Inflammatory caspases and inflammasomes: master
switches of inflammation
F Martinon1,2and J Tschopp*,1
Fifteen years have passed since the cloning and characterization of the interleukin-1b-converting enzyme (ICE/caspase-1),
the first identified member of a family of proteases currently known as caspases. Caspase-1 is the prototypical member of a
subclass of caspases involved in cytokine maturation termed inflammatory caspases that also include caspase-4 caspase -5,
caspase -11 and caspase -12. Efforts to elucidate the molecular mechanisms involved in the activation of these proteases have
uncovered an important role for the NLR family members, NALPs, NAIP and IPAF. These proteins promote the assembly of
multiprotein complexes termed inflammasomes, which are required for activation of inflammatory caspases. This article will
review some evolutionary aspects, biochemical evidences and genetic studies, underlining the role of inflammasomes and
inflammatory caspases in innate immunity against pathogens, autoinflammatory syndromes and in the biology of reproduction.
Cell Death and Differentiation (2007) 14, 10–22. doi:10.1038/sj.cdd.4402038; published online 15 September 2006
The history of caspases began with the identification of an
aspartate-specific protease activity involved in the conversion
of the 31kDa proIL-1b precursor to its active 17kDa bio-
logically active form,1,2and the identification of caspase-1 as
the protease responsible for proIL-1b maturation.3,4The
subsequent discovery of ced-3, that shares similarities with
caspase-1 and which is involved in programmed cell death
(PCD) in Caenorhabditis elegans, suggested that caspases
papers accompanying this issue of Cell Death and Differ-
entiation, the role of apoptotic caspases in C. elegans and in
development and diseases. In this review, we will focus on a
subset of caspases present only in vertebrates and known as
Inflammatory caspases (also known as group I caspases)
are encoded by three main genes in humans caspase-1,
caspase-4 and caspase-5 and three main genes in mouse,
caspase-1, caspase-11 and caspase-12.6,7In mammals,
these caspases are characterized by the presence of a CARD
domain at the N-terminus (Figure 1a). Human, chimp and
mouse inflammatory caspases share significant similarity
and are organized in a single locus (Figure 1c). Phylogenetic
analysis of the conserved CARD domain suggests that the
inflammatory caspases can be separated in evolutionary-
related clusters (Figure 1b). The caspase-1 cluster contains
caspase-1 and four other genes encoding decoy caspases:
cop, inca1, inca2 and iceberg. These decoy caspase-1-like
genes are absent in the mouse genome, suggesting that
they have arisen recently by duplication of caspase-1.
Although human and mouse caspase-1 are likely ortho-
logues, sequence analysis suggests that human caspase-4
and caspase-5 have originated from a duplication of
caspase-11.7However, the human caspase-12 gene, which
in the chimp genome contains an SHG box important for it
enzymatic activity,8evolved towards an enzymatically inac-
tive form at some stage in the recent adaptation process of
human species, probably during the out-of Africa migration of
modern humans.9,10Initial analysis of the chimp and human
genome identified 53 known or predicted genes that are found
either entirely or partially deleted in chimpanzee or in human.
Intriguingly, those 53 genes include caspase-12 and iceberg,
as well as other genes linked with inflammatory caspases
such as IL1F7 and IL1F8 (two IL-1-related genes), and
NALP12 (see below).8Additional analysis of the inflammatory
caspase locus in chimp reveals the absence of cop
(Figure 1c), further highlighting the plasticity and rapid and
recent evolution of the inflammatory caspase locus.
These caspases are termed ‘inflammatory’ as the main
caspase-1 substrates identified to date are proIL-1b and proIL-
18, two related cytokines that play critical roles in inflamma-
tion. A recent report suggests that another IL-1-related
Received 05.6.06; revised 28.7.06; accepted 31.7.06; Edited by S Kumar; published online 15.9.06
1Department of Biochemistry, University of Lausanne, BIL Biomedical Research Center, Epalinges, Switzerland
*Corresponding author: J Tschopp, Department of Biochemistry, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. Tel: þ41-21-692-5738;
Fax: þ41-21-692-5705; E-mail email@example.com
2Current address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Avenue, Boston, MA 02115, USA
Keywords: inflammasome; NLRs; interleukin-1b; sepsis; evolution; innate immunity
Abbreviations: ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase recruitment domain; ICE, interleukin-1b -converting enzyme; IL-1,
interleukin-1; IPAF, ICEprotease-activating factor; LRR, leucine-rich repeat;MDP, muramyl dipeptide; MSU, monosodium urate crystals;MyD88, myeloid differentiation
protein 88; NACHT, domain present in neuronal apoptosis inhibitory protein (NAIP), the major histocompatibility complex (MHC) class II transactivator (CIITA), HET-E
and TP1; NALP, NACHT, LRR and PYD containing proteins; NLR, NOD-like receptors; PAMP, pathogen-associated molecular patterns; PYD, pyrin domain; RI,
Cell Death and Differentiation (2007) 14, 10–22
& 2007 Nature Publishing Group All rights reserved 1350-9047/07 $30.00
caspase-11 and caspase-12 and human caspase-4 and
caspase-5 no specific substrates have been identified, there-
fore the precise function of those caspases is still an open
question. Some of the current views on the role of these
caspases will be presented in the following sections.
Caspase-1 and it Substrate IL-1b
The requirement of caspase-1 for IL-1b and IL-18 activi-
ties was revealed by the generation of mice deficient in
proIL-1b and proIL-18 and are resistant to the lethal effect
of endotoxins. IL-18 was first described as an endotoxin-
induced factor that stimulates the production of interferon-g
by splenocytes. However, IL-18 has many other functions
including induction of pro-inflammatory cytokines, upregula-
tion of adhesion molecules, and activation of natural killer cell
activity.16IL-1b is a major mediator of inflammation and, in
general, initiates and/or amplifies a wide variety of effects
associated with innate immunity and host responses to micro-
bial invasion and tissue injury.17When mice are immunized
with protein antigens together with IL-1b, serum antibody
production is enhanced, suggesting that IL-1b has adjuvant
properties.18On the other hand, the physiological role of IL-1b
adjuvanticity and costimulation of T cells has not been fully
Mouse 9 A1
Chimp (Pan Troglodyte preliminary model)
CARD p20p10 CARDCARD p20p10
ex: Caspase-1, 4, 5,11,
mouse and chimp Casp-12
ex: human Caspase-12L
ex: human Caspase-12,
INCA, COP, ICEBERG
caspase chromosomal locus. Asterisk indicates inactive caspase
Inflammatory caspases. (a) Schematic domain structure of the inflammatory caspase family. (b) Phylogenic analysis of inflammatory caspases based on the
F Martinon and J Tschopp
Cell Death and Differentiation
established. Recombinant IL-1b induces fever in experi-
mental animals, an activity shared with other cytokines
including tumor necrosis factor (TNF) and IL-6, although the
latter cytokines are much less potent than IL-1b.19In addition
to fever, IL-1b has other effects on the central nervous
system.Theseinclude induction ofslow-wave sleep,anorexia
and inflammatory pain hypersensitivity, typically associated
with infections or injury.20,21IL-1b also influences the function
of vessel wall elements, endothelial cells in particular and
may contribute to the pathogenesis of arteriosclerosis
in different ways, including by promoting coagulation and
thrombosis.22,23Importantly, IL-1b was shown to control
tumor angiogenesis and invasiveness of different tumor cells
in mice.24,25Moreover, IL-1b plays a role in destructive joint
and bone diseases. In particular, IL-1b induces production of
collagenase by synovial cells and of metalloproteinases
by chondrocytes.26Another important characteristic of IL-1b
is its toxicity for insulin-producing b-cells in Langerhans
islets, supporting a role of IL-1b in the pathogenesis of
insulin-dependent type I diabetes.27,28Similarly, IL-1b may
be toxic for neurons and is involved in acute neurodegenera-
tion and stroke.29
Although many important biological effects of IL-1b are
well described, key questions remain unresolved about the
mechanism by which the production of this cytokine is
regulated. Human proIL-1b must be proteolyticaly cleaved
between Asp116and Ala117in order to function as an active
protein, a process occurring in the cytoplasm.30,31A technical
breakthrough in the identification of the mechanisms of proIL-
1b maturation was the discovery an in vitro assay to monitor
proIL-1b maturation.2By this assay, mature IL-1b can be
generated by incubation of proIL-1b with partially fractionated
extracts from human monocytes or the monocyte-like
cell line THP-1. This assay was used to biochemically
purify and sequence the IL-1b converting enzyme (ICE) or
caspase-1.3,4,32A similar assay allowed the biochemical
identification and characterization of the inflammasome, a
molecular platform that is spontaneously activated during the
hypotonic lysis of THP-1 cells and that triggers caspase-1
activation33(see below). The biological activity of IL-1b is
cytokine maturation. A better understanding of the mechan-
isms involved in the activation of caspase-1 is therefore
crucial to appreciate the mechanisms of IL-1b regulation.
Mouse Caspase-11 and Human Caspase-4 and
Murine caspase-11 is a poorly characterized member of
the caspase-1 subfamily. Mice deficient in caspase-11
or caspase-1 show a very similar phenotype in response
to lipopolysaccharide (LPS) overdose. These mice fail to
produce mature IL-1b and are resistant to endotoxic shock
induced by bacterial endotoxins.34Moreover, caspase-11-
deficient embryonic fibroblasts are resistant to apoptosis
induced by ectopic expression of caspase-1, suggesting that
caspase-11 is an upstream activator of caspase-1.34Unlike
caspase-1, the expression of caspase-11 is LPS-inducible,
and it is reasonable to postulate that other members of
the family are regulated at the transcriptional or translational
profiles, caspase-5 was proposed to be the human functional
caspase-1 were found to be components of the NALP1
inflammasome, a complex involved in the activation of
caspase-133(see below). These findings reinforced the
hypothesis that different inflammatory caspases may co-
operate for full activity. Sequence comparison of the caspase
domain and prodomains of the inflammatory caspases
suggests that both caspase-4 and caspase-5 probably
arose following the duplication of a caspase-11 ancestor
gene.7Little is known about the second possible capase-11
that caspase-4 may play a role in endoplasmic reticulum (ER)
stress-induced apoptosis,36,37a conclusion challenged by
by extracellularstimuli.Basedon expression
Caspase-12: A Role in Sepsis and ER Stress?
Conditions interfering with the function of ER are collectively
called ER stress. ER stress is induced by accumulation
of unfolded protein aggregates or by excessive protein
trafficking usually owing to viral infection. The ER stress
possesses its own signaling pathways that ultimately may
result in cell death. Initial studies in caspase-12-deficient
mice suggested that this caspase was important for ER-
stress-induced apoptosis.39In addition, caspase-12 was
shown to participate in the development of ER-stress-related
neurodegenerative disorders such as Alzheimer’s disease or
prion associated diseases.39,40Whereas degradation or
proteolysis of caspase-12 is a well-established hallmark of
ER stress, its central role in ER-stress-induced apoptosis was
challenged by various studies.41–43The precise function of
caspase-12 in this particular pathway is yet unclear and
Human polymorphisms of caspase-12 result in the produc-
tion of either a truncated protein containing the N-terminal
(Caspase-12L), which was hypothesized to be enzymatically
inactive.44The full-length variant of caspase-12 is the less
frequent allele, confined to population of African descen-
dant and is linked to hypo-responsiveness to LPS-induced
production of cytokines such as IL-1b.44Interestingly, recent
genetic studies have suggested that the stop codon generat-
ing the CARD-only truncated form of caspase-12 was driven
by positive selection to complete fixation in the human
genome, approximately 60–100 thousand years ago.9,10This
observation suggests that loss of the C-terminus of caspase-
12 may have conferred a selective advantage, possibly
by increasing sepsis resistance in human populations that
experienced more and emergent infectious diseases as geo-
graphic expansion occurred in association with increasing
population size and density.9,10In line with this hypothesis,
caspase-12-deficient mice were shown to clear bacterial
infection more efficiently than wild-type littermates and have
an enhanced production of the pro-inflammatory cytokines
IL-1b and IL-18 but not TNF and IL-6.43Mechanistically,
caspase-12 was proposed to be a decoy caspase that
blocks caspase-1 activation resulting in enhanced vulner-
ability to bacterial infection and septic mortality, plausibly in
F Martinon and J Tschopp
Cell Death and Differentiation
the same way as cFLIP (a decoy caspase-8-like protein)
regulates caspase-8-mediated apoptosis43(see the accom-
panying review by Maya Saleh).
Activation of Inflammatory Caspases: Inflammasomes
and Other Molecular Machines
Evolutionarily caspases are linked to the Clan CD of
cysteine peptidases that includes legumain, streptopain,
separin, metacaspases and paracaspases.45The ‘c’ in the
term ‘caspase’ is intended to reflect the cysteine protease
mechanism and ‘aspase’ refers to the ability to cleave after
aspartic acid, an almost unique preference in eukaryotic
enzymes.46All caspases are produced in cells as catalytically
inactive zymogens, and generally undergo proteolytic proces-
sing during activation.47The subset of caspases that cleaves
selected substrates to produce the changes associated with
apoptosis are known as ‘executioner caspases’, which in
mammals are represented by caspase-3, caspase-6 and
caspase-7. In most instances, executive apoptotic caspases
are activated by ‘initiator caspases’ caspase-8, caspase-10,
caspase-2 or caspase-9. The mechanism of activation of
these initiator caspases depends critically on the engagement
and activation of recruitment platforms such as the death-
inducing signaling complex for caspase-8 and caspase-10,
the PIDDosome for caspase-2 and the well-described
integrate cellular signals, promote dimerization of initiator
caspases and lead to the formation of an active enzyme
proficient to initiate specific signaling cascades.51,52These
platforms are multiprotein complexes consisting of various
molecules assembled on a central scaffold protein that
characteristically possesses three main domains: a region
involved in ligand sensing, a domain driving oligomeriza-
tion and a domain involved in recruiting the caspases. The
1. Apaf-1 possesses a CARD for caspase-9 recruitment, a
NB-ARC domain for oligomerization and a WD repeat that
senses the release of cytochrome c from the mitochondria,
a signal that leads to apoptosis by apoptosome activation.
A family of intracellular receptors structurally related to Apaf-1
was described in vertebrates recently. These proteins
named NOD-like receptors (NLRs) are intracellular sensors
of pathogens and other stresses.53,54NLRs include proteins
such as NOD1 and NOD2, that sense bacterial peptidogly-
cans and activate the kinase RIP2 and nuclear factor-kB,55
and three subfamilies of proteins involved in the formation
of caspase-1-activating complexes: NALPs, IPAF and NAIPs
IPAF is a well-conserved protein that contains an
N-terminal CARD, a central NACHT domain and a C-terminal
leucine-rich repeats (LRR) region. The CARD domain
associates directly and specifically with the CARD domain of
the same complex, similarly CARDINAL and Pyrin may be involved in the formation or regulation of NALP-based inflammasomes. NALP1 has a C-terminal extension highly
paralogues are more related to CARDINAL or human NALP1. Brackets indicate interactions. The boxes identify the various units of the inflammasome, that is, the ligand
sensing domain, the oligomerization module, the possible adaptor and the caspase
Schematic models of proposed caspase-1-activating inflammasomes. The ligand-sensing motifs (LRR repeats), initiate the formation of oligomers. PYD–PYD
F Martinon and J Tschopp
Cell Death and Differentiation
The NACHT domain, related to the NB-ARC domain of
Apaf-1, induces oligomerization and promotes proximity of
the caspases, whereas the C-terminal LRR is probably
involved in ligand sensing. It may also have regulatory
properties as its absence leads to the formation of a more
Despite the absence of a CARD, the neuronal apop-
tosis inhibitor protein (NAIP) shares with IPAF the highest
sequence similarity of the NACHT and LRR domains,
suggesting that these molecules are evolutionary and
functionally related.57Instead of a CARD, NAIP harbors three
N-terminal baculovirus inhibitor-of-apoptosis repeats (BIR),58
in NAIP are associated with the development of spinal
muscular atrophy.58Mouse NAIP is mainly expressed in
genes, naip1 to naip7.60NAIP was proposed to interact with
IPAF indicating that it may be part of the same caspase-1
activating complex61(Figure 2).
by seven paralogous
NALPs, represent the largest NLRs subfamily57(Table 1).
Some of them such as NALP1, NALP2 and NALP3 were
shown to be the central scaffold of caspase-1-activating
complexes known as inflammasomes7,33(Figure 2). These
proteins harbor an NACHT and an LRR similar to IPAF and
NAIP but are characterized by an N-terminal PYD domain.
The PYD of NALPs interacts and recruits the adaptor
ASC via PYD–PYD interaction. ASC contains an N-terminal
PYD and a C-terminal CARD and is an essential component
for inflammasome formation.62The CARD domain within
ASC binds and recruits caspase-1 to the inflammasome33,63
(Figure 2). The inflammasome may also recruit other
caspases such as caspase-5 via the C-terminal CARD
of NALP1 or a second caspase-1 via the C-terminal
CARD of CARDINAL, another possible component of the
With 14 NALPs, plus Ipaf and NAIP, the repertoire
of caspase-1-activating molecular machines is potentially
very complex. This complexity can be anticipated to be
Table 1 Human and mouse NALPs, IPAF and NAIP repertoire
Other names and aliasesStructure
DEFCAP; NAC; CARD7 PYD-NACHT-NAD-LRR-FIIND-CARD
NALP2 Pypaf2; NBS1;PAN1
Cias1, Pypaf1, Mmig1
Pypaf8; Mater, PAN11
PAN5; NOD8; Pynod
Pypaf7; Monarch1; RNO2; PAN6
Birc1b, Naip-rs6, NAIP2
Birc1c, Naip-rs5, NAIP3
Birc1d, Naip-rs2, NAIP4
Birc1e, Naip-rs3, NAIP5
Birc1f, Naip-rs4, NAIP6
F Martinon and J Tschopp
Cell Death and Differentiation
even greater considering some very unusual aspects of
the NALP LRRs.
Repertoire and Genomic Organization of the NALP LRRs
Recognition modules are often organized by repeated motifs.
This is the case for example for the cysteine-rich repeats in
death receptors, the immunoglobulin domain in IL-1b recep-
tors (IL-1Rs) or WD40 repeats in Apaf-1. Another ligand
recognition motif that is frequently found in sensors of
pathogens is the LRRs, this domain being found in TLRs
and NLRs. These relatively short motifs (22–28 residues in
length) can be found in a variety of cytoplasmic, membrane
and extracellular proteins.65Although these modules are
associated with a wide range of functions, they generally are
involved in protein–protein interaction. The LRR structural
organized in such a way that all the b strands and the helices
are parallel to the same axis, resulting in a nonglobular,
horseshoe-shaped molecule with the curved b parallel sheet
lining the inner circumference of the horseshoe and the a
helices the outer circumference.65
The structures of NALP genes have a highly conserved and
intriguing intron–exon organization. Moreover, there is a
striking relationship between the intron–exon structure of the
NALPs and their modular organization. As illustrated for
NALP3 (Figure 3), all the NALPs have one exon that encodes
the entire N-terminal PYD, followed by a large exon that code
for the NACHT domain, and finally several exons, 171
nucleotides in length that form the LRR. Remarkably, the
size, the reading frame phase and the intron–exon junction
site are conserved among all NALPs LRRs throughout
evolution except for the insertion of an additional amino acid
in fugu and zebrafish. Therefore, the LRR within NALPs is
defined completely by its intron–exon structure. Why is the
NALP LRR exon organization so precise and conserved? The
phasing and position of the introns are consistent with rapid
and efficient exon amplification during evolution. Moreover,
this modular organization allows extensive alternative splicing
of the LRR region without disturbing the three-dimensional
fold of the region. Alternative splicing of the LRR region is
evident for virtually all NALPs as detected by EST analysis
and the cloning of various NALPs.66,67Moreover, it is
interesting to note that exon–exon junctions disrupt the
b-strand that is predicted in binding targets. Therefore,
alternative splicing not only reorganize the numbers of b-
strand but generate completely new b-strands allowing
maximal variability in the ligand recognition region.
The unique other protein sharing exactly the LRR modular
organization of the NALPs is the ribonuclease inhibitor, (RI).68
RI is able to bind tightly to members of the ribonuclease
(RNAse) A superfamily, and is involved in angiogenesis, RNA
degradation, cytotoxicity and host-defense responses.69The
similarity between NALPs and RI is so close that RI likely
represents a decoy form of NALPs. Further studies will be
necessary to investigate whether RI interferes with NALP
function or has acquired an independent function.
Another feature that characterizes NALPs is their strong
tendency to evolve through gene duplication events. Some
NALPs such as NALP2 and NALP7 in humans are clearly
paralogues, whereas others such as NALP4 and NALP9 are
expanded in mouse (Table 1). A similar evolutionary trend
was followed by NAIP in mouse where the locus expanded to
seven NAIP genes.
120nt 1753nt171nt171nt171nt 171nt171nt100nt
LRR B-typeLRR A-type
connected by and exon–exon junction within the b strand. The reading frame and exon–exon junction are conserved among all the NALPs in all vertebrates. The lower panel
shows an alignment of the translated exons forming the NALP3 LRR
Genomic organization of the NALP3 gene. Note that the 171 nucleotides long (nt) exons that always encode for one LRR plus two-half of two LRRs that are
F Martinon and J Tschopp
Cell Death and Differentiation
All these observations indicate that the NALP repertoire
within a species, and across vertebrates is large and made of
different genes and splice variants that mainly differ by their
LRR region. Remarkably certain NALPs, such as NALP3, are
more conserved. It is therefore tempting to speculate that
conserved NALPs are involved in recognition of conserved
pathogen-associated molecular patterns or stress signals,
whereas rapidly evolving NALPs may be involved in specific
host–pathogen interaction, similar to the innate immune
system in plants where specific resistance genes (related to
the NLRs) detect specific avirulence genes from a pathogen
in an host–pathogen, gene for gene, interaction.54,70,71In the
presence of a cognate resistance gene, a specific pathogen
elicits defense mechanisms and host resistance that lead
to a controlled infection. Conversely, in the absence of a
specific avirulence gene (of the pathogen) or resistance gene
(of the plant), the pathogen eludes specific detection
by the host plant, resulting in pathogen proliferation and
eventually death of the plant; a bad scenario for both the
pathogen and the plant. The challenge for future research
is therefore to investigate possible specific interactions
between microbial molecular patterns or specific pathogenic
factors and various types of inflammasomes. Recent
studies have shed some light on some of those interactions
Toxins and Agents Inducing Potassium Efflux Activate
the NALP3 Inflammasome
The best-studied model of caspase-1 activation is the
exposure of cells to extracellular ATP that activates P2X7
ion channel receptors. P2X7receptors belong to a family of
ion channel receptors activated by extracellular ATP.72,73
Many studies have shown the requirement of P2X7receptors
for ATP-induced caspase-1 activation and subsequent IL-1b
release.74–78However, the physiological relevance of this
mechanism, especially in the course of pathogen-induced
IL-1b maturation and release is unclear. P2X7 receptor
requires potassium efflux for caspase-1 activation,79thus
it is possible that the mechanisms, leading to the activation
of caspase-1 in the cell-free system and following ATP
stimulation, are similar. On the other hand, other models
of hypotonic stress and potassium efflux produce comparable
caspase-1 activation.79The generation of ASC (a crucial
that ATP-mediated caspase-1 activation requires ASC
and is therefore probably dependent on the activation of a
NALP protein.80This hypothesis was indeed confirmed
in studies using NALP3-deficient mice
Another study suggested that NALP3 (also known as
cryopyrin) is required for caspase-1 activation by bacterial
RNA or the small antiviral compounds R848 and R837. In
this study, caspase-1 activation was monitored in presence
of extracellular ATP, and is therefore likely to depend on
cellular potassium levels such as the potassium ionophore
nigericine and maitotoxin, a potent marine toxin, depend
on the NALP3-based inflammasome for caspase-1 activa-
tion.81NALP3 and ASC are also required for caspase-1
activation by the Gram-positive bacteria Staphylococcus
aureus and Listeria monocytogenes.81,85
genes-mediated caspase-1 activation requires the bacterial
toxin listeriolysin O (LLO). Whether this toxin and the
unidentified caspase-1-activating factor from S. aureus are
dependent on potassium efflux or NALP3 activation, requires
NALP3 Inflammasome and Autoinflammatory Disorders
Missense mutations in the NACHT domain of NALP3, also
known as Cryopyrin or CIAS1 gene, are involved in three
autosomal dominant diseases: familial cold auto-inflamma-
tory syndrome, Muckle Wells syndrome and chronic infantile
neurological cutaneous and articular syndrome/neonatal onset
multisystemic inflammatory disease(CINCA/NOMID).66,86,87
All three disorders are closely related autoinflammatory
syndromes characterized by periodic fever, skin rashes,
amyloidosis and in the case of CINCA, the eventual develop-
ment of neurological complications. Mutations in NALP3
confer a gain of function to the protein, resulting in
constitutively active NALP3 in Muckle Wells patients.64This
activation leads to an overactivation of caspase-1 in mono-
cytes, resulting in an aberrant maturation of IL-1b. Treatment
of those patients with a natural decoy IL-1 molecule (IL1ra)
rapidly and dramatically decreases disease manifesta-
tions,88,89further demonstrating that IL-1b is directly respon-
sible for the disease.
The NALP3 inflammasome and aberrant caspase-1 activa-
tion were recently linked to gout and pseudogout, two
other autoinflammatory syndromes. Here, the acute and
chronic inflammatory response is associated with the deposi-
tion of monosodium urate (MSU) or calcium pyrophosphate
dihydrate (CPPD) crystals, respectively, in joints and periarti-
cular tissues. MSU and CPPD stimulate the caspase-1-
activating NALP3 inflammasome to produce active IL-1b.82
Macrophages from mice deficient in various components of
the inflammasome such as caspase-1, ASC and NALP3
are defective in crystal-induced IL-1b activation. Moreover,
an impaired inflammation is found in an in vivo model of
crystal-induced peritonitis in inflammasome-deficient mice
or mice deficient in the IL-1R suggesting that in all the
above-mentioned autoinflammatory diseases, inflammation
is caused by overproduction of IL-1b.82Interestingly, IL-18
production is also activated by MSU;82,90nevertheless, IL-18
does not seem to play a crucial role in vivo.90
NALP3 and Activation of the Adaptive Immune System
ASC- and NALP3-deficient mice demonstrate an impaired
contact hypersensitivity response to the hapten trinitro-
phenylchloride (TNP-Cl).83Contact hypersensitivity is a
T-cell-mediated immune response to repeated exposure to
contact allergens. The response can be divided into two
phases: sensitization and elicitation. Caspase-1 and IL-1b
have beenpreviously implicated
phase.91,92Similarly, NALP3-deficient mice that receive cells
from wild-type sensitized animals develop the T-cell depen-
dent elicitor phase, suggesting that NALP3 is involved in the
sensitization phase and may bridge the TNP-Cl stress signal
in the sensitization
F Martinon and J Tschopp
Cell Death and Differentiation
with the activation of the adaptive immunity. A related agent
2,4-dinitrofluorobenzene that is able to induce contact
hypersensitivity promotes the release of IL-1b via caspase-1
in a skin dendritic cell line suggesting that the inflammasome
may directly detect such compounds.93Furthermore, uric
acid crystalsand bacterial
described activators of the NALP3 inflammasome7,82are
also well-known adjuvant that are competent in promoting
the adaptive immune response. Whether IL-1, which is also
for the adjuvantic properties of these factors remains to be
determined in vivo.
ASC Dependent but NALP3 Independent Activation
of Caspase-1 by Salmonella and Francisella
Caspase-1 plays an important role in innate immunity against
Salmonella typhimurium, as macrophages and dendritic cells
infected with S. typhimurium undergo caspase-1-mediated
cell death.94,95ASC-deficient macrophages are unable to
K+ efflux inducing agents:
ATP and P2X7 activation, Nigericine; Matotoxine..
Staphylococcus aureus and Listeria monocytogenes:(LLO)
Gout and Pseudogout crystals: MSU and CPPD
Genetic activating mutations in NALP3
Bacterial RNA; R837 and R848 (?)
apoptosis; IL-1β β
Anthrax Letal Toxin
apoptosis; IL-1β β
apoptosis; IL-1β β
(Intracellular flagellin ?)
NAIP NALP ?
apoptosis; IL-1β β
(Intracellular flagellin) ( ? )
NAIP (see the text for details). Note that direct biochemical evidences for most of these inflammasomes are missing
Activation of inflammasomes. Current models of inflammatory caspase activators and their corresponding inflammasome platforms. This figure summarize
F Martinon and J Tschopp
Cell Death and Differentiation
activate caspase-1 following S. typhimurium infection.81,83
Similarly Francisella tularensis requires ASC to activate
caspase-1 and to trigger a competent immune response.96
Activation of ASC and caspase-1 by F. tularensis is
dependent on the internalization and phagosome escape.97
NALP3-deficient mice have no apparent defect in caspase-1
NALP involved in ASC-dependent caspase-1 activation by
F. tularensis and S. typhimurium is still unknown (Figure 4).
NALP1 and Susceptibility to Anthrax
Bacillus anthracis, the causative agent of anthrax, depends for
associated with cutaneous anthrax infections, whereas LeTx,
consisting of PA and lethal factor, is believed to be responsible
for causing death in systemic anthrax infections. In mouse
macrophages, LeTx can cause rapid apoptosis that requires
caspase-1 activation.98Macrophages from inbred mice are
either susceptible or resistant to apoptosis by LeTx. This trait
difference has been mapped to a locus on chromosome 11
named Ltxs1, and was recently associated with NALP1b
gene.99TheNALP1 locus inmousecontains three paralogues,
NALP1a, NALP1b and NALP1c (Table 1). NALP1b is highly
polymorphic in mouse, and susceptibility to LeTx seems to be
associated with a functional NALP1b allele and caspase-1
activation.99Murine NALP1b does not contain a PYD; hence, it
NALP for caspase-1 recruitment. However, NALP1b pos-
sesses a CARD and a region related to CARDINAL. It is
therefore possible that this region per se is able to activate
caspase-1 in an ASC-independent manner.
Intracellular Flagellin: Detection by the NLR Proteins
NAIP and IPAF
Part of the capase-1 activation owing to the pathogen
Salmonella typhimurium infection was also shown to be IPAF
dependent,80but until recently the activating ligand of IPAF
was unknown. Two recent studies identified flagellin as the
activator for IPAF. They could show that S. typhimurium
with a deficient flagellin does not stimulate caspase-1 or IL-1b
secretion, and that intracellular flagellin activates an IPAF
inflammasome,100,101a mechanism that does not require
the other flagellin sensor TLR5.100,101Similarly, other studies
showed that NAIP5, a mouse parologue of NAIP, possibly
in combination with IPAF, recognizes intracellular flagellin
from Legionella pneumophila, in order to induce caspase-1
activation.61,102,103Moreover, early genetic studies in mice
clearly identified NAIP5 as a L. pneumophila susceptibility
locus, further delineating the importance of inflammatory
caspases in the control of bacterial pathogens.104
NALPs and the Biology of Reproduction: A Role
for the Inflammatory Caspases?
Intriguingly, the expression profile of some NALPs, together
with a few genetic studies, suggest a possible function
for these proteins in the biology of reproduction. NALP5
(also known under the name of MATER) displays oocyte-
restricted basal expression in mouse and human.105,106
NALP5-deficient female mice are sterile because of an arrest
in the development of the embryos at the two-cell stage.105
Other NALPs such as the mouse NALP4 and NALP9
paralogues, NALP4a (also termed NALP9d, see Table 1),
NALP4b, NALP4c, NALP4f, NALP9b, NALP9c and bovine
NALP5 appear to be expressed exclusively in the ovary
whereas mouse NALP9a, NALP14 and bovine NALP9 and
NALP8 seem to be essentially expressed both in the ovary
and the testis.107–111Interestingly NALP expression levels
decrease in the oocyte during maternal aging.110Moreover,
knock-down experiments performed in fertilized mouse eggs
revealed that a decrease in NALP14 expression leads to
embryo failure in mouse compared with control knock-down,
mainly owing to an early developmental arrest between
the one-cell and eight-cell stages.110The expression of most
of those NALPs decreases during the implantation phase,
suggesting that NALPs are mainly involved in the early
preimplantation phase. Genetic
allelic variants of NALP5 as possible candidates involved in
susceptibility to a mouse model of autoimmune ovarian
dysgenesis, an autoimmune disease also characterized by
bodies against NALP5.112Furthermore, NALP7 mutations
cause recurrent hydatidiform mole and reproductive failure
in humans.113Hydatidiform mole is an abnormal human
pregnancy with no embryo and cystic degeneration of
placental villi. Although it is known that inflammation and
bacterial infection causes infertility, ectopic pregnancy and
abortion, the exact function of NALP7 in this disease remains
unidentified.113Similarly, it is unknown whether the develop-
mental failure associated with NALP5 or NALP14 deficiency
in the mouse are caused by a deregulated inflammatory
caspase activation and consequent overproduction of IL-1b in
the ovary. In line with this possibility, IL-1b is known to play a
role in both ovulation and oocyte maturation.114For instance
in the mare, intrafollicular injection of IL-1b lead to increased
ovulation, but also a very low rate of embryo develop-
ment possibly owing an alteration of ovocyte maturation.115
Similarly IL-1b perfusion inthe rabbit ovaryrevealeda block in
embryo development at the four-cell stage upon IL-1b
stimulation.116It is therefore possible that NALPs and
inflammatory caspases may link some aspects of innate
immunity and reproductive biology (gamete maturation and
proposed function of Xa13, a resistance gene that play a role
both in pollen development and disease resistance in rice,117
and for TIP49a, a regulator of the resistance gene RPM1 (a
NLR-like resistance gene) that is required for both regulation
of resistance and gamete viability in Arabidopsis.118
studies also identified
Regulators of Inflammatory Caspase Activation
Although the production of IL-1b is critical for the control of
pathogenic infections, and for many physiological processes,
excessive cytokine production is harmful and needs to be
tightly controlled. Regulation of the inflammatory caspases
at the inflammasome level is an undoubtedly important
F Martinon and J Tschopp
Cell Death and Differentiation
checkpoint in the control of IL-1b biological activity. Although
little is known at the physiological level, various proteins
were proposed to interfere with inflammasome assembly and
inflammatory caspases activation. Based on their modular
structure, we can distinguish two types of inflammasome
regulators. The first type is characterized by the presence of a
CARD highly similar to the CARD of caspase-1. This group
includes the decoy caspase-1 genes present in the human
caspase-1 locus, such as iceberg, INCA, COP and caspase-
127,119–122(Figure 1). Through CARD–CARD interactions
these proteins presumably negatively regulate the processing
of proIL-1b by preventing direct recruitment and/or activation
ofthecaspasebytheadaptorASC orIPAF.Other inhibitorsof
the inflammasome are characterized by the presence of a
PYD and are believed to interfere with PYD–PYD interactions
between ASC and NALPs, blocking therefore specifically
the NALP-based inflammasomes. These PYD regulators
include Pyrin, POP (DASC) and viral PYDs (vPYDs). POP
(a human decoy ASC protein) and the poxviral gene product
M13L-PYD (vPYD) are PYD-only protein.7,123,124Viruses
deficient in vPYD induce a strong activation of caspase-1 and
secretion of IL-1b indicating that inflammasomes are not
only important for antibacterial immunity but also play a role in
immunity against viruses.124,125
Pyrin was initially identified as the product of the MEFV
gene, which is mutated in patients with familial Mediterranean
fever (FMF),126a hereditary autoinflammatory syndrome
characterized by episodic fever and serosal or synovial
inflammation. Targeted disruption of the C-terminal portion
of Pyrin in mice causes increased endotoxin sensitivity and
caspase-1 activation,127demonstrating the important role of
this protein in inflammation and IL-1b maturation. The Pyrin
protein is organized with an N-terminal PYD domain followed
by an intermediate domain that links the PYD to the TRIM
region of Pyrin. The TRIM region is found in a family of
proteins with various functions and is characterized by the
presence of a B-Box, and a conserved Coiled Coil domain.128
The B-Box is often preceded by a RING domain and a Coiled
Coil domain, and followed by a PRY and SPRY domains.
Pyrin does not contain any RING domain but contains a
B-Box, a Coiled Coil and, in humans but not in rat or mice, a
PRY and an SPRY domain (Figure 2). Most of the mutations
in Pyrin affect the PRY and SPRY domains.129The function of
these domains is not clear but a role in the regulation of
inflammasome was proposed.127This concept is supported
by evolutionary analysis and notably by the presence of this
domain as a C-terminal extension (following the LRR) in the
NALPs from zebrafish and fugu, and with the identification of
new PRY-SPRY containing proteins possibly involved in
inflammasome regulation.130,131The PYD of Pyrin was found
to interact with the PYD of ASC127suggesting that it may be
involved in blocking the recruitment of ASC to the inflamma-
some. However, another study also proposed a proinflamma-
tory role for Pyrin. In this model, Pyrin, like NALP3, would be
able to assemble an inflammasome complex with ASC and
procaspase-1 leading to ASC oligomerization, caspase-1
activation and IL-1b processing.132Clearly, the mechanism of
action of Pyrin is therefore still controversial. As FMF is an
autosomal recessive autoinflammatory disorder it probably
requires loss of function of the Pyrin protein. Apparently FMF
originated over 2000 years ago in the Middle East and from
there it spread to North Africa, Turkey, Armenia, Iraq and the
countries on the northern shores of the Mediterranean Sea
with the Sephardic expulsion of 1492.126The disease carrier
rate in some populations may be extremely high as one
in seven.133,134This may reflect a founder effect or some
unknown selective pressure, conferring an advantage to the
heterozygote carriers. This is possibly the consequence of
some environmental or localized infectious diseases that
may have affected population around the Mediterranean
Sea. Interestingly, the Pyrin mutations in the SPRY and PRY
domains often exist as wild-type Pyrin in other primates.135
For several of these human mutations, the mutant represents
and, statistical analysis revealed the presence of episodic
positive selection.135Therefore, similarly towhatproposedfor
caspases-12 (another inflammasome regulator, see above
and accompanying review by Maya Saleh), selective
pressures may have caused functional evolution of pyrin
in humans and other primates. PSTPIP1, a Pyrin inter-
acting protein is also mutated in pyogenic arthritis, pyoderma
gangrenosum, and acne (PAPA), an autoinflammatory
disease associated with overproduction of IL-1b.136More-
over,mutationsina mouse-relatedproteinPSTPIP2 causes a
macrophage autoinflammatory syndrome, further delineating
the importance of Pyrin and inflammasome regulation in
The regulation and activation of the inflammatory caspases is
a very sophisticated and fascinating system to maturate IL-1b
and IL-18 that involves many members of the NLRs family of
proteins. Both cytokines are activated by inflammasomes
and engage specific receptors (IL-1R and IL-18R) that share
similarities with the TLRs (a well-known family of innate
immune receptors) in the intracellular domain and signaling
components such as MyD88. IL-1 can therefore be
considered as a cytokine linking intracellular innate immunity
receptors to IL-1R/TLR signaling,54a mechanism that seems
to evolve rapidly in order to cop as efficiently as possible with
Although some progress has been made in the character-
ization of inflammasomes, this is an emerging field and future
studies will undoubtedly shed more light on the respective
roles of various inflammasomes in human infections and
inflammatory diseases and possibly identify new functions for
inflammasomes and/or inflammatory caspases.
Acknowledgements. We thank Daniel Muruve, Saskia Lippens, Etienne
Meylan, Virginie Pe ´trilli and Patrizia Vinciguerra for discussions and critical
reading of the manuscript. This work was supported by a Swiss National
Science Foundation fellowship (FM) and grants of the Swiss National Science
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