Interactions between Nod-Like Receptors and Intestinal Bacteria

Abstract

Nucleotide oligomerization domain (Nod)-like Receptors (NLRs) are cytosolic sensors that mediate the activation of Caspase-1 and the subsequent processing and secretion of the pro-inflammatory cytokines IL-1β and IL-18, as well as an inflammatory cell death termed pyroptosis. While a multitude of bacteria have been shown to activate one or more NLRs under in vitro conditions, the exact impact of NLR activation during the course of colonization, both of pathogenic and commensal nature, is less understood. In this review, we will focus on the role of intestinal NLRs during the various stages of infection with common gastrointestinal bacterial pathogens, as well as NLR function in controlling and shaping the microbiota.

Full text

REVIEW ARTICLE
published: 17 December 2013
doi: 10.3389/fimmu.2013.00462
Interactions between Nod-like receptors and intestinal
bacteria
Marcel R. de Zoete1and Richard A. Flavell1,2*
1Department of Immunobiology,Yale University School of Medicine, New Haven, CT, USA
2Howard Hughes Medical Institute,Yale University, New Haven, CT, USA
Edited by:
Jorg Hermann Fritz, McGill University,
Canada
Reviewed by:
Axel Lorentz, University of
Hohenheim, Germany
Juan J. Garcia-Vallejo,VU University
Medical Center, Netherlands
*Correspondence:
Richard A. Flavell, Department of
Immunobiology,Yale University
School of Medicine, 300 Cedar
Street, TAC S-569, New Haven, CT
06520, USA
e-mail: richard.flavell@yale.edu
Nucleotide oligomerization domain (Nod)-like Receptors (NLRs) are cytosolic sensors that
mediate the activation of Caspase-1 and the subsequent processing and secretion of the
pro-inflammatory cytokines IL-1βand IL-18, as well as an inflammatory cell death termed
pyroptosis. While a multitude of bacteria have been shown to activate one or more NLRs
under in vitro conditions, the exact impact of NLR activation during the course of coloniza-
tion, both of pathogenic and commensal nature, is less understood. In this review, we will
focus on the role of intestinal NLRs during the various stages of infection with common
gastrointestinal bacterial pathogens, as well as NLR function in controlling and shaping the
microbiota.
Keywords: nod-like receptors, microbiota, inflammasome, intestine, pathogen
INTRODUCTION
The human body lives in symbiosis with trillions of microbial cells,
collectively called the microbiota, with the vast majority of these
microbes being bacteria that inhabit the gastrointestinal tract (1).
This symbiosis begins with colonization of the gastrointestinal
tract at birth and then is sustained throughout life by environ-
mental exposures (2). Occasionally this microbial symbiosis is
challenged by invading bacterial pathogens, which perturb the
microbial ecosystem and cause disease.
Our ability to harbor trillions of bacteria within our intestines
relies on the maintenance of a safe distance between these bacteria
and the single layer of intestinal epithelial cells. Crucial protective
mechanisms have evolved to help ensure host-bacteria mutualism.
A major barrier bacteria encounter in the intestine is formed by
the mucus layer, a dense network of glycoproteins that most bacte-
ria are unable to breach (3). To further aid the barrier function of
the mucus layer, intestinal cells also secrete an array of antimicro-
bial proteins, like antimicrobial peptides, lectins, and lysozymes.
Furthermore, secreted IgA specifically targets bacteria for immune
exclusion (4).
At the cellular level, sensing systems continuously scan for bac-
teria that are able to actively surpass the mucus layer and attach
to and/or invade the epithelium. Two major receptor families
that detect microbes are the Toll-like Receptors (TLRs), which
control the extracellular compartment, and Nod-like Receptors
(NLRs), which sense the presence of intracellular microbes (5).
NLRs are crucial for fighting and resolving infections as many
pathogenic bacteria (and under certain conditions also mem-
bers of the commensal microbiota) attempt to exploit and enter
the cytosol for nutrients and to escape extracellular threats (6).
Here, we provide an overview of the role of NLRs in protection
against intestinal pathogenic bacteria and control of the intestinal
microbiota.
INTESTINAL NLRs
Nod-like receptors generally consist of a ligand-sensing domain in
the form of a Leucine Rich Repeat (LRR) domain, a central ATP
binding domain, and a signaling domain (often in the form of
a CARD or Pyrin domain) and are categorized by their domain
structure. While NLRs are expressed widely in a variety of tissues
in humans and mice, we will focus in this review on those that were
shown to function in the defense against bacteria in the intestine.
While Absent In Melanoma 2 (AIM2) theoretically is not part of
the NLR family, we have included it here for completeness.
NOD1 AND NOD2 (NLRC1, NLRC2)
The pattern recognition receptors NOD1 and NOD2 are amongst
the best-studied NLRs, and their ligands are well defined. Both
NOD1 and NOD2 sense cytosolic bacterial peptidoglycan frag-
ments with high specificity: NOD1 is activated by d-glutamyl-
meso-diaminopimelic acid (DAP) containing peptidoglycan frag-
ments, which are mainly found in Gram-negative bacteria (7),
whereas NOD2 was shown to bind and responds to muramyl
dipeptide (MDP), found in all bacteria (8). Despite the pres-
ence of N-terminal CARD domains, NOD1 and NOD2 are non-
inflammasome forming NLRs and do not seem to directly activate
Caspase-1. Instead, after ligand binding the CARD domain of
NOD1 and NOD2 interacts with the signaling kinase RIP2 (RIPK2,
RICK) that initiates a signaling cascade resulting in NF-κB activa-
tion, as well as the activation of ERK, p38 and mitogen-activated
protein kinases (MAPKs) (9,10). These signaling pathways result
in the expression of a variety of pro-inflammatory cytokines
and chemokines, as well as the production of reactive oxygen
species. While NOD2 expression is more restricted, both NODs
are expressed in macrophages, dendritic cells, Paneth cells, and
intestinal epithelial cells, making them highly suited to sense infec-
tions throughout the intestinal tract (10). In recent years, several
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de Zoete and Flavell NLRs and intestinal bacteria
layers of complexity were added onto the basic mechanism of
NOD1 and NOD2 sensing and signaling. For instance, NOD2
was shown to interact with NLRP1, NLRP3, and NLRP12 (11),
NOD1 and NOD2 were found to play a role in autophagy (12),
and NOD1 senses the modification of small rho GTPases injected
by Salmonella during infection (13).
NLRC4
NLRC4 is an N-terminal CARD domain containing NLR. The elu-
cidation of the NLRC4 crystal structure has revealed that, under
resting conditions, NLRC4 resides in a closed monomeric form,
kept in place by an ADP-dependent autoinhibitory mechanism
involving multiple domains including the LRR (14). Ligand bind-
ing is proposed to induce“opening” of the structure, the exchange
of ADP for ATP, and subsequent NLRC4 oligomerization. Phos-
phorylation of a conserved serine residue proximal to the LRR
was shown to be required for NLRC4 inflammasome activation in
macrophages, although the exact role in this process requires fur-
ther investigation (15). NLRC4 responds to attaching or invading
pathogens by sensing their bacterial secretion systems. So far, two
bacterial ligands are well defined: flagellin, which is co-secreted
with virulence factors either through type III or type IV secre-
tion systems (T3SS and T4SS, respectively) (16–18), and PrgJ, a
structural component of the type III secretion system that leaks
or is secreted into the host cytosol (19). Within the cytosol, fla-
gellin and PrgJ bind to the adapter proteins NLR family, apoptosis
inhibitory protein (NAIP) 2 and NAIP5, respectively (20,21),
which subsequently bind NLRC4 to initiate its oligomerization
into a ring-like inflammasome that recruits the adapter protein
apoptosis-associated speck-like protein containing a CARD (ASC)
(containing both a Pyrin and CARD domain) and Caspase-1 (22).
This complex then processes the pro-inflammatory cytokines pro-
IL-1βand pro-IL-18, and induces pyroptosis, an inflammatory
form of cell death. Interestingly, unlike mice, humans have only
one NAIP protein,which is unresponsive to both flagellin or basal
rod protein but instead binds the conserved T3SS needle protein
to activate NLRC4 (20).
NLRP3
While NLRP3 is probably the best studied of the NLRs, the mech-
anism of receptor activation remains relatively unclear. NLRP3,
or cryopyrin, was originally shown to play a key role in a collec-
tion of autoinflammatory disorders collectively termed cryopyrin-
associated periodic syndromes, which all share mutations in
NLRP3 that lead to inappropriate IL-1β-mediated inflammatory
responses (23). NLRP3 was subsequently found to “sense” a long
list of ligands or stimuli, including ATP, pore-forming toxins,
particulates like asbestos and silica, bacteria, viral, and fungal
infections (24). Initially, three main theories of the activation of
NLRP3 were proposed: potassium efflux, lysosomal rupture and
subsequent cleavage by released Cathepsin, and ROS production.
Several “second generation” unifying NLRP3 ligands were pro-
posed to combine the three, including oxidized mitochondrial
DNA released into the cytosol following mitochondrial damage
(25); thioredoxin-interacting protein (26), calcium mobilization
(27), mitochondrial cardiolipin (28), and changes in cell volume
(29). While most of these NLRP3 ligands were recently shown to
lead to potassium efflux might, suggesting this to be the common
trigger in the end (30), NLRP3 activation remains enigmatic; struc-
tural studies similar to those done for NLRC4 might eventually
elucidate the elusive NLRP3 ligand.
A new chapter for NLRP3 has been opened through the elu-
cidation of the non-canonical inflammasome pathway. Due to
the (re)discovery of the presence of a mutated, non-functional
Caspase-11 in the original Caspase-1-deficient mouse, a role
for Caspase-11 was found in NLRP3-inflammasome activation
by Gram-negative bacteria (31). After prolonged (~17h) stim-
ulation of bone-marrow macrophages with bacteria, Caspase-
11 was shown to be activated, leading to cell death and
NLRP3/ASC/Caspase-1-dependent IL-1βand IL-18 secretion. It
was subsequently shown that the TLR4-TRIF-Type I Interferon
pathway was required to induce high levels of Caspase-11 tran-
scription needed for non-canonical inflammasome activation
(32). However, it recently was shown that intracellular LPS serves
as a ligand able to activate the non-canonical inflammasome path-
way, independently of increasing levels of Caspase-11 caused by
Type I Interferon (33). Three major questions regarding non-
canonical inflammasome activation remain currently unanswered:
what is the receptor that senses intracellular LPS or poten-
tially other ligand (presumably a CARD-containing NLR), how
does this complex feed into the NLRP3 inflammasome (Caspase-
11-dependent pyroptosis resulting in potassium efflux?), and is
Caspase-11 activated by any additional receptors?
NLRP6
NLRP6 falls within the group of NLRs that was initially found
to induce NF-κB and Caspase-1 activation during overexpres-
sion in transfected tissue culture cells (34). In this system, human
NLRP6 was also shown to form punctate structures in the cyto-
plasm, but only in the presence of ASC, suggesting the ability of
NLRP6 to form inflammasomes or inflammasome-like structures.
Unlike in humans,where NLRP6 is not highly or widely expressed,
mice exhibit high NLRP6 expression throughout the intestine,kid-
neys, and liver (35,36), which can be regulated by stress factors
(37). Mechanistically, NLRP6 was shown to be a negative regula-
tor of NF-κB and MAPK in cultured bone-marrow macrophages
from NLRP6-deficient mice (38), which is the opposite of what
was initially observed in overexpression studies. NLRP6 function
as a negative regulator of NF-κB and MAPK might play a role
in the increased intestinal tissue proliferation and inflammation
observed in NLRP6-deficient mice (39,40). Furthermore, NLRP6
was shown to be involved in the production of Caspase-1 depen-
dent IL-18 (36), again suggesting the ability of NLRP6 to form an
inflammasome.
NLRP12
Since its identification, NLRP12 has been assigned a num-
ber of different functions. Like NLRP6, NLRP12 was originally
described to induce NF-κB and Caspase-1 activation when co-
expressed with ASC (41). In contrast, without ASC co-expression,
NLRP12 overexpression reduced non-canonical NF-κB activa-
tion and enhanced the expression of non-classical and classical
MHC class I genes (42–44). NLRP12-deficient mice were also
reported to have defective dendritic cell and neutrophil responses
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de Zoete and Flavell NLRs and intestinal bacteria
to chemokines and subsequent defective dendritic cell migration
to draining lymph nodes (45). In addition, NLRP12-deficiency led
to enhanced colon inflammation and colorectal cancer develop-
ment due to increased (non-canonical) NF-κB and ERK activation
(46,47), similar to what was observed previously for NLRP6.
AIM2
AIM2 was originally identified in humans as an interferon-
inducible, putative tumor suppressor protein (48), but subse-
quently found to sense cytoplasmic double stranded (ds) DNA,
form an inflammasome complex together with ASC and Caspase-
1, and trigger the processing of pro-IL-1βand pro-IL-18 (49–52).
Like NLRP receptors, AIM2 contains an N-terminal signaling
Pyrin domain; however, the C-terminal consists of a DNA-binding
HIN200 domain. AIM2 is able to sense the presence of cytosolic
non-sequence-specific dsDNA of both viral and bacterial origin.
Bacterial DNA enters the cytosol mainly by a passive process
following bacterial lysis, for instance during rapid cytosolic bac-
terial replication or after intracellular bacteria-containing vesicles
are compromised, but appears to be always preceded by bacte-
rial invasion into the host cell, making AIM2 a specific sensor
for intracellular bacteria and viruses (53–55). In a mechanism
similar to what was observed for NLRC4, the HIN200 domain
functions as a negative regulator of the signaling Pyrin domain.
Non-sequence-specific binding of dsDNA releases this inhibition,
liberating the Pyrin domain to recruit ASC and Caspase-1, and
form an inflammasome surrounding the released bacterial or viral
DNA (56,57).
ENTERIC PATHOGENS AND THEIR INTERACTIONS WITH
NLRs
Foodborne gastrointestinal pathogens are a major cause of bacte-
rial infections in humans (58). Studies on these pathogens, both
in the human host and in various murine models, have provided
great insights into microbial virulence mechanisms as well as the
immunological defense strategies of the host. For NLRs, pathogen-
host interactions have been of great value for the elucidation of the
different functions of this family of innate sensors, both in in vitro
and in vivo model systems. Below (and summarized in Table 1),
we provide an overview of the role of NLRs during infections with
the most commonly studied bacterial enteric pathogens.
SALMONELLA
In humans, infections with the Gram-negative bacterium Salmo-
nella enterica (S.enterica) generally result in one of two distinct
clinical phenotypes. S. enterica serovars Typhi and Paratyphi are
Table 1 | Role of NLRs in intestinal bacterial infections.
Bacteria Model NLR Mechanism of action Reference
S. typhimurium Systemic NLRC4 Flagellin/T3SS-induced pyroptosis, IL-1β, and IL-18 production (79–81)
NLRP3 Caspase-1 activation (79)
Systemic,T3SS-1-independent NOD1 Nitric oxide production in dendritic cells (69)
Systemic and colitis in Balb/c NLRC4 IL-1β-mediated neutrophil recruitment (84)
Colitis NLRC4 IL-1βand IL-18 production (80)
NOD1 NOD1-mediated detection of SipA (70)
Colitis, T3SS-1-independent NOD1/2 Innate CD4+T helper type 17 cell responses in the cecum (74,75)
Systemic (intraperitoneal) NLRP6 NLRP6-mediated negative regulation of NF-κB and MAPK activation (38)
C. rodentium Colitis NOD2 NOD2-activation in stromal cells, CCL2/CCR2-dependent recruitment
of inflammatory monocytes, IL-12-mediated bacterial clearance
(92)
NOD1/2 IL-6-dependent IL-17 production in the cecum (75)
NLRC4 IL-1βand IL-18 production (94)
NLRP3 IL-1βand IL-18 production (94)
H. pylori Gastritis NOD1 T4SS-mediated delivery of peptidoglycan, NF-κB-mediated
inflammatory responses
(108)
Unknown IL-18-dependent IL-17 production, T-cell-mediated antibacterial
responses
(110,112)
Unknown IL-1β-dependent impaired bacterial clearance (111,113)
Microbiota Colitis (DSS) NLRP6 IL-18/CCL5 production, increased intestinal epithelial proliferation and
tissue repair
(36,40)
NLRP3 Both increased and decreased susceptibility; microbiota-dependent? (124,125,
127,128)
NOD1/2 Induction of E-cadherin and RegIII-γexpression (120)
Colorectal cancer (DSS-AOM) NLRP6 IL-18/CCL5/IL-6 mediated increased intestinal epithelial proliferation
and tissue repair
(39,40,116)
NLRP3 Caspase-1 activation (126)
Non-alcoholic fatty liver disease NLRP3/6 IL-18-mediated control of microbiota (117)
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de Zoete and Flavell NLRs and intestinal bacteria
the causative agents of Typhoid fever, a life-threatening disease
characterized by systemic spread of ingested bacteria, high fever,
and intestinal bleeding that results in 200,000–600,000 deaths
worldwide each year (59). The more common non-typhoidal S.
enterica serovars, like S.typhimurium and S.enteritidis, cause a self-
limiting gastroenteritis characterized by (bloody) diarrhea, fever,
abdominal cramps, and vomiting that usually lasts 4–7days and
affects over 90 million people worldwide each year (60).
Two different disease models in mice represent the two clini-
cal manifestations of Salmonella infections. The classical murine
model of systemic S. typhimurium infection induces a disease simi-
lar to Typhoid fever. In this model,orally administered salmonellae
reach the distal ileum within hours of ingestion and, aided by
flagella-mediated motility and chemotaxis (61), cross the mucus
layer toward the epithelium. Here, the bacteria target the follicular-
associated epithelium overlying the Peyer’s Patches, with a strong
preference for the M-cells, as a main port of entry. To gain access
into the host cell, Salmonella employs the first of two type III secre-
tion systems (T3SS-1), which is only expressed during this initial
phase of infection, and injects an array of effector proteins into the
host’s cytosol that induce cytoskeletal rearrangements leading to
bacterial invasion (62). Other effector proteins and the activation
of pattern recognition receptors initiate inflammatory responses
that attract neutrophils, monocytes, and macrophages. Salmo-
nella replicates within the Peyer’s Patches and disseminates to
mesenteric lymph nodes (MLN), liver, and spleen within infected
monocytes and dendritic cells. In a T3SS-1-independent manner,
a small proportion of the bacteria is also taken up passively from
the lumen by CD11c+CX3CR1+dendritic cells and transported
directly to the MLN, bypassing the Peyer’s Patches (63). To survive
and replicate in the host’s cells, Salmonella resides within a vac-
uole, the Salmonella-containing Vacuole (SCV), whose integrity
is maintained by the effector proteins secreted into the cytosol
through a second T3SS (T3SS-2). During the later stages of dis-
ease, disseminated bacteria in the liver and spleen are found mostly
within macrophages, in which they rapidly replicate. Continuous
cycles of bacterial replication and dissemination eventually lead to
bacteremia from which mice succumb after a week.
In the murine colitis model, infection with Salmonella is pre-
ceded by a single dose of streptomycin, which is believed to briefly
reduce “colonization resistance” provided by the microbiota that
occupies the more distal intestinal tract and strongly competes
for nutrients. In this short window, Salmonella is able to gain a
foothold in the cecum and colon, and replicates to high numbers
within hours of infection, limiting the need for systemic spread
(64). Colonization is accompanied by mucosal penetration of bac-
teria and the development of colitis, similar to the pathology seen
in human infection with non-typhoidal serovars. For efficient col-
onization salmonellae still require T3SSs, which mediate invasion
of enterocytes and induction of (local) inflammation. A reason
for this was presented in an elegant study by Winter et al. which
revealed that T3SS-induced inflammatory responses are actively
exploited by Salmonella through the ability to utilize tetrathionate,
formed in the intestine under inflammatory conditions, to success-
fully compete with the microbiota (65). In the colitis mouse model,
Salmonella also exploits a T3SS-1-independent,“passive” route for
uptake through CD11c+CX3CR1+dendritic cells. Similar to what
is seen in the systemic model, these two invasion pathways act in
concert, although T3SS-1-mediated invasion seems much more
dominant. While bacteria are able to grow extensively in the dis-
tal intestine in the colitis model, substantial numbers of bacteria
still continue to disseminate to liver and spleen,and infected mice
usually die after 5–6 days.
As several phases of infection largely rely on cellular invasion,
NLRs appear to be the ideal sensing mechanism for Salmonella.
In vitro,Salmonella is sensed by NOD2 in cultured intestinal
epithelial cells, which enables the control of intracellular bacteria
though the induction of antimicrobial responses and autophagy
(66–68), and by NOD1 in bone-marrow-derived dendritic cells,
resulting in nitric oxide production (69). Interestingly, while Sal-
monella peptidoglycan may be involved in the activation of NOD1
and NOD2, it was recently shown that the modification of small
Rho GTPases by the T3SS-1 effector SopE, which enables bacter-
ial invasion, is a danger signal sensed by NOD1 (13). Presumably
through a similar mechanism, another T3SS-1 effector SipA acti-
vated NOD1/NOD2-dependent NF-κB responses both in vitro
and in vivo (70). In addition to NOD1 and NOD2, Salmonella
is efficiently sensed by NLRC4, both via flagellin and the basal
rod protein PrgJ, which leads to rapid pyroptosis and secretion
of IL-1βand IL-18 by cultured macrophages, dendritic cells and
B-cells (16,17,19,71,72). These responses are solely dependent
on T3SS-1, which is expressed only at the early logarithmic phase
of bacterial cultures and is believed to represent the early phase in
infection when Salmonella needs to invade host cells. The T3SS-2,
expressed only at the late logarithmic phase, does not contribute to
NLRC4 activation as flagellin expression is now repressed and the
T3SS-2 apparatus is not recognized (19). Finally, Salmonella has
been shown to activate the non-canonical NLRP3 inflammasome
through Caspase-11 in macrophages (73).
In vivo, the role of NLRs during Salmonella infection has
been rather difficult to define, mainly because of differences in
experimental models (systemic versus colitis murine models),
variations in growth phase of Salmonella at time of infection
(T3SS-1-expressing versus T3SS-2-expressing conditions), differ-
ent mouse intestinal microbiotas, and because of the redundancy
in innate receptors. These issues are clearly demonstrated when
studying NOD1 and NOD2. Both of these NLRs were shown to
be dispensable during systemic infection (69). However, under
T3SS-1-independent conditions, during which the bacteria “pas-
sively” cross the epithelial barrier through uptake and trans-
port by dendritic cells, NOD1 deficiency led to higher bacter-
ial loads and mortality. The authors show that NOD1-deficient
CD11b+CD11c+dendritic cells contain higher numbers of Salmo-
nella, likely because of a diminished NOD1-mediated nitric oxide
response. Similarly, during Salmonella colitis, NOD1/NOD2 dou-
ble knockouts or RIP2 deficient mice exhibited reduced inflamma-
tion accompanied by increased mucosal colonization and reduced
early IL-17 responses of innate CD4+T helper type 17 cells in
the lamina propria of the cecum, but again only when Salmonella
was grown under T3SS-2-expressing conditions (74,75); when
T3SS-1 was expressed at the time of oral infection in this model,
no differences as compared to wild-type mice were observed (76).
These data suggest that only one of the two major entry pathways
exploited by Salmonella is controlled by NOD1/NOD2 signaling.
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de Zoete and Flavell NLRs and intestinal bacteria
During “normal” infections, however, the T3SS-1-mediated inva-
sion seems to outweigh the alternative invasion route, leaving
NOD1 and NOD2 to play a non-significant role. Interestingly,
NOD1/NOD2-activation by the T3SS-1 effector SipA was shown
to lead to a higher gut inflammation score in the colitis model
as compared to mice lacking the receptors (70). Combined, the
above-mentioned studies suggest that NOD1 and NOD2 medi-
ated detection of Salmonella plays a specific but minor role during
salmonellosis. As a further complication, differences in micro-
biota from wild-type and knockout mice dramatically impact
Salmonella susceptibility, as demonstrated by Kaiser et al.; when
”microbiota-matched” littermate controls were used (instead of
independently bred or purchased wild-type mice) to test the role
of RIP2 during Salmonella colitis, the initially observed difference
in Salmonella-induced pathology was completely lost (64).
Caspase-1 has been shown in several publications to pro-
vide moderate protection against Salmonella infection. Without
Caspase-1, mice succumb to bacteremia sooner and have higher
bacterial loads in the MLN, liver and spleen in the typhoid model
of infection, and more colitis accompanied by increased bacte-
rial mucosal infiltration (77,78). While NLRC4 appeared to be
the main upstream candidate for caspase-1 activation, NLRC4-
deficient mice showed only minor or no defects in bacterial control
during infections (77,79,80). Two critical findings explained this
“lack” of NLRC4 function in vivo; first, Salmonella actively evades
recognition by NLRC4 by downregulating both T3SS-1 and fla-
gellin as soon as the bacteria have invaded the host cell,and T3SS-2
is not recognized by NLRC4 (19,81). The in vivo consequence
of NLRC4-evasion, and thereby the role of NLRC4 in protection
against invading pathogens, was elegantly shown by Miao et al.:
when Salmonella was forced to continuously express flagellin,100
times less bacteria were found in the spleen after 48 h during sys-
temic infection. The increased control of bacterial spreading was
attributed to NLRC4-mediated macrophage pyroptosis at periph-
eral sites, which resulted in release of the intracellular bacteria and
subsequent clearance by infiltrating neutrophils (81).
NLR redundancy is a second reason why NLRC4-deficient
mice do not phenocopy Caspase-1-deficient mice. Late logarith-
mic, non-T3SS-1 expressing salmonellae are able to activate the
non-canonical Caspase-11/NLRP3 inflammasome (73). Similar
to NLRC4, deficiency in only NLRP3 does not lead to differences
in Salmonella infection. However, deletion of both NLRC4 and
NLRP3 recapitulates the Caspase-1 phenotype completely, con-
firming a role for both NLRC4 and NLRP3 during Salmonella
infection (79). This also demonstrates that, as was predicted by
in vitro studies, NLRC4-evasion is not perfect. While pyroptosis
has a clear impact on infection, the cytokines IL-1βand IL-18
appear to play minor roles in the control of the bacteria, since
IL-1βand IL-18-deficent mice show little delay in bacteremia at
72 h (81,82).
With the realization that the Caspase-1 KO was in fact a
Caspase-1/Caspase-11 double knockout, and the elucidation of
the role of Caspase-11 in non-canonical inflammasome activa-
tion, Caspase-11-deficient mice were predicted to result in more
bacterial spread due to diminished control of infection. However,
Caspase-11-deficient mice were indistinguishable from WT mice
during Salmonella infection (79). Surprisingly, Caspase-1 single
deficient mice had even higher numbers of bacteria in liver and
spleen than the Caspase-1/Caspase-11-deficient mice, suggesting
a protective role for Caspase-11 deficiency, but only in the con-
text of Caspase-1-deficiency. A potential explanation for this may
be that, while rapid Caspase-1-mediated pyroptosis clears bacte-
ria, Caspase-11-mediated pyroptosis at later time points is actively
used by Salmonella to escape the “full”macrophage after extensive
replication. Indeed, Caspase-11 senses bacteria escaping from or
leaking out of vacuoles into the cytoplasm (83). In the absence of
Caspase-1, NLRC4 “evasion” by Salmonella is complete, resulting
in uncontrolled replication until Caspase-11 is utilized to break out
of the macrophage and invade new host cells. Why non-canonical
Caspase-11-mediated pyroptosis, like NLRC4-activation accom-
panied by IL-1βand IL-18 secretion that induces local inflam-
mation and attracts neutrophils, is less potent than Caspase-1-
mediated pyroptosis in controlling Salmonella infection remains
thus far unclear.
Unlike in C57BL/6 mice,in Balb/c mice NLRC4 appears to have
a more prominent function in controlling Salmonella infection.
In these mice, NLRC4-deficiency leads to more systemic bacter-
ial dissemination and mortality, while less inflammation-induced
pathology in the cecum was observed (84). It was subsequently
shown that Salmonella specifically activates intestinal phagocytes
that respond by producing IL-1βwhich triggered the upregulation
of endothelial adhesion molecules. The basis of the interesting dif-
ferential function of NLRC4 between C57BL/6 and Balb/c remains
to be determined.
ATTACHING AND EFFACING ENTERIC PATHOGENS: CITROBACTER,
EPEC AND EHEC
Citrobacter rodentium (C.rodentium), EnteropathogenicEscherichia
coli (EPEC), and Enterohemorrhagic Escherichia coli (EHEC) are
Gram-negative extracellular enteric pathogens that share a sim-
ilar virulence strategy, termed attaching and effacing (A/E) (85,
86). EPEC and EHEC are human pathogens; EPEC is a major
cause of diarrhea in young children, generally without major com-
plications, while EHEC infections can vary greatly in severity,
ranging from mild gastroenteritis to severe hemorrhagic colitis
and hemolytic uremic syndrome. C.rodentium is a natural mouse
pathogen resulting in self-limiting enteritis. While very little is
known about activation of NLRs by EPEC and EHEC in humans,
several studies have elucidated the role of such responses during
infection of their murine counterpart, C.rodentium.
Within a couple of hours after oral infection of mice, C.roden-
tium reaches its initial site of infection, the lymphoid tissue in the
cecum termed the cecal patch, where it reaches high density over
the following 3 days (87). The cecal patch is structurally similar
to the Peyer’s Patch and, because of their nature as antigen sam-
pling hotspots with decreased mucus layer thickness and absence
of microvilli, provide “easy access/entrance” for several intestinal
pathogens (including Salmonella, as described above). From day
3 to 4, C. rodentium starts to spread throughout the distal colon
(87). Mouse-adapted strains of C.rodentium largely skip the cecal
patch phase and colonize the colon readily, suggesting that colo-
nization of the cecal patch also serves as an adaptation phase to the
mouse intestinal environment (88). Depending on the strain of C.
rodentium used, bacterial numbers peak between day 5 and 14 with
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de Zoete and Flavell NLRs and intestinal bacteria
limited systemic spread to the MLN, liver, and spleen. The colo-
nization then slowly diminishes until bacterial clearance from the
cecum and subsequently the colon after 3–4weeks post infection.
Upon reaching the cell surface of the cecum and colon, C.
rodentium employs a T3SS which injects an array of virulence
factors into the host’s cytosol that result in the attachment of the
bacteria to the enterocytes and the accompanying local destruc-
tion of the brush border microvilli of the epithelium forming
pedestal-like structures termed A/E lesions (86). Two of these vir-
ulence proteins are central for this virulence strategy: the adhesin
Intimin expressed on the bacterial surface and the T3SS-injected
Translocated Intimin Receptor (TIR), which provides a docking
ligand for Intimin on the host epithelial surface (89,90). The
attachment of C. rodentium, in combination with the secretion of
many additional virulence proteins, leads to colonic hyperplasia,
observed readily during the peak of infection as larger intestinal
crypt length and increased colon weight.
Several reports have shown that NOD1 and NOD2 are able
to sense C. rodentium both in vitro and in vivo (75,91,92). In
the absence of NOD2, C. rodentium reaches a higher intestinal
abundance as compared to wild-type mice. At the early stages of
infection, NOD2 signaling was shown to activate the CCL2/CCR2
axis that resulted in the recruitment of inflammatory monocytes
to the site of infection, which initiated IL-12-mediated bacterial
clearance. Interestingly, NOD2-activation took place in intestinal
stromal cells and not immune cells. NOD2-deficiency led to lower
inflammation at the early stages of infection, but more severe coli-
tis later,as a result of reduced clearance and higher bacterial abun-
dance in the intestine (92). In a different study, NOD1 and NOD2
had redundant roles in the protection against C. rodentium infec-
tion and mediated IL-6-dependent IL-17 production in the cecum
at early time point (1–4 days after infection). The observed effect
on infection was similar; lower initial inflammatory responses but
increased levels of bacterial dissemination to the spleen in the sec-
ond week of infection (75). NOD1/NOD2 signaling was shown to
occur mostly in the radio-resistant compartment, but a role for
stromal cells was not further investigated. Although it is expected
that peptidoglycan is the major C. rodentium-derived ligand of
NOD1 and NOD2, T3SS-injected effector proteins may play a role
too, as was shown previously for the Salmonella effector protein
SopE (13). Indeed EspT, which targets small GTPases to induce
membrane rearrangement in a similar way as SopE, was shown
to induce NF-κB, ERK1/2 and JNK activation, common signaling
pathways activated after NOD1/2 signaling (93). Future studies
will determine to what extent effector-mediated NLR activation
contributes to colonization and bacterial clearance.
Caspase-1/Caspase-11-deficient mice were found to be hyper-
susceptible for C. rodentium infection, as determined by increased
intestinal bacterial loads, colitis,and hyp erplasia (94). Both NLRP3
and NLRC4-deficient mice,as well as mice lacking IL-1βand IL-18,
showed similar phenotypes, suggesting an important role for the
NLRP3/NLRC4/IL-1β/IL-18 axis in the control of C. rodentium. In
a different study, a similar but stronger phenotype was observed in
IL-1R-deficient mice, which mostly succumb to infection within
2 weeks (95). In contrast to what was seen in IL-18-deficient mice,
neutralizing this cytokine with antibodies had limited to no effect,
implicating IL-1βor IL-1αas the critical cytokines that mediated
protection against C. rodentium. IL-1R signaling during C. roden-
tium infection led to IFN-γand IL-6 production in the colon,
which mediated epithelial repair and maintained barrier function.
While bacterial loads remained the same, more bacteria dissemi-
nated to the liver in the absence of these cytokines. Like Salmonella
and most other Gram-negative bacteria, C. rodentium is able to
activate the non-canonical Caspase-11/NLRP3 inflammasome in
cultured bone-marrow macrophages, which occurred in a T3SS-
independent (31,94). The activation of the Caspase-11/NLRP3
non-canonical inflammasome during infection was evident when
examining Caspase-11- and TRIF-deficient mice, which were both
more susceptible for C. rodentium infection (96). Interestingly,
while NLRC4 seems to be activated by C. rodentium in vivo, bone-
marrow macrophages did not sense the T3SS of C. rodentium
during in vitro studies. Whether this is due to tightly regulated
T3SS expression or host cell tropism/specificity remains to be
determined.
HELICOBACTER PYLORI
The Gram-negative bacterium Helicobacter pylori (H.pylori ) col-
onizes the gastric mucosa of ~50% of the world’s population,
although substantial variation exists between countries (97). The
majority of people infected by H.pylori do not show any symp-
toms, despite local chronic inflammatory responses induced by
the bacterium. However, in a subset of patients, this inflammatory
response drives the formation of gastric or duodenal ulcers that
can lead to the development of mucosa-associated lymphoid tissue
lymphomas and gastric adenocarcinomas (98–100).
In order to survive in the challenging gastric niche and enable
persistent colonization, H.pylori is highly optimized to evade
host antimicrobial strategies. For instance, after ingestion H.pylori
secretes urease, which increases the gastric pH and reduces mucus
viscosity, enabling rapid penetration of the gastric mucus layer and
colonization in close proximity to the pH neutral epithelial cells
(99,101). Also, H. pylori expresses a modified LPS and flagellin to
evade the recognition of TLR4 and TLR5, and has adopted several
mechanisms to counteract the effects of host-produced reactive
oxygen species (102,103). Finally, the secreted pore-forming toxin
VacA induces epithelial cell apoptosis and inhibits T-cell activation
and proliferation (104). In contrast to immune evasion, a subset
of H. pylori strains also actively induces inflammatory responses
by means of the T4SS-mediated delivery of the effector protein
CagA. CagA modifies multiple intracellular signaling pathways of
host cells and is linked to the development of gastric cancer.
While immune evasion appears to be an important part of
the H. pylori life cycle, genetic association studies revealed that
mutations in NOD1, NOD2, and IL-1βmay be associated with
increased risk for the development of gastric cancer, suggesting
that NLRs play a role in controlling H. pylori during human infec-
tion (105–107). In addition, several NLR family members have
been shown to sense the bacterium and impact on infection or
colonization, both in in vitro cell culture and in vivo murine mod-
els. In a manner analogous to the “leakage” of flagellin through
the T3SS in Salmonella, peptidoglycan fragments were found to
enter the host cytosol through the T4SS, where they were subse-
quently sensed by NOD1 and initiated NF-κB-mediated inflam-
matory responses (108). NOD1 activation was also observed by
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de Zoete and Flavell NLRs and intestinal bacteria
peptidoglycan present in secreted bacterial outer membrane vesi-
cles that were taken up by host cells (109). H. pylori was shown to
induce the secretion of IL-1βand IL-18 both in vitro and in vivo
(110,111). As compared to wild-type mice, Caspase-1/Caspase-
11-deficient mice showed decreased numbers of Helicobacter in
the stomach, higher expression of IL-17, and aggravated gastric
immunopathology, which was phenocopied by IL-18 and IL-18R,
but not IL-1R deficient mice. Loss of IL-18 signaling in den-
dritic cells was subsequently shown to result in reduced levels
of regulatory T-cells and stronger T-cell-mediated antibacterial
responses (110,112). In contrast, different groups reported that
Caspase-1/Caspase-11, ASC, IL-1β, and IL-1R-deficient mice were
impaired in the clearance of H. pylori from the stomach, dis-
played decreased gastritis and lower levels of IL-1βand IL-18
(111,113). While the cause of the discrepancies between these
different reports is currently unknown, it appears that H. pylori
strives for the ideal level of inflammasome activation: enough IL-
18 and IL-1βto induce regulatory T-cells and decrease gastric acid
production, respectively (114), but not so much IL-1βas to lead
to T-cell mediated clearance. The nature of the inflammasome
NLR that is activated by Helicobacter remains unclear. While in
cultured dendritic cells NLRP3 was crucial for IL-1βsecretion in a
T4SS-dependent/CagA-VacA-independent manner, this NLR did
not play a role during murine infection.
THE INTESTINAL MICROBIOTA AND NLR-MEDIATED
DISORDERS
The intestinal microbiota is predicted to consist of ~100 different
bacterial species per person, and displays great variability between
individuals (115). Alterations in the composition of the microbiota
have been shown to dramatically impact disease susceptibility and
progression. Therefore, controlling and (re)shaping the “healthy”
microbiota is a crucial function of the intestinal immune system.
The role of NLRs in this process is only beginning to be unraveled.
Lack of appropriate immunological control may switch a
healthy microbiota into a pathogenic one, as exemplified by mice
lacking NLRP6. NLRP6-deficient mice show increased levels of
intestinal inflammation during DSS-induced colitis and develop
more severe colorectal cancer in a model of colitis-dependent
tumorigenesis (36,39,40,116). A potential mechanism was pro-
vided by the finding that NLRP6 acts as a negative regulator of
NF-κB and MAPK activation, and reduces the levels of cytokines
and chemokines during infections with intestinal pathogens or
epithelial barrier breach as observed during experimental mod-
els of colitis (38). More severe and prolonged inflammation in
NLRP6-deficient mice results in increased levels of intestinal
epithelial proliferation and increased tissue repair, which was
shown to be CCL5 and IL-6 dependent (36,39,40,116). The
actions of NLRP6 do not seem limited to infectious or damag-
ing episodes, as NLRP6-deficient mice already display continuous
low level inflammation in the steady state, suggesting an interac-
tion with the microbiota (36). 16S rRNA sequencing analysis of the
microbiota revealed that NLRP6-deficient mice harbor a dysbiotic,
colitogenic microbiota that showed a high relative abundance of
Prevotellaceae species, that was transmissible to wild-type control
mice. Similarly,lack of NLRP6-mediated control of the microbiota
induced non-alcoholic fatty liver disease and obesity in mice
and increased colorectal cancer, all of which were transmissible
through microbiota transfer to wild-type mice (116,117).
NLRP12 and NLRP6 may play similar roles in the control
of intestinal homeostasis. Like NLRP6, NLRP12-deficiency leads
to uncontrolled NF-κB signaling and subsequent inflammation
and intestinal cell proliferation. Although extensive analysis of
the composition of the intestinal microbiota of NLRP12-deficient
mice has not been reported, the lack of this NLR might have major
effects on the microbiota, either directly through sensing microbial
products, or indirectly through the induction of an inflammatory
environment via NF-κB dysregulation.
While systemic peptidoglycan from the intestinal microbiota
was shown to boost the development of the intestinal immune sys-
tem and prime immune responses via NOD1 in the bone-marrow
in mice (115,118), NOD1, like NOD2, does not dramatically
influence the composition of the microbiota under homeosta-
tic conditions (119). However, during DSS-induced colitis, the
murine model of inflammatory bowel disease (IBD) that is driven
by the microbiota, NOD1/NOD2-deficiency led to greater suscep-
tibility to colitis (120). Similarly, mutations in NOD2 and NOD1
in humans are associated with susceptibility to Crohn’s disease
and IBD, respectively (121–123). The role of NLRP3 in control-
ling the microbiota has been rather controversial. Initially, NLRP3
was reported to have a key role in protecting intestinal home-
ostasis, as NLRP3-deficient mice were shown to have an altered
microbiota and displayed increased susceptibility to DSS-colitis
(124,125) and tumorigenesis (126). However, NLRP3-deficiency
led to resistance to DSS-colitis in a different study (127). As the
DSS-colitis model is highly dependent on the microbiota, differ-
ential compositions of the microbiota may explain the varying
outcomes in these studies. Indeed, co-housing NLRP3-deficient
mice with wild-type mice, which equalized the intestinal micro-
biota, also equalized the inflammatory responses and disease in
both mice (128). In humans, the role of NLRP3 in Crohn’s is
equally confusing; polymorphisms associated with NLRP3 were
shown to contribute to susceptibility to Crohn’s disease (129,130),
but did not replicate in a separate study (131). More detailed
investigation of the interactions between specific members of the
microbiota and NLRs may provide deeper insights in the func-
tion of NLRs in controlling and shaping the microbiota in health
and disease.
CONCLUDING REMARKS
Nod-like receptors are crucial components of the intestinal innate
immune system, controlling both the commensal microbiota as
well as enteropathogenic bacterial infections. While a growing
body of scientific evidence now provides clear insight into the
role of NLRs in controlling intestinal bacteria, several conflicting
reports highlight the importance of precisely controlling experi-
mental conditions like bacterial growth phase and the intestinal
microbiota between wild-type and NLR-deficient mice. Several
key questions still remain unanswered, suck as the nature of the
ligands for NLRP6 and NLRP12, the interplay between NLRs and
adaptive immunity in the intestine, the potential role for other
NLRs like NLRP7 (which senses bacterial lipopeptides in human
cells), NLRP10 (which controls adaptive immune responses), and
NLRC3 (which down-regulates NF-κB), and the role of NLRs in
www.frontiersin.org December 2013 | Volume 4 | Article 462 | 7

de Zoete and Flavell NLRs and intestinal bacteria
human diseases. Future research will undoubtedly shed more light
on these interesting new subjects.
ACKNOWLEDGMENTS
We wish to thank members of the Flavell lab for critically reading
the manuscript. This work was supported by the Howard Hughes
Medical Institute (Richard A. Flavell), a Department of Defense
Grant (W81XWH-11-1-0745) (Richard A. Flavell) and a Rubi-
con Fellowship from the Netherlands Organization of Scientific
Research (NWO) (Marcel R. de Zoete).
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Conflict of Interest Statement: The authors declare that the researchwas conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 01 October 2013; accepted: 03 December 2013; published online: 17 December
2013.
Citation: de Zoete MR and Flavell RA (2013) Interactions between Nod-like receptors
and intestinal bacteria. Front. Immunol. 4:462. doi: 10.3389/fimmu.2013.00462
This article was submitted to Molecular Innate Immunity, a section of the journal
Frontiers in Immunology.
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