The inflammasome in pathogen recognition and inflammation
Fayyaz S. Sutterwala,*,† Yasunori Ogura,† and Richard A. Flavell†,‡,1
Sections of *Infectious Diseases and †Immunobiology and the ‡Howard Hughes Medical Institute, Yale University
School of Medicine, New Haven, Connecticut, USA
Abstract: The nucleotide-binding oligomerization
domain-like receptor (NLR) family of proteins is
involved in the regulation of innate immune re-
sponses and cell death pathways. Some NLR family
members promote the activation of proinflam-
matory caspases within multiprotein complexes,
called inflammasomes. Recent studies analyzing
mice deficient in various components of the inflam-
masome have provided insight into the role of these
molecules in host defense against pathogens and in
autoinflammatory disorders. Here, we review these
studies and propose that membrane disruption
leads to activation of the inflammasome. J. Leukoc.
Biol. 82: 259 –264; 2007.
Key Words: innate immunity 䡠 NLR 䡠 NALP3 䡠 IPAF 䡠 caspase-1
INTRODUCTION
The mammalian nucleotide-binding oligomerization domain
(NOD)-like receptor (NLR) family of intracellular proteins is
characterized by a unique nucleotide-binding domain called
NACHT—found in neuronal apoptosis inhibitory protein
(NAIP), Class II transactivator (CIITA), incompatibility locus
protein from Podospora anserina (HET-E), and telomerase-
associated protein (TP-1)—which is located at the center of the
molecule between an N-terminal protein-binding caspase-re-
cruitment domain (CARD), pyrin domain (PYD), or baculovirus
inhibitor of apoptosis repeat (BIR) and a C-terminal leucine-
rich repeat (LRR) domain [1– 4] (Fig. 1). The NLR family
members Nod3, Nod4, and Nod5 contain an as-of-yet unde-
fined domain at their N terminus. The structure of NLR pro-
teins resembles that of Apaf-1 of vertebrates and insects,
Ced-4 of C. elegans, and the nucleotide-binding site (NBS)-
LRR-type disease-resistance proteins (R proteins) of plants, all
of which contain another type of nucleotide-binding domain,
NB-ARC or apoptotic ATPase, which is also centrally located
between an N-terminal protein-binding domain (CARD, TIR,
or CC domain) and a C-terminal WD40 or LRR domain [5].
Our understanding of the mechanism of NLR molecules is
based on evolutionary similarities with other innate immune
receptors, which contain a LRR motif (i.e., TLRs and plant
NBS-LRR-type R proteins); NLR molecules are postulated to
function as intracellular pathogen recognition molecules. The
NACHT domain regulates activity of the molecule in a way that
is dependent on ligand binding to the LRR motif, and the
N-terminal protein-binding domain then transmits a signal to
specific downstream effectors.
0741-5400/07/0082-259 © Society for Leukocyte Biology
The plant NBS-LRR-type R proteins have evolved diver-
gently to protect the host from various pathogens, and some
NBS-LRR-type R proteins have been demonstrated to bind
pathogen-derived molecules [6, 7]. However, it remains un-
clear whether the NBS-LRR-type R proteins are direct patho-
gen recognition receptors or sensors of cellular distress. For
example, a NBS-LRR protein of Arabidopsis, RPS5, recognizes
a fragment of the host protein PBS1, which is generated by the
protease activity of the Pseudomonas syringae protein AvrPphB
following its injection into host cells by the bacteria [8]. This
finding implies that this NBS-LRR protein recognizes a con-
sequence of the activity of pathogens rather than the presence
of the pathogen itself.
THE INFLAMMASOME: A MOLECULAR
PLATFORM TO ACTIVATE CASPASE-1
Although caspases are known to play an essential role in
apoptotic cell death, a member of the caspase family,
caspase-1, regulates the secretion of the proinflammatory cy-
tokines IL-1␤ and IL-18 by cleaving the corresponding, im-
mature proforms of these cytokines, pro-IL-1␤ and pro-IL-18.
IL-33, a cytokine involved in generation of TH2 responses, is
also processed by caspase-1 [9]. Besides caspase-1, four more
caspases, which are phylogenetically related to caspase-1, are
also implicated in the regulation of inflammation and therefore,
called inflammatory caspases. These are caspase-1, caspase-4,
and caspase-5 in humans and caspase-1, caspase-11, and
caspase-12 in the mouse [10]. Although human and mouse
caspase-1 are orthologs, human caspase-4 and caspase-5 are
likely to have arisen from gene duplication of caspase-11 [10].
In contrast to murine caspase-12, human caspase-12 appears
to be inactive [11].
Based on analogy to the induced-proximity model for the
activation of the apoptotic caspases 8 and 9 by CD95/Fas-
death-inducing signaling complex (DISC) and the Apaf-1-ap-
optosome, respectively, a putative, molecular platform to acti-
vate the inflammatory caspases has been termed the “inflam-
masome” [12–14]. Tschopp and colleagues [12, 13] proposed
an inflammasome model based on biochemical analysis of three
1
Correspondence: Section of Immunobiology, Yale University School of
Medicine, 300 Cedar Street, TAC S-569, New Haven, CT 06520, USA. E-mail:
richard.flavell@yale.edu
Received December 22, 2006; revised April 3, 2007; accepted April 5,
2007.
doi: 10.1189/jlb.1206755
Journal of Leukocyte Biology Volume 82, August 2007 259

Fig. 1. Domain structure of the NLR protein family. NAD, NACHT-associ-
ated domain; FIIND, domain with function to find; IPAF, IL-1␤-converting
enzyme protease-activating factor; AD, activation domain; APAF1, apoptosis
protease activating factor-1; NB-ARC, nucleotide-binding-adaptor shared by
APAF-1, R proteins, and Caenorhabditis elegans cell death gene 4 (CED-4)
domain; WD-40, Trp-Asp 40; A. thaliana RPP5 and RPS2, Arabidopsis thali-
ana genes RPP5 and RPS2, respectively; TIR, Toll/IL-1 receptor homologous
region; CC, coiled-coil; Cys, cysteine-rich domain; TM, transmembrane do-
main; ASC, apoptosis-associated speck-like protein containing a CARD do-
main.
NLR proteins, NALP1, NALP2, and NALP3. NALP3 (also
known as cryopyrin, CIAS-1, Pypaf-1, or CLR1.1), which has a
PYD at its N terminus, binds to two kinds of adaptor proteins,
ASC and a CARD-containing protein (Cardinal); both of these
adaptors bind to caspase-1 through CARD-CARD interactions
so that a complex composed of a single NALP3, ASC, and
Cardinal molecule can attract two caspase-1 molecules to this
caspase-1 activation platform, the NALP3 inflammasome.
There is no Cardinal homologue in the mouse, and hence,
murine NALP3 is thought to recruit only a single caspase-1
molecule (Fig. 2). NALP1 and NALP2 also have been shown
to form similar caspase-1-activating platforms. It remains un-
clear whether the NALP3 inflammasome is stably present in
the cytoplasm or if it is assembled by upstream signals as in the
apoptosome or DISC.
Another NLR protein, IPAF [also known as CARD12 or
caspase-associated recruitment domain, LRR, and NAIP-,
CIIA-, HET-E-, and TP1-containing protein (CLAN)], can bind
to and activate caspase-1 through its N-terminal CARD [15]
and may form a homo-oligomeric complex—the IPAF inflam-
masome. Three other PYD-containing NLR proteins, NALP6
(Pypaf-5), NALP12 (Pypaf-7 or Monarch-1), and NALP10
(Pynod), and another PYD-containing protein, Pyrin (MEFV),
have also been reported to modulate caspase-1 activity through
an ASC-mediated pathway [16 –19]. Depending on the activat-
ing stimulus, the composition of the inflammasome can be
quite varied, and multiple, different types of inflammasome
may exist.
NALP3 AND DANGER SIGNALS
Macrophages secrete IL-1␤ in response to serial stimulation
with LPS followed by ATP. In this experimental system, LPS or
ATP alone are not sufficient to induce IL-1␤ secretion, and
LPS is required to induce pro-IL-1␤ and to prime cells for
caspase-1 activation in response to ATP [20]. The stimulation
of cells by high concentrations of ATP is thought to mimic the
rapid release of ATP from activated platelets, neurons, antigen-
stimulated T cells, and injured cells. The effect of ATP is
mediated by the P2X7 receptor, which causes a rapid K⫹ efflux

Fig. 2. Activation of the NALP3 inflamma-

some by microbes, toxins, and danger signals.
The pathogens Listeria monocytogenes and
Staphylococcus aureus, microbial toxins (mai-
totoxin, aerolysin, and nigericin), and danger
signals (ATP and uric acid) have all been
implicated in the activation of the NALP3
inflammasome. A number of these stimuli me-
diate a potassium efflux; however, it is still
unclear if NALP3 inflammasome activation by
uric acid and S. aureus is dependent on po-
tassium. These stimuli likely lead to a confor-
mational change in NALP3 by an unknown
mechanism, which allows NALP3 to oligomer-
ize. Following oligomerization, NALP3 re-
cruits ASC through a homophilic PYD-PYD
interaction. ASC, in turn, recruits pro-
caspase-1 via homophilic CARD-CARD in-
teractions, which lead to activation of
caspase-1, and active caspase-1 can then pro-
cess pro-IL-1␤ and pro-IL-18 and induce
macrophage cell death. LLO, Listeriolysin-O;
P2X7, an ionotropic ATP receptor.

260 Journal of Leukocyte Biology Volume 82, August 2007 http://www.jleukbio.org

from the cytosol upon activation [21, 22]. As potassium iono-
phores, membrane-permeablizing agents, pore-forming agents,
and K⫹ depletion from cell culture media are also known to
induce IL-1␤ secretion, the efflux of cytosolic potassium is
thought to be an essential trigger of caspase-1 activation [23–
27]. Recent studies have identified pannexin-1, a membrane
protein that can act as a nonselective pore, as being required
for caspase-1 activation in response to ATP, nigericin, and
maitotoxin. Pannexin-1 is thought to act downstream of the K⫹
efflux and may play an important role in the activation of the
NALP3 inflammasome [28, 29].
Studies using macrophages from NALP3-deficient mice have
shown that NALP3 plays a central role in caspase-1 activation
in response to TLR stimulation combined with potassium efflux
(Fig. 2). NALP3-deficient macrophages stimulated with TLR
agonists plus ATP, nigericin, or maitotoxin failed to secrete
IL-1␤ and IL-18 [30 –32]. This defect in IL-1␤ and IL-18
secretion was found to occur at the level of caspase-1 activa-
tion, as stimulation of NALP3-deficient macrophages with LPS
plus ATP did not result in the autocatalytic cleavage of pro-
caspase-1 into its p20 and p10 subunits. In vivo, mice chal-
lenged with LPS showed increased resistance to endotoxic
shock in the absence of ASC or NALP3 [30, 31]. ASC-deficient
mice, however, tolerated higher doses of LPS compared with
NALP3-deficient mice, suggesting that ASC, being a common
downstream adaptor for multiple NLR molecules, may play
additional roles in endotoxic shock, which do not overlap with
those of NALP3.
In a separate study, Martinon and colleagues [32] dem-
onstrated that monosodium urate (MSU) and calcium pyro-
phosphate dihydrate (CPPD) crystals activate caspase-1 in a
NALP3-dependent manner. Deposition of MSU and CPPD
crystals in joints is responsible for the inflammatory condi-
tions gout and pseudogout, respectively. It is unclear if MSU
and CPPD activation of the NALP3 inflammasome is depen-
dent on K⫹ effluxes, similar to other NALP3 inflammasome-
activating agents such as ATP and nigericin. This study
identifies NALP3 as playing a potentially important role in
inflammatory joint disease. In addition to its role in gout,
uric acid is a major component released into the extracel-
lular milieu by necrotic cells. Recognition of uric acid
following cell death may be an additional way in which
NALP3 detects endogenous danger signals.
BACTERIAL ACTIVATION OF THE NALP3
INFLAMMASOME
Secretion of IL-1␤ and IL-18 by macrophages in response to
infection with S. aureus and L. monocytogenes is dependent on
ASC and NALP3 [31] (Fig. 2). Mutant L. monocytogenes defi-
cient in LLO were unable to induce caspase-1 activation in
wild-type macrophages, suggesting that LLO-mediated escape
of L. monocytogenes from the phagosome into the cytosol was
crucial for activation of the NALP3 inflammasome, presumably
by allowing the detection of pathogen-associated molecular
patterns (PAMPs) now released into the cytosol. We however
found that LLO-containing supernatants, in the absence of
bacteria, added to macrophages extracellularly, result in the
NALP3-dependent activation of caspase-1 (Y. Ogura and R. A.
Flavell, unpublished observations). These data suggest that the
pore-forming capacity of LLO on the plasma membrane of the
macrophage results in a potassium efflux, which in turn leads
to the activation of the NALP3 inflammasome. Unlike LLO-
deficient L. monocytogenes, S. aureus singly deficient in ␣-, ␤-,
or ␥-hemolysin were still capable of activating caspase-1 [31].
This may be a result of redundant membrane disruptive toxins
still present when only a single hemolysin is absent.
The pore-forming toxin aerolysin from Aeromonas hydrophila
is also able to activate caspase-1 by mediating a potassium
efflux [33]. Using small interfering RNA to silence expression
of NALP3, ASC, or IPAF in HeLa cells, the NALP3 and IPAF
inflammasomes were implicated in mediating caspase-1 acti-
vation in response to aerolysin [33]. It will be informative to
determine the roles of NALP3 and IPAF in aerolysin-mediated
caspase-1 activation using primary cells from NALP3- and
IPAF-deficient mice.
Kanneganti et al. [34, 35] have suggested that NALP3 and
ASC are essential for caspase-1 activation in response specif-
ically to bacterial RNA, the imidazoquinoline compounds
R837 and R848, and viral dsRNA. Their studies found that
these PAMPs were capable of activating caspase-1 in the
absence of ATP, which is in contrast to the findings of others.
In addition, this study failed to observe ATP-mediated
caspase-1 activation in response to other TLR ligands such as
LPS, lipoteichoic acid, and Pam3CSK4. The reason for the
discrepancy between this study [34] and the others [30 –32]
remains unclear.
BACTERIAL ACTIVATION OF THE IPAF
INFLAMMASOME
Caspase-1 is important for host defense to a number of patho-
gens. Infection of macrophages with Salmonella, Shigella, Le-
gionella, and Pseudomonas all lead to caspase-1 activation,
release of IL-1␤, and rapid cell death. The caspase-1-mediated
cell death caused by infection with Salmonella typhimurium
was found to be independent of NALP3 [30, 31]. S. typhi-
murium-infected macrophages did, however, require IPAF to
activate caspase-1 [36] (Fig. 3). Recent studies in our lab
suggest that P. aeruginosa-induced activation of caspase-1 is
also dependent on IPAF (F. S. Sutterwala and R. A. Flavell,
unpublished observations). The role for ASC in IPAF-mediated
caspase-1 activation is unclear. Although IPAF can interact
directly with procaspase-1, ASC-deficient macrophages have
delayed kinetics of cell death following infection with S. typhi-
murium compared with wild-type. This suggests ASC may
serve a role stabilizing the interaction between IPAF and
procaspase-1.
Despite the dramatic in vitro effects of IPAF deficiency on S.
typhimurium-induced macrophage death, IPAF-deficient mice
infected orally with S. typhimurium did not display enhanced
susceptibility to infection [37]. However, caspase-1-deficient
mice infected with S. typhimurium were more susceptible to
infection, as demonstrated by a shorter time until death and
higher bacterial burdens in spleen and mesenteric lymph
nodes. The in vivo difference seen between caspase-1-deficient
Sutterwala et al. The inflammasome 261
Fig. 3. Activation of the IPAF inflamma-
some by Type III and Type IV secretion sys-
tems. Activation of caspase-1 following infec-
tion of macrophages with S. typhimurium,
Pseudomonas aeruginosa, or Legionella pneu-
mophila requires a functional Type III or Type
IV secretion system. Infection causes IPAF to
undergo a conformational change by an un-
known mechanism, which allows IPAF to oli-
gomerize. Following oligomerization, IPAF re-
cruits procaspase-1 via homophilic CARD-
CARD interactions, which lead to activation
of caspase-1. Bacterial-derived cytosolic
flagellin likely enhances or in the case of L.
pneumophila, is required for the activation
of the IPAF inflammasome through a possi-
ble interaction with Naip5. Activation of
caspase-1 following infection by Francisella
tularensis requires the adaptor molecule
ASC and likely activates an inflammasome
through an unknown NLR molecule. Dis-
ruption of the phagosome by F. tularensis is
required for activation of caspase-1.

mice and IPAF-deficient mice may be a result of additional,
undefined pathways, which result in caspase-1 activation, or a
synergistic effect among IPAF, NALP3, and ASC.
L. pneumophila can also mediate macrophage cell death
through a caspase-1-dependent manner [38] (Fig. 3). The ac-
tivation of caspase-1 by L. pneumophila is dependent on IPAF
but not NALP3 [38, 39]. L. pneumophila is unique in that
another NLR member, Naip5 (Birc1e), is also involved in
susceptibility to infection with L. pneumophila [40, 41]. These
findings link Naip5 to the caspase-1 pathway, which is sup-
ported by the observation that Naip5 and IPAF can interact
physically [38].
Recent studies have begun to provide insight into how the
IPAF inflammasome is activated. S. typhimurium [42, 43] and
L. pneumophila [44, 45] strains deficient in flagellin were found
to be defective in their ability to activate caspase-1. Purified
flagellin delivered to the macrophage cytosol by transfection
was also capable of activating caspase-1 [42, 43]. These find-
ings led to the hypothesis that cytosolic flagellin contributes to
the activation of caspase-1. However, the direct activation of
IPAF by cytosolic flagellin remains controversial for several
reasons. First, the P. aeruginosa mutant PAK⌬fliC, which is
deficient in flagellin, is still capable of activating caspase-1 in
an IPAF-dependent manner (F. S. Sutterwala and R. A. Fla-
vell, unpublished observations). Second, the nonflagellated
bacterium Shigella flexneri has also been suggested to activate
caspase-1 in an IPAF-dependent manner [46]. Finally, Miao et
al. [43] showed that at a high multiplicity of infection, flagellin-
deficient S. typhimurium strains were still capable of inducing
macrophage secretion of IL-1␤.
A common feature that S. typhimurium, S. flexneri, and P.
aeruginosa share is their requirement for an intact, Type III
secretion system (TTSS) to activate caspase-1 [47, 48]. L.
pneumophila-induced caspase-1 is dependent on the Dot-Icm
Type IV secretion system, which is unrelated structurally but
similar functionally to the TTSS [38]. As flagellin alone is not
sufficient to activate the IPAF inflammasome, it is possible that
rather than detecting a specific pathogen-derived molecule,
IPAF detects membrane damage induced by Type III or Type
IV secretion systems. Flagellin may serve to enhance Type III
or Type IV secretion system-dependent caspase-1 activation
through aiding bacterial adhesion to the host cell. Alterna-
tively, flagellin may interact with Naip5, which in turn associ-
ates with IPAF.
MEMBRANE DISRUPTION AS A POSSIBLE
TRIGGER FOR INFLAMMASOME ACTIVATION
Mariathasan and colleagues [49] have recently shown that F.
tularensis is also capable of mediating macrophage cell death
in a manner that is dependent on ASC and caspase-1, and
caspase-1- and ASC-deficient mice infected in vivo with F.
tularensis showed increased susceptibility to infection com-
pared with wild-type mice. NALP3 and IPAF were not found to
be responsible for mediating caspase-1 activation in response
to infection with F. tularensis, suggesting another NLR family
member plays a role in the detection of F. tularensis. It is
interesting that F. tularensis mutants, which could not escape
from the phagosome, did not induce caspase-1 activity [49].
This suggests that disruption of the phagosome membrane may
be yet another signal to activate a specific NLR pathway.
Caspase-1 has also been found to play a role in membrane
repair. Gurcel et al. [33] found that aerolysin-induced mem-
brane disruption leads to a K⫹ efflux, which is responsible for
activation of NALP3 and IPAF inflammasomes [33].
Caspase-1, which is activated in this manner, induces the
activation of sterol regulatory element-binding proteins, which
promote cell survival through membrane repair [33]. It is
unclear why activation of caspase-1 in response to pathogens
such as Salmonella, Shigella, and Pseudomonas leads to cell
death, whereas caspase-1 activation by aerolysin toxin leads to
increased cell survival. Specific activation of caspase-1 via
NALP3 or IPAF inflammasomes may be responsible for guid-
ing this process.

262 Journal of Leukocyte Biology Volume 82, August 2007 http://www.jleukbio.org

 Although rapid progress has been made in identifying how
the inflammasome is activated, many crucial questions remain.
It is unclear if NLRs serve as direct receptors of PAMPs or if
they sense the consequences of infection or inflammatory stim-
uli indirectly. We propose that NLR molecules recognize and
respond to membrane disruption itself as opposed to the con-
cept that membrane disruption serves as a portal for entry of
PAMPs into the cytosol. The NALP3 inflammasome appears to
be activated following a potassium efflux mediated by mem-
brane disruption of the plasma membrane by pore-forming
toxins or through the engagement of the P2X7 receptor by ATP.
The IPAF inflammasome, conversely, is activated by mem-
brane disruption mediated by Type III or Type IV secretion
systems. A third pathway resulting in phagosome membrane
disruption, such as that caused by the pathogen F. tularensis,
may lead to an as-of-yet undefined NLR pathway. Hence, it
appears that where the pathogen resides and what intracellular
compartments the pathogen uses are crucial to which NLR
pathway is activated. Further studies to elucidate the down-
stream events leading to activation of the inflammasome will be
required to help elucidate how the NLRs function. Multiple
NLR molecules remain, which still have unidentified functions.
Identifying activation signals as well as effector functions for
these NLRs will help in determining the role of these mole-
cules in host defense against pathogens and in autoinflamma-
tory disorders.
ACKNOWLEDGMENTS
This work was supported by an Ellison Foundation grant to
R.A.F. F.S.S. was supported by a Pfizer Fellowship in Infec-
tious Diseases. R.A.F. is an investigator of the Howard Hughes
Medical Institute. We thank Suzanne Cassel and Joao Pedra for
helpful discussion and critical review of the manuscript.
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