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Recognition of Bacteria by
Inflammasomes
Jakob von Moltke, Janelle S. Ayres, Eric M. Kofoed,
Joseph Chavarrı́a-Smith, and Russell E. Vance
Department of Molecular & Cell Biology, Division of Immunology and Pathogenesis,
University of California, Berkeley, California 94720; email: [email protected]
Annu. Rev. Immunol. 2013. 31:73–106
Keywords
First published online as a Review in Advance on
November 26, 2012
caspase, NLR, innate immunity, pyroptosis
The Annual Review of Immunology is online at
immunol.annualreviews.org
Abstract
This article’s doi:
10.1146/annurev-immunol-032712-095944
c 2013 by Annual Reviews.
Copyright !
All rights reserved
Inflammasomes are cytosolic multiprotein complexes that assemble in response to a variety of infectious and noxious insults.
Inflammasomes play a critical role in the initiation of innate immune responses, primarily by serving as platforms for the activation
of inflammatory caspase proteases. One such caspase, CASPASE-1
(CASP1), initiates innate immune responses by cleaving pro-IL1β and pro-IL-18, leading to their activation and release. CASP1
and another inflammatory caspase termed CASP11 can also initiate a rapid and inflammatory form of cell death termed pyroptosis. Several distinct inflammasomes have been described, each of
which contains a unique sensor protein of the NLR (nucleotidebinding domain, leucine-rich repeat–containing) superfamily or the
PYHIN (PYRIN and HIN-200 domain–containing) superfamily.
Here we describe the surprisingly diverse mechanisms by which
NLR/PYHIN proteins sense bacteria and initiate innate immune responses. We conclude that inflammasomes represent a highly adaptable
scaffold ideally suited for detecting and initiating rapid innate responses
to diverse and rapidly evolving bacteria.
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INTRODUCTION
Caspase: cysteine
aspartic acid–specific
protease
NBD:
nucleotide-binding
domain
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LRR: leucine-rich
repeat
NLR: nucleotidebinding domain,
leucine-rich repeat–
containing
TLR: Toll-like
receptor
CARD: caspase
activation and
recruitment domain
ASC: apoptosisassociated speck-like
protein containing a
CARD
PYHIN: PYRIN and
HIN-200 domain–
containing
Autoinhibited
NBD-LRR
NBD
PYD
NBD
PYD
The term inflammasome was first coined by
Tschopp and colleagues (1) in 2002 to describe
a high-molecular-weight protein complex that
forms in the cytosol and serves as a platform for
the recruitment and autoproteolytic activation
of certain caspases, notably CASP1, CASP4,
and CASP5 (Figure 1). These caspases, along
with CASP11 and CASP12 in the mouse,
are termed the inflammatory caspases and
are distinct from the caspases involved in the
induction of apoptosis. CASP1 is the best
characterized of the inflammatory caspases and
was first described as the protease required
for the proteolytic cleavage of pro-IL-1β
and pro-IL-18, resulting in their functional
maturation and release (2, 3).
In the decade since the initial description
of an inflammasome, it has become clear
that there are in fact multiple distinct inflammasomes, each of which is activated by
unique stimuli that can include infectious
agents as well as noninfectious stimuli. The
formation of each inflammasome is dictated
by a unique scaffolding protein (Figure 2).
Most of these scaffolding proteins contain
a nucleotide-binding domain (NBD) and
leucine-rich repeats (LRRs) and are thus members of the NBD-LRR (NLR) superfamily.
The LRRs are believed to have two functions
(Figure 1). First, LRRs are thought to be
Monomeric
CASP1
Ligand
CARD
p10
required to maintain NLRs in an autoinhibited
state because deletion of LRRs generally
leads to a constitutively active NLR. Second,
by analogy to the well-characterized ligand
binding function of LRR domains in Toll-like
receptors (TLRs), the LRRs of NLRs are
believed to mediate recognition of pathogenderived (or potentially self-derived) ligands. As
discussed below, however, evidence for ligand
recognition by NLRs is weak. The NBD is
believed to mediate the assembly of NLRs into
an oligomerized state that is critical for the
induction of downstream signaling. In NLRs,
signaling is initiated by CARDs (caspase
activation and recruitment domains) that can
recruit CASP1 directly or by PYRIN domains
that recruit CASP1 via the CARD-PYRINcontaining adaptor protein ASC (Figure 2).
Once recruited to an oligomerized inflammasome, CASP1 is activated via dimerization
and autoproteolysis (Figure 1). Interestingly,
non-NLR proteins such as PYRIN-HIN200 domain–containing (PYHIN) proteins
have also been shown to mediate CASP1
activation (4–8). Although PYHIN proteins
lack NBDs and LRRs, they may nevertheless
exhibit the properties of autoinhibition and
ligand-induced oligomerization (9).
It has become clear that inflammasomes
not only have diverse mechanisms of activation
but can also have diverse functions that extend
beyond cytokine processing. Most notably,
Inflammasome
complex
Pyroptosis
p20
CASP1 autoprocessing
ATP
Oligomerized
NBD-LRR
PYD
CARD
ASC (adaptor)
Pyroptosis
IL-1β/IL-18
Other?
Figure 1
Generic model of inflammasome activation. Cytosolic nucleotide-binding domain, leucine-rich repeat (NBD-LRR) proteins are
maintained in an autoinhibited state until ligand recognition and ATP binding drive oligomerization via the NBD domain.
Recruitment of CASP1 to form the inflammasome complex occurs directly via CARD-CARD interactions or indirectly via the adaptor
ASC. Monomeric CASP1 is believed to be activated by proximity-induced autoproteolysis (autoprocessing), leading to downstream
effector functions such as pyroptosis and processing of pro-IL-1β and pro-IL-18. In some cases, unprocessed CASP1 can also initiate
pyroptosis. Similar mechanisms are postulated for the other inflammatory caspases (CASP4, CASP5, CASP11, and CASP12) but have
not been well characterized. Abbreviations: CARD, caspase activation and recruitment domain; PYD, PYRIN domain.
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inflammasome activation is frequently associated with a rapid and lytic form of host cell
suicide called pyroptosis (10). Inflammasome
activation plays a critical role in mediating
host defense, but inappropriate or excessive
inflammasome activation can also be detrimental to host health. Inflammasomes are
therefore a complex and surprisingly diverse
set of cytosolic sensors that must be stringently
regulated. Here we focus on inflammasome activation by bacteria. We survey what is known
about how bacteria activate inflammasomes
and ask whether it is possible to discern any
general principles that govern inflammasome
activation or function in host defense.
The discovery of NLRs in mammals was
predated by work demonstrating that innate
immunity in plants depends largely on diverse
NBD-LRR proteins (reviewed in 11, 12).
Just as inflammasomes induce a pyroptosis
response, plant NBD-LRR proteins often
induce a rapid cell death response, termed
the hypersensitive response, that is believed
to restrict pathogen replication at sites of infection. Despite the strikingly similar domain
architectures and functions of mammalian and
plant NBD-LRR proteins, evolutionary analyses strongly suggest that NBD-LRR proteins
were absent in the common ancestor of plants
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 2
Murine inflammasome proteins and their domains.
NBD-LRR family members can be categorized
according to the unique domains they encode:
! NLRP1: FIIND; " all other NLRP proteins:
PYRIN domain (PYD); # NLRC: CARD;
$ NAIP: BIR domains. % AIM2 belongs to the
PYHIN family of proteins and uses the HIN-200
domain for ligand recognition. & NLRP10 is the
only family member lacking an LRR domain.
' ASC is an adaptor required by NLRP proteins
for recruitment of CASP1. ( CASP1 contains a
CARD and a protease domain that autoprocesses
into p10 and p20 subunits. CASP11 and CASP12
are additional inflammatory caspases in mice;
CASP4 and CASP5 are additional inflammatory
caspases in humans. Other important differences
between murine and human proteins are listed
(below). (See text for abbreviations.)
and animals and thus evolved independently
(convergently) in each lineage (13, 14). The
independent evolution of plant and animal
NBD-LRR proteins raises a key question:
What about the conjunction of an NBD and
an LRR is so fundamentally advantageous
that evolution would strike at least twice upon
this protein architecture as a solution to the
problem of effective innate immune defense?
NLRP1A-C
1
NBD
FIIND
CARD
LRR
NLRP2, 3, 4A-G, 5, 6, 9A-C, 12, 14
2
PYD
NBD
LRR
NLRC1-5*
3
LRR
CARD
NBD
* NLRC1 = NOD1; NLRC2 = NOD2 and has 2× CARD
NAIP1, 2, 5, 6
4
BIR
BIR BIR
NBD
LRR
AIM2
5
PYD
HIN-200
NLRP10
6
PYD
NBD
ASC
7
PYD
CARD
CASP1
8
CARD
p10
p20
Humans versus mice:
• Humans express only one NLRP1 and it has
• both a PYD and a CARD
• Humans express only one NAIP
• Humans express NLRP7, 8, 11, and 13, which
• are absent in mice
• Humans express only one NLRP4
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NLRC: NLR family,
CARD-containing
T3SS: bacterial type
III secretion system
T4SS: bacterial type
IV secretion system
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NAIP: NLR family,
apoptosis inhibitory
protein (formerly
known as BIRC)
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By articulating the key principles underlying
inflammasome activation by bacteria, this
review attempts to address this question.
RECOGNITION OF BACTERIA
BY INFLAMMASOMES
The NAIP/NLRC4 Inflammasome
Activation of NLRC4. NLRC4 (also known
as IPAF, CLAN, and CARD12) was identified on the basis of similarity to apoptotic
protease-activating factor-1 (APAF-1) and was
shown to recruit and activate CASP1 (15–17).
Initial studies of Nlrc4−/− mice demonstrated
that Nlrc4 is essential for CASP1 activation
by Salmonella enterica serovar Typhimurium
(S. Typhimurium) (18). Since then, CASP1
activation by a large number of additional
pathogens, including Legionella pneumophila
(19–21), Pseudomonas aeruginosa (22–24), and
Shigella flexneri (25), has been shown to
depend on NLRC4. Activation of the NLRC4
inflammasome by bacteria depends largely
[e.g., S. Typhimurium (26–28)] or almost
entirely [e.g., L. pneumophila (19, 20, 27, 29)]
on bacterial flagellin expression. Indeed, the
cytosolic presence of the flagellin protein—or
just its C-terminal 35 amino acids—is sufficient
to activate NLRC4 (26–29). C-terminal truncation of the flagellin protein (20) or mutation
of three conserved C-terminal hydrophobic
amino acids (27) abolished recognition of
flagellin by NLRC4 (20, 26, 27, 30–33). The
C terminus comprises part of the D0 region
of flagellin and is distinct from the D1 region,
which is recognized by the TLR5 cell-surface
flagellin receptor (34, 35). Importantly, direct
binding of flagellin to NLRC4 has not been
demonstrated, and in fact, flagellin activation
of NLRC4 appears to be indirect (see below).
Activation of NLRC4 by flagellated bacteria
also requires bacterial expression of specialized
secretion systems, such as the S. Typhimurium
SPI-1 type III secretion system (T3SS) or the L.
pneumophila Dot/Icm type IV secretion system
(T4SS). The physiological role of these secretion systems is to translocate bacterial virulence
von Moltke et al.
factors into the host cell cytosol, but considerable evidence suggests they can also translocate flagellin (27, 36). Cytosolic translocation
of flagellin is presumably inadvertent, because
flagellin has no known function in the host cell
cytosol. The natural hosts of L. pneumophila are
amoebae, not mammals. Thus L. pneumophila
may not have experienced selective pressure
to evade NLRC4, whereas other mammalianadapted pathogens (e.g., S. Typhimurium and
Listeria monocytogenes) appear to have evolved
regulatory mechanisms that limit flagellin expression in host cells (see below).
Interestingly, several flagellin-deficient
bacteria, most notably Shigella, are also capable
of activating NLRC4 (22, 25, 31, 32, 37). Activation of NLRC4 by these pathogens requires
expression of a T3SS (22, 25, 31, 32, 37). Miao
and colleagues (31) provided an explanation for
the flagellin-independent activation of NLRC4
by demonstrating that the inner rod proteins of
several bacterial T3SSs are capable of activating
NLRC4. Like the D0 domain of flagellin that
activates NLRC4, the T3SS inner rod proteins
are believed to adopt an α-helical secondary
structure and oligomerize to form a proteintranslocation channel (31, 38). However,
despite analogous functional and structural
properties, flagellin and T3SS inner rod proteins share little primary amino acid sequence
similarity. The question of how NLRC4 could
respond to structurally diverse ligands has
recently been resolved by the discovery that
cytosolic recognition of flagellin and inner rod
proteins is mediated not directly by NLRC4
but instead by the NAIP subfamily of NLRs
(32, 37). According to the current model,
NAIP5 and NAIP6 directly recognize flagellin,
whereas NAIP2 recognizes T3SS rod proteins
(Figure 3). Epistasis analysis indicates that
activated NAIPs induce downstream oligomerization and activation of NLRC4 (37), which
appear to be required for the downstream
recruitment and activation of CASP1 by
NLRC4. In addition, CASP1 activation by
mouse NLRC4 requires phosphorylation of
NLRC4 on serine 533. PKCδ may be the
kinase that phosphorylates NLRC4, but other
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Phagosome
S. Typhimurium
L. pneumophila
T4SS
IL-1β/IL-18,
pyroptosis
CASP1
NAIP5/6
NLRC4
NAIP5/6
CASP1
processing
ASC (adaptor)
CASP1
P. aeruginosa
SS
Pyroptosis
S. flexneri
T3
NAIP2
NLRC4
NAIP2
= Flagellin
= T3SS rod
T3SS
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Pyroptosis
T3SS
Figure 3
Activation of NAIP/NLRC4 inflammasomes by bacteria. NAIP/NLRC4 inflammasomes are activated
following detection of flagellin or T3SS rod proteins secreted into the host cytosol during bacterial infection
by Legionella pneumophila, Salmonella enterica serovar Typhimurium, Shigella flexneri, and Pseudomonas
aeruginosa. Ligand specificity is determined by the NAIP proteins: NAIP5 and NAIP6 recognize flagellin,
and NAIP2 recognizes the T3SS rod. The CARD-containing NLRC4 serves as an adaptor for recruitment
of CASP1 downstream of the NAIPs. Phosphorylation of NLRC4 is required for NLRC4 function. NAIP,
NLRC4, and CASP1 are sufficient to initiate pyroptosis, but processing of the IL-1β and IL-18 cytokines
requires recruitment of the ASC adaptor into the complex. Abbreviations: T3SS, type III secretion system;
T4SS, type IV secretion system; CARD, caspase activation and recruitment domain.
kinases may also be involved (39). It remains
unclear how NLRC4 kinases are activated
and whether this occurs downstream of ligand
recognition by NAIPs.
NAIPs. Naip5 was originally identified as a
gene that mediates resistance of B6 mouse
macrophages to intracellular replication
of L. pneumophila (40, 41). By contrast,
macrophages from the A/J mouse strain carry
a partially defective Naip5 allele and are thus
highly permissive to L. pneumophila replication.
Naip5 is one of several tandemly repeated Naip
genes in mice, but Naip5 is the only Naip gene
essential for the restriction of L. pneumophila
replication, despite the expression in B6 mice
of functional Naip1, 2, 5, and 6 transcripts (40,
41). Among different inbred mouse strains, the
Naip locus contains variable numbers of Naip
paralogs. Individual Naip genes also vary from
strain to strain; for example, the A/J Naip5
allele encodes 14 amino acid polymorphisms
compared with the B6 allele and appears to be
expressed at lower levels (41). However, the
precise polymorphism(s) in Naip5 responsible
for the permissiveness of A/J remains unknown
(42). The requirement for Naip5 in restriction
of L. pneumophila replication was confirmed
by the generation of Naip5−/− mice on the B6
background (27).
NAIP proteins contain a central NBD and
C-terminal LRRs, but they differ from other
NLRs in that they contain three N-terminal
baculovirus inhibitor-of-apoptosis repeats
(BIRs). The BIRs appear critical for NAIPs to
activate NLRC4 (37), but the mechanism of
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BIR: baculovirus
inhibitor-of-apoptosis
repeat
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NLRP: NLR family,
PYRIN domain–
containing (formerly
known as NALP)
11:58
activation is completely unknown. Consistent
with the proposed role of the NBD in mediating protein-protein oligomerization, mutation
of the NBD abolishes the ability of NAIP5
to induce NLRC4 oligomerization (37). The
LRRs of NAIPs appear to be responsible for
ligand recognition (E. Kofoed, J. Tenthorey,
and R. Vance, unpublished observations).
Physical association of bacterial flagellin to
NAIP5 and NAIP6 (but not NAIP2) has been
shown recently using a variety of biochemical
approaches (32, 37). Similarly, T3SS inner
rod proteins associate with NAIP2 (but not
NAIP5). These biochemical studies were supported by knockout and knockdown studies.
For example, Naip5−/− macrophages exhibit
a selective defect in the response to flagellin
but respond normally to inner rod proteins
(37, 43). Conversely, knockdown of Naip2
selectively affects NLRC4 activation in response to inner rod proteins but does not affect
flagellin recognition (32, 37). Taken together,
these observations suggest a receptor-ligand
model for activation of the NAIP/NLRC4
inflammasomes (Figure 3) but do not rule out
the involvement of additional proteins.
Human NAIP. In humans, L. pneumophila
is the causative agent of a severe pneumonia
known as Legionnaires’ disease (44). Human
alveolar macrophages and monocytes cultured
ex vivo generally support robust L. pneumophila
replication, which contrasts with the restricted
replication seen in most mouse macrophages
(45, 46). This difference between human and
mouse macrophages is likely explained by the
recent report that human NAIP (hNAIP) does
not respond to flagellin or T3SS inner rod proteins but instead responds specifically to the
T3SS needle protein from Chromobacterium violaceum (32). Because C. violaceum is not a significant human pathogen, it is important that T3SS
needle proteins from a variety of clinically important pathogens, such as Shigella, Salmonella,
and Burkholderia, are also detected by hNAIP
(32). Whether hNAIP detection of T3SS needle proteins is relevant during infection by these
78
von Moltke et al.
or other common bacterial pathogens warrants
further study.
It is striking that the needle proteins recognized by hNAIP are, like the mouse NAIP
ligands, α-helical proteins that oligomerize to
form a protein-translocation channel. Because
these ligands share little in the way of primary sequence, a tempting hypothesis is that all
NAIP ligands share a unique secondary structural characteristic that renders them suitable as
targets of innate immune recognition. Unlike
the tandemly duplicated arrays of Naip genes in
mice, the human NAIP gene locus is believed
to harbor only a single full-length functional
ortholog; however, copy number variation has
been reported between geographic human populations (47, 48). It is therefore possible that in
humans, as in mice, NAIP function is variable
among individuals.
The NLRP1 Inflammasome
NLRP1 was a key constituent of the originally
identified inflammasome (1), but the physiological signals that activate NLRP1 were initially unknown. It has since become apparent
that many mammals have NLRP1 orthologs,
and some species, such as mice, harbor multiple paralogs exhibiting high allelic variation
(49, 50). Here we focus on rodent NLRP1 proteins because their activation and in vivo roles
are best understood.
Activation of NLRP1B by anthrax lethal
toxin. One of the main virulence factors
of Bacillus anthracis, the causative agent of
anthrax, is a bipartite protein toxin termed
lethal toxin (LeTx). LeTx is composed of
protective antigen (PA) and lethal factor (LF).
PA forms a channel that translocates LF, a zinc
metalloprotease, into the host cell cytosol (51).
The best-characterized proteolytic targets of
LF are the MAP kinase kinases (MAPKKs)
1–4 and 6–7. LF-mediated cleavage of
MAPKKs results in their inactivation, thereby
impairing numerous host defense pathways
including neutrophil chemotaxis, cytokine and
chemokine production, antigen presentation,
and costimulation (reviewed in 52). In addition
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to disrupting MAPKK signaling, LeTx can also
cause pyroptosis of macrophages from specific
strains of mice (50, 53, 54); this responsiveness
to LeTx was genetically mapped to the Nlrp1b
gene (50). Although NLRP1B is sufficient to
confer LeTx sensitivity, its requirement was
only recently verified through the creation
of an Nlrp1b knockout mouse line (55). Rat
macrophages also undergo pyroptosis in
response to LeTx; this responsiveness was
genetically mapped to a locus containing the
Nlrp1 gene, suggesting that NLRP1 activation
is mechanistically conserved between mice and
rats (56, 57). It is worth noting that LeTx can
also induce a slower cell death in murine and
human macrophages that is independent of the
NLRP1B inflammasome and is instead related
to disruption of p38 MAP kinase signaling
downstream of TLRs (58, 59).
The mechanism of NLRP1B activation by
LF is not fully resolved. NLRP1B activation
requires the proteolytic activity of LF and is
not due to recognition of LF protein alone, as
catalytically dead LF does not induce pyroptosis (54, 60). As mentioned above, the known
proteolytic substrates of LF are MAPKKs 1–4
and 6–7. Although plant NLRs respond to
disrupted kinase signaling pathways, no strong
connection between MAPKK cleavage and
NLRP1B activation has been made. Recently
it was shown that LF cleaves the N terminus
of the responsive rat NLRP1 allele (from CDF
rats) but does not cleave the nonresponsive
allele (from Lewis rats). Importantly, cleavage
of NLRP1 was suggested to be required for
its activation, as mutations that made NLRP1
cleavage resistant abolished its ability to activate CASP1 (61). Although it is likely that the
LeTx-sensitive alleles of mouse and rat NLRP1
share a conserved mechanism of activation, the
site cleaved in rat NLRP1 does not appear to
be conserved in any mouse NLRP1B allele.
Thus it remains to be determined whether
mouse NLRP1B is also directly cleaved by LF.
Additionally, it is unknown whether cleavage
by LF is sufficient for rat or mouse NLRP1B
activation; it is possible that other LF substrates
participate. Supporting this latter possibility,
several groups have shown that proteasome
activity is required for NLRP1B activation and
that this activity is a specific requirement for
activation of NLRP1B but not of NLRP3 or
NLRC4 (54, 62, 63). In addition, inhibition
of the N-end rule degradation pathway blocks
NLRP1B activation (64). Taken together,
these observations suggest a model in which
LF cleaves cellular substrates that act as
negative regulators of NLRP1B, and these
substrates become destabilized according to the
N-end rule degradation pathway (Figure 4).
Human NLRP1. Human NLRP1 does not
respond to LeTx, and human macrophages do
not undergo pyroptosis in response to LeTx
(58, 65). Use of a cell-free reconstituted system suggested that muramyl dipeptide (MDP),
a fragment of bacterial peptidoglycan, might be
an agonist of human NLRP1 (66). However,
although MDP has been reported to activate
CASP1 in human macrophages, the requirement for NLRP1 is only partial, and the effect of
MDP may result primarily from its established
ability to activate NOD2-dependent priming
of pro-IL-1β and NLRP3 (67–69). Thus, the
function of human NLRP1 remains poorly understood.
A unique domain: FIIND. Both the human
and mouse NLRP1 have a unique domain,
termed the FIIND (domain with function to
find), that is not found in other NLRs (70).
Recently, investigators suggested that this domain resembles the ZU-5 and UPA domains
found in proteins such as PIDD and UNC5B.
In PIDD, the ZU-5/UPA domains exhibit an
unusual ability to undergo autoproteolysis at a
conserved HFS site (49, 71). Similar spontaneous cleavage was observed in the FIINDs of
human NLRP1 and murine NLRP1B (49, 72).
Mutations that inactivate this self-cleavage disrupt the ability of NLRP1B to respond to LeTx
(72). Thus, self-cleavage of NLRP1B within the
FIIND, which occurs independently of LeTx,
appears to be a maturation step required for
NLRP1B function.
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B. anthracis
Toxin
secretion
PA
LF
TLR
Toxin receptor
H+
H+
H+
LF translocation
into the cytosol
MAPKKK
Unprocessed FIIND
NBD
FIIND
MAPKK
CARD
Inactive
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LRR
Autoprocessed mature form
MAPK
Other
substrates?
?
?
Cleavage of rat
NLRP1 N terminus
Host defense
Proteasome
degradation
Sufficient?
CASP1
Pyroptosis
ASC
CASP1
processing
IL-1β/IL-18,
pyroptosis
Figure 4
Activation of the NLRP1B inflammasome by anthrax lethal toxin. Bacillus anthracis secretes lethal factor (LF)
and protective antigen (PA), which together form lethal toxin (LeTx). PA binds to the toxin receptor on host
cells, leading to PA processing, PA oligomerization, and binding of LF. LeTx is then endocytosed, and in an
acidified endosome LF is translocated into the host cell cytosol. LF rapidly cleaves and inactivates MAP
kinase kinases (MAPKKs), interfering with many host defense pathways. NLRP1 must undergo
autoproteolytic processing in the FIIND domain prior to LF activation. LF cleaves the N terminus of
sensitive rat NLRP1 alleles, but it is unknown whether this cleavage is sufficient for activation. As the activity
of the proteasome is required for NLRP1 activation, LF may also cleave unidentified substrates that become
destabilized and degraded by the proteasome and can therefore no longer act as negative regulators of
NLRP1. Once NLRP1B is activated, it can recruit CASP1 directly and induce pyroptosis; upon further
recruitment of ASC, the NLRP1B inflammasome can also process IL-1β and IL-18 cytokines.
The NLRP3 Inflammasome
NLRP3 agonists. Unlike the Nlrp1b, Nlrc4,
and the Naip genes, Nlrp3 is not constitutively
expressed in most resting cells. Activation of the
80
von Moltke et al.
NLRP3 inflammasome therefore requires two
signals: (a) NF-κB-dependent transcriptional
induction downstream of TLR or NOD ligands
or inflammatory cytokines and (b) an agonist
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to induce oligomerization. The first signal, the
transcriptional priming, represents a critical
checkpoint: Once primed, the NLRP3 inflammasome is activated by an astonishing array of
stimuli, at least in vitro. These stimuli include,
but are not limited to, crystalline or particulate
matter (e.g., uric acid crystals, asbestos, alum)
(73–75), ATP signaling through the P2X7
receptor (76), viral infections (77), fungal products (78), bacterial RNA and DNA (79–81), and
numerous bacterial toxins and effector proteins
(Supplemental Table 1; see the Supplemental Material link in the online version of this
article or at http://www.annualreviews.org).
NLRP3 also appears to be activated in response to the gut microbiota (see below).
Although structurally distinct, many of the
bacterial products that activate NLRP3 (e.g.,
nigericin, listeriolysin O, hemolysins) share
pore-forming activity, suggesting that this
“pattern of pathogenesis” (82) is somehow
detected by the NLRP3 inflammasome.
In evaluating NLRP3 agonists, it is important to distinguish their relative contributions
to priming versus oligomerization of NLRP3.
In some contexts, notably human blood monocytes and THP-1 cells, secretion of autocrine
ATP may cause NLRP3 to oligomerize spontaneously once primed (83). Accordingly, stimuli such as lipopolysaccharide (LPS) and MDP,
which in most cells [e.g., murine macrophages
(76)] merely prime NLRP3, appear sufficient
for NLRP3 oligomerization in human monocytes (84, 85).
Mechanism of activation. Given the variety
of NLRP3 agonists, it is generally accepted that
they do not act as direct ligands for NLRP3
but instead all converge on one or more disruptions of host physiology that are sensed by
the NLRP3 inflammasome. A unifying mechanism for NLRP3 activation remains elusive
(Figure 5). In the case of crystalline and particulate NLRP3 stimuli, the common mechanism
may be lysosomal destabilization resulting
in cytosolic release of lysosomal enzymes,
such as cathepsin B, that somehow activate
NLRP3 (86). For noncrystalline NLRP3
stimuli—including bacterial pathogens, ATP,
and nigericin—guanylate-binding protein
5 (GBP5) is required in interferon (IFN)γ-primed macrophages for robust NLRP3
oligomerization, although the mechanism of
GBP5 and NLRP3 interaction remains unclear
(87). Importantly, NLRP3 is readily activated
in the absence of IFN-γ priming; therefore,
it seems GBP5 mostly functions to amplify
NLRP3 signaling.
Another unifying model of NLRP3 activation postulates a role for reactive oxygen
species (ROS) generated by ATP signaling
through the P2X7 receptor (88), by NADPHoxidase during phagocytosis of particulate stimuli (75), or by mitochondria (89, 90). How
ROS might promote NLRP3 oligomerization remains unresolved. One report suggested that ROS can dissociate thioredoxininteracting protein (TXNIP) from thioredoxin,
allowing TXNIP to act as a ligand for NLRP3
(91); however, more recent data demonstrating that NLRP3 signaling is unchanged in
Txnip−/− macrophages strongly contradict this
model (92). The ROS hypothesis also remains controversial, as ROS inhibitors may
only block NLRP3 priming (93). Furthermore,
macrophages deficient in NADPH-oxidase
subunits respond normally to NLRP3 agonists
(94–96). A model of NLRP3 ligand delivery
through large membrane hemichannels formed
by pannexin-1 (PANX1) (97) was similarly undermined by data from knockout mice demonstrating that Panx1−/− macrophages activate
the NLRP3 inflammasome normally (98).
NLRP3 activation by all agonists can be
blocked by high extracellular potassium (K+ ),
suggesting that efflux of intracellular K+ is a
downstream convergence point (99); however,
high extracellular K+ also blocks NLRP1B
oligomerization (62). It seems, therefore, that
additional mechanisms must exist to explain
the activation of these distinct inflammasomes
by distinct stimuli; the characterization of
these mechanisms remains a central challenge
for the field. One emerging model suggests
that NLRP3 agonists converge on the mobilization of calcium (Ca2+ ), which in turn causes
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Supplemental Material
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TLR
Pore-forming
toxins
Phagosome
Nucleus
Cathepsin B,
etc.
Nlrp3
Signal 1
?
K+
efflux?
Ca2+ influx?
Secreted
effector
?
?
?
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ROS
?
Mitochondrion
IL-1β/IL-18,
pyroptosis
Signal 2
Inactivated
endogenous
ligand?
CASP1
ASC
IFN-γ
NLRP3
?
?
GBP5
Autoprocessing
Figure 5
Activation of the NLRP3 inflammasome by bacteria. Activation of the NLPR3 inflammasome requires two
signals: Transcriptional induction of Nlrp3 (Signal 1) and an agonist to drive NLRP3 oligomerization (Signal
2). The mechanism underlying the second signal remains unresolved. The model shown here postulates an
endogenous ligand for NLRP3 that is activated downstream of one or more disruptions in host physiology,
including generation of reactive oxygen species (ROS), potassium (K+ ) efflux, calcium (Ca2+ ) influx, and/or
phagolysosomal rupture or leakage. It is unclear how bacteria cause these disruptions, although in many
cases activation of NLRP3 requires secreted bacterial effectors or toxins. In IFN-γ-stimulated macrophages,
guanylate-binding protein 5 (GBP5) acts to promote NLRP3 oligomerization. Because NLRP3 lacks a
CARD, the adaptor ASC is required for recruitment and activation of CASP1. Abbreviation: TLR, Toll-like
receptor.
release of mitochondrial ligand(s) that activate
NLRP3 (100). Further experiments are needed
to confirm these findings and to address many
remaining questions. In particular, what is the
difference (if any) between NLRP3 agonists
and all other signals that mobilize Ca2+ ?
Although not the focus of this review,
numerous noninfectious disease processes—
including type II diabetes, atherosclerosis, and
macular degeneration—have been linked to
the NLRP3 inflammasome. Point mutations in
Nlrp3 underlie the inheritable familial cold autoinflammatory syndrome, neonatal onset multisystem inflammatory disease, and Muckle-
AIM2: absent in
melanoma 2
82
von Moltke et al.
Wells syndrome. For reviews, see References
101 and 102.
The AIM2 Inflammasome
Like the NLRP3 inflammasome, the AIM2 inflammasome requires transcriptional priming,
but this priming occurs downstream of type
I IFN signaling rather than NF-κB signaling. This pathway was first uncovered in studies of Francisella tularensis activation of CASP1
(103), which required signaling through the
IFN receptor but not through any of the known
NBD-LRR inflammasomes. Around the same
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time, a DNA-sensing inflammasome was hypothesized on the basis of CASP1 activation in
macrophages after double-stranded DNA (dsDNA) is delivered to the cytosol (81). These
two observations were unified by the identification of AIM2 as the inflammasome that detects
DNA released into the cytosol upon lysis of several pathogens, including F. tularensis (4–8).
AIM2 belongs to a family of IFN-inducible
PYHIN proteins that contain PYRIN and
HIN-200 DNA-binding domains. Like the
NLRP proteins, AIM2 includes a PYRIN
domain for recruitment of CASP1 via ASC;
however, AIM2 lacks an NBD, and a HIN-200
domain replaces the LRR. Thus, AIM2 is
structurally distinct from the other known inflammasomes. Despite these differences, AIM2
can nonetheless orchestrate the oligomerization of a large inflammasome complex (8), and
the downstream pathways (cytokine processing
and pyroptosis) are indistinguishable from
those of NBD-LRR proteins. Another member
of the PYHIN superfamily, IFI16, has also
been proposed to function in CASP1 activation
in response to Kaposi’s sarcoma–associated
herpesvirus (104).
The generation of Aim2−/− mice confirmed
that AIM2 is indeed the F. tularensis–sensing inflammasome (8, 105, 106) and that AIM2 can
also detect L. monocytogenes (105, 107–110). The
marked susceptibility of Aim2−/− mice to F. tularensis provides one of the best examples of the
critical role inflammasomes can play in host
defense against bacteria. In response to both
L. monocytogenes and F. tularensis, activation of
AIM2 results from bacterial lysis and release of
DNA that occur upon bacterial entry into the
cytosol (8, 106, 107, 111) (Figure 6). Recently,
it was suggested that L. pneumophila that lacks its
effector protein SdhA might also activate AIM2
via release of bacterial DNA into the host cell
cytosol (112).
AIM2 is the only inflammasome for which
direct interaction of sensor and ligand has been
visualized in an infected cell (8, 106) or analyzed
crystallographically (9). The optimal ligand for
AIM2 activation is dsDNA of a sufficient length
(>80 bp). Modeling suggests an 80-bp dsDNA
molecule could accommodate up to 20 AIM2
HIN-200 domains (9). It was therefore proposed that instead of using an NBD to mediate oligomerization and downstream signaling,
AIM2 multimerizes on a DNA template. Thus,
oligomerization may be a conserved principle
of inflammasome signaling despite the significant differences in the protein domains found
in NLR versus PYHIN inflammasomes.
Other NBD-LRR Proteins
Most members of the NBD-LRR protein family remain poorly characterized. NLRP12 was
found to activate CASP1 even before the term
inflammasome was coined (113), and polymorphisms in NLRP12, like those in NLRP3, are
associated with hereditary inflammatory disorders in humans (114). Studies of Nlrp12−/−
mice have uncovered diverse roles for NLRP12
in immunity and disease. Two groups described
a role for NLRP12 in colonic inflammation
(see below) (115, 116); additionally, in a model
of contact hypersensitivity, NLRP12 appears
to be required for migration of myeloid cells
(117). There are also reports of a role for
NLRP12 in negative regulation of NF-κB
(115). Interestingly, a recent report also found
a clear role for NLRP12 in immune defense
against bacterial pathogens. Nlrp12−/− mice
show increased bacterial load and decreased
survival when infected with Yersinia pestis
(118). NLRP12 appears to provide protection
via induction of IL-18, which in turn induces
IFN-γ; accordingly, Il18−/− and Ifnγ −/− mice
are also susceptible to Y. pestis. Although the
immune defect in Nlrp12−/− mice appears selective for Yersinia, it remains unclear whether
NLRP12 detects the pathogen directly or by
monitoring disruptions in host homeostasis.
NLRP7, which is absent in mice, assembles
an inflammasome in response to microbial
acylated lipopeptides (119). Lastly, it should be
noted that several NBD-LRR proteins (e.g.,
CIITA and NLRC5) seem to have diverse
non-inflammasome-related functions (e.g., in
regulation of gene expression). One further
example may be NLRP6, which has been
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Phagosome
L. monocytogenes
F. tularensis
Type I IFN
Bacterial lysis
Nucleus
Signal 1
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Aim2
DNA
Signal 2
AIM2
ASC
CASP1
IL-1β/IL-18,
pyroptosis
Autoprocessing
Figure 6
Activation of the AIM2 inflammasome. Activation of the AIM2 inflammasome requires two signals:
Transcriptional induction of Aim2 downstream of type I IFN signaling (Signal 1) and cytosolic
double-stranded DNA (Signal 2). In the case of bacterial infection, e.g., by Listeria monocytogenes or
Francisella tularensis, DNA is released if invading bacteria undergo lysis in the cytosol. Because AIM2 lacks a
CARD, the adaptor ASC is required for recruitment and activation of CASP1.
suggested to negatively regulate NF-κB and
MAP kinase signaling downstream of TLR
activation in a manner apparently independent
of CASP1 (120). At the same time, the role of
NLRP6 in regulating intestinal inflammation
appears to be IL-18-, CASP1-, and ASCdependent (121, 122), suggesting that NLRP6
can form an inflammasome. Although it is
surprising that NLRP6 exhibits such diverse
functions, it is important not to assume that
all NBD-LRR proteins act via inflammasome
formation.
Noncanonical Inflammasomes
CASP1 is not the only inflammasome effector
caspase. After confirming earlier reports (123)
that the available Casp1−/− mice are actually
Casp1/11−/− double knockouts, investigators
showed that pyroptosis induced by Escherichia
coli and Vibrio cholerae is CASP11 dependent
84
von Moltke et al.
and CASP1 independent (124). Interestingly,
IL-1β processing and secretion downstream
of E. coli and V. cholerae require NLRP3 and
CASP1 in addition to CASP11 (123, 124),
suggesting that CASP11 can induce pyroptosis
directly but can only induce IL-1β via downstream activation of NLRP3 and CASP1 (125,
126, 127).
Surprisingly, pyroptosis induced by the
noncatalytic B subunit of cholera toxin (CTB)
also activates CASP11, although it is not clear
how CTB would be sensed (124). One possible
explanation is that CTB’s ability to activate
CASP11 may result from LPS contamination
of the B-subunit preparation, though the trafficking function of the B subunit may also play
a role. The idea that LPS can activate CASP11
is supported by recent reports that CASP11
activation by gram negative bacteria requires
TLR4, TRIF, and the type I IFN receptor
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(125, 126). CASP11 can also be activated by
purified LPS (125, 126) or type I IFN (125), and
Casp11−/− mice are resistant to LPS-induced
toxic shock (124, 127). Interestingly, mice
deficient in both Casp1 and Casp11 were more
resistant to S. Typhimurium compared with
Casp1 singly deficient mice (126). As a result,
Casp11-dependent pyroptosis was proposed to
be effective at restricting bacterial replication
only if accompanied by a Casp1-dependent
recruitment of neutrophils, which eliminate
extracellular bacteria.
It remains unclear how CASP11 is activated. Pyroptosis downstream of known inflammasomes (NAIP/NLRC4, AIM2, NLRP1B)
does not appear to require CASP11 (124, 128).
Mechanistically, it is possible that a novel noncanonical inflammasome serves as a platform
for CASP11 activation. Alternatively, because
CASP11 expression is inducible (125–127),
CASP11 may autoactivate upon expression,
though one report suggests expression alone is
insufficient for CASP11 activation (126). There
is no direct ortholog of CASP11 in humans;
instead, humans express CASP4 and CASP5
(which are absent in mice). Although CASP5
associates with the NLRP1 inflammasome in
humans (1), it is not clear whether its functional
role in the response to LPS in humans is similar
to that of CASP11 in mice.
Accumulating evidence also suggests that
inflammasome complexes include more than
just NLR/PYHIN proteins, ASC, and CASP1.
For example, the inhibitor of apoptosis (IAP)
proteins cIAP1 and cIAP2 regulate inflammasomes, although published reports disagree
about whether this interaction is activating
or inhibitory (129, 130). The double-stranded
RNA–dependent protein kinase PKR has also
been proposed to regulate multiple inflammasomes (131).
EFFECTOR FUNCTIONS
OF INFLAMMASOMES
The development of knockout mice deficient for key inflammasome components has
been critical for studying the contribution of
inflammasomes to host defense and disease
pathogenesis in vitro and in vivo. In some cases
[notably Mycobacterium tuberculosis (132, 133)],
in vitro activation of an inflammasome by a
bacterial pathogen does not clearly correlate
with an in vivo phenotype, but overwhelmingly,
disruption of the relevant inflammasome leads
to an increase in bacterial replication (colonyforming units) and a decrease in survival following bacterial infection of mice. Conversely,
forced expression of inflammasome ligands or
deletion of inflammasome inhibitors in bacterial pathogens that normally evade inflammasome detection leads to bacterial restriction (22,
30, 134, 135). Therefore, inflammasomes are
an important component of innate immunity to
bacterial pathogens, as summarized in Table 1.
The control by inflammasomes of bacterial
infections results from the inflammasomes’
downstream effector functions, of which pyroptosis and the processing and secretion of proIL-1β/pro-IL-18 are the best characterized.
Pyroptosis
Pyroptosis is thought to result from osmotic
pressure generated by CASP1-dependent
formation of membrane pores (136) and is
characterized by the rapid release of cytosolic
contents. Pyroptosis is not associated with the
formation of characteristic apoptotic membrane blebs and does not require the apoptotic
caspases (e.g., CASP3, 7, 9). Pyroptosis has
been described in macrophages and dendritic
cells but may also occur in other cell types.
Surprisingly, cell lysis downstream of some
NLRP3 agonists is largely undetectable or
occurs hours after CASP1 autoproteolysis
and IL-1β/IL-18 processing (74, 137). It is
unclear why CASP1 activated via NLRP3
activates pyroptosis only inefficiently, whereas
CASP1 activated downstream of NLRC4 or
NLRP1B induces pyroptosis within minutes
of inflammasome oligomerization in nearly
all cells. The CASP1 substrate(s) required for
pyroptosis remains elusive (138–140).
Autoproteolysis has long been considered
an essential step in activation of CASP1. Yet,
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Table 1 Contribution of inflammasome responses to bacterial restriction and host survivala
Bacteria
CASP1
ASC
CFU+
CFU+
Aeromonas veronii
CFU+
CFU+
Bacillus anthracis
SVL−
Anaplasma
phagocytophilum
NBD/PYHIN
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CFU+, SVL−
Burkholderia
thailandensis
CFU+
CFU = , SVL−
CFU = /+
Chlamydia
muridarum
Chlamydia
pneumoniae
CFU+, SVL−
Chlamydia
trachomatis
CFU =
Citrobacter
rodentium
CFU+
Escherichia coli
(O21:H+)
CFU = ,
SVL+
Francisella
tularensis
CFU+, SVL−
Klebsiella
pneumoniae
Legionella
pneumophila
CFU+
Listeria
monocytogenes
CFU = /+,
SVL−
Mycobacterium
tuberculosis
CFU = /+,
SVL = /−
CFU+, SVL−
CFU = ,
SVL = /−
CFU =
CFU = ,
SVL = /−
199
Nlrp1b: CFU+, SVL−
Il1R: SVL−
149, 150
Il1R: CFU+
201
Nlrc4: CFU+, SVL−
Nlrp3: CFU = , SVL−
Il1R: CFU = , SVL+
Il18: CFU+, SVL−
Il1β/Il18: CFU+,
SVL−
153
Il1β/Il18: CFU =
30
Il1β: CFU = /+
202
Nlrc4: CFU =
Nlrp3: CFU =
Salmonella enterica
serovar
Typhimurium
CFU+, SVL−
Shigella flexneri
CFU+, SVL−
CFU = /−,
SVL = /−
CFU+
Staphylococcus
aureus
200
Il1R: CFU =
203, 204
Il18: CFU =
205, 206
Nlrc4: CFU+
Nlrp3: CFU+
Il1β: CFU+
Il18: CFU+
207
Nlrc4: CFU = , SVL+
Naip5: CFU = , SVL+
Il1β: CFU = , SVL+
196
Nlrc4: CFU = , SVL =
Aim2: CFU+, SVL−
Nlrc4: CFU =
Nlrp3: CFU = , SVL = /−
Nlrc4: CFU+
GBP5: CFU+
Nlrp6: SVL+, CFU =
Nlrc4: SVL =
Nlrp3: CFU = , SVL =
Aim2: CFU+, SVL =
Nlrc4: CFU+, SVL =
Pseudomonas
aeruginosa
References
Il18: CFU+
Bordetella pertussis
Burkholderia
pseudomallei
Cytokines
Nlrc4: CFU+
Nlrp3: CFU =
Nlrc4: CFU = /+, SVL−
Nlrp3: CFU = , SVL =
Nlrc4; Nlrp3: CFU+
Nlrp6: CFU =
Nlrp3: CFU = , SVL−
8, 106, 208
209
Il1R: CFU+
Il18: CFU =
19, 21, 30,
143, 210, 211
Il1R: CFU = , SVL =
87, 120, 135,
212, 213
Il1β: CFU+, SVL−
Il1R: CFU+, SVL−
Il18: CFU = ,
SVL = /−
132, 133, 147,
214, 215, 216
Il1R: CFU+, SVL−
Il1β: CFU = /+,
SVL = /−
Il18: CFU+, SVL−
Il1β/Il18: CFU+,
SVL−
120, 157,
218–221c
Il1R: CFU−
Il1β: CFU+
22,b 24, 148,
217
Il1β: CFU =
Il18: CFU+
154
Il1R: SVL =
Il1β: CFU+
222, 223
(Continued)
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Table 1 (Continued)
Bacteria
Streptococcus
agalactiae
(Group B)
CASP1
CFU+, SVL−
Streptococcus
pneumoniae
ASC
Nlrp3: CFU+, SVL−
CFU+, SVL−
Nlrp3: CFU = /+, SVL−
Nlrp12: CFU+, SVL−
Nlrp3: CFU+, SVL−
Yersinia pestis
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Yersinia
pseudotuberculosis
NBD/PYHIN
CFU+, SVL−
CFU+
CFU+
Cytokines
References
224
225, 226
118d
Il1R: SVL−
Il1β: SVL−
Il1β/Il18: CFU+,
SVL−
134e
Nlrp3: CFU =
a
This table summarizes changes in colony-forming units (CFU) and/or host survival (SVL) from in vivo models of bacterial pathogenesis in mice deficient
for inflammasome-related genes (column headings). (+) increase; (−) decrease; ( = ) no change.
b
CFU difference in Reference 22 seen only in the absence of ExoU.
c
Salmonella enterica serovar Typhimurium results vary depending on status of Nramp1 gene and route of infection (intraperitoneal versus oral).
d
CFU and survival differences observed only with Y. pestis expressing hexaacylated lipopolysaccharide.
e
CFU differences enhanced in absence of inhibitory Y. pseudotuberculosis effectors.
in the absence of the adaptor protein ASC,
CASP1 fails to undergo autoproteolysis (18,
25, 141) but can nevertheless induce pyroptosis
in a manner requiring CASP1 protease activity
(128). Induction of CASP1-dependent, ASCindependent pyroptosis occurs in response to
activation of NLRC4 and NLRP1B (which
contain CARDs) but not AIM2 or NLRP3
(which lack CARDs). The CARDs were proposed to mediate direct recruitment, dimerization, and activation of CASP1 without a
requirement for ASC, which appears necessary
only for CASP1 autoproteolysis and IL-1β/IL18 processing. In fact, pyroptosis is accelerated
in the absence of ASC, suggesting an inhibitory
role for this adaptor (141). The molecular
mechanisms that allow uncleaved CASP1 to
induce pyroptosis but not cytokine processing
remain uncertain. Inflammasomes lacking
CARDs (e.g., NLRP3, AIM2) require ASC for
both pyroptosis and cytokine processing (4, 76).
Interestingly, in many cases, innate restriction of bacterial replication is completely
IL-1β/IL-18 independent. For example,
replication of L. pneumophila in Il-1β/Il-18−/−
bone marrow–derived macrophages is indistinguishable from that in wild-type cells
(142). Similarly, L. pneumophila, Burkholde-
ria thailandensis, and S. Typhimurium or
L. monocytogenes overexpressing flagellin are all
restricted by the inflammasome in vivo in the
absence of IL-1β/IL-18 (30, 135, 143). The
nature of this cytokine-independent bacterial
restriction remains uncertain, but the simplest
model is that pyroptotic cell death eliminates
the niche that intracellular pathogens must
exploit for replication (21, 144). However,
studies of Casp11-deficient mice suggest that
pyroptosis alone is insufficient to restrict
bacterial replication and that an additional
IL-1β/IL-18-independent function of CASP1
is required to recruit neutrophils that eliminate
the extracellular bacteria ejected from the
intracellular niche by pyroptosis (126).
Cytokine Processing
Despite appearing dispensable for innate
restriction in some in vivo models, the CASP1dependent cytokines IL-1β and IL-18 are
critical inflammasome effectors in other models, although each cytokine contributes very
differently. IL-18 is known principally for its
ability to induce IFN-γ production, which in
turn helps to restrict intracellular pathogens.
By contrast, IL-1β is critical for inflammation
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(e.g., neutrophil recruitment) and helps shape
adaptive immune responses. Signaling through
the widely distributed IL-1 receptor rapidly
induces the expression of hundreds of inflammatory genes—including those encoding IL-6,
IL-8, IL-12, COX-2, numerous chemokines,
and antimicrobial peptides—as well as the
IL-1β transcript itself. In the liver, IL-1β,
together with IL-6, drives the acute phase
response, and on CD4+ T cells, IL-1 can
promote Th17 or Th2 differentiation depending on the cytokine context (145). Although
IL-1β production often depends on CASP1,
CASP1-independent IL-1β processing has
been reported (146, 147, 148).
The NLRP1B-mediated response to B. anthracis provides an interesting case study for the
role of cytokines and neutrophils in bacterial
restriction downstream of inflammasomes. B6
mice carrying a responsive Nlrp1b allele exhibit
increased resistance to B. anthracis spore challenge compared with nonresponsive nontransgenic B6 mice, even though macrophages in
the transgenic mice are susceptible to LeTxmediated lysis (149). Similar results were obtained in B6 mice congenic for a responsive allele of Nlrp1b; these mice survived spore and
vegetative bacterial challenges in a manner dependent on Nlrp1b and Casp1. This restriction
required the IL-1 receptor and recruitment of
neutrophils to the site of infection (150), suggesting that inflammasome-dependent maturation of IL-1β is critical for restriction of B. anthracis. Therefore, although LeTx is important
for B. anthracis virulence (151), likely through
its inactivation of MAPKKs, the activity of
LeTx is counteracted by NLRP1B-mediated
detection of the toxin. Indeed, the release of
intracellular pathogens by pyroptosis and the
recruitment of neutrophils by IL-1β likely synergize to restrict bacterial replication in vivo.
The recruitment of neutrophils also carries
with it the risk of significant damage to
host tissues. Therefore, in circumstances in
which neutrophils fail to restrict the microbe,
neutrophil recruitment is associated with
severe pathology and even increased bacterial
dissemination (30, 152–154). For example,
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Il1R−/− mice infected with Burkholderia pseudomallei, which can replicate in neutrophils, are
protected compared with wild-type mice (153).
Signaling through the IL-18 receptor induces proinflammatory cytokines and, depending on the cytokine context, can drive CD4+ T
cells to Th1 and perhaps even Th2 and Th17
phenotypes (145). However, IL-18 was first
identified as an IFN-γ-inducing factor, and this
remains its primary contribution to innate restriction of bacterial pathogens. For example,
restriction of B. pseudomallei is lost in Il18−/−
mice but can be restored by exogenous IFN-γ
(153). In the spleen, memory CD4+ T cells secrete IFN-γ in response to IL-18 produced by
CD8α+ dendritic cells that activate the NLRC4
inflammasome in response to S. Typhimurium
(155). Surprisingly, even purified flagellin can
activate this response, so it was suggested that
cross-presentation pathways in these CD8α+
cells deliver flagellin to the cell cytosol.
The contribution of IL-1β and IL-18 to T
cell differentiation and the potential adjuvanticity of endogenous factors released during pyroptosis suggest a link between inflammasome
activation and the adaptive immune response.
Although evidence for this link exists in influenza infections (77), relatively little work has
examined the requirement of inflammasomes
for adaptive immunity to bacterial pathogens.
Protective immunity to Helicobacter infections
requires IL-1 receptor but is independent of
IL-18 (156). Conversely, mice infected with a
strain of L. monocytogenes engineered to hyperinduce the NAIP5/NLRC4 inflammasome fail
to develop protective immunity (135). Further
work is needed to clarify how inflammasome
effector functions, such as cytokine processing,
regulate adaptive immune responses.
Other Effector Functions
Although certain inflammasomes (notably
NAIP/NLRC4 and NLRP1B) can be activated
within minutes without priming, IL-1β and to
a lesser extent IL-18 require de novo transcription and translation before they can be processed by CASP1. Although this transcriptional
priming could occur constitutively (157), some
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data suggest that inflammasomes are connected
to additional fast-acting signaling outputs. Indeed, activation of NAIP5/NLRC4 by systemic
cytosolic delivery of flagellin can kill mice in less
than 30 min (158). This rapid response appears
not to require IL-1β/IL-18 or pyroptosis but
instead depends on the biosynthesis of inflammatory signaling lipids termed eicosanoids—
e.g., prostaglandins and leukotrienes—that
induce vascular leakage and massive fluid
loss into the gut and peritoneum (158). Of
note, NAIP5/NLRC4-dependent eicosanoid
biosynthesis occurs specifically in resident peritoneal macrophages and is completely absent in
bone marrow–derived macrophages commonly
used to study inflammasome responses (158).
Interestingly, administration of purified LeTx
into rats can also cause rapid (<1 h) death
that is related to vascular shock and pleural
(rather than peritoneal) fluid accumulation;
this death appears to require NLRP1, though
a role for eicosanoids is not established (56). In
mice, activation of NLRP1B by purified LeTx
also induces rapid (but nonlethal) vascular
leakage that is ameliorated in the absence of
prostaglandin biosynthesis (149, 158). During
infection, localized vascular leakage resulting
from inflammasome-induced eicosanoids
produced by tissue-resident macrophages
might provide complement, antibodies, and
leukocytes access to the site of infection,
though this process remains to be shown.
Like the cytosolic flagellin model described
above, the LPS-induced model of sepsis is
also inflammasome dependent [requiring ASC,
CASP11, and at some doses NLRP3 (124,
159)] but IL-1β/IL-18 independent (160). Although LPS-mediated lethality requires days,
not minutes, it is interesting that lethality is delayed when prostaglandin synthesis is decreased
(161). High-mobility group B1 (HMGB1) protein, which is released from cells during pyroptosis and has proinflammatory effects, has
also been linked to LPS-mediated inflammation and pathology downstream of the inflammasome (160).
HMGB1, IL-1β, and IL-18 are all members
of a group of proteins lacking canonical secre-
tion signals that rely on CASP1 for their release
(162). In addition, IL-1α—a cytokine that signals through the same receptor as IL-1β and
thus has essentially the same in vivo effects—
lacks a signal sequence and can in some cases be
secreted in a CASP1-dependent manner (163).
These proteins are passively released during pyroptotic cell lysis, but there is also evidence for
an active secretion process that occurs before or
even in the absence of readily detectable cell lysis (74, 137). Current models for this pathway,
which is calcium dependent (164–166), include
microvesicle shedding and lysosome exocytosis (165–167). The relative contributions of active and passive processes to CASP1-dependent
protein release likely vary by cell type, inflammasome, and agonist; it will be particularly important to investigate these processes in vivo.
Another suggested effector function of
CASP1 is the transcriptional induction of
lipogenic genes to promote membrane biosynthesis and counteract cell death (168). This
transcriptional response requires several hours
and is apparently inadequate to counteract
pyroptosis by CASP1, which can occur in
1–2 h. The in vivo relevance of the link
between CASP1 and lipogenic genes is uncertain, and the response may be unique to the
nonhematopoietic cells used in the study.
In a departure from the view that pyroptosis
is the dominant mechanism that restricts
intracellular bacterial replication via elimination of the intracellular niche, some studies
have suggested that pyroptosis-independent
pathways may also restrict bacterial replication.
For example, Naip5-defective A/J macrophages
are highly permissive to L. pneumophila replication (see above) despite the fact that some
studies (169) (but not all; see Reference 20)
show normal induction of pyroptosis in A/J
macrophages in response to L. pneumophila.
Restriction of L. pneumophila replication in
Naip5-sufficient B6 macrophages may be due
to delivery of L. pneumophila phagosomes to
lysosomes for degradation (19, 144, 170, 171).
This activity has been reported downstream
of CASP1 through its processing of CASP7
(142) and also downstream of CASP11 (172).
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Whether pyroptosis or altered phagosome
trafficking causes bacterial restriction has been
difficult to determine because there is currently
no way to selectively eliminate either of these
effector functions of the inflammasome.
In summary, inflammasomes seem to trigger
more downstream outputs than previously appreciated, although many of these outputs need
further evaluation. In particular, it will be important to understand their individual contributions to host immunity and pathology in vivo.
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INHIBITION/EVASION OF
INFLAMMASOMES BY BACTERIA
If an innate immune response plays an important role in host defense, then it is likely that
bacterial pathogens will evolve to evade the
response. The inflammasome is no exception
to this general rule. L. monocytogenes and
S. Typhimurium, for example, downregulate
flagellin expression inside cells to evade detection by NAIP5/NLRC4 (173, 174). Enforced
expression of flagellin by these bacteria in
vivo results in their severe attenuation; this
attenuation is reversed in Nlrc4−/− mice,
demonstrating that the attenuation is indeed
due to inflammasome activation (30, 135).
Staphylococcus aureus meanwhile modifies its cell
wall to prevent degradation by lysozymes, and
this modification indirectly reduces detection
by NLRP3 (175).
Examples of bacterial inflammasome inhibitors include the M. tuberculosis–secreted
metalloprotease Zmp1 and the P. aeruginosa–
secreted phospholipase ExoU (22, 176). The
various Yersinia species are perhaps best
equipped with an armament of inflammasome
inhibitors. Y. enterocolitica, for example, blocks
CASP1 activation through the inhibitory effect of its secreted effector YopE on Rho
GTPases, in particular Rac-1 (177). Which inflammasome is targeted by this activity and
how endogenous Rho GTPases contribute to
CASP1 activation remain unresolved. Using a
YopK-deficient strain of Y. pseudotuberculosis,
Brodsky et al. (134) uncovered the activation of
both the NLRC4 and NLRP3 inflammasomes
by the Yersinia T3SS. YopK secreted by wild90
von Moltke et al.
type bacteria normally blocks this host pathway by binding the T3SS, and this inhibition is
critical for host colonization. Bacterial load is
attenuated in wild-type mice infected with the
YopK-deficient Y. pseudotuberculosis strain compared with that in mice infected with the wildtype strain, but this attenuation is reversed in
mice that are deficient for components of the inflammasome. Meanwhile, a YopJ isoform with
enhanced MAP kinase and NF-κB inhibitory
activity that is expressed in the Y. pestis KIM
strain also leads to enhanced CASP1 activation and IL-1β secretion. Y. pestis CO92 and
Y. pseudotuberculosis, in contrast, secrete a less
potent YopJ isoform, suggesting another possible mechanism of inflammasome evasion (178).
Lastly, a study (118) recently demonstrated
that production of a nonstimulatory tetraacylated LPS allows Y. pestis to evade TLR4- and
inflammasome-dependent production of protective IL-18. Evasion or inhibition of the inflammasome by bacterial pathogens means that,
in many cases, inflammasome deficiency should
not be expected to result in heightened susceptibility to infection. Indeed, dramatic in
vivo phenotypes are not always observed in
inflammasome-deficient mice (Table 1).
INFLAMMASOME RECOGNITION
OF THE MICROBIOTA
The mammalian intestine harbors a complex
microbial ecosystem, termed the microbiota,
that is composed of trillions of microbes representing hundreds of species. The microbiota
provides numerous benefits to its host but also
poses serious challenges to the mucosal immune
system in the intestinal tract. On the one hand,
the immune system must detect and provide
a barrier against the microbiota and incoming
pathogens. On the other hand, the immune system must avoid inappropriate pathological responses to the microbiota. Disruption of the
host-microbiota equilibrium can lead to severe
pathology, including inflammatory bowel disease (IBD) and sepsis.
The first evidence suggesting that inflammasomes may in part mediate host-microbiota
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interactions stemmed from genome-wide
association studies of IBD patients. Polymorphisms in the Nlrp3 gene have been linked
to an increased risk of developing Crohn’s
disease (CD), a form of IBD (179, 180).
However, this link was not confirmed in a
larger study of ∼13,000 CD patients, a finding
that may be explained by regional differences
in genetic backgrounds (181). Additionally,
polymorphisms in the genes encoding IL-1β,
IL-18, and IL-18 receptor accessory protein
have been associated with an increased risk of
developing CD (182–184). Together, these
studies, and others discussed below, suggest
that inflammasomes may contribute to the
pathogenesis of IBD and, more broadly, may
play a variety of roles in regulating homeostasis
in the intestinal tract (Figure 7).
The Inflammasome, Inflammation,
and Tissue Repair
Oral administration of dextran sulfate sodium
(DSS), which causes mucus erosion and cytotoxicity to colonic epithelial cells, provides a
useful model of colitis-like disease in mice. Initial studies examining a role for the inflammasome in intestinal homeostasis found that
CASP1-deficient mice were less susceptible to
DSS-induced colitis compared with wild-type
mice, suggesting that the inflammasome contributes to disease pathogenesis (185). Consistent with this notion, administration of an IL18 neutralization antibody to wild-type mice
protected against disease (185). However, more
recent studies demonstrated that the inflammasome may actually provide protective responses to DSS-mediated colitis. Three groups
demonstrated that mice deficient for functional
CASP1 or ASC exhibit greater disease severity (186–188). Disease was associated with increased epithelial barrier permeability, resulting in increased extraintestinal dissemination
of the microbiota (188). The increased barrier
permeability was suggested to result from defective cell proliferation and repair of the epithelium. Il18−/− mice exhibit pathologies similar to those in Casp1−/− mice, and administra-
tion of recombinant IL-18 to Casp1−/− mice
reduced disease severity (187–189). Which inflammasomes control CASP1 in the context of
DSS-induced colitis? Two independent studies (121, 190) reported that Nlrp3−/− mice
are partially protected from DSS. By contrast,
other studies have reported that Nlrp3−/− mice
exhibit increased disease severity, suggesting
that NLRP3 may protect against the development of DSS-induced colitis. Inconsistencies among DSS studies may result from differences in the microbiota among mouse strains
and/or colonies. Hirota et al. (191) showed that
Nlrp3 deficiency is associated with reduced βdefensin antimicrobial peptide production in
the colon. Zaki et al. (188) demonstrated that
disease in Nlrp3−/− mice is associated with increased gut barrier permeability, defective epithelial proliferation, and increased extraintestinal invasion of the microbiota. In support of a
role for the microbiota in triggering disease, antibiotic administration to Nlrp3−/− mice attenuates disease progression. Thus, NLRP3 may
be required for the tissue-regenerative function
of IL-18 in response to DSS-induced intestinal injury. However, it remains to be shown
whether the levels of colonic IL-18 in Nlrp3−/−
mice are impaired and whether administration of recombinant IL-18 to Nlrp3−/− mice is
protective. Additional reports have suggested
that NLRP6 and NLRP12 are also important
for attenuation of colonic inflammation. Impaired NLRP12 signaling in both hematopoietic and nonhematopoietic cells results in increased inflammation in the DSS model (115,
116). NLRP6-deficient mice exhibit increased
intestinal permeability and inflammation and
impaired IL-18 production (192, 193). Interestingly, one study (121) proposed that NLRP6
also regulates responses to DSS via regulation of the microbiota composition (see below).
These alternate roles for NLRP6 are not mutually exclusive.
Chronic inflammation associated with
IBD is recognized as a major risk factor for
the pathogenesis of colitis-associated colon
cancer (CAC). As in colitis, the inflammasome
reportedly plays a negative regulatory role in
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Mucus erosion
and damaged
epithelium
1
Repair
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Lumen
?
Microbial
translocation
3
Mucus
Regulation
of microbiota
composition
IL-18
2
Inflammation
(localized and systemic)
Epithelium
Lamina propria
IL-1β
IL-18
?
Hematopoietic
cell
Figure 7
Proposed roles for inflammasomes in host-microbiota interactions. ! Repair: Activation of inflammasomes
by unknown signals leads to production of IL-18, which in turn leads to regeneration of epithelial cells by an
unknown mechanism. Deficiencies in inflammasome signaling and IL-18 production are associated with
impaired repair of the colonic epithelial barrier. " Inflammation: Disruption of the epithelial barrier and
microbial translocation result in localized and systemic inflammasome activation and inflammation driven by
IL-18 and IL-1β. # Regulation of the microbiota composition: Upon recognition of unknown ligands,
NLRP3 and NLRP6 lead to production of IL-18, contributing to regulation of the microbiota composition
through an unknown mechanism. Disruption of inflammasome signaling is associated with a dysbiotic
microbiota with proinflammatory properties.
the development of CAC. In a mouse model of
CAC, Casp1−/− and Asc−/− mice present with
increased tumor burdens and reduced levels
of IL-18. Furthermore, roles for NLRP3,
NLRP6, and NLRP12 in defense against CAC
have also been established (186, 188, 192, 193).
92
von Moltke et al.
The Inflammasome and
Intestinal Dysbiosis
It is now well recognized that maintaining
a properly balanced microbial composition is
crucial for intestinal homeostasis. Disruption of
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this composition (dysbiosis) can result in the expansion of pathobionts—constituents of a normal microbiota that are typically harmless to the
host but can become pathogenic when homeostasis is disrupted (194, 195). Recent studies
have revealed complex roles for the inflammasome in regulating the microbiota composition.
For example, Nlrp6−/− mice, which are more
sensitive to DSS than are wild-type mice, were
found by 16S rRNA deep sequencing of the intestinal microbiota to harbor an overrepresentation of bacteria from the phyla Bacteroidetes
(Prevotellaceae) and TM7 (121). An expansion of these phyla was also found in Casp1−/− ,
Asc−/− , and Il18−/− mice but not in Nlrc4−/− ,
Aim2−/− and Nlrp3−/− mice (121).
Dysbiosis associated with Nlrp6 deficiency
appears to be transmissible (121). Wild-type
mice cohoused with Nlrp6−/− mice became
colonized with Prevotellaceae and TM7 and
developed more severe DSS-induced colitis (121). Cohousing wild-type mice with
Casp1−/− , Asc−/− , or Il18−/− mice produced
similar results. Furthermore, both antibiotictreated Nlrp6−/− and wild-type mice cohoused
with antibiotic-treated mutant mice exhibited
reduced colitis severity that correlated with reduced levels of Prevotellaceae and TM7 (121).
Colonization with these colitogenic-associated
microbes correlated with increased production of intestinal CCL5 (121). Ccl5−/− mice
cohoused with Nlrp6−/− mice did not become more sensitive to DSS-induced colitis
despite having increased colonization of Prevotellaceae and TM7. Bone marrow chimera
experiments suggested that NLRP6 and IL-18
are required in radioresistant (perhaps epithelial) cells. The authors proposed a model in
which the NLRP6 inflammasome regulates the
colonization of these procolitogenic-associated
bacteria through a mechanism dependent on
IL-18 production by intestinal epithelial cells
(121). The mechanism by which IL-18 contributes to the shaping of the microbiota composition and the ligand that activates NLRP6
remain unknown. Importantly, it remains to be
shown whether Prevotellaceae and TM7 are the
causative agents of disease in this model. An al-
ternative model is that their expansion is the
indirect consequence of the host response to
other members of the microbiota.
In addition to producing local pathogenic
effects, intestinal dysbiosis associated with
impaired inflammasome signaling can trigger
inflammatory disorders in distal organs. For
example, the inflammasome is critical for protection against diet-induced liver inflammation,
and this protection is associated with the intestinal microbiota composition (122). Asc−/− and
Casp1−/− mice fed a methionine- and cholinedeficient diet (MCDD) developed more severe
liver damage compared with wild-type mice, as
well as enhanced hepatic steatosis and increased
levels of immune cells populating the liver.
Disease progression appears to depend in part
on the NLRP3 inflammasome, as MCDD-fed
Nlrp3−/− mice and Il18−/− mice exhibit increased liver damage and hepatic inflammation
(122). Consistent with previous findings (121),
Asc−/− mice also exhibited increased susceptibility to MCDD-induced liver inflammation,
and this susceptibility was associated with
specific alterations in the microbiota of Asc−/−
mice (122). It remains to be shown whether
MCDD-fed Nlrp3−/− , Nlrp6−/− , Casp1−/− , and
Il18−/− mice exhibit microbiota compositions
similar to those in Asc−/− mice and what microbial species are the causative agents of disease.
The above studies demonstrate that the
inflammasome plays an important beneficial
role in controlling the microbiota composition. A recent study (196) provides evidence
that inflammasomes can also mediate disease
pathogenesis triggered by intestinal dysbiosis.
Colony-born mice treated with a broadspectrum antibiotic cocktail followed by a
DSS challenge developed a sepsis-like syndrome, rather than colitis, that was associated
with extraintestinal bacterial dissemination.
Antibiotic treatment induced the expansion
of an antibiotic-resistant E. coli O21:H+
pathobiont in the intestinal tracts of mice.
Genome sequencing of the E. coli O21:H+
isolate revealed that this strain encodes several
potential virulence factors associated with
septicemic clinical E. coli isolates, including a
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functional flagellin and a T3SS. The authors
demonstrated that the flagellin from this E. coli
can activate the NAIP5/NLRC4 inflammasome. Furthermore, mice deficient for Naip5,
Nlrc4, Casp1, and Il1β function exhibited an attenuated disease phenotype when treated with
antibiotics/DSS or systemically challenged
with E. coli O21:H+, despite having similar
levels of microbial colonization of extraintestinal tissues (196), leading to the proposal
that aberrant activation of the NAIP5/NLRC4
inflammasome by E. coli O21:H+ triggers
IL-1β-mediated immunopathology. Thus,
taken together, the studies on recognition of
the microbiota by inflammasomes indicate that
inflammasomes can have both beneficial and
harmful regulatory effects, and the question of
how appropriate responses are maintained will
be important to address in future studies.
Inflammasome recognition of the microbiota remains a nascent field of research, and as
such, the best practices and research tools are
still emerging. One particular challenge is analysis of the complex microbiota. Although cost
effective, basic laboratory culturing techniques
fail to uncover this complexity, as the majority
of the microbiota cannot be cultured by standard laboratory techniques. The commonly applied technique of 16S rDNA qPCR also has
important caveats because 16S rDNA is a variable multicopy gene, and its abundance therefore does not correlate directly with bacterial
colonization. Techniques such as microarraybased applications, 16S full-length sequencing,
and pyrosequencing provide better alternative
approaches to characterize the microbiota ecology with greater taxonomic resolution. Furthermore, novel culturing techniques or in vivo
imaging are needed to identify how and where
inflammasome activation occurs in the gut.
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IMPLICATIONS AND
PERSPECTIVE
Although the diversity both of inflammasomes
and of the mechanisms by which they detect and respond to infection resists simplification, we propose that inflammasomes ex94
von Moltke et al.
hibit several essential properties that may account for their broad roles in host defense. First,
inflammasomes exhibit the important property of autoinhibition. This prevents the unwanted initiation of inappropriate and potentially harmful inflammatory signals when no
pathogen is present. For NLRs, autoinhibition
is thought to be mediated by the LRR domain,
which blocks NBD-mediated oligomerization
through an unknown mechanism. Interestingly,
a conceptually similar autoinhibitory function
has been proposed for the HIN-200 domain
of the non-NLR AIM2 inflammasome (9), implying that autoinhibition might be a general
and essential feature of inflammasomes. Second, inflammasome activation appears highly
cooperative, capable of transitioning from a
fully off state to a fully on state across a narrow range of agonist concentration. This binary
mode of activation contrasts with most signaling through cell surface receptors such as the
TLRs, in which signaling outputs scale across a
wide range of ligand concentration. The binary
behavior of inflammasomes is a consequence of
their highly cooperative oligomerization into
a single cytosolic complex. For example, timelapse movies of fluorophore-tagged ASC strikingly illustrate its complete oligomerization
within minutes (197). Functionally, cooperative inflammasome oligomerization allows for
rapid and decisive high-amplitude responses;
such responses may be critical in host defense.
Interestingly, although the non-NLR protein
AIM2 lacks an NBD, it has nevertheless been
proposed to be activated by a DNA-dependent
multimerization step (9), suggesting that cooperative oligomerization is a common feature
of inflammasomes. Third, inflammasomes must
be able to evolve rapidly to keep pace with
pathogen evasion mechanisms. The LRR domain appears to be a structurally robust domain
that can tolerate mutations, particularly on its
concave β-sheet surface, without loss of hydrophobic core stability (198). A final unifying
property of inflammasomes is that they are all
localized in the host cell cytosol. This localization implies that inflammasomes will respond
to pathogens, which generally access the cytosol
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(directly or indirectly) as part of their virulence
strategy, but will ignore harmless microbes,
which are generally relegated to the extracellular space (82). Cytosolic localization, and the
consequent ability to discriminate pathogens
from nonpathogens, is a critical feature of
inflammasomes that distinguishes them from
TLRs, which detect extracellular-derived ligands from both pathogens and nonpathogens.
Indeed, we suggest that the above properties
of the NBD-LRR architecture, taken together,
may explain why proteins with this architecture
have been maintained alongside TLRs in animal evolution for the purposes of host defense.
The unique properties of the NBD-LRR architecture may also explain why it has been
deployed as the basis for much of the immune system of plants. Superimposed upon the
above-mentioned core properties, inflammasomes have evolved numerous and surprisingly
diverse mechanisms of activation and action,
many of which we likely have yet to appreciate.
FUTURE ISSUES
1. Other than IL-1β and IL-18, what are the relevant protein substrates of CASP1 that
mediate pyroptosis and other potential functions of the inflammasome?
2. What are the self or microbial ligands that interact with inflammasomes? With the
exception of AIM2 and the NAIPs, ligands for inflammasomes remain unknown.
3. What are the molecular mechanisms of inflammasome activation? The biochemical
mechanism of activation of even the intensively studied NLRP3 inflammasome remains
poorly understood. How are inflammasomes maintained in an autoinhibited state and
how does ligand binding, nucleotide binding, and hydrolysis regulate inflammasome
assembly?
4. Do specific cell types in vivo exhibit unique inflammasome functions? Inflammasomes have been studied primarily in hematopoietic cells (particularly monocytes and
macrophages), although recent work has hinted that inflammasomes can also have important roles in nonhematopoietic cells in vivo. Even different macrophage populations can
have unique inflammasome signaling outputs (e.g., eicosanoid production). How many
additional functions may be discovered in vivo?
5. What microbial constituents of the microbiota are recognized by inflammasomes? How
and where are they recognized?
6. Do inflammasome responses play an important role in the induction of adaptive
immunity?
7. How are inflammasome responses terminated? Most studies have focused on the activation of inflammasomes, but presumably termination of inflammasome activation is also
critical for the maintenance of homeostasis.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
This work was supported by investigator awards from the Cancer Research Institute (R.E.V.)
and the Burroughs Wellcome Fund (R.E.V.), by a grant from the University of California Cancer
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Research Coordinating Committee ( J.v.M.), by a National Science Foundation Graduate Research
Fellowship ( J.C.S.), by National Institutes of Health Ruth L. Kirschstein National Research
Service Award Fellowship AI091068 ( J.S.A.), and by National Institutes of Health R01 grants
AI063302, AI075039, and AI080749 (R.E.V.).
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Contents
Annual Review of
Immunology
Volume 31, 2013
Annu. Rev. Immunol. 2013.31:73-106. Downloaded from www.annualreviews.org
by University of California - Berkeley on 05/08/13. For personal use only.
Years in Cologne
Klaus Rajewsky ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1
The Biology of Recent Thymic Emigrants
Pamela J. Fink ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !31
Immunogenic Cell Death in Cancer Therapy
Guido Kroemer, Lorenzo Galluzzi, Oliver Kepp, and Laurence Zitvogel ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !51
Recognition of Bacteria by Inflammasomes
Jakob von Moltke, Janelle S. Ayres, Eric M. Kofoed, Joseph Chavarrı́a-Smith,
and Russell E. Vance ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !73
The Immunology of Fibrosis
Georg Wick, Cecilia Grundtman, Christina Mayerl, Thomas-Florian Wimpissinger,
Johann Feichtinger, Bettina Zelger, Roswitha Sgonc, and Dolores Wolfram ! ! ! ! ! ! ! ! ! 107
Memory T Cell Subsets, Migration Patterns, and Tissue Residence
Scott N. Mueller, Thomas Gebhardt, Francis R. Carbone, and William R. Heath ! ! ! ! ! 137
Control of Human Viral Infections by Natural Killer Cells
Stephanie Jost and Marcus Altfeld ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 163
Functional T Cell Immunodeficiencies (with T Cells Present)
Luigi D. Notarangelo ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 195
Controlling Natural Killer Cell Responses: Integration of Signals for
Activation and Inhibition
Eric O. Long, Hun Sik Kim, Dongfang Liu, Mary E. Peterson,
and Sumati Rajagopalan ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 227
Metabolic Regulation of T Lymphocytes
Nancie J. MacIver, Ryan D. Michalek, and Jeffrey C. Rathmell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 259
Mesenchymal Stem Cell: Keystone of the Hematopoietic Stem Cell Niche
and a Stepping-Stone for Regenerative Medicine
Paul S. Frenette, Sandra Pinho, Daniel Lucas, and Christoph Scheiermann ! ! ! ! ! ! ! ! ! ! ! 285
Interleukin-4- and Interleukin-13-Mediated Alternatively Activated
Macrophages: Roles in Homeostasis and Disease
Steven J. Van Dyken and Richard M. Locksley ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 317
Brain-Reactive Antibodies and Disease
B. Diamond, G. Honig, S. Mader, L. Brimberg, and B.T. Volpe ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 345
v
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7:16
Immunology of the Maternal-Fetal Interface
Adrian Erlebacher ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 387
Regulation of Ligands for the NKG2D Activating Receptor
David H. Raulet, Stephan Gasser, Benjamin G. Gowen, Weiwen Deng,
and Heiyoun Jung ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 413
Pathways of Antigen Processing
Janice S. Blum, Pamela A. Wearsch, and Peter Cresswell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 443
The Immune Response in Tuberculosis
Anne O’Garra, Paul S. Redford, Finlay W. McNab, and Chloe I. Bloom,
Robert J. Wilkinson, and Matthew P.R. Berry ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 475
Annu. Rev. Immunol. 2013.31:73-106. Downloaded from www.annualreviews.org
by University of California - Berkeley on 05/08/13. For personal use only.
The Adaptable Major Histocompatibility Complex (MHC) Fold: Structure
and Function of Nonclassical and MHC Class I–Like Molecules
Erin J. Adams and Adrienne M. Luoma ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 529
The Dendritic Cell Lineage: Ontogeny and Function of Dendritic Cells and
Their Subsets in the Steady State and the Inflamed Setting
Miriam Merad, Priyanka Sathe, Julie Helft, Jennifer Miller, and Arthur Mortha ! ! ! 563
T Cell–Mediated Host Immune Defenses in the Lung
Kong Chen and Jay K. Kolls ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 605
Human Hemato-Lymphoid System Mice: Current Use and Future Potential
for Medicine
Anthony Rongvaux, Hitoshi Takizawa, Till Strowig, Tim Willinger,
Elizabeth E. Eynon, Richard A. Flavell, and Markus G. Manz ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 635
Signaling by the Phosphoinositide 3-Kinase Family in Immune Cells
Klaus Okkenhaug ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 675
Broadly Neutralizing Antiviral Antibodies
Davide Corti and Antonio Lanzavecchia ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 705
Molecular Control of Steady-State Dendritic Cell Maturation and Immune
Homeostasis
Gianna Elena Hammer and Averil Ma ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 743
Indexes
Cumulative Index of Contributing Authors, Volumes 21–31 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 793
Cumulative Index of Articles Titles, Volumes 21–31 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 800
Errata
An online log of corrections to Annual Review of Immunology articles may be found at
http://immunol.annualreviews.org/errata.shtml
vi
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