Annurev.pathol NOD

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NOD-Like Receptors:Role in Innate Immunityand Inflammatory Disease

Grace Chen, Michael H. Shaw, Yun-Gi Kim,and Gabriel Nunez

Departments of Pathology and Internal Medicine and the Comprehensive Cancer CenterUniversity of Michigan Medical School, Ann Arbor, Michigan 48109;email: [email protected]; [email protected]; [email protected]; [email protected]

Annu. Rev. Pathol. Mech. Dis. 2009. 4:36598

First published online as a Review in Advance onOctober 17, 2008

The Annual Review of Pathology: Mechanisms ofDisease 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 intracellulareceptorsthatrepresentakeycomponentofthehostinnateimmunesys

tem. Since the discovery of the first NLR almost 10 years ago, the studof this special class of microbial sensors has burgeoned; consequently,

better understanding of the mechanism by which these receptors recog

nize microbes and other danger signals and of how they activate inflammatory signaling pathways has emerged. Moreover, in addition to theiprimary role in host defense against invading pathogens, their ability t

regulate nuclear factorkappa B (NF-B) signaling, interleukin-1-bet(IL-1) production, and cell death indicates that they are crucial to th

pathogenesis of a variety of inflammatory human diseases.

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PAMPs: pathogen-associated molecularpatterns

PRRs: pathogenrecognition receptors

LPS:lipopolysaccharide

PGN: peptidoglycan

TLRs: Toll-likereceptors

INTRODUCTION

Essential to human survival is the ability toeradicate pathogenic microorganisms. To en-

sure the efficient detection and removal ofharmful microbes, two effector mechanisms

have evolved: the innate and adaptive im-mune systems. The adaptive immune response

is characterized by a delayed response involv-ing gene rearrangements for the clonal se-lection and expansion of T cell and B cell

lymphocytes with antigen-specific receptors.This process results in the generation of a di-

verse, yet specific repertoire of immune effec-tors that also contribute to immunologic mem-

ory. The innate immune system, however, israpidly activated and does not require the so-

matic gene rearrangements that form the cor-nerstone of adaptive immunity. It represents

one of the first lines of defense against microor-ganisms, whereby conserved microbial struc-

tures known as pathogen-associated molecularpatterns (PAMPs) are recognized by germline-

encoded innate immune receptors, also known

as pathogen recognition receptors (PRRs). Ex-amples of PAMPs include lipopolysaccharides

(LPS), peptidoglycan (PGN), flagellin, and mi-crobial nucleic acids. Recognition of these

PAMPs by PRRs results in the activation ofsignaling pathways, which promotes an inflam-

matory, antimicrobial response. Emerging datahave also demonstrated a link between the in-

nate PRRs and the activation of the adaptiveimmune response that can act in concert in de-

fense against invasive organisms (1). Moreover,it has also become apparent that these recep-

tors are involved in sensing not only invading

pathogens, but also endogenous nonmicrobialdanger or stress signals, both of which re-

sult in the activation of inflammatory signal-ing pathways such as nuclear factorkappa B

(NF-B) and mitogen-activatedproteinkinases(MAPKs). This upstream link to complexeffec-

tor pathways highlights the importance of theseimmune receptors in both microbial defense

and the pathogenesis of noninfectious, inflam-matory diseases when signaling becomes dys-

regulated.

Three major classes of PRRs have b

identified: (a) the Toll-like receptors (TLR which are transmembrane proteins with

extramembranous domain involved in land recognition on either the extracellu

surface or within endosomes and a cytopl

mic domain involved in signal transducti(b) the NOD-like receptors (NLRs), which

intracellular, cytoplasmic sensors; and (c) tretinoid acidinducible gene1 (RIG-1)-l

receptors (RLRs), which are cytosolic helicathat primarily sense viruses. Historically,

TLRs were first recognized for their rolehost defense, but the importance of NLRs

complementing the functions of the TLRs become increasingly clear. The high evoluti

ary conservation of the NLRs attests to theirficacy and vitality in host defense. Homologs

the NLRs (e.g., R genes) have been discove

throughout the plant and animal kingdomincluding phylogenetically primitive organis

such as the zebrafish (2) and the sea urchwhich has at least 203 identified putative NL

(3).

DEFINING FEATURES OF THENOD-LIKE RECEPTOR FAMILY

In humans, the NLR family is composed of

proteins ( Table 1), and there are at least NLR genes in mice. Although primarily

pressed in immune cells, including both lyphocytes and antigen-presenting cells (APC

such as macrophages and dendritic cells, NL

can also be expressed in nonimmune cells, cluding epithelial and mesothelial cells. T

family of proteins is defined by a triptite structure consisting of (a) a variable

terminal protein-protein interaction domadefined by the caspase recruitment dom

(CARD), pyrin domain (PYD), acidic transtivating domain, or baculovirus inhibitor

peat (BIR); (b) a central nucleotide-bindoligomerization (NOD) domain, which me

ates self-oligomerization that occurs during tivation (4); and (c) a C-terminal leucine-r

repeat (LRR) that detects PAMPs.

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Table 1 NOD-like receptor (NLR) family members

HGNC-approved symbol

Human Mouse Other names and aliases Domain organization NLR family

CIITA Cllta NLRA; MHC2TA; C2TA

Nlra; MHC2TA; C2TA NLRA

NAIP NLRB1; BIRC1; CLR5.1

Naip1 Birc1a

Naip2 Birc1b Naip3 Birc1c

NLRB Naip4 Birc1d

Naip5 Birc1e

Naip6 Birc1f

Naip7 Birc1g

NOD1 NLRC1; CARD4; CLR7.1NLRC

Nod1 Nlrc1; Card4

NOD2 NLRC2; CARD15; CD;

BLAU; IBD1; PSORAS1;

CLR16.3

NLRC

Nod2 Nlrc2; Card15

NLRC3 NOD3; CLR16.2

NLRC Nlrc3 CLR16.2

NLRC4 CARD12; CLAN; CLR2.1;

IPAF NLRC

Nlrc4 Card12; CLAN; Ipaf

NLRC5 NOD27; CLR16.1NLRC

NIrc5

NLRP1 NALP1; DEFCAP; NAC;

CARD7; CLR17.1

Nlrp1a NALP1

NLRPNlrp1b

Nlrp1c NLRP2 NALP2; PYPAF2; NBS1;

PAN1; CLR19.9 NLRP

Nlrp2 Pypaf2; Nbs1; Pan1

NLRP3 CIAS1; PYPAF1; Cryopyrin;

NALP3; CLR1.1 NLRP

Nlrp3 Cias1; Pypaf1; Cryopyrin;

Nalp3; Mmig1

NLRP4 NALP4; PYPAF4; PAN2;

RNH2; CLR19.5

Nlrp4a Nalp4a; Nalp-eta; Nalp9D

Nlrp4b Nalp4b; Nalp-gamma; Nalp9ENLRP Nlrp4c Nalp4c; Nalp-alpha; Rnh2

Nlrp4d Nalp4d; Nalp-beta

Nlrp4e Nalp4e; Nalp-epsilon

Nlrp4f Nalp4f; Nalp-kappa; Nalp9F

Nlrp4g Nalp4g

(Continued

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Table 1 (Continued)

HGNC-approved symbol

Human Mouse Other names and aliases Domain organization NLR famil

NLRP5 NALP5; PYPAF8; MATER;

PAN11; CLR19.8 NLRP

Nlrp5 Mater; Op1

NLRP6 NALP6; PYPAF5; PANS;

CLR11.4NLRP

Nlrp6 NLRP7 NALP7; PYPAF3; NOD12;

PAN7; CLR19.4 NLRP

NLRP8 NALP8; PAN4; NOD16;

CLR19.2 NLRP

NLRP9 NALP9; NOD6; PAN12;

CLR19.1

Nlrp9a Nalp9a; Nalp-thetaNLRP Nlrp9b Nalp9b; Nalp-delta

Nlrp9c Nalp9c; Nalp-zeta

NLRP10 NALP10; PAN5; NOD8;

PYNOD; CLR11.1 NLRP

Nlrp10 Nalp10; Pynod NLRP11 NALP11; PYPAF6; NOD17;

PAN10; CLR19.6 NLRP

NLRP12 NALP12; PYPAF7; Monarch1;

RNO2; PAN6; CLR19.3 NLRP

Nlrp12 Nalp12


CLR19.7 NLRP


CLR11.2 NLRP

Nlrp14 Nalp14; Nalp-iota; GC-LRR

NLRX1 NOD9; CLR11.3

NLRX

The N-terminal domain of the NLRsis critical for downstream signaling. CARDdomains were originally associated with pro-

teins involved in apoptosis and inflammationsuch as many of the caspases, including caspase-

1; however, CARDs have also been shownto mediate caspase-independent interactions.

The structure of PYD is homologous to tof CARD and promotes homophilic intactions with other PYD-containing prote

that are important for downstream signalevents. Both CARD and PYD are members

the death domainfold superfamily, membof which are involved in both apoptosis a

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inflammation. Finally, the BIR-containing pro-

teins can be classified into two major groups,

inhibitor of apoptosis proteins (IAPs) and neu-ronal apoptosis inhibitor proteins (NAIPs),

both of which are NLR family members. Be-cause of the variety in domain structures for

each NLR, the nomenclature of the differentNLRs has been inconsistent and confusing. Re-

cently, in order to promote uniformity in thenomenclature of the various NLRs, a new sys-

tem approved by the Human Genome Orga-nization Gene Nomenclature Committee was

established (5). This new annotation systemrecognizes the evolutionarily conserved NOD

and LRR domains that are the central defin-

ing features of these PRRs, but it further sub-divides the NLR family into four subfamilies

(indicated by letters A through C and P) basedon the type of N-terminal effector domain

(Table 1). An additional subfamily, NLRX, hasno strong homology to the N-terminal domain

of any of the other four subsets and currentlyconsists of only one member, NLRX1 (Nod9),

which appears to be unique in its localization tothe mitochondria (6, 7).

NOD-LIKE RECEPTORSIGNALING: DIVERSE ANDCOMPLEX PATHWAYS WITH

MULTIPLE LEVELSOF REGULATION

In general, the primary function of PRRs is theactivation of inflammatory signaling pathways.

In some respects, NLR signaling is very sim-ilar to that of the TLRs, with shared down-

stream targets (Figure 1). For example, uponrecognition of their respective PAMPs, the pri-

marily membrane-bound TLRs recruit adaptorproteins such as MyD88 and TRIF, which acti-

vate the MAPK and NF-B signaling pathways,

resulting in the induction of proinflammatoryand antimicrobial mediators such as interleukin

(IL)-6, tumor necrosis factor alpha (TNF-),and IL-1 (for a review, see Reference 8). Sim-

ilarly, stimulation of the intracellular NLRs ac-tivates downstream signaling pathways for the

production of proinflammatory mediators to

defend the host against infection. However, the

end targets of NLR signaling are not the samefor all NLRs. Three major activation targets of

NLR signaling after PAMP recognition have

been identified: (a) NF-B, (b) MAPKs, and(c) caspase-1 (Figure 1).

Nuclear FactorKappaB/Mitogen-ActivatedProtein Kinase Signaling

The earliest-identified and best-characterized

NLRs are NOD1 and NOD2, which are pro-totypical of NLR activation of both the NF-

B and MAPK pathways (912). Upon recog-

nition of their respective agonists, both NOD1and NOD2 self-oligomerize to recruit and ac-

tivate the adaptor protein RICK (also knownas RIP2), which is essential for the activation

of both NF-B and the MAPKs (4, 10, 1316). RICK is a serine-threonine kinase that be-

comes polyubiquitinated upon interaction withNOD1 or NOD2 through homotypic CARD-

CARD interactions (17). This RICK K63linked ubiquitination step is essential for re-

cruitment of the kinase TAK1, which activatesthe NF-B-activating complex and is inhibited

by the deubiquitinase A20 (17). RICK itself

recruits and promotes the K63-linked polyu-biquitination 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 aubiquitin-dependent manner (18). The colo-

calization of NEMO and TAK1 promotes thesubsequent phosphorylation of the IK sub-

unit of IK by TAK1 and results in the phos-phorylation and degradation of IB, a criti-

cal step that allows the cytoplasmic release andnuclear translocation of NF-B (19). NF-B

subsequently activates transcription of inflam-

matory cytokines and chemokines such asTNF-, IL-6, IL-8, and membrane cofactor

protein 1, which are important for stimulationand recruitment of additional effector cells dur-

ing host defense.As has been demonstrated for both NOD1

and NOD2 signaling, ubiquitination plays a



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a

b

c

Lipoprotein

Flagellin

Viral

dsRNAT

RIF

Endosome

TLR1

or

TLR6

TLR5

TLRs

TLR2

TLR

4

LPS

MyD88

Imiquimod

IRA

K1

IRAK4

TRAF6

TAK1T

AB1

TAB2/3

NEMO

TLR3

TLR7

TLR9

CpGDNA

MKKs

Nucleus

ERK

JNK

IKK

IKK

NF-B

NF-B

R1

r R6

TLR5

7

TLR9

C

TLRs NNN

FNF-FBBB

NF-B

TAK1

TAK1

iE-DAP

MDP

NOD1

N

OD2

RICK

CA

RD9

RICK

CARD9

NLR

inflammasome

Ub

Ub

TAB1 T

AB2/3

TAB1

TAB2/3

NEMO

MKKs

MKKs

p38

JNK

ERK

p3

8

JNK

ERK

IKK

IKK

Pro-IL-1

IL-1

Figure1

Toll-likereceptor(TLR)andNod-likereceptor(NLR)signalingpathways.(a)Extracellularpathogen-associatedmolecularpatterns(PAMPs)

arerecognizedbyTLRsat

theplasmamembranean

dendosomes,whichsignalthroughtheadaptorsMyD88andToll/interleukin-1receptordomain-containingadapter-inducinginterferonbeta

(TRIF),aswellasthroughinterleukin-1receptorassociatedkin

ase(IRAK)proteinsandtumornecrosi

sfactorreceptorassociatedfactor6(TRAF6).(b)TheNLR

proteinsNOD1andNO

D2senseintracellularD--glutyamyl-meso-DAP(iE-DAP)andmuramyldipeptide(MDP),respectively,

leadingtor

ecruitmentoftheadaptor

proteinsRICKandcaspaserecruitmentdomain9(CARD9).Su

bsequently,bothTLRsandNOD1/NO

D2signalingpathwaysrecruitTAK1,whichmediatesthe

activationofnuclearfactorkappaB(NF-B)andmitogen-activ

atedproteinkinases(MAPKs),resultinginthetranscriptionalupregulationof

proinflammatorygenes.

(c)ActivationofNLRsb

ymicrobialorendogenousmoleculesinthecytosolresultsintheformationof

caspase-1-activatinginflammasomes.Activationofcaspase-1

inducesprocessingoftheinterleukin-1-beta(IL-1)precursorandsecretionofthematurecytokine.Abbreviations:ERK,extracellularsignal

regulatedproteinkinase;

IKK,

I-kappa-Bkinase;JNK,c-JunN-terminalkinase;MKK,M

APkinasekinase;NEMO,

NF-Bessentialmodulator.

370 Chen et al.


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key role in activation of downstream signaling.

For NOD2-mediated activation of NF-B in

particular, the E3 ligase tumor necrosis factorreceptorassociated factor 6 (TRAF6) was sug-

gested to be importantbut dispensableforNOD1 signaling, whereas TRAF2 and TRAF5

were essential (17, 20). The significance of thedifferential utilization of E3 ligases for ubiqui-

tination is not clear, but this ligase may provideadditional points of regulation during the in-

duction of inflammatory cytokine production.NOD2 is also additionally regulated by Erbin,

which interacts with NOD2 and is capable ofdownregulating NOD2-dependent activation

of NF-B. However, whether Erbin also reg-

ulates signaling of other NLRs remains to bedetermined (21).

Stimulation of NOD1 and NOD2 also re-sults in the activation of MAPKs, including

the p38, extracellular signalregulated proteinkinase (ERK), and c-Jun N-terminal kinase

( JNK) pathways (Figure 1) (15). In contrastto NF-B, the molecular events that occur for

activation of these pathways are not as well de-fined, but they involve similar upstream signal-

ing 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 ac-tivation of MAPK pathways and less so for

NF-B (24).Importantly, there are NLR members that,

instead of promoting NF-B activation, mayhave a primarily negative regulatory role. These

members include NLRP12 (25, 26), NLRC3 inT cells (27), and NLRP2 in macrophages (28).

As the studies identifying these members wereall performed in vitro and typically involved

overexpression of proteins or the use of tumorcell lines, the physiologic roles of these NLRs

in vivo remain to be determined.

Biological Responses to NOD-1and NOD-2 Signaling

A consequence of NOD1- and NOD2-mediated activation of NF-B and MAPK is

the upregulatedtranscription and productionof

MDP: muramyldipeptide

inflammatory mediators, including cytokines,

chemoattractants, adhesive molecules, and in-

ducible molecules (e.g., iNOS, Cox-2). ForNOD1 specifically, which is expressed in ep-

ithelial and mesothelial cells, its stimulation caninduce chemokine production and recruitment

of effector immune cells, including neutrophilsin vivo (29, 30). Equally important is the upreg-

ulation of antimicrobial peptide productionespecially in epithelial cells, which may be NF-B dependent, by both NOD1 and NOD2 sig-naling. Disruption of this function may con-

tribute to the pathogenesis of inflammatorydiseases in the bowel (3136). NOD1 signal-

ing is also important in the coordination of an

adaptive immune response by both T and Bcells through synergistic antigen presentation

with TLRs (1). Similarly, a role for NOD2 inmounting adaptive responses is implicated by

the observation that the NOD2 agonist, mu-ramyl dipeptide (MDP), can act as an effective

adjuvant for antigen-specific T cell responsesand antibody production (37, 38).

Aside from the induction of proinflamma-tory mediators, NOD1 and NOD2 have also

been shown to induce apoptosisin in vitro over-expression systems; in fact, these proteins were

originally identified by their structural homol-

ogy to Apaf-1 and CED-4, known regulatorsof apoptosis (13, 14). The specific pathways in-

volved in the induction of apoptosis are not en-tirely clear, but for NOD1 this process involves

both caspase-8 and caspase-9 and requiresRICK. Note, however, that these studies were

all performed in vitro and that functional rele-vanceremainstobeinvestigatedinvivo(13,23).

Interleukin-1 Productionand the Inflammasome

In addition to NF-B and MAPKs, the third

important pathway activated by NLR signalinginvolves ASC (adaptor protein apoptosis speck

protein with caspase recruitment) to activatecaspase-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



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into their mature, biologically active forms. Re-

cruitment of ASC by these NLRs is believed to

occur through homophilic PYD-PYD interac-tions. Via its CARD domain, ASC, in turn, in-

teracts with caspase-1. These protein-proteininteraction domains promote oligomerization,

recruitment,andapproximationofeffectorpro-teins essential for activationof caspase-1 (4, 39).

Indeed, a hallmark of this caspase-1-dependentpathway is the assembly of large macromolec-

ular complexes through CARD-CARD andPYD-PYD protein-protein interactions that

function to form a scaffold for procaspase-1 recruitment and activation. This molecu-

lar platform, of which NLR family members

are the cornerstone, has been termed the in-flammasome (40) as an analogy to the apopto-

some, an activator of caspase-9 duringapoptosis(41).

Currently there are three well-described in-flammasomes named after the NLR involved:

NLRP1, NLRP3, and NLRC4. Common tothese inflammasomes is the role of ASC as the

adaptor protein that bridges these NLRs tocaspase-1 (40, 42). The NLRP1 inflammasome

was the first to be characterized and is the onlyinflammasome that has been reconstituted in

vitro with purified proteins (40, 43). The ex-

istence of the NLRP3 and NLRC4 inflamma-somes, the activities of which are dictated by

the specific PRRs recognized by the NLRs, hasbeen implicated primarily by the ability of both

NLRP3 and NLRC4 to activate caspase-1 in anASC-dependent manner (4450).

OtherNLRmembersthatcanassociatewithprocaspase-1 and promote IL-1 production

such as NLRP2have only been demonstratedin vitro, but whether the latter is a bona fide in-

flammasome that functions to activate caspase-1 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 caspase-

1 at least for the murine isoform Naip5; how-ever, the relevance of its ability to interact with

caspase-1 and whether it acts alone or togetherwith NLRC4 remain controversial. We discuss

this issue in more detail below.

Another important consequence of caspa

1 activation is the induction of a newly rognized process of programmed cell de

termed pyroptosis, which is distinct from aptosis and necrosis. Pyroptosis is typically

duced in macrophages infected with cert

intracellular bacteria such as Salmonella andassociated with a proinflammatory response

volving caspase-1-mediated IL-1 and IL-production (51). This response causes rapid f

mation of plasma membrane pores, cellular sis, and release of intracellular inflammato

contents to fuel additional inflammatory snaling pathways (52). Caspase-1 activation a

the release of IL-1 and IL-18 have bshown to be particularly important in h

defense against such pathogens as Shig(53), Legionella (48), Francisella (54), Liria (55), Yersinia (56), and Bacillus anthr

(52). How the different inflammasomes enlisted to defend against specific pathoge

or stimuli is described in the followsections.

NOD-LIKE RECEPTORSRECOGNIZE SPECIFICBACTERIAL ANDENDOGENOUS MOLECULES

Although numerous studies have delineated

importance of individual NLRs against specpathogens, a direct interaction between a p

tative ligand and its corresponding NLR hnot been demonstrated for most of the NL

Therefore, the possibility that the interactibetween NLR and PRR is indirect and invol

an intermediary host factor or activity cannbe precluded. In this section, we describe w

is known about the sensing of pathogens by various NLRs (Table 2).

NOD1

Originally, LPS preparations were demostrated to activate both NOD1 and NO

through RICK, but this was recently shownbe due to contamination of LPS preparatio

with PGN moieties (10, 15, 57). Both NO

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Table 2 NOD-like receptor (NLR) agonists and downstream signaling pathwaysa

Receptor Agonist Bacteria Signaling pathway

Nod1 GM-tripeptide Helicobacter pylori NF-B

meso-lanthionine, meso-DAP Shigella flexneri MAPK

-d-Glu-DAP(iEDAP) Listeria monocytogenes

Campylobacter jejuni

d-lactyl-l-Ala--Glu-meso-DAP-Gly (FK156) Enteropathogenic Escherichia coli

heptanolyl--Glu-meso-DAP-Ala (FK565) Chlamydia pneumoniae

Pseudomonas aeruginosa

Bacillusspp.

Nod2 MDP Streptococcus pneumoniae NF-B

MurNAc-l-Ala-g-d-Glu-l-Lys (M-TRILys) Listeria monocytogenes MAPK

Mycobacterium tuberculosis

Salmonella Typhimurium

Staphylococcus aureus

Shigella flexneri

Nlrc4 flagellin (Salmonella, Legionella, Pseudomonas) Salmonella Typhimurium caspase-1

unknown (Shigella) Legionella pneumoniae


Shigella flexneri

Listeria monocytogenesb

Naip unknown Legionella pneumophila caspase-1


Salmonella

Listeria

Nlrp1b anthrax lethal toxin Bacillus anthracis caspase-1

Nlrp3 bacterial RNA Staphylococcus aureus caspase-1

viral RNA and DNA Listeria monocytogenesb

uric acid crystals

LPS

LTA

MDPsilica

asbestos

aAbbreviations: LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MDP, muramyl dipeptide; NF-B, nuclear factorkappa B.bBoth 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 bybacteria (5861). PGN provides structure and

rigidity to bacteria and is found in virtually

all bacteria, although the amount, location,and specific composition may vary (62).PGN consists of sugar chains of alternating

N-acetylglucosamine (GlcNAc) and N-acetyl

muramic acid (MurNAc), which are cross-linked by short peptide chains (Figure 2).

These peptide chains contain unique aminoacids that are differentially found in gram-

negative and gram-positive bacteria and that

are also differentially recognized by NOD1 andNOD2. Specifically, NOD1 can sense PGN

moieties containing meso-diaminopimelic

acid, an amino acid that is found predomi-nantly in gram-negative bacteria but also insome gram-positive bacteria such as Listeriamonocytogenes and Bacillus spp. The minimalstructure recognized by NOD1 is the dipeptide

D--glutyamyl-meso-diaminolimelic acid (iE-

DAP) (59, 60). Studies defining synthetic iE-DAP derivatives that stimulate NOD1 revealed



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Plasma membrane

PGN

Lipoteichoic acid

Polysaccharide

Cytosol

Cytosol

LPS Porin

Lipopeptide

Plasma membrane

PGN

Outer membrane

L-alanine

D-glutamic acid

Mesodiaminopimelic acid

D-alanine

Tetra-peptidechain (amino acids)

MDP(Nod2 ligand)

iE-DAP

(Nod1 ligand)Pep

tidoglycan(PGN)

Glycan chain

Gram-negativ

ebacteria

Gram-positivebacteria

NAGNAM NAG NAM

NAG NAMNAG NAM

374 Chen et al.


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that the attachment of hydrophobic acyl

residues enhanced stimulation of NOD1 up to

severalhundredfold(63).Astheseresiduescon-tained fatty acid chains similar to those found

in the phospholipids that make up the hostcell membrane, it is possible that the increased

NOD1-stimulatory activity is due to an in-creased interaction with the cellmembrane that

facilitates translocation into the intracellularcompartment of the host cell (63). The

lipophilicity of the NOD1 ligand appears im-portant for recognition by NOD1, butprecisely

how NOD1 interacts with these lipophilicmolecules 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 activ-ity, with the strongest activity associated with

the genus Bacillus (58). Moreover, the pres-ence of bacteria has not always been necessary

for NOD1 stimulation, as water-soluble ex-tracts from food and soil as well as supernatant

from overnight bacterial cultures are capable ofstimulating NOD1 (58). This suggests that

physical contact with live bacteria is not nec-essary and that NOD1 agonists can be pro-

duced and released by bacteria. Consistent

with this observation, Shigella mutants that in-crease release of PGN fragments upregulated

NOD1-dependent NF-B activation in vitro(64). In addition, where and how NOD1 in-

teracts with bacteria are not clear. Localiza-tion of bacteria to the intracellular compart-

ment has been shown to not necessarily berequired for NOD1 stimulation, as the Liste-ria streptolysin O mutant, which is requiredfor Listeria to escape from the phagosome into

the cytosol, is still capable of activating NF-B

in a NOD1-dependent manner (58). Localiza-tion studies of NOD1 through overexpression

have shown that NOD1 becomes membrane

bound, at least during infection with Shigellaflexneri at sites of bacterial entry. However,

whether this occurs in vivo remains to be de-termined, as endogenous NOD1 localization

during infection has been difficult to visualize(65).

Despite the identification of the PGN moi-ety sensed by NOD1, the role of NOD1 dur-

ing in vivo infection remains unclear, with thepartial exception ofHelicobacter pylori, a bacte-

rial organism associated with the developmentof gastritis and duodenal ulcers. Specifically,

Nod1-deficient mice have shown increased sus-ceptibility to H. pylori infection, with an as-

sociated impairment of -defensin-4 produc-

tion (34, 66). In vitro studies, on the otherhand, have shownNOD1-dependent activation

by other pathogenic bacteria such as L. mono-cytogenes (15), S. flexneri (9), Campylobacter je-

juni(36, 67), enteroinvasive Escherichia coli(68),

Chlamydophila pneumonia (69), and Pseudomonasaeruginosa (70). However, these findings werebased on in vitro studies only and may not nec-

essarily reflect requirements in vivo. For exam-ple, NOD1 was originally implicated in pro-

tection against Chlamydia trachomatis infectionin vitro, where Nod1-deficient mouse embry-

onic fibroblasts (MEFs) exhibited decreased cy-

tokine production after C. trachomatisinfection;however, Nod1-deficient mice did not have

increased bacterial load or clinical symptoms,suggesting either that NOD1 is not directly in-

volved or that other redundant signaling path-ways can compensate (71).

Figure 2

Generation of NOD1 and NOD2 ligands from bacterial peptidoglycan (PGN). In this simplified diagram,the bacterial cell wall and PGN structure from Escherichia coliare depicted. Parallel PGN strands composedof the alternating amino sugars N-acetylglucosamine (NAG) and N-acetyl muramic acid (NAM) are cross-linked to each other by stem peptides. Notice that E. coliPGN lacks bridging amino acids linking stempeptides 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.



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NOD2

Separate studies have also demonstrated thatNOD2 can detect pathogenic bacteria. In

contrast to NOD1, which appears to beprimarily involved in sensing gram-negative

bacterial pathogens, NOD2 senses the spe-cific MDP motif that is found in a broader

range of bacteria, with some overlap withthose recognized by NOD1 (Figure 1). These

bacteria include Streptococcus pneumoniae (72),

L. monocytogenes(33, 73), Mycobacterium tuber-culosis (74), Salmonella (75), and Staphylococcus

aureus (76). Again, most of these studies wereperformed in vitro; therefore, the in vivo rel-

evance of these findings remains to be deter-mined. One exception, however, is L. monocyto-

genes, in which increased bacterial burdens wereobserved in Nod2-deficient mice when infected

orally rather than intravenously or intraperi-toneally (33). This immunity is associated with

decreased production of-defensins in Panethcells (33). In addition, both Nod1 and Nod2

have been shown to be important for bacterialrecognition and host defense against L. mono-cytogenesafter exposure of macrophages or an-

imals to LPS or E. coliin vivo (77). These ob-servations suggest that the intracellular sensors

NOD1 and NOD2 play a critical role in hostdefense when TLRsignaling is reduced, such as

within the intestine due to low expression levelsof TLRs (78, 79) or after induction of toleriza-

tion by exposure to TLR ligands.

NLRC4

As mentioned above, a subset of NLRs partic-ipate in the formation of inflammasomes that

ultimately leads to activation of caspase-1 and

maturation of IL-1. To date, these NLRs in-clude NLRC4, NLRP1, and NLRP3. Studies

have shown that the NLRC4 inflammasomecan activate caspase-1 in response to infection

by S. Typhimurium, an activity not shared bythe NLRP3 inflammasome (49, 50). Salmonella

infection can lead to gastroenteritis as well as totyphoid fever, which canbe associated with high

morbidity and mortality. What is recognized

by NLRC4 is flagellin, as flagellin-defici

Salmonella mutants elicit substantially redulevels of caspase-1 activation (49). Similarly,

tosolic detection ofL. monocytogenesby Ipaf

quires flagellin (80). A direct interaction btween flagellin and NLRC4 has not yet be

demonstrated, however, and therefore recogtion is likely indirect. TLR5, a member of t

TLR family, also senses flagellin; howeverhas been shown that the cytoplasmic delivery

flagellin requires NLRC4 but not TLR5, cosistent withits cellular location.As an additio

consequence of caspase-1 activation, caspa1 can lead to early macrophage cell death,

NLRC4-dependent process in the host defeagainst Salmonella (47).

In vivo, NLRC4 has also demonstrated

tivity against Legionella pneumophila, an intcellular pathogen that causes Legionnaires d

ease, which can be deadly. The pathogenesisLegionella requires successful entry and repli

tion inside host macrophages and is dependon the formation of a specialized vacuole th

blocks the fusion of the phagosome to the lysome, a process dependent on an intact fun

tional type IV secretion system (TFSS). Athecasewith Salmonella, Legionella also conta

flagellin, and NLRC4-dependent caspase-1 tivation requires the presence of intact flage

and its intracellular delivery (48, 81). As a

sult of NLRC4-induced caspase-1 activatiLegionella growth is restricted by promoti

the fusion of the Legionella-containing phagsome to thelysosome fordegradation(48).H

caspase-1 activation controls phagosome muration in response to Legionella infection

not known, but it may involve interactioncaspase-1 with host proteins involved in pha

some formation and transport or direct targing ofLegionella virulence factors required

lysosomal evasion.In vitro, NLRC4 is important in respond

to Shigella, an intestinal pathogen that cau

dysentery and, as mentioned above, can aelicit NOD1-dependent MAPK activation a

secretion of IL-8 (9). Like Legionella, Shigcan escape from within membrane vacuo

and enter the cytosol. Interestingly, Shig

376 Chen et al.


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does not express flagellin genes, yet it can ac-

tivate caspase-1 macrophages and induce py-

ropoptosis in an NLRC4-dependent process(82). This suggests that NLRC4 also rec-

ognizes an as-yet-unidentified PAMP or sig-nal associated with Shigella. Shigella also in-

duces autophagy, which is facilitatedin NLRC4but not in ASC-deficient macrophages, sug-

gesting that NLRC4 provides an additionalhost-defense measure independent of ASC and

IL-1 production by inhibiting autophagy (amembrane-trafficking system in which cyto-

plasmic components are sequestered for de-livery to lysosomes, where cytoplasmic ma-

terial is degraded). This downregulation of

autophagy is specific to Shigella-induced au-tophagy, as autophagy from serum starvation or

rapamycin treatment does not require NLRC4or caspase-1 (82). The reason for the inhibi-

tion of autophagy by NLRC4 is not clear, but ithas been proposed that this inhibition allows

the induction of pyroptosis, which may bet-ter promote an effective inflammatory response

(82).

NAIP

One of the mouse homologs of human NAIP,

Naip5, is also important in the host defenseagainst Legionella. This finding arose from

observations that different mouse strainsshow varying permissiveness to intracellular

replication of Legionella and that this permis-siveness is genetically controlled (83, 84). The

genetic locus responsible for the increasedsusceptibility to Legionella infection in the A/J

mouse strain in particular has been determinedto be Naip5 (85). Naip5 promotes the fusion

of the Legionella-containing phagosomes tothe lysosome, thereby preventing intracellu-

lar replication (86). Studies in vitro have also

suggested that Naip5 activation requires the in-tracellular delivery of flagellin, which results in

caspase-1 activation and in promotion of earlycell death, thereby controlling intracellular

replication ofLegionella (81, 87, 88). However,these studies used large numbers of Legionellabacteria, which can lead to nonspecific early

cell death of macrophages; they also used

an indirect, nonspecific method of detectingcaspase-1 activation. In a separate study using

low bacterial load and caspase-1-specific anti-

bodies, Legionella-induced caspase-1 activationand IL-1 production occurred independently

of Naip5 but required Nlrc4 as well as flagellin(89). Naip5, however, was still required for in-

hibition ofLegionella replication intracellularly,irrespective of Nlrc4 signaling; this suggests

a caspase-1-independent pathway regulatedby 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 requireflagellin. In addition, Naip5like Nlrc4has

been suggested to regulate autophagy: A/J

mice that harbor mutant Naip5 exhibitedslower rates of autophagy induction after

Legionella infection of macrophages comparedwith Legionella-resistant C57BL/6 that have

intact Naip5 (90). This observation furtherdemonstrates the complexity of intracellular

Legionella replication by different NLRs, and itremains an active area of investigation.

Based on in vitro studies using A/J mice thatare considered Naip5 deficient compared with

C57BL/6, which have intact Naip5 and are re-sistant toLegionella replication, Naip5 also con-

tributes to IL-1 production by bone marrow

derived macrophages after infection withP. aeruginosa, an opportunistic bacteria that can

cause pneumonia and sepsis (91). The decreasein IL-1 production in A/J mice, although

statistically significant, may reflect inherentlydifferent genetic susceptibility differences be-

tween the two mouse strains that are indepen-dent of Naip5; therefore, this decrease may

be physiologically irrelevant in vivo. There-fore, additional studies examining a true

Naip5-knockout strain should provide addi-tional insight into and confirmation of these

findings.

NLRP1

Recently discovered mutations in the Nlrp1

(Nalp1b) gene in various mouse strains have



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been found to influence the susceptibility of

mice to Bacillus anthracislethal toxin (LT) (92),

the major virulence factor in the pathogen-esis of anthrax. This was the first demon-

stration of an NLR that responds to avirulence factor, as opposed to a specific bacte-

rial component. LT results in the formation ofpores allowing lethal factor, a protease, to enter

cells, resulting in proteolytic cleavage of hostsubstrates and subsequent cell death. Suscepti-

bility to LT-induced macrophage lysis/necrosisand death requires caspase-1 activation and is

associated with Nalp1b polymorphisms. Five ofthese polymorphisms have been identified, ex-

plaining the variability in susceptibility to LT-

induced cell death in different mouse strains(92). Caspase-1 activation by LT results in

pyroptosis similar to that seen in NLRC4-mediated cell death induced by Salmonella

infection (52).The human NLRP1 inflammasome, recon-

stituted with purified proteins including ASC,NLRP1, andprocaspase-1, hasalso beenshown

to respond to MDP and to induce the matura-tion of IL-1. Because only purified proteins

were used in this cell-free system (40), it is pos-sible that, in this case, a direct interaction be-

tween the inflammasome and MDP occurred

under physiological conditions.

NLRP3

NLRP3 is an NLR that also participates ininflammasome formation through the recruit-

ment of ASC and subsequent activation ofcaspase-1 and secretion of IL-1 and IL-18.

NLRP3 detects PAMPs such as LPS, MDP, andbacterial and viral RNA, including the double-

stranded RNA analog poly I:C (4446) as wellas the imidazoquinoline antiviral compounds

R837 and R848 (93). Consistently, NLRP3

has been found to respond to infection withthe Sendai and influenza viruses, resulting in

caspase-1 activation (44). Subsequently, it wasshown that NLRP3 can detect viral DNA (94).

In vitro, NLRP3 has also been demonstrated tobe required for caspase-1 activation/IL-1 se-

cretion in response to the bacterial pathogens

S. aureusand L. monocytogenes(46). However

the case ofL. monocytogenes, the role of NLRhas been controversial, as there are oppos

data to suggest that caspase-1 activation byL

teria can occur independently of NLRP3 (102). This discrepancy may be explained

redundant contributions by both NLRP3 aNLRC4 in caspase-1 activation in response

Listeria (80).Characteristic of NLRP3, however, is its

pability to respond to a broad repertoire of onists that are not necessarily microbial in o

gin, but rather are endogenous signals. NLRhas been suggested to respond to changes

cellular ion concentrations, particularly potsium, that are produced, for example, by

marine toxin maitotoxin and the K+/H+

tiport ionophore nigericin (46). NLRP3 actition is also observed with uric acid crystals (

and reactive oxygen species that are generain response to, for example, asbestos and sil

(7).Significant insight into the mechanism

which NLRP3 recognizes its various ligacame from the observation that the addition

ATP to macrophages prestimulated with bterial molecules such as LPS can significan

enhance caspase-1 activation and IL-1 p

duction. Identifying the role of ATP in NLRactivation has been controversial, as high no

physiological concentrations of ATP are tycally required for enhanced IL-1 producti

An important function of ATP is the stimlation of the P2X7 receptor, which, in tu

results in the opening of a pore mediatedthe hemichannel protein pannexin-1 (96, 9

Therefore, a consequence of ATP additionthe ability of bacterial products to enter the

tosol through the pore and to subsequently tivate NLRP3 and caspase-1. Indeed, with

addition of ATP, several bacterial product

including LPS, PGN, and lipoteichoic acas well as heat-killed bacteriacan activ

caspase-1inanNLRP3-dependent(butaTLand RICK-independent) manner (44, 45,

98). Moreover, the requirement for ATP canbypassed when other methods of cytosolic

livery are implemented; such methods inclu

378 Chen et al.


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the use of pore-forming bacterial toxins (e.g.,

streptolysin O) or the lipophilic DOTAP deliv-

ery system (98, 99). However, not all bacterialpathogens that can invade the cytosol require

NLRP3 for caspase-1 activation as Salmonellaand Francisellawhich are capable of deliver-

ing bacterial products into the cytosol throughtype III secretion systems (TTSS) or through

pore-forming molecules without ATPcanactivate caspase-1 independently of NLRP3

(98). Thus, NLRP3 recognition of PAMPs ap-pears to be linked to pannexin-1-dependent

pore formation. MDP, a NOD2 agonist, hasalso been shown to activate NLRP3 (100) so

as to activate caspase-1. This activation is in-

dependent of NOD2 and involves the internal-ization of MDP into acidified vesicles that are

released into the cytosol upon addition of ATPin a pannexin-1-dependent manner (101).

An increasingly recognized consequence ofactivation of the P2X7 receptor and pannexin-

1 by ATP is the alteration in potassiumconcentrations in the cell (i.e., intracellular

potassium depletion), which has been sug-gested to be important for NLRP3 but not

for NLRC4 signaling (102, 103). Treatmentof macrophages with the pore-forming tox-

ins nigericin and maitotoxin is also associ-

ated with intracellular potassium depletionand results in NLRP3-induced activation

of caspase-1 (46). Therefore, in additionto facilitating entry of NRLP3-responsive

PAMPs, pore-forming toxins also promotechanges in intracellular potassium concen-

trations that may be required for NLRP3activation. However, the concentration of

potassium itself may not regulate caspase-1 ac-tivation, as changes in potassium concentration

in the cell are associated with other ionic fluxesor cellular events that may be critically involved

in NLRP3 signaling. Moreover, in the absence

of PAMP prestimulation, ATP does not acti-vate caspase-1, even though it alone can trigger

potent efflux of intracellular potassium (102).Thus, the true significance of potassium con-

centration changes associated with NLR sig-naling 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 surro-gate indicators of cellular stress or injury; there-

fore, NLRP3 andperhaps other NLRs mayalso

function to recognize and respond to endoge-nous danger signals besides microbial infection

(95, 104).

MECHANISMS OFINTRACELLULAR DELIVERYOF MICROBIAL PRODUCTSFOR DETECTION BY

NOD-LIKE RECEPTORS

A defining feature of the NLRs is their in-

tracellular localization. With the exception ofNLXR1, which has been shown to be im-

portant within the mitochondria (6, 7), andCIITA, which resides in part in the nucleus,

all NLRs characterized to date are locatedwithin the cytoplasm. This raises the question

of how these receptors recognize pathogens,or how pathogens are delivered into the intra-

cellular compartment. Based on their cytoso-lic localization, NLRs may respond primarily

to (a) bacteria that escape extracellular detec-tion by the TLRs and invade directly into the

cell, (b) bacterial components that are deliv-

ered into the cytosol through secretion sys-tems or through pore-forming molecules, or

(c)bacterialproductsthatareuptakenbytheim-mune cell through phagocytosis or pinocytosis

(Figure 3). Consistent with the first possibil-ity, NOD1 activation depends upon the intra-

cellular localization of directly invasive bacteriasuch as S. flexneri(9, 64, 105) and S. pneumoniae(72). Also, only live and not paraformaldehyde-fixed C. jejunielicit NOD1-dependent NF-B

activation (36).Bacteria with active secretion systems pro-

vide an additional mechanism for intracellu-

lar entry that is important for NLR recog-nition. Two secretion systems, type III and

type IV, are involved in the injection of viru-lence proteins into the host cell to stimulate

NLR signaling. These secretion systems arefound in gram-negative bacteria and are en-

coded within regions of the bacterial genome



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P2X7R

ATPPannexin-1a

-BNF

NLRinflammasome

Pore-formingtoxin

Pro-IL-1

Nucleus

IL-1

Caspase-1

Phagosome

Bacteria type III or IVsecretion system

P2X7R

PAMPs

Figure 3

Modes of intracellular entry of microbes for Nod-like receptor (NLR)activation. Microbes and microbial molecules enter the cytosol viapore-forming toxins, type III or IV secretion systems, or ATP-mediatedactivation of the pannexin-1 pore. Sensing of microbial molecules in the cytosolby several NLRs results in the formation of inflammasomes. The mechanismwhereby NLRs recognize microbial molecules appears indirect and remainspoorly 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 secre-tion systems allow NLR detection by non-

phagocytosing cells such as epithelial cells. ForTTSS, 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 asintestinal epithelial cells and (b) their ability

to survive as well as replicate within phago-cytes. Both of these features depend upon

intact TTSS. TTSS consists of a complexassembly of proteins that form transporter ap-

paratuses; these contain approximately 20 pro-tein subunits for the trafficking of bacterial

proteins involved in bacterial virulence. As

scribed above, the delivery of flagellin to tcytosol for NLRC4 inflammasome activat

has been proposed to occur via TTSS, a

Salmonella mutants with nonfunctional TTare unable to activate NLRC4 in macropha

(50).InthecaseofLegionella, TFSSis essential

the survival of the pathogen within the phagsome. In a not-fully-understood mechanis

Legionellaonce phagocytosed by the APsuch as the macrophagereplicates within

phagosome and avoids fusion with the lysome through secretion of virulence prote

by the TFSS, which causes pore formati

Legionella is recognized by NLRC4 most lik

as a result of translocation of bacterial liga

(flagellin) through the TFSS into the cyto(107). As with Legionella, delivery of PG

derived molecules into the cytosol by H. pyforNOD1signalingrequiresanintactTFSS

sociated with the cag pathogenicity island, a

H. pyloristrains that lackcaghave reduced lev

of NF-B activation (66).Bacterial secretion systems also ena

pathogens to form pores to escape phasomes after phagocytosis, as well as

transport bacterial products. For example, pore-forming toxin listeriolysin produced

Listeria permits the delivery of PGN tha

recognizable by NOD1 and NOD2 (12, 108). Other bacteria that secrete pore-form

toxins, such as S. pneumoniae, which produpneumolysin, and B. anthraciswith anthroly

O, can also allow internalization of PGN frments for recognition by NLRs, especially

nonphagocytosing cells such as epithelial c(109).

With certain bacteria, activation of NLcan occur with heat-killed bacteria alone, su

gesting that neither an active secretion stem nor direct invasion is required for reco

nition. In these cases, dead bacteria are lik

directly uptaken into the cell by phagocyto(73). For example, inhibition of macropha

phagocytosis of heat-killed S. aureusresulteddecreased inflammatory cytokine producti

which was partly dependent on both NO

380 Chen et al.


17/36

and NOD2 (76). The entry of MDP has also

been proposed to occur in intestinal epithe-

lial cells through a host-dependent rather thanbacterial-dependent mechanism, in which the

active peptide transporter hPepT1 transportsMDP in vitro (110).

NOD-LIKE RECEPTORSIGNALING SYNERGIZES ANDCOMPLEMENTS TOLL-LIKERECEPTOR SIGNALING

There is great overlap and an apparent redun-dancy in the natureof thepathogensrecognized

by the various NLRs and TLRs, and many of

the receptors converge to common pathways(e.g., NF-B and IL-1 activation). An obvi-

ous reason for the existence of multiple recep-tors channeling into common end targets is

the potentiation of the inflammatory responseto infection. This is consistent with the syn-

ergy observed with TLR and NLR signalingin response to PGN (74, 111, 112) and be-

tween TLR and NOD2 agonists (33). In addi-tion, a segregation of function between NLRs

and TLRs is naturally imposed by their cellu-lar localization, such that NLRs may acquire

greater importance in the detection of intracel-

lularly located bacteria. Regardless, NLRs andTLRs also play nonredundant roles, as is re-

flected in the observations of TLR- and NLR-specific transcriptomes associated with Listeria

infection andof synergistic activation of NF-B(113).

Additional evidence for complementaryroles of NLRs and TLRs in the inflamma-

tory response has been demonstrated with IL-1 secretion (see Figure 4a). The release

of IL-1 involves two distinct steps: (a) in-duction of pro-IL-1, which is NF-B de-

pendent, and (b) cleavage of pro-IL-1 by

caspase-1 to the mature, active IL-1. The firststep requires TLR activation of NF-B (98);

however, activation of caspase-1 occurs inde-pendently of TLRs and requires the inflam-

masome (98). This two-part pathway, differen-tially regulated by TLRs and NLRs, may serve

as a safeguard against excessive production of

IL-1, which can cause pathology (see In-flammatory Diseases Associated with Excessive

Interleukin-1 Production, below). Similarly,

multiple NLRs or TLRs may recognize simi-lar stimuli but regulate different aspects of the

same process. Using the same example of reg-ulation of IL-1 production, caspase-1 activa-

tion by MDP stimulation requires NLRP3 andATP but not NOD2; however, the induction

of IL-1 messenger RNA, on the other hand,requires NOD2 but not NLRP3 (101). There-

fore, similar to therequirement for TLRsignal-ing to promote pro-IL-1 production through

NF-B,inthecaseofIL-1 induction by MDP,NOD2 mediates the transcription of pro-IL-

1byNF-B, whereas NLRP3 mediates IL-1

processing through caspase-1 activation. Also,as described above, studies have suggested that

Legionella recognition by both NLRC4 andNaip5 can result in alternate activities to re-

strict intracellular replication ofLegionella andthat recognition of flagellin in either Legionella

or Salmonella by TLR5 and NLRC4 can resultin the activation of separate pathways, NF-B

and caspase-1 activation, respectively (4850,89, 114). Thus, the multiplicity of receptors in-

volved in the recognition of the same ligandallows not only multiple levels of control to

prevent excessive production of cytokines (e.g.,IL-1) that can harm the host, but it also pro-

motes a concerted response against infection

through the activation of separate, but coop-erative pathways.

In addition to having a synergistic effect with TLR signaling, NLRs may provide the

host with a backup defense mechanism thatmay be required under conditions when TLR

signaling has been tolerized (Figure 4b). Awell-recognized phenomenon associated with

TLR signaling is the induction of tolerance: After an initial LPS exposure to, for exam-

ple, macrophages, cytokine responses are sup-pressed upon secondary exposure to LPS.

Possible physiologic explanations for this phe-

nomenon are (a) to prevent excessive cytokineproduction by bacterial stimuli, which can lead



18/36

TLRs

NF-B

Pro-IL-1 IL-1

Caspase-1

NLRinflammasome

TLRs

Invasion ofintracellular bacteria

TLR activation

Reduced TLR signaling (tolerance)(prevent overproduction of proinflammatory cytokine)

+Enhanced NLR signaling

Prepare to fight invasive pathogen

a b

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 ofnuclear factorkappa B (NF-B) signaling, leading to transcription of the IL-1 gene. In contrast, the presence of microbial molecuin the cytosol is sensed by the NLRs, resulting in inflammasome formation and caspase-1 activation. Activated caspase-1 cleavespro-IL-1 and results in secretion of mature IL-1. (b) Intracellular bacteria such as Listeria are recognized by TLRs, leading to TLactivation and production of proinflammatory and antimicrobial molecules. Persistent TLR stimulation leads to TLR tolerization anto reduced TLR signaling. Cytosolic invasion of the bacteria activates NLRs (e.g., NOD1/NOD2 and inflammasome-inducing NLR

resulting in enhanced NLR signaling and in production of antimicrobial molecules.

to the clinical syndrome of sepsis and to mul-tiorgan failure in the host, and (b) to stymie

an excessive immune response against com-mensal bacteria, which can lead to patholog-

ical inflammation. Although theoretically this

is a good safeguard mechanism, a potenrisk in the development of hyporesponsiven

with serial LPS challenge in APCs is an creased susceptibility to bacterial superinf

tion (115). Because TLR-induced tolerizat

382 Chen et al.


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is limited to TLR agonists that act in the

same pathway, NLRs may play a protective role

against bacterial infection in the TLR-tolerizedstate (Figure 4b) (77). Indeed, it has recently

been shown that cross-tolerization does not oc-cur with the combination of the NLR ago-

nist MDP and the TLR agonist LPS, as pres-timulation with either LPS or MDP does not

abrogate cytokine responses to MDP or LPS,respectively (77). Rather, enhancement in NF-B and MAPK activation as well as in cytokineproduction occurred after stimulation of MDP

in macrophages made refractory to TLR lig-ands, and vice versa (77).

A functional consequence of this phe-

nomenon is the heightened importance ofNOD1 and NOD2 response to intracellular

bacterial infection in macrophages tolerizedto previous exposure with TLR agonists (77).

Consistently, it was demonstrated that afterprevious exposure to E. colior LPS, response to

subsequent infection with Listeria was greatlycompromised 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 capableofIL-1productionthrough NLRC4signaling

(49). Thus, NLR signaling can potentiate TLR

signaling and can provide additional protectionto the host against bacterial invasion. One im-

plicationfor this model is that immune cells canremain sensitive to infection by pathogenic bac-

teria through NLR signaling in cells that havebecome tolerized to TLR ligands, such as com-

mensal bacteria in the gut.Note, however, that several groups (116

118) have also shownboth in humans andin micethat cross-tolerization still does oc-

cur with stimulation of immune cells by NLRagonists followed by TLR agonists. This dis-

crepancy may be due in part to differences inexperimental methods, including the timing of

pretreatment (early versus late second expo-

sure) or cell type (macrophages versus den-dritic cells), and it suggests that the regulation

of tolerization and synergy between NLR andTLRs may be specific to both context and cell

type.

IBD: inflammatorybowel disease

UC: ulcerative colit

CD: Crohns diseas

DYSREGULATED NOD-LIKERECEPTOR SIGNALING ISINVOLVED IN THEPATHOGENESIS OF MULTIPLEHUMAN DISEASES

The physiologic significance of the variousNLRs lies primarily in their roles in host de-

fense against microbial infection. However, itis becoming increasingly clear that NLRs alsofunction in organ homeostasis, which is un-

derscored by the fact that many inflammatoryand noninflammatory disease processes can

be attributed to dysregulated NLR signaling(Figure 5). The following subsections de-

scribe certain defined NLR roles in humandiseases.

Inflammatory Bowel Disease

The role of NLRs in intestinal homeostasis was

highlighted by the observations of mutationsin NOD2 that are associated with the devel-

opment of inflammatory bowel disease (IBD).IBD encompasses two different diseases, ulcer-

ative colitis (UC) and Crohns disease (CD);these diseases cause inflammation of the intes-

tine, leading to the common clinical presenta-

tion of abdominal pain, bloody diarrhea, andweight loss. Despite their similar clinical symp-

toms, UC and CD have distinguishing clinicaland histologic features. In UC, inflammation is

limited to the mucosal layer of the colon, mostcommonly therectum. In CD,however, inflam-

mation is transmural and can involve any part ofthe gastrointestinal tract. Because of the trans-

mural inflammation in CD, complications nottypically seen in UC can occur; these include

bowel-wall perforation and fistula formation.CD also differs from UC with regard to the af-

fected areas of the bowel: CD most commonly

involves the lower part of the small intestine,the terminal ileum.

The underlying pathogenesis of IBD is un-clear, but studies suggest that both environ-

mental and genetic factors contribute to theetiology of CD. Genetic linkage analyses of

affected families have identified eight genetic



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Blau syndrome/early-onsetsarcoidosis

NOD2(skin, eyes, joints)

Cryopyrinopathies

(MWS, FCAS, NOMID)NLRP3

(skin, eyes, joints)

Asthma

NOD1

(lungs)

Barelymphocyte

syndrome

CTIIA(lymphocytes)

Inflammatory bowel disease

NOD2, NOD1?(intestine)

Sarcoidosis

NOD1(lungs)

Vitiligo

NLRP1(skin)

Gain-of-functionmutations

Loss-of-functionmutations

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 ofNLRP3 and NOD2 cause autoinflammatodisorders: The former cause Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome

(FCAS), and neonatal-onset multisystem inflammatory disease (NOMID), and the latter cause Blausyndrome/early-onset sarcoidosis. Consistently, the latter diseases are inherited in a Mendelian-dominantfashion. In contrast, loss-of-function mutations ofNOD1, NOD2, and CTIIA are associated with thedevelopment of asthma, adult sarcoidosis, bare lymphocyte syndrome, and Crohns disease. Genetic variatin NLRP1 is associated with vitiligo. The type of functional alteration of the disease-associated NLRP1alleles remains to be determined.

loci(IBD1IBD8) containingCD-susceptibility

genes (119). The risk allele for IBD1 has beenidentified as NOD2 by genetic and functional

studies (120, 121). Although multiple variantsof NOD2 have been found to be associated

with CD, approximately 40% of CD patientsof North American or Western European de-

scent 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 thesemutations have a 20- to 40-fold increased risk

for disease development, whereas heterozyg

subjects have only a two- to fourfold increarisk (120, 121). These CD-associated NO

variants exhibit reduced capacity to activNF-B following MDP stimulation (61), co

sistent with the finding that these mutationsnear or within the LRR domain of NOD2. T

impaired sensing of PGN and/or MDP sugests that these CD-associated mutations res

in a loss-of-function phenotype. Furthermo

monocytes isolated from CD patients harbing the L1007fsins mutation exhibit reduc

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3

34

4

Reduced NOD2-dependentexpression of bactericidal-defensins in Paneth cells

Impaired recognition andclearance of bacteria byintestinal phagocytes

Dysregulation of TLR2-mediatedinflammation in intestinalmacrophages and/or dendritic cells

Enhanced production of IL-1

Macrophage

Nod2

IL-1

Nod2

Proinflammatorycytokines

TLR2

4

innflflaaammmmmmaammmmaaaccrrooopphphh

EEnEnnnhhhaaanncncced

11LL

-defensins

Paneth cellsNod2

4 ILIh

1-1L-L4 LIL

222R2R2TLRRRTLR

Dendritic cell

1

1

2

2

Figure 6

Proposed models for the role of NOD2 in Crohns disease. Shown are four nonexclusive models of the contribution of mutant NODalleles to Crohns disease. In the first two models, impaired NOD2 function is associated with either increased invasion of intestinalbacteria by deficient production of-defensins (1) or reduced clearance of bacteria by intestinal phagocytes, leading to inappropriateactivation of NOD2-independent pathogen-recognition receptor (PRR)-signaling pathways (2). In the third model, NOD2 acts as abrake of commensal bacteriadriven inflammation, and the presence of deficient 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 NODmutations function as gain-of-function alleles, resulting in inappropriate interleukin-1-beta (IL-1) production (4).

production of proinflammatory cytokines such

as TNF-, IL-6, and IL-8, as well as the anti-inflammatory cytokine IL-10 (122, 123). How-

ever, the loss of NF-B inducibility by the CD-associated NOD2 variants is inconsistent with

the occurrence of increased NF-B-dependentinflammation observed in clinical samples iso-

lated from CD patients. In order to reconcilethese observations and provide a mechanistic

explanation, two broad hypotheses regardingthe role of NOD2 in the pathogenesis of CD

have been advanced (Figure 6). The first hy-pothesisisthatmutationsinNOD2resultinde-

ficiencies in epithelial-barrier function and/orin immune cells required for limiting bacterial

invasion or clearance, which subsequently leads

to increased inflammation at intestinal sites.The second contends that primary dysregula-

tion of the mucosal immune system leads to ex-

cessive activation of proinflammatory signalingpathways.

Evidence for a role for NOD2 in regulatingepithelial-barrier function comes from trans-

fection experiments demonstrating that wild-type (but not mutant) NOD2 can restrict pro-

liferation of Salmonella in intestinal epithelialcells (75). The ability of NOD2 to restrict in-

vasive bacterial growth may be related to itsability to activate NF-B-dependent produc-

tion of the -defensins by Paneth cells, whichare specialized epithelial cells located in the

crypts of the ileal mucosa. Consistent with this



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hypothesis is the observation that NOD2 is ex-

pressedconstitutively in Paneth cells (124, 125).

In addition, CD patients with mutant NOD2have reduced expression of two -defensins

in the ileal mucosa, human -defensin-5 and-defensin-6 (31). Furthermore, mice genet-

ically deficient for Nod2 displayed impairedprotection against oral, but not intravenous or

intraperitoneal, administration of L. monocyto-

genes. The increased susceptibility of Nod2-

deficient mice was correlated with diminishedexpression of Panethcellderived 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 bea plausible link between NOD2 and suscepti-

bility to CD. However, the observation of de-creased -defensins should be interpreted with

caution, as this may merely reflect Paneth cellloss from inflamed, damaged epithelium (126).

Another possibility is that NOD2 mutationsresult in impaired clearance of locally inva-

sive bacteria due to defective recognition byintestinal phagocytes. This alternative loss-of-

function hypothesis is consistent with observa-tions of impaired production of antimicrobial

molecules and of IL-1, induced by MDP in

monocytes, expressing CD-associated NOD2mutations (61, 123). The defective removal of

intestinal bacteria may drive inflammation viaactivation of NOD2-independent PRRs, in-

cluding NOD1 and TLRs.The second hypothesis regarding the patho-

genesis of CD contends that the disease re-sults from inappropriate hyperresponsiveness

to commensal bacteria in the normal intestinemicroflora (127). This hyperreactive hypoth-

esis is supported by evidence demonstratingthat (a) NOD2 functions as a negative regu-

lator of IL-12 production mediated by PGNthroughTLR2and(b)intheabsenceofNOD2-

mediated regulation, PGN elicits a heightened

NF-B-dependent IL-12 or IL-23 responseby intestinal APCs, thereby promoting an ex-

uberant adaptive Th1 or Th17 type of im-mune response often observed in CD patients

(128, 129). In this model, it is assumed that in

normal intestine, APCs are constantly expo

to commensal bacteriaderived PGN withany overt immune response. It is then pos

lated that in normal individuals, NOD2 futions as a brake, dampening any potential

nate immune response to PGN. In contr

PGN-mediated immune responses in indivuals with NOD2 mutations are refractory

NOD2 modulation, thus resulting in high lels of IL-12/IL-23 andcreating an environm

capable of generating a pathological Th1/Thimmune response. Consistent with the ideat

dysregulated IL-12 promotes colitis, treatmof CD patients with antibody against IL-12

been shown to be effective (130). Alternativedata derived from the analysis of knockin m

expressing Nod2, which mimics L1007fsinsuggest that macrophages from these knoc

mice had increased levels of IL-1 product

(131). In addition to increased cytokine prodtion, theseknockin mice were shown to be m

susceptible to dextran sodium sulfateinduccolitis, suggesting that the frame-shiftmutat

associated with CD is a gain-of-function mtation. However, unlike the published knoc

Nod2 mouse model, monocytes from healtor CD patients homozygous for NOD2 mu

tions exhibit loss-of-function phenotypes (123). Thus, the relevance of the knoc

Nod2 mouse model remains uncertain.The hpotheses presented here may not be mutua

exclusive; further, they suggest that NOD2 m

contribute to CD development via multimechanisms.

Due to the similarity in signaling and struture between NOD1 and NOD2, research

are interested in determining whether NOis also associated with susceptibility to IB

Studies have been conflicting and may be poulation dependent; however, two studies h

demonstrated an association of NOD1 wIBD (132, 133). The first study to identify an

sociation between NOD1 polymorphisms aIBD involved two independent cohorts co

prising over 1000 IBD patients in England

these patients the common deletion allelea complex polymorphism (ND1+326561) w

significantly associated with IBD and wit

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such as BS, researchers have attempted

to determine whether NOD2 variants are

also associated with disease susceptibility forsarcoid. However, unlike with NOD1, no

correlation between NOD2 mutations and sar-coid has been observed (162164). Interest-

ingly, however, NOD2 has been associated with juvenile-onset sarcoid (EOS). This disease is

distinct from adult sarcoidosis in that it usuallyis seen in individuals younger than four years

with the clinical triad of skin, joint, and eyeinvolvementsimilar to that seen in BS, but

lacking pulmonary findings. In a small studyof 10 Japanese patients diagnosed with EOS,

nine had heterozygous missense mutations in

the NOD domain of NOD2, totaling six dif-ferent variants (165). Only one variant was

identical to that reported with Blau syndrome,while the remaining five were novel. Regard-

less, EOS-associated NOD2 variants, like theirBS-associated NOD2 counterparts, exhibited

increased basal NF-B activity compared towild type that was further enhanced with the

addition of MDP in a reporter assay in vitro(165).

Allergic diseases. Allergies are associated

with hyperreactivity to antigens; they are char-acterized by increased immunoglobin E re-

sponse and are therefore inflammatory innature. Certain NOD1 genetic variants are

associated with an increased risk of develop-ing asthma and atopic eczema, and therefore

NOD1 may play a role in the modulation of amucosal allergic response (166, 167). Because

asthma results from gene-environment inter-actions, its association with NOD1 may impli-

cate bacterial signaling through NOD1 in thepathogenesis of asthma. This would be consis-

tent with the prevailing hygiene hypothesis

of asthma risk: Studies have demonstrated thatindividuals with reduced exposure to bacteria

during childhood have an increased risk of de-veloping asthma (168170). Moreover, NOD1

polymorphisms can also modify the protectiveeffect of early exposure to allergens (171), pro-

vidingadditional evidence for a possible role for

NOD1 in modulating the response to environ-

mental bacteria and development of asthma.

Cancer. Recent studies have suggested thatTLR signaling plays an important role in car-

cinogenesis, especially within the gastrointesti-

nal tract (172, 173). A role for NLRs has not yetbeen investigated; however, a tumor-suppressor

function in NOD1 has been suggested in breastcancer xenograft models (174). This phenotype

was attributed to a functional role for NOD1in regulating apoptosis, in which retrovirally

infected MCF-7 cells with defective NOD1expression were more sensitive to TNF--

induced apoptosis in vitro in the presence ofcyclohexamide, which facilitates apoptosis by

inhibiting expression of antiapoptotic factors.In vivo, this mutant cell line also showed in-

creased growth potential as xenografts; this po-

tential was not associated with decreased apop-tosis, but rather with a lack of responsiveness

to estrogen-induced proliferation, suggestingan additional role for NOD1 in regulating es-

trogen receptor expression. However, sponta-neous growth of tumors has not been observed

in Nod1-deficient mice, and whether NOD1influences tumor growth in other organs that

are not necessarilyhormone responsive remainsto be determined.

CONCLUSIONS ANDPERSPECTIVES

The identification and initial characterization

of NLR proteins have yielded new insights intothe host recognition system involved in micro-

bial detection and host-defense mechanisms.The involvement of NLRs in the pathogenesis

of several genetic diseases indicates that theseproteins play an important role in the regula-

tion of immune and inflammatory responses.

There is conclusive evidence that several NLRssense 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 andTLRs in the regulation of the inflammatory re-

sponse against microbes.



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