3 nod

Available online at www.sciencedirect.com

Nod-like receptors: sentinels at host membranesDana J Philpott1 and Stephen E Girardin2

Innate immune detection of danger signals and microbial motifs

is achieved by distinct families of pattern recognition

molecules. These include the membrane-anchored Toll-like

receptors (TLRs), as well as cytosolic Nod-like receptors

(NLRs) and Rig-I-like receptors (RLRs). The precise mode of

NLR activation in the host cytosol remains poorly defined, as

evidence of direct interaction between NLRs and danger- or

microbial-associated molecular patterns remains elusive.

However, a number of convergent observations now suggest

that activation of some NLRs occurs at the level of host

membranes or as a consequence of membrane damage. This

review focuses on this emerging theme and discusses the

functional consequences of innate immune sensing at the

vicinity of the membrane.

Addresses1 Department of Immunology, University of Toronto, Toronto, Canada2 Department of Laboratory Medicine and Pathobiology, University of

Toronto, Toronto, Canada

Corresponding authors: Girardin, Stephen E

([email protected])

Current Opinion in Immunology 2010, 22:428–434

This review comes from a themed issue on

Host pathogens

Edited by Adolfo Garcia-Sastre and Philippe Sansonetti

Available online 3rd June 2010

0952-7915/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.coi.2010.04.010

Innate immunity relies on the detection of danger- and

microbial-associated molecular patterns (DAMPs and

MAMPs, respectively) by several families of secreted

or cellular pattern-recognition molecules (PRMs). Several

classes of cellular PRMS are directly responsible for the

induction of signal transduction pathways that shape the

innate immune response, and these include the type I

transmembrane molecules of the Toll-like receptor

(TLR) family as well as the cytosolic Nod-like receptors

(NLRs) and Rig-I-like receptors (RLRs) [1].

The NLR familyNLRs are defined by the juxtaposition of a central

(NACHT) domain and C-terminal leucine-rich repeat

(LRR) domain [2,3]. Twenty-two NLR proteins are pre-

sent in the human genome, which are further grouped

into subfamilies on the basis of their N-terminal region.

Upon activation, NLR proteins trigger a number of signal


transduction cascades, which include the pro-inflamma-

tory NF-kB (for Nod1 and Nod2) [2,3] and the caspase-1

inflammasome (for NLRC4, NLRP3 and NLRP1) path-

ways [4] as well as activation of autophagy (Nod1, Nod2

and NLRC4) [5,6] and cell death (including NLRC4,

NLRP1 and NLRP3) [5,7,8].

In the past few years, the identification of the nature of

the molecular patterns detected by NLRs has been the

subject of intense investigations, and the key role of

NLRs in the cytosolic detection of various MAMPs

and DAMPs is clearly emerging. Nod1 and Nod2 detect

specific structures within bacterial peptidoglycan [2], and

NLRC4 senses bacterial flagellin [9] as well as the bac-

terial type III secretion apparatus [10]. The inflamma-

some-triggering protein NLRP3 was shown to detect a

wide array of molecules, including ATP, potassium efflux,

muramyl dipeptide (MDP), bacterial toxins, xenocom-

pounds (silica, asbestos, and aluminium hydroxide), viral

nucleic acids, b-amyloid fibrils and malarial hemozoin

(reviewed in [11,12]). NLRP1 was also found to be a

pleiotropic sensor, mediating the detection of anthrax

lethal toxin, MDP, and potassium efflux. The extremely

diverse repertoire of the molecular triggers activating the

caspase-1 inflammasome led to the hypothesis that

NLRP proteins must in fact be activated by common

upstream cellular events [12–14]. The NADPH-depend-

ent generation of reactive oxygen species (ROS) or rup-

ture of the lysosomal membrane could represent such

common activators of the caspase-1 inflammasome. In

support of the former, recent evidence has shown that

NLRP3 interacts with thioredoxin-interacting proteins

(TXNIP) through the ROS-induced liberation of this

protein from thioredoxin [15].

The interest in NLR biology is also driven by the striking

association between mutations or polymorphisms in NLR

or NLR-associated genes and human inflammatory dis-

orders, which underscores the importance of this family in

the control of inflammation [16,17]. In particular, Nod2has been identified as the first susceptibility gene for

Crohn’s disease (CD) and Blau syndrome, and mutations

in Nlrp3 (also known as Cryopyrin or Cias1) are associated

with several rare autoinflammatory disorders, including

Muckle–Wells syndrome, chronic infantile neurological

cutaneous and articular syndrome (CINCA) and familial

cold urticaria (FCU), and common variants in Nlrp3 have

also been recently associated with CD [16,17]. In

addition, polymorphisms in Nod1 have been associated

with asthma and atopic eczema, and Nlrp1 was identified

as a susceptibility gene for vitiligo, Addison’s disease and

type I diabetes [16,17].

www.sciencedirect.com

mailto:[email protected]

http://dx.doi.org/10.1016/j.coi.2010.04.010

Sentinels at host membranes Philpott and Girardin 429

Signal transduction cascades triggered by NLR engage-

ment have been studied in detail. A general common

mechanism of activation likely requires the (homo- or

hetero-) oligomerization of NLR proteins, resulting in the

recruitment of adaptor proteins, such as Rip2 for Nod1

and Nod2, or ASC for NLRC4, NLRP1 and NLRP3 [2,3].

Rip2 is essential for mediating Nod1- and Nod2-depend-

ent activation of NF-kB and MAPK signaling [18], but is

dispensable for the Nod-driven recruitment of the autop-

hagosome at the site of bacterial entry in murine embryo-

nic fibroblasts [6]. The exact requirement of ASC for

NLRP and NLRC4 signaling is more complex, since this

adaptor protein was found to be either essential or dis-

pensable in multiple models of NLR-dependent acti-

vation of the caspase-1 inflammasome [12].

The nature of the events occurring upstream of NLR

engagement, and in particular the exact mode of the

detection of MAMPs or DAMPs by NLRs, remains less

understood. In the vast majority of cases, direct detection

or interaction between NLRs and either MAMPs or

DAMPs has not been demonstrated. An exception to this

was a recent study in which the authors performed an invitro reconstitution of the NLRP1 inflammasome and

demonstrated oligomerization of NLRP1 and caspase-1

through addition of MDP [19], therefore suggesting the

existence of a direct interaction between NLRP1 and

MDP. In the case of NLRP3, as discussed above, it is

believed that secondary cellular events such as ROS-

induced modifications or lysosomal damage might play

a key role in activation [12–14]. The cytosolic sub-cellular

localization where NLR engagement occurs is also not

clearly established, because of the lack of sensitive tools

to probe NLR activation by microscopy techniques.

However, recent biochemical evidence points to a tight

relation between NLR activation and sub-cellular vicinity

to plasma or internal membranes. An intriguing possib-

ility would suggest that NLR localization at membranes is

not coincidental with NLR activation but actually a

prerequisite for physiological activation. The following

discussion reviews this hypothesis.

Evidence for the activation of NLRs at hostmembranesNod1 and Nod2

The first indication that NLRs function at host mem-

branes came from a study by Barnich et al. who demon-

strated that Nod2-dependent responses to MDP

correlated with the capacity of the protein to localize

to the plasma membrane [20]. Importantly, the authors

identified that the C-terminal end of the protein was

responsible for membrane targeting, therefore explaining

the defective MDP sensing capacity of the CD-associated

Nod2 variant Nod2 3020insC, which lacks the last 33

COOH-terminal amino acids of the protein. A recent

study further validated these observations by showing

that Rip2 was also found at the plasma membrane, and


demonstrated that enforced targeting of Rip2 to the

plasma membrane was sufficient to trigger NF-kB [21].

Similar results were also reported for Nod1, as both Nod1

and the IKKg subunit of the IKK complex, critical for

NF-kB activation, were found enriched at the plasma

membrane, at the site of bacterial entry [22].

The consequence of targeting Nod protein to the plasma

membrane is likely complex and dynamic. Indeed, relo-

calization of Nod2 from a Triton-X-100 insoluble to

soluble fractions with cytochalasin D, or exclusion of

Nod2 from membrane ruffles, were found to correlate

with enhanced capacity to trigger NF-kB pathways [23],

suggesting that, in basal conditions, Nod-dependent

activity might be locked out by membrane- or actin-

associated cofactors. In agreement for this, two indepen-

dent studies identified the membrane-associated protein

Erbin as a Nod2-interacting protein, and demonstrated

that Erbin acted as a negative modulator of Nod2-de-

pendent pathways [24,25]. Other membrane-associated

proteins have been shown to interact with Nod proteins

and to modulate their function. The Rho GTPase Rac1,

which is essential for the regulation of membrane ruffling

and is also a component of the NADPH oxidase complex,

was found to interact with Nod2 and to negatively

regulate its function [23,26]. In contrast, the guanine

exchange factor GEF-H1, which plays important roles

in RhoA activation in the context of Shigella invasion in

non-myeloid cells, was found to interact with Nod1 and

positively modulate its function [27]. Finally, the protein

Duox2 of the NADPH oxidase complex was found to

interact with Nod2 and to potentiate its function [28].

The opposite effect of two members of the NADPH

oxidase complex (Rac1 and Duox2) on Nod2-dependent

pathway remains unclear, but might reflect the more

general impact of membrane targeting on Nod activity,

acting as a negative or positive modulator of Nod function

in resting versus ROS-generating or membrane-remodel-

ing conditions, respectively (Figures 1 and 2).

NLRP1 and NLRP3

The functional relation between the activity of NLRP3

and NLRP1 and host membranes is indirectly inferred by

the nature of the MAMPs or DAMPs that they detect.

Indeed, the fact that potassium efflux at the plasma

membrane is sufficient to trigger NLRP3 and NLRP1

activation [29,30] is a strong indication that these NLRs

are activated at the vicinity of the plasma membrane,

where the dynamic variation of [K+] is the greatest.

Similarly, NADPH oxidase-dependent ROS levels likely

diffuse poorly into the cytosolic space because of their

unstable nature and the presence of a number of buffering

cellular antioxidants. Therefore, NADPH-generated

ROS concentration must display a dynamic gradient

resulting from the flux of their generation, with the

highest concentration found at the vicinity of the plasma

or phagocytic membranes. Another interesting possibility


430 Host pathogens

Figure 1

Activation of Nod1 and Nod2 at host membranes. Nod proteins can detect peptidoglycan fragments produced in host phagosome or phagolysosome

during degradation of phagocytosed bacteria. The nature of the transporters involved in the translocation of peptidoglycan from the phagolysosome to

the cytosol remains unknown. Peptidoglycan fragments in the extracellular milieu can also enter cells by endocytosis. In HEK293T cells, peptidoglycan

fragments translocate to the cytosol at least in part through the oligopeptide transporter SLC15A4. Nod proteins interact with membrane-associated

proteins GEF-H1, Erbin, as well as Rac1 and Duox2 from the NADPH oxidase complex. Nod1 and Nod2 also detect invading bacteria and trigger the

recruitment of the autophagic machinery (see also Figure 3).

would be that NLRP proteins could directly detect

membrane damage or leakage. This idea is consistent

with the fact that several membrane toxins that insert into

host membranes, such as listeriolysin O, streptolysin O, a-

hemolysin or the anthrax lethal toxin were found to

trigger NLRP-dependent caspase-1 inflammasomes

[12,13]. In addition, multiple lines of evidence point to

a role of lysosomal damage and cytosolic leakage of

cathepsin B in NLRP3 activation [13], suggesting that

NLRP protein may act as intracellular sensors of host

membrane integrity.

NLRC4

NLRC4 was found to detect intracellular bacterial fla-

gellin [31,32]. While this detection could in theory occur

anywhere in the host cytosol in the case of flagellated

bacteria that escape the phagocytic vacuole, it is inter-

esting to note that NLRC4 activation by Salmonella and

Legionella requires both flagellin and functional type III or

Type IV secretion systems, which insert into host mem-

branes [31,32]. Together, these observations suggest that

the detection of flagellin by NLRC4 might be functional

in the context of membrane damage. In agreement with


this, NLRC4 has been recently shown to detect directly

the basal body rod component of the type III secretion

system apparatus of several Gram-negative bacteria, in-

cluding Salmonella typhimurium, Shigella flexneri and Pseu-domonas aeruginosa. It must be noted that Naip5, another

NLR protein previously identified as a critical protein

implicated in the restriction of Legionella growth in macro-

phages, was also shown to participate in intracellular

flagellin detection, together with NLRC4 [33].

NLRX1

NLRX1 localizes to mitochondria and is the only known

NLR protein that targets specifically a cellular organelle

[34,35]. Recent evidence demonstrated that NLRX1

translocates to the mitochondrial matrix via its N-terminal

addressing sequence and biochemical studies identified

that the protein associates with the mitochondrial inner

membrane, at least in part, through its interaction with

UQCRC2, a matrix-exposed core component of the com-

plex III of the respiratory chain [36]. It is possible that this

interaction contributes to the capacity of NLRX1 to

regulate mitochondrial ROS generation, as previously

demonstrated by over-expression studies. Finally, it has



Figure 2

Activation of the inflammasome-triggering NLR proteins at host membranes. Bacterial processing in phagosome or phagolysosome generates

molecules (including peptidoglycan and flagellin) that translocate to the cytosol and trigger the caspase-1 inflammasome through NLRP3 and NLRC4/

NAIP5, respectively. NLRC4 also detects the structure of the type three secretion system (TTSS) inserted into the host membrane. A large variety of

molecules can also traffic through the host endocytic machinery to trigger NLRP3 and NLRP1 (see text for details). In this case, the mechanism

underlying inflammasome activation is not fully elucidated but is thought to involve NADPH oxidase-generated reactive oxygen species (ROS) or the

damage to the lysosomal membrane and the release of cathepsin B (cat B). Finally, potassium efflux (that can be triggered by the binding of ATP to the

purinergic receptors of the P2X family) also triggers NLRP3 through a mechanism likely involving ROS generation.

been proposed that NLRX1 could interact with the

antiviral protein MAVS, on the cytosolic side of the

mitochondrial outer membrane [34]. The capture of

NLRX1 by MAVS on its way to the mitochondrial matrix

in certain conditions, such as viral infection, is an inter-

esting hypothesis that awaits experimental evidence.

Functional consequences of NLR signaling athost membranesNLRs as gatekeepers of endosomal or phagosomal

trafficking

The intimate relation between host membrane dynamics

and NLR activation has been recently illustrated by the

observation that Nod ligands are internalized by clathrin-

mediated endocytosis and seem to be exported to the

cytosol in a specific manner. In the case of the Nod2

ligand MDP, studies in macrophages suggest that the

molecule travels through the endocytic machinery up to

lysosomes, where MDP is exported to the cytosol and is


detected by NLR proteins [37,38]. It is likely that this site

of translocation to the cytosol is physiologically critical for

phagocytic cells; indeed, two recent reports demonstrate

that the progressive degradation and processing of bac-

teria or bacterial cell walls in the phagolysosome results in

the generation of peptidoglycan fragments that can trig-

ger either Nod2 [37] or NLRP3 [39] after cytosolic

translocation. However, the nature of the transporters

for MDP or other peptidoglycan motifs in the phagolyso-

some remains unknown. In HEK293T epithelial cells,

recent evidence demonstrated that Nod1 ligands are also

internalized by clathrin-dependent endocytosis and are

exported to the cytosol from early endosomes, in part

through the oligopeptide transporter SLC15A4 [40]. Stu-

dies in other human epithelial cells similarly identified

roles for other related transporters in mediating the trans-

port of Nod ligands. Indeed, SLC15A1 and SLC15A2

were shown to transport MDP (in Caco-2 cells) [41,42]

and iE-DAP (in upper airway epithelial cells) [43],


432 Host pathogens

respectively. However, in the latter cases, the precise

mechanism of internalization and location of cytosolic

export was not identified. Together, these recent findings

demonstrate that Nod-dependent activation is physio-

logically coupled with endocytic/phagocytic maturation

and bacterial cell wall degradation processes. It is con-

ceivable that the topological restriction of the cytosolic

entry site (plasma membrane, early endosomes, and pha-

golysosome) for NLR ligands might direct the nature of

the host response, such as NF-kB, caspase-1 inflamma-

some or type I interferon pathways. This concept was

indeed recently put forward in the case of TLR stimu-

lation, since the engagement of TLR4/MyD88/NF-kB

versus TLR4/TRAM/type I interferon pathways were

found to occur at the level of the plasma membrane

versus early endosomes, respectively [44].

Induction of bacterial autophagy by Nod proteins

Autophagy is a cellular process through which defective

organelles, protein aggregates or foreign material

(including microbes) are sequestered into double mem-

branes coated by proteins of the autophagic machinery, and

directed to lysosomes for destruction. Recently, the NLR

Figure 3

Nod proteins trigger the recruitment of the autophagic machinery at the

bacterial entry site. (A) Nod proteins detect invading bacteria likely

following local delivery of peptidoglycan fragments. This results in the

recruitment of ATG16L1, which in turn directs the coalescence of the

autophagic machinery at the site of bacterial entry. (B) Schematic

representation of the Nod1/2-dependent signaling cascade. While the

adaptor protein Rip2 is essential for Nod-dependent activation of NF-kB

as well as MAP kinase cascades, autophagy triggered by bacterial

invasion seems to operate in a Rip2-independent manner, likely through

the complex formed between Nod1/2 and ATG16L1.


proteins Nod1 and Nod2 have been shown to play a key

role in mediating bacterial autophagy [6]. Indeed, these

NLR proteins were found to interact with the autophagy-

nucleator protein ATG16L1, resulting in the recruitment

of the proteins of the autophagic machinery at the site of

bacterial entry (Figure 3). These results link Nod2 and

ATG16L1, two proteins whose genes have been associated

with CD susceptibility, therefore strongly suggesting that

improper targeting of bacteria by the autophagic machin-

ery plays a key role in CD pathogenesis. The fact that

bacteria could be targeted by Nod-dependent autophagy at

the plasma membrane before cell invasion suggests that

these NLR proteins likely detect minute amounts of

peptidoglycan delivered to the cytosol by the type III

secretion system. Moreover, the rupturing of the mem-

brane during bacterial invasion seems to provide a signaling

platform that potentially initializes both autophagy and

NLR activation [45]. This illustrates another important

role of Nod-dependent activation at host membranes. In

agreement with these observations, Nod2-dependent

induction of autophagy in human dendritic cells has been

shown to be critical for bacterial targeting to the lysosome

and for subsequent optimal antigen presentation on the

major histocompatibility (MHC) complex II [46].

Concluding remarksThe functional importance of NLRs as cytosolic sentinels

of the innate immune system at the vicinity of host

membranes is an emerging concept that is supported

by numerous experimental evidence, as presented in this

review. Linking NLR activation to host membrane

patrolling likely confers two main advantages: (i) it allows

safe and rapid response to microbes and danger, since

cytosolic threats necessarily need to cross at least one host

membrane, and membrane rupture, damage or electro-

chemical alteration are common events triggered by

bacteria, viruses, parasites or danger signals. (ii) It allows

NLR activation in a restricted area of the cell where

signals to be detected (ROS, K+ efflux, microbial motifs

translocated through a specific secretion system, material

released from the lysosome) display their highest con-

centration, thus ensuring the activation threshold is effi-

ciently met. This last point likely explains how Nod1 can

induce innate immune response to extracellular bacterial

pathogens [47,48], or direct the recruitment of the autop-

hagic machinery at the site of bacterial entry, when

bacteria are still extracellular [6]. In the case of Helico-bacter pylori, it was demonstrated that peptidoglycan is

translocated to the host cell cytosol via the type IV

secretion system, resulting in Nod1-dependent inflam-

matory signaling [48].

Finally, are there data to show NLR activation at sites

distant from host membranes? Paradoxically, for these

cytosolic sensors, direct evidence for this mode of acti-

vation is lacking. Indeed, it remains unknown if for

instance intracellular bacteria, moving freely in the host



cytosol (as is the case for Shigella or Listeria), can be

efficiently detected by NLRs, to trigger inflammatory

signaling or autophagy induction. Answering this crucial

question will require the development of reactive probes

to allow visualization and measurement of NLR acti-

vation in live cells.

AcknowledgementsWe apologize to those whose original work was not cited in this review,because of space limitations. Research in the laboratories of D.J.P. andS.E.G. is supported by funding from the Canadian Institutes of HealthResearch (to S.E.G. and D.J.P.), Crohn’s and Colitis Foundation of Canada(S.E.G.), Burrows Wellcome Fund (S.E.G.) and Howard Hughes MedicalInstitutes (D.J.P.).

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