsets and currently consists of only one member, NLRX1 (Nod9), which appears to be unique in its localization to the mitochondria (6, 7).
defend the host against infection. However, the end targets of NLR signaling are not the same for all NLRs. Three major activation targets of NLR signaling after PAMP recognition have been identied: (a) NF-B, (b) MAPKs, and (c) caspase-1 (Figure 1).
Nuclear FactorKappa B/Mitogen-Activated Protein Kinase Signaling

The earliest-identied and best-characterized NLRs are NOD1 and NOD2, which are prototypical of NLR activation of both the NFB and MAPK pathways (912). Upon recognition of their respective agonists, both NOD1 and NOD2 self-oligomerize to recruit and activate the adaptor protein RICK (also known as RIP2), which is essential for the activation of both NF-B and the MAPKs (4, 10, 13 16). RICK is a serine-threonine kinase that becomes polyubiquitinated upon interaction with NOD1 or NOD2 through homotypic CARDCARD interactions (17). This RICK K63 linked ubiquitination step is essential for recruitment of the kinase TAK1, which activates the NF-B-activating complex and is inhibited by the deubiquitinase A20 (17). RICK itself recruits and promotes the K63-linked polyubiquitination of the I-kappa-B kinase gamma (IK) regulator subunit of the IK complex, or NF-B essential modulator (NEMO), which can also facilitate recruitment of TAK1 in a ubiquitin-dependent manner (18). The colocalization of NEMO and TAK1 promotes the subsequent phosphorylation of the IK subunit of IK by TAK1 and results in the phosphorylation and degradation of IB, a critical step that allows the cytoplasmic release and nuclear translocation of NF-B (19). NF-B subsequently activates transcription of inammatory cytokines and chemokines such as TNF-, IL-6, IL-8, and membrane cofactor protein 1, which are important for stimulation and recruitment of additional effector cells during host defense. As has been demonstrated for both NOD1 and NOD2 signaling, ubiquitination plays a
NOD-LIKE RECEPTOR SIGNALING: DIVERSE AND COMPLEX PATHWAYS WITH MULTIPLE LEVELS OF REGULATION
In general, the primary function of PRRs is the activation of inammatory signaling pathways. In some respects, NLR signaling is very similar to that of the TLRs, with shared downstream targets (Figure 1). For example, upon recognition of their respective PAMPs, the primarily membrane-bound TLRs recruit adaptor proteins such as MyD88 and TRIF, which activate the MAPK and NF-B signaling pathways, resulting in the induction of proinammatory and antimicrobial mediators such as interleukin (IL)-6, tumor necrosis factor alpha (TNF-), and IL-1 (for a review, see Reference 8). Similarly, stimulation of the intracellular NLRs activates downstream signaling pathways for the production of proinammatory mediators to
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LPS
TLR4 TLRs iE-DAP NOD1 TLR7 7 IRAK4 RICK CARD9 Ub TAB2/3 TAK1 Ub TAK1 TAB2/3 p38 MKKs JNK IKK IKK TAK1 NEMO IKK IKK ERK JNK ERK NEMO MKKs p38 RICK CARD9 TRAF6 TLR9 TAB1 C CpGDNA TAB1 TAB1 NOD2 MDP MyD88 Imiquimod
Lipoprotein Flagellin TLR1 R1 or r TLR2 TLR6 TLR5 R6
Chen et al.
TRIF
Viral dsRNA
IRAK1
TLR3
NLR inammasome
Endosome
TAB2/3
MKKs
Pro-IL-1
IL-1
JNK NF-B Nucleus NF-B
ERK NF- B NF B NF-B
Figure 1
Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling pathways. (a) Extracellular pathogen-associated molecular patterns (PAMPs) are recognized by TLRs at the plasma membrane and endosomes, which signal through the adaptors MyD88 and Toll/interleukin-1 receptordomain-containing adapter-inducing interferon beta (TRIF), as well as through interleukin-1 receptorassociated kinase (IRAK) proteins and tumor necrosis factor receptorassociated factor 6 (TRAF6). (b) The NLR proteins NOD1 and NOD2 sense intracellular D--glutyamyl-meso-DAP (iE-DAP) and muramyl dipeptide (MDP), respectively, leading to recruitment of the adaptor proteins RICK and caspase recruitment domain 9 (CARD9). Subsequently, both TLRs and NOD1/NOD2 signaling pathways recruit TAK1, which mediates the activation of nuclear factorkappa B (NF-B) and mitogen-activated protein kinases (MAPKs), resulting in the transcriptional upregulation of proinammatory genes. (c) Activation of NLRs by microbial or endogenous molecules in the cytosol results in the formation of caspase-1-activating inammasomes. Activation of caspase-1 induces processing of the interleukin-1-beta (IL-1) precursor and secretion of the mature cytokine. Abbreviations: ERK, extracellular signalregulated protein kinase; IKK, I-kappa-B kinase; JNK, c-Jun N-terminal kinase; MKK, MAP kinase kinase; NEMO, NF-B essential modulator.
key role in activation of downstream signaling. For NOD2-mediated activation of NF-B in particular, the E3 ligase tumor necrosis factor receptorassociated factor 6 (TRAF6) was suggested to be importantbut dispensablefor NOD1 signaling, whereas TRAF2 and TRAF5 were essential (17, 20). The signicance of the differential utilization of E3 ligases for ubiquitination is not clear, but this ligase may provide additional points of regulation during the induction of inammatory cytokine production. NOD2 is also additionally regulated by Erbin, which interacts with NOD2 and is capable of downregulating NOD2-dependent activation of NF-B. However, whether Erbin also regulates signaling of other NLRs remains to be determined (21). Stimulation of NOD1 and NOD2 also results in the activation of MAPKs, including the p38, extracellular signalregulated protein kinase (ERK), and c-Jun N-terminal kinase ( JNK) pathways (Figure 1) (15). In contrast to NF-B, the molecular events that occur for activation of these pathways are not as well dened, but they involve similar upstream signaling molecules such as RICK and TAK1 (15, 22, 23). Recent evidence also suggests that the adaptor protein CARD9 may be particularly important in NOD2- and RICK-mediated activation of MAPK pathways and less so for NF-B (24). Importantly, there are NLR members that, instead of promoting NF-B activation, may have a primarily negative regulatory role. These members include NLRP12 (25, 26), NLRC3 in T cells (27), and NLRP2 in macrophages (28). As the studies identifying these members were all performed in vitro and typically involved overexpression of proteins or the use of tumor cell lines, the physiologic roles of these NLRs in vivo remain to be determined.
inammatory mediators, including cytokines, chemoattractants, adhesive molecules, and inducible molecules (e.g., iNOS, Cox-2). For NOD1 specically, which is expressed in epithelial and mesothelial cells, its stimulation can induce chemokine production and recruitment of effector immune cells, including neutrophils in vivo (29, 30). Equally important is the upregulation of antimicrobial peptide production especially in epithelial cells, which may be NFB dependent, by both NOD1 and NOD2 signaling. Disruption of this function may contribute to the pathogenesis of inammatory diseases in the bowel (3136). NOD1 signaling is also important in the coordination of an adaptive immune response by both T and B cells through synergistic antigen presentation with TLRs (1). Similarly, a role for NOD2 in mounting adaptive responses is implicated by the observation that the NOD2 agonist, muramyl dipeptide (MDP), can act as an effective adjuvant for antigen-specic T cell responses and antibody production (37, 38). Aside from the induction of proinammatory mediators, NOD1 and NOD2 have also been shown to induce apoptosis in in vitro overexpression systems; in fact, these proteins were originally identied by their structural homology to Apaf-1 and CED-4, known regulators of apoptosis (13, 14). The specic pathways involved in the induction of apoptosis are not entirely clear, but for NOD1 this process involves both caspase-8 and caspase-9 and requires RICK. Note, however, that these studies were all performed in vitro and that functional relevance remains to be investigated in vivo (13, 23).
MDP: muramyl dipeptide
Interleukin-1 Production and the Inammasome

In addition to NF-B and MAPKs, the third important pathway activated by NLR signaling involves ASC (adaptor protein apoptosis speck protein with caspase recruitment) to activate caspase-1 (Figure 1). NLRs that participate in caspase-1 activation include NLRP1, NLRP3, and NLRC4. Caspase-1 activation is required for the cleavage of pro-IL-1 and pro-IL-18
Biological Responses to NOD-1 and NOD-2 Signaling

A consequence of NOD1- and NOD2mediated activation of NF-B and MAPK is the upregulated transcription and production of
into their mature, biologically active forms. Recruitment of ASC by these NLRs is believed to occur through homophilic PYD-PYD interactions. Via its CARD domain, ASC, in turn, interacts with caspase-1. These protein-protein interaction domains promote oligomerization, recruitment, and approximation of effector proteins essential for activation of caspase-1 (4, 39). Indeed, a hallmark of this caspase-1-dependent pathway is the assembly of large macromolecular complexes through CARD-CARD and PYD-PYD protein-protein interactions that function to form a scaffold for procaspase1 recruitment and activation. This molecular platform, of which NLR family members are the cornerstone, has been termed the inammasome (40) as an analogy to the apoptosome, an activator of caspase-9 during apoptosis (41). Currently there are three well-described inammasomes named after the NLR involved: NLRP1, NLRP3, and NLRC4. Common to these inammasomes is the role of ASC as the adaptor protein that bridges these NLRs to caspase-1 (40, 42). The NLRP1 inammasome was the rst to be characterized and is the only inammasome that has been reconstituted in vitro with puried proteins (40, 43). The existence of the NLRP3 and NLRC4 inammasomes, the activities of which are dictated by the specic PRRs recognized by the NLRs, has been implicated primarily by the ability of both NLRP3 and NLRC4 to activate caspase-1 in an ASC-dependent manner (4450). Other NLR members that can associate with procaspase-1 and promote IL-1 production such as NLRP2have only been demonstrated in vitro, but whether the latter is a bona de inammasome that functions to activate caspase1 during host defense in vivo remains to be determined (28). NAIP with its BIR domain, rather than its CARD or PYD domain, has also been shown to be capable of activating caspase1 at least for the murine isoform Naip5; however, the relevance of its ability to interact with caspase-1 and whether it acts alone or together with NLRC4 remain controversial. We discuss this issue in more detail below.
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Another important consequence of caspase1 activation is the induction of a newly recognized process of programmed cell death termed pyroptosis, which is distinct from apoptosis and necrosis. Pyroptosis is typically induced in macrophages infected with certain intracellular bacteria such as Salmonella and is associated with a proinammatory response involving caspase-1-mediated IL-1 and IL-18 production (51). This response causes rapid formation of plasma membrane pores, cellular lysis, and release of intracellular inammatory contents to fuel additional inammatory signaling pathways (52). Caspase-1 activation and the release of IL-1 and IL-18 have been shown to be particularly important in host defense against such pathogens as Shigella (53), Legionella (48), Francisella (54), Listeria (55), Yersinia (56), and Bacillus anthracis (52). How the different inammasomes are enlisted to defend against specic pathogens or stimuli is described in the following sections.
NOD-LIKE RECEPTORS RECOGNIZE SPECIFIC BACTERIAL AND ENDOGENOUS MOLECULES

Although numerous studies have delineated the importance of individual NLRs against specic pathogens, a direct interaction between a putative ligand and its corresponding NLR has not been demonstrated for most of the NLRs. Therefore, the possibility that the interaction between NLR and PRR is indirect and involves an intermediary host factor or activity cannot be precluded. In this section, we describe what is known about the sensing of pathogens by the various NLRs (Table 2).
NOD1
Originally, LPS preparations were demonstrated to activate both NOD1 and NOD2 through RICK, but this was recently shown to be due to contamination of LPS preparations with PGN moieties (10, 15, 57). Both NOD1
Table 2 Receptor Nod1
NOD-like receptor (NLR) agonists and downstream signaling pathwaysa Agonist GM-tripeptide meso-lanthionine, meso-DAP -d-Glu-DAP(iEDAP) d-lactyl-l-Ala--Glu-meso-DAP-Gly (FK156) heptanolyl--Glu-meso-DAP-Ala (FK565) Bacteria Helicobacter pylori Shigella exneri Listeria monocytogenes Campylobacter jejuni Enteropathogenic Escherichia coli Chlamydia pneumoniae Pseudomonas aeruginosa Bacillus spp. Streptococcus pneumoniae Listeria monocytogenes Mycobacterium tuberculosis Salmonella Typhimurium Staphylococcus aureus Shigella exneri Salmonella Typhimurium Legionella pneumoniae Pseudomonas aeruginosa Shigella exneri Listeria monocytogenesb Legionella pneumophila Pseudomonas aeruginosa Salmonella Listeria Bacillus anthracis Staphylococcus aureus Listeria monocytogenesb Signaling pathway NF-B MAPK
Nod2
MDP MurNAc-l-Ala-g-d-Glu-l-Lys (M-TRILys)
NF-B MAPK
Nlrc4
agellin (Salmonella, Legionella, Pseudomonas) unknown (Shigella)
caspase-1
Naip
unknown
caspase-1
Nlrp1b Nlrp3
anthrax lethal toxin bacterial RNA viral RNA and DNA uric acid crystals LPS LTA MDP silica asbestos
caspase-1 caspase-1
Abbreviations: LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MDP, muramyl dipeptide; NF-B, nuclear factorkappa B. Both Nlrc4 and Nlrp3 may play redundant roles in caspase-1 activation in response to this pathogen, as both have been shown to contribute to IL-1 production in vitro.
and NOD2 recognize PGN moieties found in the bacterial cell that are secreted by bacteria (5861). PGN provides structure and rigidity to bacteria and is found in virtually all bacteria, although the amount, location, and specic composition may vary (62). PGN consists of sugar chains of alternating N-acetylglucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc), which are crosslinked by short peptide chains (Figure 2). These peptide chains contain unique amino acids that are differentially found in gram-
negative and gram-positive bacteria and that are also differentially recognized by NOD1 and NOD2. Specically, NOD1 can sense PGN moieties containing meso-diaminopimelic acid, an amino acid that is found predominantly in gram-negative bacteria but also in some gram-positive bacteria such as Listeria monocytogenes and Bacillus spp. The minimal structure recognized by NOD1 is the dipeptide D--glutyamyl-meso-diaminolimelic acid (iEDAP) (59, 60). Studies dening synthetic iEDAP derivatives that stimulate NOD1 revealed
LPS
Porin Outer membrane

Lipopeptide
Gram-negative bacteria
PGN Plasma membrane Cytosol
Polysaccharide Lipoteichoic acid
Gram-positive bacteria
PGN
Plasma membrane Cytosol
MDP (Nod2 ligand)
NAG
NAM
NAG
NAM
Peptidoglycan (PGN)
L-alanine D-glutamic
acid
Mesodiaminopimelic acid
D-alanine
Tetra-peptide chain (amino acids) Glycan chain NAM NAG NAM
iE-DAP (Nod1 ligand)
NAG
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that the attachment of hydrophobic acyl residues enhanced stimulation of NOD1 up to several hundred fold (63). As these residues contained fatty acid chains similar to those found in the phospholipids that make up the host cell membrane, it is possible that the increased NOD1-stimulatory activity is due to an increased interaction with the cell membrane that facilitates translocation into the intracellular compartment of the host cell (63). The lipophilicity of the NOD1 ligand appears important for recognition by NOD1, but precisely how NOD1 interacts with these lipophilic molecules and whether this actively contributes to its transfer and recognition into the host cell remain to be determined. In vitro studies have demonstrated that many bacteria have NOD1-stimulatory activity, with the strongest activity associated with the genus Bacillus (58). Moreover, the presence of bacteria has not always been necessary for NOD1 stimulation, as water-soluble extracts from food and soil as well as supernatant from overnight bacterial cultures are capable of stimulating NOD1 (58). This suggests that physical contact with live bacteria is not necessary and that NOD1 agonists can be produced and released by bacteria. Consistent with this observation, Shigella mutants that increase release of PGN fragments upregulated NOD1-dependent NF-B activation in vitro (64). In addition, where and how NOD1 interacts with bacteria are not clear. Localization of bacteria to the intracellular compartment has been shown to not necessarily be required for NOD1 stimulation, as the Listeria streptolysin O mutant, which is required for Listeria to escape from the phagosome into
the cytosol, is still capable of activating NF-B in a NOD1-dependent manner (58). Localization studies of NOD1 through overexpression have shown that NOD1 becomes membrane bound, at least during infection with Shigella exneri at sites of bacterial entry. However, whether this occurs in vivo remains to be determined, as endogenous NOD1 localization during infection has been difcult to visualize (65). Despite the identication of the PGN moiety sensed by NOD1, the role of NOD1 during in vivo infection remains unclear, with the partial exception of Helicobacter pylori, a bacterial organism associated with the development of gastritis and duodenal ulcers. Specically, Nod1-decient mice have shown increased susceptibility to H. pylori infection, with an associated impairment of -defensin-4 production (34, 66). In vitro studies, on the other hand, have shown NOD1-dependent activation by other pathogenic bacteria such as L. monocytogenes (15), S. exneri (9), Campylobacter jejuni (36, 67), enteroinvasive Escherichia coli (68), Chlamydophila pneumonia (69), and Pseudomonas aeruginosa (70). However, these ndings were based on in vitro studies only and may not necessarily reect requirements in vivo. For example, NOD1 was originally implicated in protection against Chlamydia trachomatis infection in vitro, where Nod1-decient mouse embryonic broblasts (MEFs) exhibited decreased cytokine production after C. trachomatis infection; however, Nod1-decient mice did not have increased bacterial load or clinical symptoms, suggesting either that NOD1 is not directly involved or that other redundant signaling pathways can compensate (71).

Figure 2 Generation of NOD1 and NOD2 ligands from bacterial peptidoglycan (PGN). In this simplied diagram, the bacterial cell wall and PGN structure from Escherichia coli are depicted. Parallel PGN strands composed of the alternating amino sugars N-acetylglucosamine (NAG) and N-acetyl muramic acid (NAM) are crosslinked to each other by stem peptides. Notice that E. coli PGN lacks bridging amino acids linking stem peptides and that cross-linking occurs via a direct link between a meso-diaminolimelic acid (meso-DAP) residue and the d-alanine residue in position four from a peptide anchored on the parallel glycan strand. Minimal motifs required for NOD1 and NOD2 (dashed boxes) recognition are also shown. Abbreviations: iE-DAP, d--glutyamyl-meso-DAP; LPS, lipopolysaccharide; MDP, muramyl dipeptide.
NOD2
Separate studies have also demonstrated that NOD2 can detect pathogenic bacteria. In contrast to NOD1, which appears to be primarily involved in sensing gram-negative bacterial pathogens, NOD2 senses the specic MDP motif that is found in a broader range of bacteria, with some overlap with those recognized by NOD1 (Figure 1). These bacteria include Streptococcus pneumoniae (72), L. monocytogenes (33, 73), Mycobacterium tuberculosis (74), Salmonella (75), and Staphylococcus aureus (76). Again, most of these studies were performed in vitro; therefore, the in vivo relevance of these ndings remains to be determined. One exception, however, is L. monocytogenes, in which increased bacterial burdens were observed in Nod2-decient mice when infected orally rather than intravenously or intraperitoneally (33). This immunity is associated with decreased production of -defensins in Paneth cells (33). In addition, both Nod1 and Nod2 have been shown to be important for bacterial recognition and host defense against L. monocytogenes after exposure of macrophages or animals to LPS or E. coli in vivo (77). These observations suggest that the intracellular sensors NOD1 and NOD2 play a critical role in host defense when TLR signaling is reduced, such as within the intestine due to low expression levels of TLRs (78, 79) or after induction of tolerization by exposure to TLR ligands.
NLRC4
As mentioned above, a subset of NLRs participate in the formation of inammasomes that ultimately leads to activation of caspase-1 and maturation of IL-1. To date, these NLRs include NLRC4, NLRP1, and NLRP3. Studies have shown that the NLRC4 inammasome can activate caspase-1 in response to infection by S. Typhimurium, an activity not shared by the NLRP3 inammasome (49, 50). Salmonella infection can lead to gastroenteritis as well as to typhoid fever, which can be associated with high morbidity and mortality. What is recognized
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by NLRC4 is agellin, as agellin-decient Salmonella mutants elicit substantially reduced levels of caspase-1 activation (49). Similarly, cytosolic detection of L. monocytogenes by Ipaf requires agellin (80). A direct interaction between agellin and NLRC4 has not yet been demonstrated, however, and therefore recognition is likely indirect. TLR5, a member of the TLR family, also senses agellin; however, it has been shown that the cytoplasmic delivery of agellin requires NLRC4 but not TLR5, consistent with its cellular location. As an additional consequence of caspase-1 activation, caspase1 can lead to early macrophage cell death, an NLRC4-dependent process in the host defense against Salmonella (47). In vivo, NLRC4 has also demonstrated activity against Legionella pneumophila, an intracellular pathogen that causes Legionnaires disease, which can be deadly. The pathogenesis of Legionella requires successful entry and replication inside host macrophages and is dependent on the formation of a specialized vacuole that blocks the fusion of the phagosome to the lysosome, a process dependent on an intact functional type IV secretion system (TFSS). As is the case with Salmonella, Legionella also contains agellin, and NLRC4-dependent caspase-1 activation requires the presence of intact agellin and its intracellular delivery (48, 81). As a result of NLRC4-induced caspase-1 activation, Legionella growth is restricted by promoting the fusion of the Legionella-containing phagosome to the lysosome for degradation (48). How caspase-1 activation controls phagosome maturation in response to Legionella infection is not known, but it may involve interaction of caspase-1 with host proteins involved in phagosome formation and transport or direct targeting of Legionella virulence factors required for lysosomal evasion. In vitro, NLRC4 is important in responding to Shigella, an intestinal pathogen that causes dysentery and, as mentioned above, can also elicit NOD1-dependent MAPK activation and secretion of IL-8 (9). Like Legionella, Shigella can escape from within membrane vacuoles and enter the cytosol. Interestingly, Shigella
does not express agellin genes, yet it can activate caspase-1 macrophages and induce pyropoptosis in an NLRC4-dependent process (82). This suggests that NLRC4 also recognizes an as-yet-unidentied PAMP or signal associated with Shigella. Shigella also induces autophagy, which is facilitated in NLRC4 but not in ASC-decient macrophages, suggesting that NLRC4 provides an additional host-defense measure independent of ASC and IL-1 production by inhibiting autophagy (a membrane-trafcking system in which cytoplasmic components are sequestered for delivery to lysosomes, where cytoplasmic material is degraded). This downregulation of autophagy is specic to Shigella-induced autophagy, as autophagy from serum starvation or rapamycin treatment does not require NLRC4 or caspase-1 (82). The reason for the inhibition of autophagy by NLRC4 is not clear, but it has been proposed that this inhibition allows the induction of pyroptosis, which may better promote an effective inammatory response (82).
NAIP
One of the mouse homologs of human NAIP, Naip5, is also important in the host defense against Legionella. This nding arose from observations that different mouse strains show varying permissiveness to intracellular replication of Legionella and that this permissiveness is genetically controlled (83, 84). The genetic locus responsible for the increased susceptibility to Legionella infection in the A/J mouse strain in particular has been determined to be Naip5 (85). Naip5 promotes the fusion of the Legionella-containing phagosomes to the lysosome, thereby preventing intracellular replication (86). Studies in vitro have also suggested that Naip5 activation requires the intracellular delivery of agellin, which results in caspase-1 activation and in promotion of early cell death, thereby controlling intracellular replication of Legionella (81, 87, 88). However, these studies used large numbers of Legionella bacteria, which can lead to nonspecic early
cell death of macrophages; they also used an indirect, nonspecic method of detecting caspase-1 activation. In a separate study using low bacterial load and caspase-1-specic antibodies, Legionella-induced caspase-1 activation and IL-1 production occurred independently of Naip5 but required Nlrc4 as well as agellin (89). Naip5, however, was still required for inhibition of Legionella replication intracellularly, irrespective of Nlrc4 signaling; this suggests a caspase-1-independent pathway regulated by Naip5 that restricts Legionella replication and acts in concert with Nlrc4 signaling (89). The PAMP involved in this process has yet to be determined, as it does not require agellin. In addition, Naip5like Nlrc4has been suggested to regulate autophagy: A/J mice that harbor mutant Naip5 exhibited slower rates of autophagy induction after Legionella infection of macrophages compared with Legionella-resistant C57BL/6 that have intact Naip5 (90). This observation further demonstrates the complexity of intracellular Legionella replication by different NLRs, and it remains an active area of investigation. Based on in vitro studies using A/J mice that are considered Naip5 decient compared with C57BL/6, which have intact Naip5 and are resistant to Legionella replication, Naip5 also contributes to IL-1 production by bone marrow derived macrophages after infection with P. aeruginosa, an opportunistic bacteria that can cause pneumonia and sepsis (91). The decrease in IL-1 production in A/J mice, although statistically signicant, may reect inherently different genetic susceptibility differences between the two mouse strains that are independent of Naip5; therefore, this decrease may be physiologically irrelevant in vivo. Therefore, additional studies examining a true Naip5-knockout strain should provide additional insight into and conrmation of these ndings.
NLRP1
Recently discovered mutations in the Nlrp1 (Nalp1b) gene in various mouse strains have
been found to inuence the susceptibility of mice to Bacillus anthracis lethal toxin (LT) (92), the major virulence factor in the pathogenesis of anthrax. This was the rst demonstration of an NLR that responds to a virulence factor, as opposed to a specic bacterial component. LT results in the formation of pores allowing lethal factor, a protease, to enter cells, resulting in proteolytic cleavage of host substrates and subsequent cell death. Susceptibility to LT-induced macrophage lysis/necrosis and death requires caspase-1 activation and is associated with Nalp1b polymorphisms. Five of these polymorphisms have been identied, explaining the variability in susceptibility to LTinduced cell death in different mouse strains (92). Caspase-1 activation by LT results in pyroptosis similar to that seen in NLRC4mediated cell death induced by Salmonella infection (52). The human NLRP1 inammasome, reconstituted with puried proteins including ASC, NLRP1, and procaspase-1, has also been shown to respond to MDP and to induce the maturation of IL-1. Because only puried proteins were used in this cell-free system (40), it is possible that, in this case, a direct interaction between the inammasome and MDP occurred under physiological conditions.
NLRP3
NLRP3 is an NLR that also participates in inammasome formation through the recruitment of ASC and subsequent activation of caspase-1 and secretion of IL-1 and IL-18. NLRP3 detects PAMPs such as LPS, MDP, and bacterial and viral RNA, including the doublestranded RNA analog poly I:C (4446) as well as the imidazoquinoline antiviral compounds R837 and R848 (93). Consistently, NLRP3 has been found to respond to infection with the Sendai and inuenza viruses, resulting in caspase-1 activation (44). Subsequently, it was shown that NLRP3 can detect viral DNA (94). In vitro, NLRP3 has also been demonstrated to be required for caspase-1 activation/IL-1 secretion in response to the bacterial pathogens
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S. aureus and L. monocytogenes (46). However, in the case of L. monocytogenes, the role of NLRP3 has been controversial, as there are opposing data to suggest that caspase-1 activation by Listeria can occur independently of NLRP3 (98, 102). This discrepancy may be explained by redundant contributions by both NLRP3 and NLRC4 in caspase-1 activation in response to Listeria (80). Characteristic of NLRP3, however, is its capability to respond to a broad repertoire of agonists that are not necessarily microbial in origin, but rather are endogenous signals. NLRP3 has been suggested to respond to changes in cellular ion concentrations, particularly potassium, that are produced, for example, by the marine toxin maitotoxin and the K+ /H+ antiport ionophore nigericin (46). NLRP3 activation is also observed with uric acid crystals (95) and reactive oxygen species that are generated in response to, for example, asbestos and silica (7). Signicant insight into the mechanism by which NLRP3 recognizes its various ligands came from the observation that the addition of ATP to macrophages prestimulated with bacterial molecules such as LPS can signicantly enhance caspase-1 activation and IL-1 production. Identifying the role of ATP in NLRP3 activation has been controversial, as high nonphysiological concentrations of ATP are typically required for enhanced IL-1 production. An important function of ATP is the stimulation of the P2X7 receptor, which, in turn, results in the opening of a pore mediated by the hemichannel protein pannexin-1 (96, 97). Therefore, a consequence of ATP addition is the ability of bacterial products to enter the cytosol through the pore and to subsequently activate NLRP3 and caspase-1. Indeed, with the addition of ATP, several bacterial products including LPS, PGN, and lipoteichoic acid, as well as heat-killed bacteriacan activate caspase-1 in an NLRP3-dependent (but a TLRand RICK-independent) manner (44, 45, 47, 98). Moreover, the requirement for ATP can be bypassed when other methods of cytosolic delivery are implemented; such methods include
the use of pore-forming bacterial toxins (e.g., streptolysin O) or the lipophilic DOTAP delivery system (98, 99). However, not all bacterial pathogens that can invade the cytosol require NLRP3 for caspase-1 activation as Salmonella and Francisellawhich are capable of delivering bacterial products into the cytosol through type III secretion systems (TTSS) or through pore-forming molecules without ATPcan activate caspase-1 independently of NLRP3 (98). Thus, NLRP3 recognition of PAMPs appears to be linked to pannexin-1-dependent pore formation. MDP, a NOD2 agonist, has also been shown to activate NLRP3 (100) so as to activate caspase-1. This activation is independent of NOD2 and involves the internalization of MDP into acidied vesicles that are released into the cytosol upon addition of ATP in a pannexin-1-dependent manner (101). An increasingly recognized consequence of activation of the P2X7 receptor and pannexin1 by ATP is the alteration in potassium concentrations in the cell (i.e., intracellular potassium depletion), which has been suggested to be important for NLRP3 but not for NLRC4 signaling (102, 103). Treatment of macrophages with the pore-forming toxins nigericin and maitotoxin is also associated with intracellular potassium depletion and results in NLRP3-induced activation of caspase-1 (46). Therefore, in addition to facilitating entry of NRLP3-responsive PAMPs, pore-forming toxins also promote changes in intracellular potassium concentrations that may be required for NLRP3 activation. However, the concentration of potassium itself may not regulate caspase-1 activation, as changes in potassium concentration in the cell are associated with other ionic uxes or cellular events that may be critically involved in NLRP3 signaling. Moreover, in the absence of PAMP prestimulation, ATP does not activate caspase-1, even though it alone can trigger potent efux of intracellular potassium (102). Thus, the true signicance of potassium concentration changes associated with NLR signaling remains to be fully elucidated. However, one can interpret changes in cellular potassium
concentrations associated with pore formation, uric acid release, and ATP generation as surrogate indicators of cellular stress or injury; therefore, NLRP3 and perhaps other NLRs may also function to recognize and respond to endogenous danger signals besides microbial infection (95, 104).
MECHANISMS OF INTRACELLULAR DELIVERY OF MICROBIAL PRODUCTS FOR DETECTION BY NOD-LIKE RECEPTORS

A dening feature of the NLRs is their intracellular localization. With the exception of NLXR1, which has been shown to be important within the mitochondria (6, 7), and CIITA, which resides in part in the nucleus, all NLRs characterized to date are located within the cytoplasm. This raises the question of how these receptors recognize pathogens, or how pathogens are delivered into the intracellular compartment. Based on their cytosolic localization, NLRs may respond primarily to (a) bacteria that escape extracellular detection by the TLRs and invade directly into the cell, (b) bacterial components that are delivered into the cytosol through secretion systems or through pore-forming molecules, or (c) bacterial products that are uptaken by the immune cell through phagocytosis or pinocytosis (Figure 3). Consistent with the rst possibility, NOD1 activation depends upon the intracellular localization of directly invasive bacteria such as S. exneri (9, 64, 105) and S. pneumoniae (72). Also, only live and not paraformaldehydexed C. jejuni elicit NOD1-dependent NF-B activation (36). Bacteria with active secretion systems provide an additional mechanism for intracellular entry that is important for NLR recognition. Two secretion systems, type III and type IV, are involved in the injection of virulence proteins into the host cell to stimulate NLR signaling. These secretion systems are found in gram-negative bacteria and are encoded within regions of the bacterial genome
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Figure 3 Modes of intracellular entry of microbes for Nod-like receptor (NLR) activation. Microbes and microbial molecules enter the cytosol via pore-forming toxins, type III or IV secretion systems, or ATP-mediated activation of the pannexin-1 pore. Sensing of microbial molecules in the cytosol by several NLRs results in the formation of inammasomes. The mechanism whereby NLRs recognize microbial molecules appears indirect and remains poorly understood. Activation of caspase-1 induces processing of the interleukin-1-beta (IL-1) precursor and secretion of the mature cytokine. Abbreviation: PAMPs, pathogen-associated molecular patterns.
known as pathogenicity islands. These secretion systems allow NLR detection by nonphagocytosing cells such as epithelial cells. For TTSS, the mechanism by which gram-negative bacteria invade the cell is well illustrated by Salmonella (106). Two important features of Salmonella pathogenesis are (a) the bacterias ability to invade nonphagocytic cells such as intestinal epithelial cells and (b) their ability to survive as well as replicate within phagocytes. Both of these features depend upon intact TTSS. TTSS consists of a complex assembly of proteins that form transporter apparatuses; these contain approximately 20 protein subunits for the trafcking of bacterial
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proteins involved in bacterial virulence. As described above, the delivery of agellin to the cytosol for NLRC4 inammasome activation has been proposed to occur via TTSS, and Salmonella mutants with nonfunctional TTSS are unable to activate NLRC4 in macrophages (50). In the case of Legionella, TFSS is essential for the survival of the pathogen within the phagosome. In a not-fully-understood mechanism, Legionellaonce phagocytosed by the APC, such as the macrophagereplicates within the phagosome and avoids fusion with the lysosome through secretion of virulence proteins by the TFSS, which causes pore formation. Legionella is recognized by NLRC4 most likely as a result of translocation of bacterial ligand (agellin) through the TFSS into the cytosol (107). As with Legionella, delivery of PGNderived molecules into the cytosol by H. pylori for NOD1 signaling requires an intact TFSS associated with the cag pathogenicity island, and H. pylori strains that lack cag have reduced levels of NF-B activation (66). Bacterial secretion systems also enable pathogens to form pores to escape phagosomes after phagocytosis, as well as to transport bacterial products. For example, the pore-forming toxin listeriolysin produced by Listeria permits the delivery of PGN that is recognizable by NOD1 and NOD2 (12, 73, 108). Other bacteria that secrete pore-forming toxins, such as S. pneumoniae, which produces pneumolysin, and B. anthracis with anthrolysin O, can also allow internalization of PGN fragments for recognition by NLRs, especially in nonphagocytosing cells such as epithelial cells (109). With certain bacteria, activation of NLRs can occur with heat-killed bacteria alone, suggesting that neither an active secretion system nor direct invasion is required for recognition. In these cases, dead bacteria are likely directly uptaken into the cell by phagocytosis (73). For example, inhibition of macrophage phagocytosis of heat-killed S. aureus resulted in decreased inammatory cytokine production, which was partly dependent on both NOD1
and NOD2 (76). The entry of MDP has also been proposed to occur in intestinal epithelial cells through a host-dependent rather than bacterial-dependent mechanism, in which the active peptide transporter hPepT1 transports MDP in vitro (110).
NOD-LIKE RECEPTOR SIGNALING SYNERGIZES AND COMPLEMENTS TOLL-LIKE RECEPTOR SIGNALING

There is great overlap and an apparent redundancy in the nature of the pathogens recognized by the various NLRs and TLRs, and many of the receptors converge to common pathways (e.g., NF-B and IL-1 activation). An obvious reason for the existence of multiple receptors channeling into common end targets is the potentiation of the inammatory response to infection. This is consistent with the synergy observed with TLR and NLR signaling in response to PGN (74, 111, 112) and between TLR and NOD2 agonists (33). In addition, a segregation of function between NLRs and TLRs is naturally imposed by their cellular localization, such that NLRs may acquire greater importance in the detection of intracellularly located bacteria. Regardless, NLRs and TLRs also play nonredundant roles, as is reected in the observations of TLR- and NLRspecic transcriptomes associated with Listeria infection and of synergistic activation of NF-B (113). Additional evidence for complementary roles of NLRs and TLRs in the inammatory response has been demonstrated with IL1 secretion (see Figure 4a). The release of IL-1 involves two distinct steps: (a) induction of pro-IL-1, which is NF-B dependent, and (b) cleavage of pro-IL-1 by caspase-1 to the mature, active IL-1. The rst step requires TLR activation of NF-B (98); however, activation of caspase-1 occurs independently of TLRs and requires the inammasome (98). This two-part pathway, differentially regulated by TLRs and NLRs, may serve
as a safeguard against excessive production of IL-1, which can cause pathology (see Inammatory Diseases Associated with Excessive Interleukin-1 Production, below). Similarly, multiple NLRs or TLRs may recognize similar stimuli but regulate different aspects of the same process. Using the same example of regulation of IL-1 production, caspase-1 activation by MDP stimulation requires NLRP3 and ATP but not NOD2; however, the induction of IL-1 messenger RNA, on the other hand, requires NOD2 but not NLRP3 (101). Therefore, similar to the requirement for TLR signaling to promote pro-IL-1 production through NF-B, in the case of IL-1 induction by MDP, NOD2 mediates the transcription of pro-IL1 by NF-B, whereas NLRP3 mediates IL-1 processing through caspase-1 activation. Also, as described above, studies have suggested that Legionella recognition by both NLRC4 and Naip5 can result in alternate activities to restrict intracellular replication of Legionella and that recognition of agellin in either Legionella or Salmonella by TLR5 and NLRC4 can result in the activation of separate pathways, NF-B and caspase-1 activation, respectively (4850, 89, 114). Thus, the multiplicity of receptors involved in the recognition of the same ligand allows not only multiple levels of control to prevent excessive production of cytokines (e.g., IL-1) that can harm the host, but it also promotes a concerted response against infection through the activation of separate, but cooperative pathways. In addition to having a synergistic effect with TLR signaling, NLRs may provide the host with a backup defense mechanism that may be required under conditions when TLR signaling has been tolerized (Figure 4b). A well-recognized phenomenon associated with TLR signaling is the induction of tolerance: After an initial LPS exposure to, for example, macrophages, cytokine responses are suppressed upon secondary exposure to LPS. Possible physiologic explanations for this phenomenon are (a) to prevent excessive cytokine production by bacterial stimuli, which can lead
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Invasion of intracellular bacteria
Reduced TLR signaling (tolerance) (prevent overproduction of proinammatory cytokine) + Enhanced NLR signaling
Prepare to ght invasive pathogen

Figure 4 The cooperative interplay between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in response to intracellular bacteria. (a) Stimulation of TLRs or NOD1/NOD2 induces the production of pro-interleukin-1-beta (pro-IL-1) through the activation of nuclear factorkappa B (NF-B) signaling, leading to transcription of the IL-1 gene. In contrast, the presence of microbial molecules in the cytosol is sensed by the NLRs, resulting in inammasome formation and caspase-1 activation. Activated caspase-1 cleaves pro-IL-1 and results in secretion of mature IL-1. (b) Intracellular bacteria such as Listeria are recognized by TLRs, leading to TLR activation and production of proinammatory and antimicrobial molecules. Persistent TLR stimulation leads to TLR tolerization and to reduced TLR signaling. Cytosolic invasion of the bacteria activates NLRs (e.g., NOD1/NOD2 and inammasome-inducing NLRs), resulting in enhanced NLR signaling and in production of antimicrobial molecules.
to the clinical syndrome of sepsis and to multiorgan failure in the host, and (b) to stymie an excessive immune response against commensal bacteria, which can lead to pathological inammation. Although theoretically this
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is a good safeguard mechanism, a potential risk in the development of hyporesponsiveness with serial LPS challenge in APCs is an increased susceptibility to bacterial superinfection (115). Because TLR-induced tolerization
is limited to TLR agonists that act in the same pathway, NLRs may play a protective role against bacterial infection in the TLR-tolerized state (Figure 4b) (77). Indeed, it has recently been shown that cross-tolerization does not occur with the combination of the NLR agonist MDP and the TLR agonist LPS, as prestimulation with either LPS or MDP does not abrogate cytokine responses to MDP or LPS, respectively (77). Rather, enhancement in NFB and MAPK activation as well as in cytokine production occurred after stimulation of MDP in macrophages made refractory to TLR ligands, and vice versa (77). A functional consequence of this phenomenon is the heightened importance of NOD1 and NOD2 response to intracellular bacterial infection in macrophages tolerized to previous exposure with TLR agonists (77). Consistently, it was demonstrated that after previous exposure to E. coli or LPS, response to subsequent infection with Listeria was greatly compromised in the absence of NOD1 and NOD2 signaling both in vitro and in vivo (77). Similarly, in the case of Salmonella infection, TLR-tolerant macrophages were still capable of IL-1 production through NLRC4 signaling (49). Thus, NLR signaling can potentiate TLR signaling and can provide additional protection to the host against bacterial invasion. One implication for this model is that immune cells can remain sensitive to infection by pathogenic bacteria through NLR signaling in cells that have become tolerized to TLR ligands, such as commensal bacteria in the gut. Note, however, that several groups (116 118) have also shownboth in humans and in micethat cross-tolerization still does occur with stimulation of immune cells by NLR agonists followed by TLR agonists. This discrepancy may be due in part to differences in experimental methods, including the timing of pretreatment (early versus late second exposure) or cell type (macrophages versus dendritic cells), and it suggests that the regulation of tolerization and synergy between NLR and TLRs may be specic to both context and cell type.
DYSREGULATED NOD-LIKE RECEPTOR SIGNALING IS INVOLVED IN THE PATHOGENESIS OF MULTIPLE HUMAN DISEASES
The physiologic signicance of the various NLRs lies primarily in their roles in host defense against microbial infection. However, it is becoming increasingly clear that NLRs also function in organ homeostasis, which is underscored by the fact that many inammatory and noninammatory disease processes can be attributed to dysregulated NLR signaling (Figure 5). The following subsections describe certain dened NLR roles in human diseases.
IBD: inammatory bowel disease UC: ulcerative colitis CD: Crohns disease
Inammatory Bowel Disease

The role of NLRs in intestinal homeostasis was highlighted by the observations of mutations in NOD2 that are associated with the development of inammatory bowel disease (IBD). IBD encompasses two different diseases, ulcerative colitis (UC) and Crohns disease (CD); these diseases cause inammation of the intestine, leading to the common clinical presentation of abdominal pain, bloody diarrhea, and weight loss. Despite their similar clinical symptoms, UC and CD have distinguishing clinical and histologic features. In UC, inammation is limited to the mucosal layer of the colon, most commonly the rectum. In CD, however, inammation is transmural and can involve any part of the gastrointestinal tract. Because of the transmural inammation in CD, complications not typically seen in UC can occur; these include bowel-wall perforation and stula formation. CD also differs from UC with regard to the affected areas of the bowel: CD most commonly involves the lower part of the small intestine, the terminal ileum. The underlying pathogenesis of IBD is unclear, but studies suggest that both environmental and genetic factors contribute to the etiology of CD. Genetic linkage analyses of affected families have identied eight genetic
Gain-of-function mutations
Loss-of-function mutations
Asthma NOD1 (lungs) Cryopyrinopathies (MWS, FCAS, NOMID) NLRP3 (skin, eyes, joints) Annu. Rev. Pathol. Mech. Dis. 2009.4:365-398. Downloaded from www.annualreviews.org by Department of Primary Industries - New South Wales on 01/31/11. For personal use only. Blau syndrome/ early-onset sarcoidosis NOD2 (skin, eyes, joints) Sarcoidosis NOD1 (lungs) Bare lymphocyte syndrome CTIIA (lymphocytes) Inammatory bowel disease NOD2, NOD1? (intestine)
Vitiligo NLRP1 (skin)

Figure 5 Genetic variation in Nod-like receptors (NLRs) is associated with development of human disease. Gain-of-function point mutations within the NOD domains of NLRP3 and NOD2 cause autoinammatory disorders: The former cause Muckle-Wells syndrome (MWS), familial cold autoinammatory syndrome (FCAS), and neonatal-onset multisystem inammatory disease (NOMID), and the latter cause Blau syndrome/early-onset sarcoidosis. Consistently, the latter diseases are inherited in a Mendelian-dominant fashion. In contrast, loss-of-function mutations of NOD1, NOD2, and CTIIA are associated with the development of asthma, adult sarcoidosis, bare lymphocyte syndrome, and Crohns disease. Genetic variation in NLRP1 is associated with vitiligo. The type of functional alteration of the disease-associated NLRP1 alleles remains to be determined.
loci (IBD1IBD8) containing CD-susceptibility genes (119). The risk allele for IBD1 has been identied as NOD2 by genetic and functional studies (120, 121). Although multiple variants of NOD2 have been found to be associated with CD, approximately 40% of CD patients of North American or Western European descent carry at least one of the three major disease-associated variants: G908R, R702W, and a frame-shift deletion mutation at L1007 (L1007fsinsC). Patients homozygous for these mutations have a 20- to 40-fold increased risk
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for disease development, whereas heterozygous subjects have only a two- to fourfold increased risk (120, 121). These CD-associated NOD2 variants exhibit reduced capacity to activate NF-B following MDP stimulation (61), consistent with the nding that these mutations are near or within the LRR domain of NOD2. The impaired sensing of PGN and/or MDP suggests that these CD-associated mutations result in a loss-of-function phenotype. Furthermore, monocytes isolated from CD patients harboring the L1007fsins mutation exhibit reduced
Dendritic cell Nod2
1 Reduced NOD2-dependent expression of bactericidal -defensins in Paneth cells 2 Impaired recognition and clearance of bacteria by intestinal phagocytes
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Figure 6
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Proposed models for the role of NOD2 in Crohns disease. Shown are four nonexclusive models of the contribution of mutant NOD2 alleles to Crohns disease. In the rst two models, impaired NOD2 function is associated with either increased invasion of intestinal bacteria by decient production of -defensins (1) or reduced clearance of bacteria by intestinal phagocytes, leading to inappropriate activation of NOD2-independent pathogen-recognition receptor (PRR)-signaling pathways (2). In the third model, NOD2 acts as a brake of commensal bacteriadriven inammation, and the presence of decient NOD2 alleles results in enhanced Toll-like receptor (TLR)-induced activation (3). In the fourth model, which is based on a mouse Nod2-knockin model, Crohns diseaseassociated NOD2 mutations function as gain-of-function alleles, resulting in inappropriate interleukin-1-beta (IL-1) production (4).
production of proinammatory cytokines such as TNF-, IL-6, and IL-8, as well as the antiinammatory cytokine IL-10 (122, 123). However, the loss of NF-B inducibility by the CDassociated NOD2 variants is inconsistent with the occurrence of increased NF-B-dependent inammation observed in clinical samples isolated from CD patients. In order to reconcile these observations and provide a mechanistic explanation, two broad hypotheses regarding the role of NOD2 in the pathogenesis of CD have been advanced (Figure 6). The rst hypothesis is that mutations in NOD2 result in deciencies in epithelial-barrier function and/or in immune cells required for limiting bacterial invasion or clearance, which subsequently leads
to increased inammation at intestinal sites. The second contends that primary dysregulation of the mucosal immune system leads to excessive activation of proinammatory signaling pathways. Evidence for a role for NOD2 in regulating epithelial-barrier function comes from transfection experiments demonstrating that wildtype (but not mutant) NOD2 can restrict proliferation of Salmonella in intestinal epithelial cells (75). The ability of NOD2 to restrict invasive bacterial growth may be related to its ability to activate NF-B-dependent production of the -defensins by Paneth cells, which are specialized epithelial cells located in the crypts of the ileal mucosa. Consistent with this
hypothesis is the observation that NOD2 is expressed constitutively in Paneth cells (124, 125). In addition, CD patients with mutant NOD2 have reduced expression of two -defensins in the ileal mucosa, human -defensin-5 and -defensin-6 (31). Furthermore, mice genetically decient for Nod2 displayed impaired protection against oral, but not intravenous or intraperitoneal, administration of L. monocytogenes. The increased susceptibility of Nod2decient mice was correlated with diminished expression of Paneth cellderived antimicrobial peptides, Defcr4 and Defcr-rs10 (33). Thus, impaired production of -defensins in Paneth cells due to NOD2 mutations, which results in a compromise in barrier function, might be a plausible link between NOD2 and susceptibility to CD. However, the observation of decreased -defensins should be interpreted with caution, as this may merely reect Paneth cell loss from inamed, damaged epithelium (126). Another possibility is that NOD2 mutations result in impaired clearance of locally invasive bacteria due to defective recognition by intestinal phagocytes. This alternative loss-offunction hypothesis is consistent with observations of impaired production of antimicrobial molecules and of IL-1, induced by MDP in monocytes, expressing CD-associated NOD2 mutations (61, 123). The defective removal of intestinal bacteria may drive inammation via activation of NOD2-independent PRRs, including NOD1 and TLRs. The second hypothesis regarding the pathogenesis of CD contends that the disease results from inappropriate hyperresponsiveness to commensal bacteria in the normal intestine microora (127). This hyperreactive hypothesis is supported by evidence demonstrating that (a) NOD2 functions as a negative regulator of IL-12 production mediated by PGN through TLR2 and (b) in the absence of NOD2mediated regulation, PGN elicits a heightened NF-B-dependent IL-12 or IL-23 response by intestinal APCs, thereby promoting an exuberant adaptive Th1 or Th17 type of immune response often observed in CD patients (128, 129). In this model, it is assumed that in
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normal intestine, APCs are constantly exposed to commensal bacteriaderived PGN without any overt immune response. It is then postulated that in normal individuals, NOD2 functions as a brake, dampening any potential innate immune response to PGN. In contrast, PGN-mediated immune responses in individuals with NOD2 mutations are refractory to NOD2 modulation, thus resulting in high levels of IL-12/IL-23 and creating an environment capable of generating a pathological Th1/Th17 immune response. Consistent with the idea that dysregulated IL-12 promotes colitis, treatment of CD patients with antibody against IL-12 has been shown to be effective (130). Alternatively, data derived from the analysis of knockin mice expressing Nod2, which mimics L1007fsinsC, suggest that macrophages from these knockin mice had increased levels of IL-1 production (131). In addition to increased cytokine production, these knockin mice were shown to be more susceptible to dextran sodium sulfateinduced colitis, suggesting that the frame-shift mutation associated with CD is a gain-of-function mutation. However, unlike the published knockin Nod2 mouse model, monocytes from healthy or CD patients homozygous for NOD2 mutations exhibit loss-of-function phenotypes (61, 123). Thus, the relevance of the knockin Nod2 mouse model remains uncertain. The hypotheses presented here may not be mutually exclusive; further, they suggest that NOD2 may contribute to CD development via multiple mechanisms. Due to the similarity in signaling and structure between NOD1 and NOD2, researchers are interested in determining whether NOD1 is also associated with susceptibility to IBD. Studies have been conicting and may be population dependent; however, two studies have demonstrated an association of NOD1 with IBD (132, 133). The rst study to identify an association between NOD1 polymorphisms and IBD involved two independent cohorts comprising over 1000 IBD patients in England; in these patients the common deletion allele of a complex polymorphism (ND1 +32656 1) was signicantly associated with IBD and with a
stronger association with Crohns disease (133). This nding, however, was not upheld in a study of IBD patients in Germany (134), nor in a study of over 2000 Scottish and Swedish patients (135), which was afrmed by a second study of the same group of patients in Scotland and Scandinavia that used a gene-wide haplotypetagging approach (136). Nonetheless, a second positive study of 434 CD patients in Hungary identied a different polymorphism, G796A, which increased disease susceptibility (132). For the most part, these studies are observational, so a mechanistic link between NOD1 signaling and the pathophysiology of IBD remains to be investigated.
Inammatory Diseases Associated with Excessive Interleukin-1 Production

A number of inammatory and autoimmune disorders are related to dysregulated IL-1 production. IL-1 can cause fever and hypotension and can stimulate production of other proinammatory cytokines. Excessive IL-1 production has been associated with familial cases of inammatory disorders such Muckle-Wells syndrome (MWS), familial cold autoinammatory syndrome (FCAS), and neonatal-onset multisystem inammatory disease (NOMID), which are rare autosomal dominant diseases characterized by periodic fevers, skin rashes, painful joints, and sensineural decits (137, 138). IL-1 production has also been associated with nonhereditary inammatory diseases such as gout or pseudogout. Thus, it is not surprising that mutations in NLR proteins associated with the inammasome underlie the pathology of many of these disorders. Hereditary autoinammatory syndromes. The best examples of dysregulated inammasome signaling forming the basis of the pathogenesis of inammatory disorders are the cryopyrinopathies, wherein mutations in NLRP3 were discovered to be associated with the hereditary periodic fever syndromes MWS, FCAS, and NOMID. In particular, the disease-
associated NLRP3 gain-of-function mutations result in upregulated caspase-1 activation and IL-1 secretion by causing constitutive activation of the NLRP3 inammasome through interaction with ASCeven in the absence of any stimuli (137, 139). The functional relevance of IL-1 in these pathologies was demonstrated by the efcacy of the IL-1 receptor antagonist for reversing symptoms (140, 141). Not all patients with these syndromes respond to IL-1 receptor antagonism, suggesting that there are additional mechanisms through which inammasomes cause disease. An additional NLRP3mediated process that may contribute to disease pathogenesis was suggested by a study that demonstrated that NLRP3 mutations associated with the cryopyrinopathies promote necrotic cell death in vitro (142). Interestingly, the observed necrotic cell death was independent of caspase-1 activation, although it still required ASC. How this phenomenon may contribute to the pathogenesis of the cryopyrinopathies is unclear, but it has been proposed that necrotic cells may release additional danger signals that can also promote IL-1 release (142). Familial Mediterranean fever. Familial Mediterranean fever (FMF) is also part of the spectrum of autoinammatory, periodic fever syndromes, but it involves a different NLR. FMF is autosomal recessive and is characterized by recurrent fevers, abdominal pain, arthritis, and skin rashes. Mutations in the MEFV gene, which encodes Pyrin, have been associated with this disease. Pyrin has been demonstrated to have a negative regulatory role, as it can inhibit NLRP3-mediated apoptosis and NF-B activation when overexpressed in vitro (143). Although Pyrin can physically interact with the PYD of ASC (39), it has been shown to directly interact with caspase-1 through a different domain, known as the SPRY domain. This interaction, however, is inhibitory, and abrogation of this interaction results in enhanced IL-1 processing (144, 145). However, it has not been consistently shown that Pyrin mutations associated with
FMF reduce the interaction between Pyrin and caspase-1 to enhance caspase-1 activity (144146). Potential treatment with IL-1 receptor blockade remains a potential option, but it has not been routinely used as rst-line treatment in place of the anti-inammatory agent colchicine (147). This may be due to the fact that excessive IL-1 secretion in patients with FMF has not always been consistently demonstrated, which suggests that other cytokines may be involved (148).
matous disease with arthritis, iritis, and skin rashes (154). Mutations in NOD2 within the NOD domain, e.g., 334Q, R334W, and L469F, are associated with BS (155). These mutations result in a gain of function and in constitutive activation of NF-B (156, 157).
Other Disorders Involving NOD-Like Receptors

Vitiligo. Vitiligo is an autoimmune phenomenon that involves the destruction of melanocytes within the epidermis, resulting in patches of depigmented skin. Individuals with vitiligo have an increased frequency of other autoimmune disorders, such as autoimmune thyroid disease, rheumatoid arthritis, diabetes, and lupus (158). In large-scale genomic association studies in the United States and in Europe, NLRP1 variants were shown to be a susceptibility factor for vitiligo (158, 159). How NLRP1 variants mechanistically contribute to disease pathogenesis is not known, but it may be related to dysregulated caspase-1 activation, as serum IL-1 levels have been found to be elevated in patients with vitiligo (160). Sarcoidosis. Sarcoidosis is an autoimmune disorder characterized by noncaseating granulomas that can affect any organ, but most often the lungs and lymph nodes. Patients present with fatigue, joint pains, shortness of breath, and other symptoms directly related to specic organ involvement. The pathophysiology of sarcoid is not clear. However, in an analysis of 50 Japanese sarcoidosis patients, a genetic variant of NOD1 associated with increased disease susceptibility was found. This variant was associated with reduced NOD1 expression and the ability to activate NF-B in response to both iEDAP and infection with Propionibacterium acnes. P. acnes can be isolated in sarcoid-involved lymphoid tissues as well as in involved organs; therefore, the impaired response to P. acnes by NOD1 may contribute to the pathogenesis of this disease (161). Because of the demonstrated involvement of NOD2 in other granulomatous diseases,
Gout. As mentioned above, NLRP3 can also detect monosodium urate crystals generated from uric acid and therefore may play a critical role in the pathophysiology of gout, which involves the deposition of uric acid crystal in joints. In a mouse model of monosodium urate crystalinduced inammation with induction of peritonitis, IL-1 blockade resulted in impaired neutrophil inux in Nlrp3 inammasomedecient mice (95). A promising treatment option derived from these results came from a small clinical trial in which anakinra, an IL-1 receptor antagonist, was administered to patients with gout who were refractory to standard anti-inammatory agents. All 10 patients on the study had an effective response (149).
Bare Lymphocytic Syndrome

Bare lymphocytic syndrome (BLS) is an autosomal recessive congenital immunodeciency syndrome in which affected individuals become highly susceptible to infection due to defects in cellular and humoral immunity from defective major histocompatibility complex (MHC)II expression (150, 151). BLS is associated with loss-of-function mutations in CIITA, a unique NLR that acts as a transcriptional activator and results in decreased expression and decreased numbers of CD4+ T cells, consistent with the demonstrated function of CIITA (152, 153).
Blau Syndrome
Blau syndrome (BS) is an autosomal dominant disease characterized by early-onset granulo388 Chen et al.
such as BS, researchers have attempted to determine whether NOD2 variants are also associated with disease susceptibility for sarcoid. However, unlike with NOD1, no correlation between NOD2 mutations and sarcoid has been observed (162164). Interestingly, however, NOD2 has been associated with juvenile-onset sarcoid (EOS). This disease is distinct from adult sarcoidosis in that it usually is seen in individuals younger than four years with the clinical triad of skin, joint, and eye involvementsimilar to that seen in BS, but lacking pulmonary ndings. In a small study of 10 Japanese patients diagnosed with EOS, nine had heterozygous missense mutations in the NOD domain of NOD2, totaling six different variants (165). Only one variant was identical to that reported with Blau syndrome, while the remaining ve were novel. Regardless, EOS-associated NOD2 variants, like their BS-associated NOD2 counterparts, exhibited increased basal NF-B activity compared to wild type that was further enhanced with the addition of MDP in a reporter assay in vitro (165).
NOD1 in modulating the response to environmental bacteria and development of asthma. Cancer. Recent studies have suggested that TLR signaling plays an important role in carcinogenesis, especially within the gastrointestinal tract (172, 173). A role for NLRs has not yet been investigated; however, a tumor-suppressor function in NOD1 has been suggested in breast cancer xenograft models (174). This phenotype was attributed to a functional role for NOD1 in regulating apoptosis, in which retrovirally infected MCF-7 cells with defective NOD1 expression were more sensitive to TNF-induced apoptosis in vitro in the presence of cyclohexamide, which facilitates apoptosis by inhibiting expression of antiapoptotic factors. In vivo, this mutant cell line also showed increased growth potential as xenografts; this potential was not associated with decreased apoptosis, but rather with a lack of responsiveness to estrogen-induced proliferation, suggesting an additional role for NOD1 in regulating estrogen receptor expression. However, spontaneous growth of tumors has not been observed in Nod1-decient mice, and whether NOD1 inuences tumor growth in other organs that are not necessarily hormone responsive remains to be determined.
Allergic diseases. Allergies are associated with hyperreactivity to antigens; they are characterized by increased immunoglobin E response and are therefore inammatory in nature. Certain NOD1 genetic variants are associated with an increased risk of developing asthma and atopic eczema, and therefore NOD1 may play a role in the modulation of a mucosal allergic response (166, 167). Because asthma results from gene-environment interactions, its association with NOD1 may implicate bacterial signaling through NOD1 in the pathogenesis of asthma. This would be consistent with the prevailing hygiene hypothesis of asthma risk: Studies have demonstrated that individuals with reduced exposure to bacteria during childhood have an increased risk of developing asthma (168170). Moreover, NOD1 polymorphisms can also modify the protective effect of early exposure to allergens (171), providing additional evidence for a possible role for
CONCLUSIONS AND PERSPECTIVES

The identication and initial characterization of NLR proteins have yielded new insights into the host recognition system involved in microbial detection and host-defense mechanisms. The involvement of NLRs in the pathogenesis of several genetic diseases indicates that these proteins play an important role in the regulation of immune and inammatory responses. There is conclusive evidence that several NLRs sense conserved microbial molecules to activate discrete signaling pathways including NF-B, MAPK, and caspase-1 activation. There is also clear evidence for interplay between NLRs and TLRs in the regulation of the inammatory response against microbes.
However, many questions remain, including the identication of the signaling pathways activated by several NLRs, the mechanism whereby NLRs sense microbial molecules, the interplay between NLRs and other PRRs, and the role of NLRs in host defense in vivo. In addition, we need a better understanding of
the mechanism whereby mutations of NLRs including NOD2 and NLRP3associate with susceptibility to inammatory disease. The answers to these questions may lead to the development of more rational therapies for NLRassociated diseases and for inammatory disease in general.
SUMMARY POINTS
1. A broad repertoire of NLRs is involved in the recognition of conserved microbial components as well nonmicrobial signals, including uric acid crystals and silica. 2. NLR signaling results in the activation of NF-B and MAPK pathways, which in turn result in the induction of proinammatory cytokines and chemokines. NLR signaling also causes the inammasome to activate caspase-1, which leads to pro-IL-1 maturation and pyropoptosis. 3. NLRs are strategically positioned in the cytoplasm, allowing them to respond to PAMPs derived from bacteria that directly invade the cell or enter the cell through phagocytosis, type III or IV secretion systems, pore-forming toxins, or host-derived channels (e.g., pannexin-1). 4. NLR signaling has complementary functions to TLR signaling, as has been demonstrated for IL-1 secretion. 5. Dysregulated NLR signaling can lead to either gain-of-function or loss-of-function phenotypes that result in human diseases, such as inammatory bowel disease, that are associated predominantly with NOD2. The cryopyrinopathies are associated with NLRP3 mutations and excessive IL-1 signaling.
FUTURE ISSUES 1. Do NLRs interact directly or indirectly with the microbes they sense? What other entities, be they microbial- or host-derived, are specically recognized by individual NLRs? 2. How are NLR and TLR signals integrated during the host response? 3. How precisely does NOD2 in the intestine protect against the development of IBD? How can we resolve discrepancies in loss-of-function and gain-of-function phenotypes observed in mice models and in patients with IBD? 4. What are the functions of the less-characterized NLRs in vivo, and do they have a role in disease?
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.
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ACKNOWLEDGMENTS
We apologize to our colleagues whose work was not cited here due to space limitations. Work on NLR proteins in our laboratory is supported by grants from the National Institutes of Health (NIH). G.C. is supported from NIH NCI grant number T32 CA009357. Y.K. is supported by a postdoctoral fellowship from the University of Michigan Comprehensive Cancer Center. M.S. is supported by a postdoctoral training grant from the NIH. LITERATURE CITED
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Contents
Annual Review of Pathology: Mechanisms of Disease Volume 4, 2009
The First Fifty Years in Research Peter A. Ward p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Graft Vascular Disease: Immune Response Meets the Vessel Wall Richard N. Mitchell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p19 Molecular Pathology of Head and Neck Cancer: Implications for Diagnosis, Prognosis, and Treatment Sara I. Pai and William H. Westra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p49 Mechanisms of Endothelial Dysfunction, Injury, and Death Jordan S. Pober, Wang Min, and John R. Bradley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71 The Pathogenesis of Pituitary Tumors Sylvia L. Asa and Shereen Ezzat p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p97 PTEN and the PI3-Kinase Pathway in Cancer Nader Chalhoub and Suzanne J. Baker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 127 Pathogenesis of Classical and Lymphocyte-Predominant Hodgkin Lymphoma Roland Schmitz, Jens Stanelle, Martin-Leo Hansmann, and Ralf Kppers p p p p p p p p p p p p p 151 Molecular Genetics of Acute Lymphoblastic Leukemia Michael A. Teitell and Pier Paolo Pandol p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 175 MicroRNAs in Cancer Yong Sun Lee and Anindya Dutta p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 199 Epigenetic Changes in Cancer Christine A. Iacobuzio-Donahue p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 229 Molecular Pathogenesis and Diagnostics of Bladder Cancer Anirban P. Mitra and Richard J. Cote p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 251 Ovarian Cancer Kathleen R. Cho and Ie-Ming Shih p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287
Drosophila Models of Neurodegenerative Diseases Bingwei Lu and Hannes Vogel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 315 Serrated Polyps and Colorectal Cancer: New Pathway to Malignancy Amy E. Noffsinger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 343 Nod-Like Receptors: Role in Innate Immunity and Inammatory Disease Grace Chen, Michael H. Shaw, Yun-Gi Kim, and Gabriel Nunez p p p p p p p p p p p p p p p p p p p p p p p 365 Tumor Suppressors, Chromosomal Instability, and Hepatitis C VirusAssociated Liver Cancer David R. McGivern and Stanley M. Lemon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 399 The Immunopathogenesis of Rheumatoid Arthritis John B. Imboden p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 417 The Pathology of Chronic Obstructive Pulmonary Disease James C. Hogg and Wim Timens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 435 Linking the Cellular Functions of BRCA Genes to Cancer Pathogenesis and Treatment Ashok R. Venkitaraman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 461 Regulation of Hepcidin and Iron-Overload Disease Pauline L. Lee and Ernest Beutler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489 The Brainstem and Serotonin in the Sudden Infant Death Syndrome Hannah C. Kinney, George B. Richerson, Susan M. Dymecki, Robert A. Darnall, and Eugene E. Nattie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 517 Molecular Pathogenesis of Cutaneous Melanocytic Neoplasms Nageatte Ibrahim and Frank G. Haluska p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 551 Indexes Cumulative Index of Contributing Authors, Volumes 14 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 581 Cumulative Index of Chapter Titles, Volumes 14 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 583 Errata An online log of corrections to Annual Review of Pathology, Mechanisms of Disease articles may be found at http://pathol.annualreviews.org
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ANNUAL REVIEWS

NOD-Like Receptors: Role in Innate Immunity and Inammatory Disease

DEFINING FEATURES OF THE NOD-LIKE RECEPTOR FAMILY

HGNC-approved symbol Human CIITA Mouse Cllta

Nlrp3 NLRP4 Nlrp4a Nlrp4b Nlrp4c Nlrp4d Nlrp4e Nlrp4f Nlrp4g

NLRP NLRP NLRP

Nuclear FactorKappa B/Mitogen-Activated Protein Kinase Signaling

Lipoprotein Flagellin TLR1 R1 or r TLR2 TLR6 TLR5 R6

JNK NF-B Nucleus NF-B

ERK NF- B NF B NF-B

MDP: muramyl dipeptide

Interleukin-1 Production and the Inammasome

Biological Responses to NOD-1 and NOD-2 Signaling

NOD-LIKE RECEPTORS RECOGNIZE SPECIFIC BACTERIAL AND ENDOGENOUS MOLECULES

Table 2 Receptor Nod1

MDP MurNAc-l-Ala-g-d-Glu-l-Lys (M-TRILys)

agellin (Salmonella, Legionella, Pseudomonas) unknown (Shigella)

Porin Outer membrane

PGN Plasma membrane Cytosol

Polysaccharide Lipoteichoic acid

Plasma membrane Cytosol

MDP (Nod2 ligand)

Tetra-peptide chain (amino acids) Glycan chain NAM NAG NAM

iE-DAP (Nod1 ligand)

MECHANISMS OF INTRACELLULAR DELIVERY OF MICROBIAL PRODUCTS FOR DETECTION BY NOD-LIKE RECEPTORS

P2X7R Phagosome PAMPs

Bacteria type III or IV secretion system

Caspase-1 Pro-IL-1 IL-1

NOD-LIKE RECEPTOR SIGNALING SYNERGIZES AND COMPLEMENTS TOLL-LIKE RECEPTOR SIGNALING

www.annualreviews.org NLRs: Role in Immunity and Disease

Invasion of intracellular bacteria

Prepare to ght invasive pathogen

Inammatory Bowel Disease

Vitiligo NLRP1 (skin)

Dendritic cell Nod2

4 IL-1 IL-1 L IL-1 h Macrophage 2 Nod2

TLR2 R2 R TLR2 3 Proinammatory cytokines

Nod2 1 Paneth cells

Inammatory Diseases Associated with Excessive Interleukin-1 Production

Other Disorders Involving NOD-Like Receptors

Bare Lymphocytic Syndrome

CONCLUSIONS AND PERSPECTIVES

Annual Review of Pathology: Mechanisms of Disease Volume 4, 2009

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