Nucleotide-Binding Oligomerization Domain-Like
Receptors: Intracellular Pattern Recognition Molecules
for Pathogen Detection and Host Defense1
Luigi Franchi, Christine McDonald, Thirumala-Devi Kanneganti, Amal Amer, and
Gabriel Nu ´n ˜ez2
The nucleotide binding oligomerization domain-like re-
ceptor (NLR) family of pattern recognition molecules is
involved in a diverse array of processes required for host
immune responses against invading pathogens. Unlike
TLRs that mediate extracellular recognition of microbes,
several NLRs sense pathogens in the cytosol and upon ac-
TLRs and NLRs differ in their mode of pathogen recogni-
tion and function, they share similar domains for micro-
bial sensing and cooperate to elicit immune responses
against the pathogen. Genetic variation in several NLR
genes is associated with the development of inflammatory
Further understanding of NLRs should provide critical
genesis of inflammatory diseases. The Journal of Immu-
nology, 2006, 177: 3507–3513.
ence of infection is through pathogen recognition molecules
that detect the presence of unique microbial and viral compo-
nents called pathogen-associated molecular patterns (PAMPs)3
(i.e., LPS, lipoteichoic acid, and peptidoglycan), specialized
bacterial proteins (i.e., flagellin), as well as nucleic acid struc-
tures unique to bacteria and viruses (i.e., CpG DNA, dsRNA)
(2). Three main families of pathogen recognition molecules co-
operate in host defense and include TLRs, nucleotide-binding
oligomerization domain (Nod)-like receptors (NLRs), and ret-
inoid acid-inducible gene 1-like receptors (2). The detection of
PAMPs by TLRs, NLRs, and retinoid acid-inducible gene
n effective immune response against microbial infec-
tion requires both the ability to sense the presence of
the infectious agent, as well as the ability to destroy
1-like receptors stimulates the activation of proinflammatory
signaling pathways and caspases, as well as antiviral and bacte-
ricidal responses (2, 3). The coordination and cooperation of
responses triggered by these pathogen sensors tailor the im-
mune response to effectively abrogate the specific infection (2).
Genetic mutations that cause alterations in these signaling
pathways frequently result in inflammatory disease or immune
disorders (4). Recent advances have been made in our under-
standing of the role of NLRs and their cooperation with TLRs
in innate immunity and are the focus of this review.
NLR family of pathogen sensors
The NLR family (NLRs, also called Nod-leucine-rich repeats
(LRRs), NACHT-LRRs, or CATEPILLER proteins) is com-
conserved Nod (3). The general domain structure of these pro-
teins include an amino-terminal effector binding region that
consists of protein-protein interaction domains such as caspase
recruitment domains (CARD), pyrin, or baculovirus inhibitor
repeat domains, a central Nod that acts to oligomerize these
proteins, and carboxyl-terminal LRRs that are required to de-
tect specific PAMPs and is involved in autoregulation of NLR
activity (3). These proteins have a remarkable structural simi-
to play key roles in pathogen defense through sensing bacteria
and generating immune responses (5).
Recognition of microbes by NLRs
Nod1/CARD4 senses the presence of bacterial pathogens, such
as Shigella flexneri (6), enteroinvasive Escherichia coli (7),
Pseudomonas aeruginosa (8), and Helicobacter pylori (9, 10),
through the recognition of peptidoglycan (PGN) molecules
that contain meso-diaminopimelic acid (meso-DAP) (11).
Gram-negative and only specific Gram-positive bacteria (12).
Department of Pathology and Comprehensive Cancer Center, University of Michigan
Medical School, Ann Arbor, MI 48109
Received for publication May 22, 2006. Accepted for publication June 20, 2006.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI063331, AI064748,
DK61707, and DK067628 and a grant from the Eli and Edythe L. Broad Foundation (to
G. N.). Other support includes a fellowship from Fondazione Italiana Ricerca sul Cancro (to
L.F.), a Career Development Award from the Crohn’s and Colitis Foundation of America
(to C.M.), and Grant T32/HL007517 from the National Institutes of Health (T.-D.K.).
2Address correspondence and reprint requests to Dr. Gabriel Nu ´n ˜ez, Department of Pa-
East Medical Center Drive, Ann Arbor, MI 48109. E-mail address: email@example.com
3Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; Nod,
nucleotide-binding oligomerization domain; CARD, caspase-recruitment domain; LRR,
leucine-rich repeat; ASC, apoptosis-associated speck-like protein containing a CARD;
MDP, muramyl dipeptide; NLR, Nod-like receptor; PGN, peptidoglycan; DAP, diamin-
opimelic acid; iE-DAP, ?-D-glutamyl-meso-DAP; IKK, I?B kinase; CD, Crohn’s disease;
BS, Blau syndrome; FCAS, familial cold autoinflammatory syndrome.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
Analysis of synthetic compounds revealed that the dipeptide
?-D-glutamyl-meso-DAP (iE-DAP) is sufficient to trigger
Nod1 activation (11, 13). However, these iE-DAP-contain-
ing PGN molecules must be delivered intracellularly
through either invasion of the cytosol by intracellular bacte-
ria, as in S. flexneri infection (6), or transport of these mol-
ecules through bacterial secretion systems, like the type IV se-
cretion system of H. pylori (9).
Another NLR family member, Nod2/CARD15, also rec-
ognizes the presence of bacteria through sensing a compo-
nent of PGN. Nod2 is activated by muramyl dipeptide
(MDP), which is a conserved structure in virtually all types
of PGN (12). Like iE-DAP, MDP must be delivered intra-
cellularly either by bacteria that invade the cell or through
other cellular uptake mechanisms to be detected by Nod2.
However, in contrast to Nod1 that recognizes only a subset of
general sensor of bacteria (12).
Ipaf/CLAN/CARD12 is critical for generating an immune
response to Salmonella typhimurium and Legionella pneumo-
phila through sensing a component of these bacteria distinct
from PGN (14, 15). Recent studies have revealed that intra-
cellular flagellin is the PAMP recognized by Ipaf indepen-
dently of TLR5, which senses extracellular flagellin (16, 17).
The recognition of flagellin by Ipaf upon infection with S.
typhimurium or L. pneumophila is dependent on a functional
bacterial secretion system (type III or type IV, respectively),
suggesting that monomeric flagellin delivered to the host
cytosol through these secretion systems activates Ipaf
Cryopyrin/PYPAF1/NALP3 not only detects the presence
of microbes, but also mediates activation of the immune sys-
tem in response to endogenous danger signals that are re-
leased by injured cells (18, 19). Several recent articles have
demonstrated that Cryopyrin senses bacterial RNA, syn-
thetic purine analogs R837 (imiquimod) and R848 (re-
dium urate or calcium pyrophosphate dehydrate crystals (18,
19). Both R838 and R848 structurally resemble uric acid,
suggesting that Cryopyrin senses purine-like structures. In
addition, other results indicate that Cryopyrin regulates
caspase-1 activation in response to factors that induce intra-
cellular K?efflux, such as certain toxins and high extracel-
lular concentrations of ATP (20, 21).
Finally, the susceptibility of certain mouse strains to in-
fection by specific bacteria has been shown to be dependent
on other NLR family members. Mouse susceptibility to Ba-
cillus anthracis lethal toxin-induced macrophage cell death is
controlled by Nalp1b, indicating that this NLR protein is
involved in sensing lethal toxin (22). A mutation in the
mouse NLR protein, Naip5, has been associated with host
susceptibility to the intracellular pathogen L. pneumophila
(23), suggesting that Naip5 functions as a cytosolic sensor of
L. pneumophila. However, the specific component of L.
pneumophila recognized by Naip5 remains to be identified.
activated by specific PAMPs, the mechanism involved remains
unclear. Several studies have demonstrated that the LRRs of
NLR proteins are required for PAMP sensing (24–26). How-
ever, there is no clear evidence that mammalian NLRs or their
plant R protein homologs directly interact with their cognate
microbial activators (5). This suggests that the sensing of mi-
crobial agonists by NLRs might be indirect through adaptor
molecules or induced host factors that are recognized by NLRs.
The identification of the molecular mechanism by which mi-
lenge for future studies.
Activation of NLR intracellular responses
Molecular studies have revealed that stimulation of NLRs by
microbial components results in the activation of two distinct
proinflammatory intracellular responses. The current model of
NLR activation is based on studies of one family member, the
apoptosis regulator Apaf-1 (27), that revealed how this factor
activates caspase-9 upon oligomerization.
Once activated, one group of NLRs, which includes Nod1
and Nod2, activates gene transcription through the NF-?B
transcription factor and the MAPK signaling pathway. Stimula-
tion of Nod1 or Nod2 by specific components of bacterial PGN
(Fig. 1). RICK has been demonstrated to be an essential compo-
nent of both the Nod1 and Nod2 signaling pathways, since cells
that do not express RICK are unable to activate an Nod1- or
Nod2-mediated NF-?B response (28). The binding of RICK to
Nod1 or Nod2 and its oligomerization activates downstream sig-
cruitment of the I?B kinase (IKK) complex to RICK (29). RICK
directly binds to IKK? and activates the IKK complex through
promoting the ubiquitinylation of IKK? and stimulating the ki-
nase activity of the two other components of the IKK complex,
IKK? and IKK? (29, 30). Activation of the IKK complex causes
hibitor I?B? (31). The activated NF-?B translocates to the nu-
cleus where it binds to target gene promoters and stimulates gene
transcription. In addition to the NF-?B pathway, MAPKs are ac-
tivated in response to stimulation of Nod1 or Nod2 and result in
the activation of the kinases p38, Erk, and Jnk (32). These two
pathways are thought to work together to up-regulate the expres-
sion of proinflammatory molecules to stimulate both innate and
adaptive immune responses (Fig. 1).
A second group of NLRs, which include Ipaf and Cryopyrin,
respond to microbial components through proteolytic activa-
tion of caspase-1 to generate the proinflammatory cytokines
caspase-5 in humans and caspase- 11 in mice (33). Caspase-1 is
synthesized as an inactive zymogen that becomes activated by
cleavage at aspartic residues to generate an enzymatically active
heterodimer composed of a 10- and a 20-kDa chain (33).
Cryopyrin and Ipaf can form multiprotein complexes termed
quent processing of pro-IL-1? (34).
Recent studies have shown that Cryopyrin and Ipaf, as well as
the adaptor ASC, are required for the activation of caspase-1 in
tivation of caspase-1 in response to a broad range of PAMPs and
togenes (35), Francicella tularensis (36), and Staphylococcus aureus
(21). In contrast, the functions of Ipaf and Cryopyrin are more
restricted. Ipaf is required for caspase-1 activation induced by S.
3508BRIEF REVIEWS: NLRs: ROLE IN HOST DEFENSE AND DISEASE
eral NLRs may use the adaptor ASC to activate caspase-1 in re-
sponse to bacterial infection.
Functions of NLRs in the epithelium
The functions of NLRs in the immune defenses of the epithe-
lium are beginning to emerge. Several studies have implicated
Nod1-dependent NF-?B activation in the induction of ?-de-
fensin and chemokine expression in response to H. pylori (9,
10), enteroinvasive E. coli (7), and S. flexneri infection (6) (Fig.
are largely deficient in TLR signaling and must rely on alterna-
tive systems, such as NLRs, for the detection of pathogens. H.
pylori is an extracellular bacterium, but it injects muropeptides
into gastric epithelial cells through its type IV secretion appa-
ratus, providing a mechanism for the sensing of noninvasive
bacteria intracellularly by host epithelial cells. Nod1 is required
for the induction of ?-defensin-2 in human gastric epithelial
?-defensin-2 is also induced in vivo by H. pylori in a Nod1-
dependent manner, this may explain the increased gastric col-
onization of cag-positive H. pylori strains in Nod1-null mice
(9). In addition, induction of chemokine expression in epithe-
is largely dependent on Nod1 (Fig. 2B) (6, 7). A role for Nod1
in early immune responses is supported by the finding that in-
jection of mice with synthetic Nod1 activators induces high levels
signals harmful infection by pathogenic bacteria in intestinal tis-
sues and the activation of inflammatory immune responses.
Nod2 has been implicated in the expression of antimicro-
bial peptides in epithelial barriers. One such barrier includes
Paneth cells, which are specialized intestinal cells located in
the crypts of the small intestine, that secrete antimicrobial
peptides such as ?- and ?-defensins (Fig. 2C) (38). Studies
in mice have revealed an important role for Nod2 in bacterial
clearance after oral challenge, but not infection via i.v. or i.p.
routes, with L. monocytogenes (32). This defect is associated
with reduced expression of a subgroup of ?-defensins
(known as cryptdins in mice) in Nod2- deficient mice. The
Nod2-dependent regulation of ?-defensins is consistent
with the presence of Nod2 in Paneth cells (39), but this ac-
tivity is intriguing in that there is no evidence that ?-defen-
sin expression is regulated via NF-?B-dependent pathways
(39). In addition, production of human ?-defensin-2 in ker-
atinocytes of the skin was shown to be regulated by bacterial
products in a Nod2-dependent manner (40). Interestingly,
up-regulation of this gene was found to be dependent on NF-?B
(40). Clearly, further work is needed to understand the role of
Nod2 in the regulation of antimicrobial peptides at mucosal
responses by NLRs in response to specific PAMPs.
Stimulation of Nod1 or Nod2 by intracellular iE-
DAP or MDP, respectively, causes NLR oligomer-
ization and recruitment of the protein kinase
RICK. Oligomerization of RICK causes the activa-
tion of NF-?B and MAPK signaling and the tran-
scriptional induction of genes involved in immune
responses. Activation of two other NLRs, Ipaf and
the formation of inflammasomes of oligomerized
NLRs, the adaptor ASC, and the protease
caspase-1. Recruitment of these proteins to the in-
flammasome causes the proteolytic activation of
caspase-1 and cleavage of the pro-forms of the in-
ture forms for secretion.
Activation of intracellular signaling
3509The Journal of Immunology
Cooperation between NLR and TLR signaling
Both MDP- and iE-DAP-containing molecules synergize
with TLR ligands to induce the secretion of multiple cyto-
kines and induction of costimulatory molecules in macro-
phages and dendritic cells (32, 41–43). Cooperation be-
tween MDP and LPS stimulation can be observed in whole
animals where it enhances the severity of LPS-induced shock
in a Nod2-dependent manner (32). This synergism between
NLRs and TLRs could be explained by cross-induction of
critical signaling molecules upon microbial stimulation (44,
45). The enhancement of TLR-mediated responses by Nod1
and Nod2 stimulatory molecules has been widely used to
study Nod1 and Nod2 in mouse systems because, in contrast
to human cells, mouse cells respond poorly to MDP and iE-
DAP stimulation (32).
Cooperative signaling occurs between NLRs and TLRs to
control the secretion of IL- 1?. Secretion of IL-1? is regulated
by two distinct processes. Short-term stimulation of macro-
phages with TLR ligands including LPS, which is referred to as
The second step involves activation of caspase-1, which is re-
quired for processing of pro-IL-1? into the biologically active
mature IL-1? (33). Studies using macrophages deficient in
MyD88, an essential adaptor for TLR signaling, revealed that
MyD88 signaling is critical for the expression of pro-IL- 1? via
NF-?B activation, but is dispensable for caspase- 1 activa-
tion in response to microbial stimuli (46). In the case of the
NLR proteins, Ipaf and Cryopyrin play a critical role in
caspase-1 activation independently of TLR signaling (16,
17, 19, 35), and, conversely, do not play a role in the acti-
vation of NF-?B and in the up-regulation of pro- IL-1? (14,
21). Thus, it appears that cooperative signaling occurs be-
tween NLRs and TLRs to control the secretion of IL-1?
(Fig. 2D). Because both Ipaf and Cryopyrin can be activated
by PAMPs that also activate TLRs (i.e., flagellin (16, 17) or
microbial RNA (19)), it is hypothesized that secretion of
IL-1? and other proinflammatory cytokines, whose expres-
sion is induced via TLR signaling (e.g., IL-6 or TNF-?), is
coupled during bacterial infection. However, the require-
ment of a functional bacterial secretion system for caspase-1
activation in response to S. typhimurium infection (14, 16,
17) indicates that only virulent intracellular bacteria induce
IL-1? secretion. These findings suggest that activation of cy-
tosolic NLR proteins function to produce a more robust im-
mune response to control invasive bacteria.
Prolonged exposure of macrophages to PAMPs (such as
LPS) induces a state of cell tolerance to secondary TLR and
bacterial challenge that is thought to protect the host from
the harmful effects associated with overproduction of proin-
flammatory cytokines (47). Notably, macrophages tolerized
with LPS secrete high levels of IL-1? which correlates with
activation of Ipaf-dependent caspase-1 in response to S. typhi-
murium infection (17) (Fig. 2D). Cytosolic signaling and IL-1?
alert the immune system to the presence of invasive pathogens in-
side the macrophage.
NLR genetic variants and human disease susceptibility
A remarkable finding has been the discovery that genetic
variation in several NLRs is associated with susceptibility to
Secretion of human ?-defensin-2 by gastric epithelial cells in response to H.
iE-DAP-containing molecules into the host cytosol. Nod1 is then activated,
leading to induction of the human ?-defensin-2 gene via the induction of NF-
?B. B, The presence of bacterial infection is sensed by epithelial cells from tis-
sues, such as the intestine and lung, through the recognition of iE-DAP
molecules in the host cytosol. Recognition leads to the induction of che-
mokine genes and recruitment of neutrophils to the infection site. C, Elim-
ination of commensal and pathogenic bacteria through the secretion of an-
timicrobial peptides by Paneth cells. Nod2 is thought to contribute to this
process through the regulation of ?-defensin expression and secretion. D,
Stimulation of TLRs induces the production of pro-IL-1?, whereas the ac-
tivation of Ipaf by flagellin induces the activation of caspase-1 in response to
Salmonella. Tolerization of TLR pathways does not affect Ipaf-induced
caspase-1 activation because macrophages tolerant to TLR stimulation re-
tain their ability to produce high levels of IL-1?.
Function of NLRs at epithelial barriers and macrophages. A,
3510BRIEF REVIEWS: NLRs: ROLE IN HOST DEFENSE AND DISEASE
several inflammatory or infectious diseases in human popu-
lations. Loss-of-function mutations in CIITA, an NLR fam-
ily member, that result in MHC class II deficiency are re-
sponsible for the type II bare lymphocyte syndrome. CIITA
mutations impair the transcriptional activity of CIITA, re-
sulting in decreased MHC class II expression, immunodefi-
ciency, and increased susceptibility to infection by a wide
variety of pathogens (48).
Genetic variation in Nod2 is associated with susceptibility to
several inflammatory diseases. Crohn’s disease (CD), a chronic
inflammatory disorder of the intestinal wall, is associated with
three common mutations (R702W, G908R, and L1007insC)
(49, 50). Biochemical and functional studies in cell lines
revealed that the human CD-associated Nod2 variants exhibit
reduced or loss of activity when compared with the wild-type
protein (24, 50). Similarly, the induction of TNF-?, IL-6, IL-
10, and IL-1? by monocytes in response to MDP, but not to
TLR agonists, is specifically impaired in patients and healthy
51). The defective function of CD-associated Nod2 mutations
is consistent with genetic studies that revealed that homozygos-
ity for the common mutations is required for increased disease
susceptibility (52), but is at odds with a study in mice showing
that the L1007insC Nod2 mutation confers enhanced IL-1?
secretion (53). The reason for the discrepancy between the hu-
man and mouse studies is unclear. The observation that the re-
sponse to MDP is impaired in human cells homozygous for
CD-associated Nod2 mutations suggests that a deficit in bacte-
rial sensing triggers an abnormal inflammatory response to un-
clear bacteria or bacterial product in the intestinal tissue. Al-
though several nonexcluding hypotheses have been proposed
to explain the link between Nod2 mutations and CD, the
precise mechanism(s) remains poorly understood. These
mechanisms include reduced expression of ?-defensins in
Paneth cells, impaired production of inflammatory mole-
cules by intestinal macrophages and/or dendritic cells, neu-
trophil dysfunction, and dysregulated TLR2 signaling (32,
54). The hypothesis that Nod2 mutations leads to decreased
Paneth cell function is particularly attractive in that similar
findings have been found in Nod2-deficient mice and CD
patients (32, 55). However, there is no direct evidence that
reduced production of ?-defensins leads to impaired clear-
ance of bacteria and intestinal inflammation. Clearly, fur-
ther studies are needed to understand the link between Nod2
mutations and the development of CD.
The CD-associated Nod2 variants have been also linked to
susceptibility to graft-vs-host disease and mortality in patients
can regulate, by mechanisms that remain unclear, the response
of allogeneic T cells causing graft-vs-host disease.
Several missense mutations involving amino acid residues in
the Nod domain of Nod2 cause two autosomal dominant dis-
orders characterized by granulomatous inflammation at multi-
ple organ tissues, called Blau syndrome (BS) and early-onset
sarcoidosis (58, 59). In contrast to CD, the Nod2 mutations
activity (25), which is consistent with the dominant mode of
inheritance of these diseases.
Several Nod1 polymorphisms have been associated with
the development of atopic eczema and asthma, as well as
with increased levels of serum IgE in several human populations
microbial exposure in childhood is known to protect against the
ognition of bacterial products via Nod1 in the skin and mucosal
surfaces regulates directly or indirectly Th2 polarization and IgE
Missense mutations in the CIAS1 gene, which encodes
Cryopyrin, are the cause of familial cold autoinflammatory
syndrome (FCAS), Muckle-Wells syndrome, and neonatal-
onset multisystem inflammatory disease. These diseases are
characterized by spontaneous attacks of systemic inflamma-
tion without an apparent infectious or autoimmune etiology
and represent a spectrum of related disorders of different se-
verity, where FCAS patients are the least affected and neo-
natal-onset multisystem inflammatory disease patients the
most severely affected (4). Notably, the mutations associated
main, suggesting that they affect the activation state of Cryopyrin
and Muckle-Wells syndrome corresponds to the R334W Nod2
mutation found in BS (25, 63). Consistent with these observa-
tions, functional studies revealed that the Cryopyrin mutants ex-
hibit enhanced activity to induce IL-1? secretion (64). Further-
more, mononuclear cells from patients with autoinflammatory
servations suggest that the disease-associated mutations confer a
state of constitutive activation to Cryopyrin, leading to increased
caspase-1 activity. Notably, disease activity is greatly reduced after
matory syndromes, indicating a critical role for IL-1? in disease
There is now conclusive evidence that several members of the
NLR family play important roles in the immune response
against invading pathogens and that NLR genetic variation
causes or contributes to human disease. However, several ques-
tions remain, including the mechanism involved in microbial
recognition through the LRRs of NLRs, the mechanism re-
sponsible for delivery of PAMPs to the cytosol, and a clearer
understanding of the link between NLR and disease. Further
studies including in-depth analyses of mutant mice deficient in
NLR genes and further biochemical characterization of NLR
signaling pathways are required to understand the function of
NLRs in immune responses.
The authors have no financial conflict of interest.
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3511 The Journal of Immunology