REVIEWS

Converging roles of caspases in

inflammasome activation, cell death
and innate immunity
Si Ming Man and Thirumala-Devi Kanneganti
Abstract | Inflammatory and apoptotic caspases are central players in inflammation and
apoptosis, respectively. However, recent studies have revealed that these caspases have functions
beyond their established roles. In addition to mediating cleavage of the inflammasome-
associated cytokines interleukin1 (IL1) and IL18, inflammatory caspases modulate distinct
forms of programmed cell death and coordinate cell-autonomous immunity and other
fundamental cellular processes. Certain apoptotic caspases assemble structurally diverse and
dynamic complexes that direct inflammasome and interferon responses to fine-tune
inflammation. In this Review, we discuss the expanding and interconnected roles of caspases
thathighlight new aspects of this family of cysteine proteases in innate immunity.

Inflammasomes
Multi-protein complexes
that activate caspase 1
toinduce processing of
pro-interleukin1 and caspases are broadly divided into two groups based
prointerleukin18 and to
drive pyroptosis.
Pyroptosis
An inflammatory and lytic
formof programmed cell
deathmediated by
inflammatory caspases.
Apoptosis
A non-inflammatory form
ofprogrammed cell death
mediated by apoptotic
caspases. Apoptotic cells
exhibit membrane blebbing
(but have an intact cell
membrane), DNA
fragmentation and
cellshrinkage.
Department of Immunology,
St Jude Childrens Research
Hospital, Memphis, Tennessee
38105, USA.
Correspondence to T.-D.K.
thirumala-devi.kanneganti@
stjude.org
doi:10.1038/nri.2015.7
Published online 14 Dec 2015
The term caspase was introduced in 1996 to embody two
functional roles of this group of enzymes. The c refers
to cysteine proteases, and the aspase refers to their abil-
ity to cleave after aspartic acid residues1. Mammalian
on their functions (TABLE1). Inflammatory caspases
include caspase 1, caspase 4, caspase 5, caspase11
and caspase12. With the exception ofcaspase 12, all
inflammatory caspases participate in the activation
of inflammasomes to initiate inflammation and the
induction of an inflammatory form of programmed
cell death known as pyroptosis24. The human genome
encodes the inflammatory caspases caspase1, caspase4,
caspase 5 and caspase 12.By contrast, the mouse
genome encodes caspase 1, caspase 11 and caspase 12;
caspase11 being the homologue of humancaspase 4
and caspase5(REF.5).
Unlike inflammatory caspases, apoptotic caspases
initiate and execute an immunologically silent form of
programmed cell death known as apoptosis. Members
of the apoptotic caspase family include the initiator
caspases caspase 2, caspase 8, caspase 9 and caspase10,
and the effector caspases caspase 3, caspase 6 and
caspase7 (TABLE1). Unlike humans, rodents do not have
a gene encoding caspase 10. Caspase 8 and caspase 9
initiate extrinsic and intrinsic apoptosis, respectively 6.
The extrinsic pathway of apoptosis is induced by death
receptors, including FAS (also known as TNFRSF6, CD95
or APT1), tumour necrosis factor receptor 1 (TNFR1),
TNFR2 and TNF-related apoptosis-inducing ligand
(TRAIL; also known as TNFSF10). Signalling through
FAS induces formation of the death-inducing signalling
complex, which results in the recruitment of caspase 8
(and caspase 10 in human cells) by the adaptor protein
FAS-associated death domain (FADD). Dimerization of
caspase 8 triggers the apoptotic cascade7. The engage-
ment of TNFR1 by its cognate ligand TNF promotes the
formation of a complex comprised of receptor-interacting
serine/threonine-protein kinase 1 (RIPK1), FADD and
caspase 8. The RIPK1FADDcaspase 8 complex initi-
ates apoptosis in the cell when there are low levels of the
long isoform of FLICE-like inhibitory protein (FLIPL; FLIP is
also known as CFLAR)811.
The intrinsic pathway of apoptosis requires the
induction of mitochondrial outer membrane permeabi-
lization, which mediates the release of the pro-apoptotic
factor cytochromec for binding by the cytosolic pro-
tein apoptotic protease-activating factor 1 (APAF1).
APAF1 and caspase 9 assemble a multi-protein complex
known as the apoptosome1216. Activation of initiator
caspases by either the extrinsic or intrinsic apoptosis
pathway engages subsequent activation of caspase 3,
caspase6 and caspase 7. Caspase 14 cannot be classified
as either an apoptotic or inflammatory caspase and has a
specialized role in the differentiation of keratinocytes17,18.
Caspases are expressed by immune cells and non-
immune cells and in many tissues and organs. They
are produced as inactive zymogens, which consist of a
carboxy-terminal protease effector domain comprised of
a large subunit and a small subunit (TABLE1). Caspase1,
caspase 2, caspase 4, caspase 5, caspase 9, caspase 11
and caspase 12 also have an amino-terminal prodomain

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Table 1 | Classification of caspases

Caspase Classification Initiator or Genomic location Domain structure
effector DED DED Protease
Caspase domain
Human Mouse Prodomain domain
Caspase 1 Inflammatory Initiator 11q23 9 A1 CARD Caspase domain
DED DED Caspase domain
DED DED Caspase domain
Caspase 2 Apoptotic Initiator 7q347q35 6 B2.1 CARD Caspase domain
Caspase domain
CARD Caspase domain

Caspase 3 Apoptotic Effector 4q34 8 B1.1 Caspase domain

DEDNature Caspase
DED Reviews domain
| Immunology
Caspase domain

Caspase 4 Inflammatory Initiator 11q22.211q22.3 Absent CARD Reviews

Caspase domain
DEDNature
DED | Immunology
Caspase domain
DEDNature
DED Reviews | Immunology
Caspase domain
Caspase 5 Inflammatory Initiator 11q22.211q22.3 Absent CARD Caspase domain
Caspase domain
CARD Caspase domain
DED DED Caspase domain
Caspase 6 Apoptotic Effector 4q25 3 H1 Caspase domain
Nature Caspase
CARD Reviews domain
| Immunology
Caspase domain

Caspase 7 Apoptotic Effector 10q25 19 D2 Caspase domain

Nature Reviews | Immunology
Nature Reviews | Immunology
Caspase 8 Apoptotic Initiator 2q332q34 1B DED
DED DED
DED Caspase domain
Caspase domain
Nature Reviews | Immunology
Caspase 9 Apoptotic Initiator 1p36.21 4 E1 CARD
CARD Caspase
Caspase domain
domain

Caspase
Caspase domain
domain
Caspase 10 Apoptotic Initiator 2q332q34 Absent DED DED
DED Caspase domain
domain
DED Caspase

Caspase 11 Inflammatory Initiator Absent 9 A1 CARD Caspase domain

Death receptors
A subset of receptors within
the tumour necrosis factor
receptor superfamily that, on
binding to their ligands, convey
cell death signals.
FLICE-like inhibitory protein
(FLIP). The long isoform (FLIPL)
heterodimerizes with
pro-caspase 8 to inhibit
apoptosis and potentially
inhibits necroptosis. By
contrast, the short isoform
(FLIPS) promotes necroptosis.
Apoptosome
A multi-protein complex
comprised of apoptotic
protease-activating factor 1
(APAF1) and caspase 9, which
induces the intrinsic pathway
of apoptosis.
Microorganism-associated
molecular patterns
(MAMPs). Conserved
structures that constitute
microorganisms (for example,
lipopolysaccharide or flagellin),
which are recognized by
pattern-recognition receptors.
Damage-associated
molecular patterns
(DAMPs). Molecules that are
released by injured cells (for
example, ATP or host DNA)
and are recognized by
pattern-recognition receptors.
DEDNature
CARD Reviews
Nature || Immunology
Caspase domain
DED Reviews Immunology
Caspase domain
DED DED Caspase domain
Caspase domain
Caspase domain
domain
Caspase 12* Inflammatory Initiator 11q22.3 9 A1 CARD Caspase
CARD Caspase domain
Caspase 14 Keratinocyte Effector 19p13.1 10 C1 Caspase domain
Nature Reviews
Nature Reviews Immunology
|| Immunology
Caspase domain
differentiation
CARD, caspase activation and recruitment domain; DED, death effector domain. *The human genome encodes 12 caspases. Two
Nature
isoforms of human caspase 12 have been identified. Caspase 12S is an inactive truncated isoform (not Reviews
shown), | Immunology
whereas caspase
Nature Reviews | Immunology
12L is an active full-length isoform.
known as a caspase activation and recruitment domain (NOD), leucine-rich repeat (LRR)-containing protein
(CARD), whereas caspase 8 and caspase 10 contain (NLR) family or the pyrin and HIN domain-containing
death effector domains (DEDs). Effector caspases, protein (PYHIN) family 21. The NLR and PYHIN sensors
caspase 3, caspase 6 and caspase 7, have a short pro mediate recognition of microorganism-associatedmolecular
domain and exist as inactive homodimers until they are patterns (MAMPs) or damage-associatedmolecularpatterns
cleaved andactivated by initiator caspases19. (DAMPs) (BOX1). So far, five receptors have been estab-
The established roles for inflammatory caspases lished to form an inflammasome, including NOD-,
and apoptotic caspases in inflammation and apopto- LRR- and pyrin domain-containing protein 1 (NLRP1),
sis, respectively, have been previously reviewed (see NLRP3, NOD-, LRR- and CARD-containing protein4
REFS24,6,11,20) and are not the focus of this article. (NLRC4), absent in melanoma 2 (AIM2) and pyrin
However, recent work has shown that inflammatory (FIG.1). Furthermore, interferon- (IFN)-inducible pro-
caspases control fundamental cellular processes inde- tein 16 (IFI16), retinoic acid-inducible gene I (RIGI),
pendently of their abilities to induce an inflammatory NLRP6, NLRP7 and NLRP12 have been suggested
response. Conversely, apoptotic caspases support the to activate caspase1 (REF.4). The caspase 1 inflamma
initiation or inhibition of inflammation. The identifi- some orchestrates antimicrobial effector functions, tis-
cation of new functionalities associated with caspases sue repair, tumour suppression, metabolic regulation,
pushes the boundaries of the traditional classification of autoinflammatory disease and cell survival through
caspases and prompts a new paradigm for understanding membranebiogenesis2227.
their contributions to health and disease. Here, we pro- Recent studies have identified a role for caspase 4,
vide an overview of the converging and newly described caspase 5 and caspase 11 in inflammasome signalling
functions of inflammatory and apoptotic caspases in the (BOX2). However, our understanding of the biological
context of inflammation, cell death and immunity. functions attributed to caspase 1 and caspase 11 gained
from mouse studies will probably be reevaluated in
Inflammatory caspases and the inflammasome the years ahead in light of findings showing that con
Caspase 1 is activated in inflammasomes that are initi ventional Casp1/ mouse strains lack caspase11 expres-
ated by a member of the nucleotide-binding domain sion28,29. Caspase 4, caspase 5 and caspase 11 have been

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Box 1 | Discovery of caspase 1 and inflammasomes

Caspase 1 was identified in 1989 and named interleukin1 (IL-1)converting enzyme (ICE) owing to its ability to convert
the 31 kDa proIL1 into an active 17.5 kDa fragment182,183. Characterization of purified caspase 1 in 1992 revealed that it
is a heterodimeric cysteine protease composed of two subunits, p10 and p20 (REF.184). Caspase 1 mediates proteolytic
processing of IL18 with similar efficiency185. In 2002, the existence of a macromolecular protein complex comprised of
caspase 1, caspase 5, an adaptor protein known as apoptosis-associated speck-like protein containing a CARD (ASC) and
NOD-, LRR- and pyrin domain-containing protein 1 (NLRP1) was described in an overexpression and cell free system21.
This elegant study provided evidence that caspase 1 is activated in a large
multi-protein complex initiated by a member of the NOD, LRR-containing
protein (NLR) family. The term inflammasome was then coined to describe
its multi-protein composition. In 2004, mutant mouse strains were
generated with a genomic deletion of ASC and NOD-, LRR- and
CARD-containing protein 4 (NLRC4); macrophages from these mice failed
to engage caspase 1 activation and did not process proIL1 in response to
infection by the intracellular bacteria Salmonella enterica subsp. enterica
serovar Typhimurium140, thereby providing the first line of genetic evidence
for the role of an NLR in the modulation of inflammasome activities under
endogenous settings. In addition, three groups independently showed in
2006 that another member of the NLR family, NLRP3, assembles the
inflammasome to activate caspase 1 in response to ATP, bacterial RNA
molecules and uric acid crystals141143. The absent in melanoma 2 (AIM2)
andpyrin inflammasomes were subsequently identified in later
studies146149,186,187. Inflammasomes can be visualized as a distinct speck
byfluorescence or confocal microscopy95,111,144,145,188 (see figure; arrows
indicate ASCcaspase 1 inflammasomes in macrophages transfected with
double-stranded DNA. Scale bar represents 10m).
Nature Reviews | Immunology

Alarmin
A damage-associated
molecular pattern that is
released by damaged or
necrotic cells in response to
infection or injury. Alarmins
arerecognized by pattern-
recognition receptors to
furtheractivate immune cells.
shown to interact with and activate caspase 1 (REFS30,31).
Further studies have shown that caspase 4, caspase 5
and caspase11 activate the NLRP3 inflammasome in
response to Gram-negative bacteria and intracellu-
larly delivered lipopolysaccharide (LPS), suggesting
that caspase 4, caspase 5 and caspase 11 are activators
ofcaspase 1 that promote caspase 1dependent cleav-
age of interleukin1 (IL1) and IL18 (REFS29,3241).
Direct cleavage of IL1 and IL18 by caspase 4 has been
proposed42,43, but further study is required to confirm
this observation. Activation of caspase 4, caspase 5 and
caspase 11 also leads to pyroptosis and the release of IL1
and the alarmin high-mobility group box 1 (HMGB1)
independently of caspase 1 (REFS 29,3239,44,45) .
Emerging evidence now indicatesthat inflammatory
caspases orchestrate distinct functions that do not rely
on the effects of IL1 and IL18.
Pyroptosis and other effector functions
Inflammatory caspases orchestrate physiological func-
tions that are independent of their ability to induce the
secretion of IL1 and IL18. Studies have shown that
wild-type mice and mice lacking IL1 and IL18 are
sensitive to LPS-induced endotoxaemia or Escherichia
coli-induced septic shock, whereas mice lacking only
caspase 11 or both caspase 1 and caspase 11 are rela-
tively resistant 31,4648. The contribution of caspase 1 and
caspase11 to LPS-induced endotoxaemia was revisited
recently using mice with a genomic deletion of caspase1
alone. A bacterial artificial chromosome transgene
encoding caspase 11 was microinjected into Casp1/
Casp11/ mouse embryos to reestablish caspase11
expression in this mouse line so that it only lacks
caspase 1 expression (referred to as Casp1/Casp11Tg
mice)29. This study found that caspase 11 largely drives
LPS-induced mortality, but that caspase 1 contributes
in part to this process29. In agreement, a mutant mouse
strain with a gene encoding human caspase 4 introduced
into its genome has increased sensitivity to LPS-induced
endotoxaemia49. Interestingly, antibody-mediated neu-
tralization of caspase 11induced HMGB1 largely pro-
tects mice from LPS-induced mortality 47. In another
mouse model of lethal systemic inflammation, activation
of caspase 1 via the NLRC4 inflammasome by systemic
delivery of cytosolic flagellin causes the production of
inflammatory mediators known as eicosanoids, which
drive inflammation and mortality independently of
IL1 and IL18 (REF.50). In keratinocytes, caspase 1
can mediate the export of proteins that are unrelated
to inflammation, including proangiogenic fibroblast
growth factor2 (REF.51).
A well-known effect that is mediated by inflam-
mator y caspases is pyroptosis 35,36,5255 (BOX 3) .
Although the molecular signatures of caspase 1- and
caspase11induced cell death are likely to differ, recent
studies have revealed that pyroptosis triggered by either
caspase is an effective antimicrobial defence against cer-
tain intracellular pathogens even in the absence of IL1
and IL18 release43,53,5658. Caspase1induced pyropto-
sis releases intracellular bacteria from infected macro
phages for uptake by neutrophils, whereby bacteria are
killed through a mechanism requiring reactive oxygen
species56. Indeed, neutrophils are resistant to pyroptosis
in response to some inflammasome activators59, which
makes them a suitable cell type to facilitate the clearance
of infectious agents that normally replicate in macro
phages. Similarly, caspase 11dependent pyroptosis is
protective against infection by cytosolic bacteria, and

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a B. anthracis b c S. Typhimurium d CMV or e

vaccinia virus
F. novicida

Protective ATP, pore-forming T3SS Vacuole

antigen Toxin-induced
toxins, ion ux and IRF1 modications
particulate matters
of RHO GTPases
Needle Inner rod Flagellin GBPs
Lethal factor

Caspase 11-
independent
(canonical)
dsDNA

NAIP1

NAIP2

NAIP5

NAIP6
LRR

NAIP
Pyrin

NLRP1B AIM2
CARD

ASC
NLRP1B

PYD NLRC4
NLRP3

Pro-caspase 1

AIM2 Pyrin
NLRP1B inammasome NLRP3 inammasome NAIPNLRC4 inammasome inammasome inammasome

Active caspase 1

Gasdermin D N-terminal fragment Pro-IL-1 IL-1

Pyroptosis

Figure 1 | Caspase 1 and the canonical inflammasomes. a | Release of the
Bacillus anthracis anthrax lethal toxin, composed of a protective antigen and a
lethal factor, activates the mouse NOD-, leucine-rich repeat (LRR)- and pyrin
domain (PYD)-containing protein 1B (NLRP1B) inflammasome by inducing
cleavage of NLRP1B190,191. b | NLRP3 responds to numerous activators
and physiological aberrations 141143 . Engagement of the NLRP3
inflammasomeindependently of caspase 11 is known as the canonical
NLRP3inflammasome pathway. c | Certain pathogenic bacteria such as
Salmonella enterica subsp. enterica serovar Typhimurium inject proteins into
host cells via the typeIII secretion system (T3SS). These effector proteins are
detected by neuronal apoptosis inhibitory proteins (NAIPs) in the cytoplasm,
where they engage activation of the NOD-, LRR- and caspase activation and
recruitment domain (CARD)-containing protein 4 (NLRC4) inflammasome150156.
d | The cytosolic bacteria Francisella tularensis subsp. novicida and the
double-stranded DNA (dsDNA) mouse cytomegalovirus (CMV) or vaccinia virus
release DNAduringinfection to activate the absent in melanoma 2 (AIM2)
inflammasome146149,192,193. The transcription factor interferon (IFN)-regulatory
Nature Reviews
factor 1 (IRF1) induces expression of IFN-inducible | Immunology
GTPases known as
guanylate-binding proteins (GBPs), which mediate the lysis of F.novicida and
expose its DNA194,195. e | Pyrin senses modifications of RHO GTPases caused by
RHO-inactivating toxins from bacteria including Vibrio parahaemolyticus,
Histophilus somni, Clostridium botulinum and Burkholderia cenocepacia186,187.
Apoptosis-associated speck-like protein containing a CARD (ASC) is an
adaptor protein that carries a PYD and CARD and is used to bridge the
PYD-bearing inflammasome sensors NLRP3, AIM2 and pyrin with the CARD of
pro-caspase 1. Inflammasome sensors carrying a CARD, such as NLRP1B or
NLRC4, may engage pro-caspase 1 directly by CARDCARD interactions. In
all cases, caspase 1 drives prointerleukin1 (pro-IL1) and proIL18
processing. Inflammatory caspases cleave gasdermin D, causing pyroptosis,
which allows the release of mature IL1 and IL18 from the cell6264. The
aminoterminal fragment of gasdermin D conveys a signal by caspase 4,
caspase 5 and caspase 11 to activate the non-canonical NLRP3 inflammasome.

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Non-canonical NLRP3 during intraperitoneal infection by the pathogenic
inflammasome bacteria Burkholderia thailandensis the effect is largely
An inflammasome containing independent of IL1 and IL18 (REF.53). Epithelial cells
NLRP3 that is activated by infected by Salmonella enterica subsp. enterica serovar
Gram-negative bacteria or
lipopolysaccharide, requiring
Typhimurium also undergo caspase1dependent pyro
activation of caspase 4 or ptosis mediated by the neuronal apoptosis inhibitory
caspase 5 in human cells protein (NAIP)NLRC4 inflammasome, leading to
andcaspase 11 in mouse cells. physical extrusion of infected cells from the gut epithe-
lium to reduce overall intraepithelial bacterial burden57.
Canonical inflammasomes
Inflammasomes that do not
A similar study showed that caspase4dependent pyro
require caspase 4, caspase 5 ptosis controls the amount of S.Typhimurium in human
and caspase 11 for their intestinal cell monolayers43. In another infection model,
activation, including the the marked susceptibility of mice lacking caspase1 and
canonical NLRP3, NLRC4
andAIM2 inflammasomes.
caspase11 to subcutaneous infection by Francisella
tularensis subsp. novicida is not fully recapitulated in
Guanylate-binding proteins wild-type mice injected with neutralizing antibodies
A group of interferon-inducible specific for IL1 and IL18 (REF.60), which highlights
GTPases produced by the
a crucial role for pyroptosis in the control of F.novi
hostcell that often target
pathogen-containing vacuoles,
cida infection in addition to IL1 and IL18 release.
contributing to the release of However, a detrimental role for caspase 1dependent
pathogens from the vacuole pyroptosis has been observed during HIV1 infec-
and mediating pathogen killing. tion, whereby pyroptosis depletes quiescent lymphoid
CD4+ Tcells and c ontributes to chronic inflammation
andimmunodeficiency 61.
Biochemical analyses have revealed that caspase4,
caspase 5 and caspase 11 directly cleave the substrate
gasdermin D, yielding an Nterminal fragment ofgas-
dermin D that drives pyroptosis and activation of
the non-canonical NLRP3 inflammasome 62,63 (FIG. 2) .
Caspase1 also cleaves gasdermin D to mediate pyro
ptosis by canonical inflammasomes6264 (FIG.1). However,
caspase1dependent gasdermin Dindependent pyro
ptosis can be observed following prolonged inflamma
some activation, which raises the possibility that other
pro-pyroptotic factors exist 62. Importantly, mice with
a deletion of the gene encoding gasdermin D are less
susceptible to LPS-induced mortality compared with
wild-type mice62, providing further support for a role of
pyroptosis in the pathogenesis of endotoxaemia.
In addition to infectious diseases, caspase 1 and
caspase 11 orchestrate protective responses against
colorectal cancer independently of IL1 and IL18
in a context-specific manner. Activation of caspase1
and release of IL18 via the NLRP3 inflammasome
Box 2 | Cross-species functionality of caspase 4, caspase 5 and caspase 11
Mouse caspase 11 was identified in 1996 (REF.30). It shares 46% amino acid sequence
identity with mouse caspase 1 and 45% sequence identity with human caspase 1
(REF.30). Human caspase 4 and caspase 5 share 78% nucleotide sequence identity79,
indicating that these two genes evolved from a common ancestor by gene
multiplication. The expression of caspase 5 and caspase 11 can be upregulated in
response to lipopolysaccharide (LPS), other Toll-like receptor (TLR) agonists,
interferon (IFN) or IFN in a cell-type specific manner, whereas caspase 4 is
constitutively and highly expressed in many cell types5,30,37,189. In addition, caspase 4,
caspase 5 and caspase 11 are activated following recognition of cytoplasmic LPS from
Gram-negative bacteria, leading to activation of the non-canonical NOD-, LRR- and
pyrin domain-containing protein 3 (NLRP3) inflammasome3234,41. Caspase 4, caspase 5
and caspase 11 have cross-species activities and are functionally interchangeable in
human and mouse cells. Stably expressed caspase 4 or caspase 5 induces pyroptosis in
mouse macrophages lacking caspase 11, and caspase 11 induces pyroptosis when
introduced into human 293T cells49,73.
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contribute to the protection against colorectal tumori-
genesis and subsequent growth of metastasized cancer
cells in mice6567. By contrast, activation of the NLRC4
inflammasome prevents cancer through a mechanism
that suppresses overt proliferation of colonic crypt
cells68. How NLRC4 and NLRP3 differentially coordi-
nate caspase 1 functions to achieve IL18dependent
and -independent effects during colorectal tumori
genesis is largely unknown. Recent studies have also
demonstrated a requirement for caspase 11 in medi
ating resistance against dextran sulfate sodium-
induced intestinal inflammation in mice6971. Whether
IL1 and IL18 are contributing factors in this case is
contentious, with both similar and reduced levels of
IL1 and IL18 being reported in caspase 11deficient
micetreated with dextran sulfate sodium compared
with treated wild-type mice 70,71. Differences in gut
microbiota harboured by mice housed in different
facilities, together with differences in experimen-
tal conditions, could account for these discrepan-
cies. Nevertheless, there is clear evidence to indicate
that caspase 1- and caspase 11mediated cellular and
pathological effects might occur independently of the
concomitant release of IL1 and IL18.
Inflammatory caspases and LPS sensing
Although the membrane-bound Toll-like receptor4
(TLR4) has been recognized as the bonafide LPS sen-
sor for nearly two decades72, recent studies have identi
fied a selective role for caspase 11 in the recognition
of LPS. Caspase 11 induces the release of IL1, IL1
and IL18, and mediates pyroptosis when LPS is trans-
fected or electroporated into the cytoplasm or when
Gram-negative bacteria expose their LPS in the cyto-
plasm35,36,53 (FIG.2). Similarly, caspase 4 drives IL1
and IL1 secretion and cell death in human mono-
cyte-derived macrophages, monocytes, keratinocytes
or epithelial cell lines in response to transfection of
LPS3739,73. In addition, the vacuole-restricted patho
gens S. Typhimurium and Legionella pneumophila
have been shown to activate caspase 4 and caspase11
(REFS33,37,53,73,74), probably owing to aberrant entry of
a small proportion of bacteria into the cytoplasm follow-
ing rupture of the pathogen-containing vacuole caused
by guanylate-bindingproteins54,75.
The presence of a CARD in caspase 11 suggests that a
CARD-containing protein might function as an upstream
receptor that directly binds LPS. However, a search for a
potential binding partner failed to identify an interaction
between any CARD-containing proteins and LPS73.
Instead, caspase 4, caspase 5 and caspase 11 were found
to directly bind LPS and its lipid A moiety 73. Specifically,
the full-length 43kDa form, but not the 38kDa form,
of caspase 11 is precipitated by LPS73. Unlabelled LPS
competes with biotinylated LPS for caspase 11 bind-
ing, whereas biotinylated muramyl dipeptide (MDP)
and Pam3CysSerLys4trihydrochloride (Pam3CSK4)
do not precipitate caspase 11. The surprising observa-
tion that certain members of the inflammatory caspase
family are sensors of cytosolic LPS clearly broadens the
traditional scope of caspase functions.
REVIEWS
Box 3 | Pyroptosis
The term pyroptosis was first proposed in 2001 to describe the pro-inflammatory and
caspase 1dependent nature of Salmonella-induced cell death and to distinguish it from
non-inflammatory apoptosis52. Pyro describes fire or fever and ptosis signifies a
pro-inflammatory form of programmed cell death. Pyroptosis is characterized by
rupture of the cell membrane and release of cellular contents, cell swelling, positivity
for annexin V and TUNEL staining, evidence of chromatin condensation and absence of
DNA laddering55,188. Furthermore, the mitochondria of pyroptotic cells lose membrane
potential and release their contents188. Several investigators have also referred to
caspase 11induced cell death as pyroptosis35,36,53,54. Caspase 11 activates pyroptosis in
response to Escherichia coli and other triggers of the non-canonical NOD-, LRR- and
pyrin domain-containing protein 3 (NLRP3) inflammasome29,33. It is important, however,
to note that caspase 11 induces cell death independently of caspase 1 and does not
directly cleave interleukin1 (IL1) and IL18. From this standpoint, it is likely that
caspase 1 and caspase 11dependent pyroptosis have their own set of molecular
signatures, some of which might be common. Both caspase 1 and caspase 11 cleave
a53kDa substrate known as gasdermin D, generating a 31kDa amino-terminal
fragment that initiates pyroptosis and a 22kDa carboxy-terminal fragment that has
unknown functions6264.
Enigmatic roles of caspase 12
Caspase 12 is a member of the inflammatory caspase
family and is expressed in many organs76,77. An early
study indicated that caspase 12 is localized to the endo-
plasmic reticulum (ER) and is activated by ER stress
signals but not by other apoptotic stimuli78. The human
genome encodes either or both the full-length and
truncated isoforms of caspase 12, whereas the mouse
and rat genomes encode only a full-length caspase 12
(REFS79,80). The gene that encodes full-length human
caspase 12 protein (caspase 12L; also known as the
ancestral or active form) spans 8 exons. The inactive,
truncated form of human caspase 12 (caspase 12S; also
known as the derived or inactive form) consists of only
the prodomain owing to a TGA stop codon at amino
acid position 125 in exon 4 of the gene. Analysis of
the global human population revealed that only 28%
ofsub-Saharan African populations and less than 1% of
populations outside of Africa carry at least one allele for
caspase 12L81. Further studies revealed that 1.712.5% of
individuals in certain regions of the Middle East, South
Asia and East Asia carry at least one allele encoding
caspase 12L82,83. Homozygosity for caspase 12L is rare in
humans and is found in only 2% of individuals with an
African ancestry 80,84.
The role of caspase 12 in the immune system and
the explanation for its loss of function in populations
outside of Africa are unclear. Several human and mouse
studies suggested that caspase 12 could have a role in
the negative regulation of inflammation and caspase 1
activity, as well as confer resistance to bacterial sepsis
and infection80,85. This is exemplified by a study show-
ing that whole blood cells derived from individuals of
African descent expressing caspase 12L produce less
IL1 and granulocytemacrophage colony-stimulating
factor (also known as CSF2) when stimulated with LPS
Necroptosis compared with individuals expressing caspase 12S
A type of necrosis and a form (REF.80). By contrast, another study found no correlation
of non-apoptotic cell death
driven by the kinases RIPK1
between caspase 12 variants and cytokine production84.
and RIPK3 under conditions in There is some evidence to suggest that homozygosity for
which caspase 8 is inhibited. caspase 12L is linked to lower baseline joint-narrowing
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2015 Macmillan Publishers Limited. All rights reserved
scores in AfricanAmericans with rheumatoid arthri-
tis86. However, no association was found between a par-
ticular variant of caspase 12 and susceptibility to sepsis
induced by the fungal pathogen Candida albicans or to
community-acquired pneumonia87,88. There is also lim-
ited evidence to suggest that malaria contributed to the
persistence of caspase 12L in African populations89. The
reason for the apparent discrepancy between these stud-
ies is unclear. Nevertheless, it has been proposed that the
loss of caspase 12 function in many human populations
is indicative of a positive selection event that probably
occurred within the past 100,000years81. Consistent with
this idea, a loss of caspase 12 function in Europe has
been predicted to predate animal domestication and be
unrelated to zoonotic infections from livestock90.
Biochemical analyses have found that rat
caspase 12 cleaves itself and does not induce the
cleavage ofcaspase1, caspase 3, caspase 4, caspase 5,
caspase8, caspase 9, the apoptotic substrates poly-
ADP-ribose polymerases (PARPs) and inhibitor of
caspase-activated DNase (ICAD; also known as DFFA),
proIL1, proIL18 or amyloid precursor proteins91.
However, the full-length or a catalytically inactive form
of rat caspase 12 interacts with caspase 1 and partially
withcaspase 5, but not with caspase 9 (REF.85), and
prevents caspase 1 activity and the release of IL1 in
macrophages stimulated with TLR ligands85,91. The
inhibitory activity of rodent caspase 12 does not seem
to be restricted to caspase 1. Overexpression of rat
caspase 12 in HEK293T cells dampens the activation
of nuclear factorB (NFB) signalling induced by
MDP92. Caspase12 displaces the E3 ubiquitin ligase
TNFR-associated factor 6 (TRAF6) in the NOD2
RIPK2 complex and interacts with the kinase RIPK2
(REF.92). The biochemical functions exhibited by rodent
caspase 12 raise the possibility that it might be an anti-
inflammatory caspase. However, further studies are
required to fully elucidate the immunological repertoire
of this understudiedcaspase.
Caspase 8 and inflammation
Caspase 8 is recognized for its role in apoptosis.
However, ongoing investigations have uncovered
essential functions for this protease in the regulation
of inflammation (FIG.3). Caspase 8 is required for the
activation of NFB signalling 93100 and for direct or indi-
rect cleavage of proIL1 or proIL18 together with or
independently of the caspase 1 inflammasome95,96,101119.
By contrast, there is also evidence suggesting that
caspase8 contributes to the inhibition of inflammation,
including its ability to block inflammasome activities120,
IFN responses121,122 and a type of inflammatory pro-
grammed cell death known as necroptosis123,124 (FIG.3). We
discuss below the diverse characteristics of caspase8 in
the context of inflammation.
Modulating NFB signalling. Caspase 8 can regulate
NFB activation. Studies have identified two patients
with a homozygous arginine-totryptophan mutation in
the p18 subunit of caspase 8 that impairs the activation
and apoptosis of Tcells, Bcells and natural killer (NK)

LPS
Gram-negative
bacteria
TLR4
TRIF
GBPs Non-canonical
NLRP3 inammasome
LRR
Vacuole
LPS
NLRP3
PYD
Caspase 11
ASC
CARD
Pro-caspase 1
Active caspase 1
Gasdermin D N-terminal fragment Pro-IL-1 IL-1
Pyroptosis
Figure 2 | Inflammatory caspases and the non-canonical NLRP3 inflammasome.
Nature Reviews | Immunology
Lipopolysaccharide (LPS) released by Gram-negative bacteria activates Toll-like
receptor4 (TLR4) and its adaptor TIR-domain-containing adaptor protein inducing
interferon- (TRIF), inducing caspase 11 expression via typeI interferon signalling.
Cytoplasmic LPS that enters the cytoplasm owing to rupture of the pathogen-containing
vacuole by guanylate-binding proteins (GBPs) binds mouse caspase 11 and activates the
non-canonical NOD-, leucine-rich repeat (LRR)- and pyrin domain (PYD)-containing
protein 3 (NLRP3) inflammasome29,3241,73. Cytoplasmic LPS also binds human caspase 4
and caspase 5. ASC, apoptosis-associated speck-like protein containing a caspase
activation and recruitment domain (CARD).
cells93. These patients have normal levels of total T cells
and Bcells, but a reduced proportion of CD4+ Tcells93.
They also have a range of clinical symptoms, including
splenomegaly, lymphadenopathy, eczema, airway con-
ditions and susceptibility to recurrent herpes simplex
virus infection and pneumonia, and they have a poor
response to immunization93. Further examination of the
lymphocyte and NK cell populations revealed defective
activation of NFB in these patients98. Caspase8 is
also required for NFB signalling and production of
pro-inflammatory cytokines in mouse macrophages
and Bcells9497 (FIG.3). Mouse macrophages that lack
caspase 8 and the kinase RIPK3 and that are infected
with S. Typhimurium, Citrobacter rodentium or E.coli
or are stimulated with LPS, Pam3CSK4, polyinosinic
polycytidylic acid (poly(I:C)) or MDP fail to induce
robust expression of proIL1, IL6 and TNF compared
with wild-type macrophages or macrophages lacking
RIPK3 (REFS 9597). Additional studies confirmed
that the caspase 8 adaptor FADD is required for NFB
NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 7
2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
activation in response to a range of stimuli96,97,99,102. It is
worth noting that genomic deletion of Casp8 or Fadd
is lethal in embryonic mice125128, a phenotype that is
rescued by genomic deletion of Ripk3 (REFS129131).
Therefore, studies have used macrophages deficient in
both caspase 8 and RIPK3 or FADD and RIPK3. Bcells
derived from mice lacking caspase 8 only in the Bcell
population exhibit impaired phosphorylation and
nuclear translocation of the p65 subunit of NFB and
decreased expression of genes encoding IL6, TNF, IFN
and CXC-chemokine ligand 10 (CXCL10) in response to
LPS stimulation94.
So far, there is no consensus on the molecular mech-
anism by which caspase 8 mediates NFB signalling.
One report found that full-length caspase 8 and its cata
lytic activity are crucial for driving NFB activities98,
whereas another showed that the DEDs of caspase 8
are sufficient 132. Further studies have reported that
the catalytic activity of caspase 8 is dispensable for or
even reduces NFB signalling 99,132. A dual and oppos-
ing role for caspase 8 has also been proposed, whereby
pro-caspase 8 contributes to TNFR1induced activa-
tion of NFB whereas activated caspase 8 engages
cleavage ofNFBinducing kinase (NIK; also known
as MAP3K14) to prevent TNFR1induced NFB activa-
tion99. Further investigation demonstrated that caspase 8
targets and cleaves FLIPL, which generates an Nterminal
p43 fragment and a Cterminal p12 fragment100. Thep43
fragment of FLIPL, TRAF2 and caspase8 form a tri-
partite complex that subsequently activates the NFB
signalling pathway 100. Despite a lack of clarity with
respect to the specific mechanism involved, it is clear
that caspase8 is essential for the activation of NFB
signalling that leads to the production of a range of
pro-inflammatorycytokines.
Direct cleavage of IL1. Another pathway by which
caspase 8 contributes to inflammation is the conversion
of proIL1 to its mature bioactive form. Recombinant
caspase 8 directly cleaves human and mouse proIL1
at the Asp117 site the same site that is targeted by
caspase 1 whereas caspase 3, caspase 7 and caspase9
or a catalytically inactive mutant of caspase 8 fail to
do so101103. Depending on the stimuli received by the
cell, caspase 8 assembles macromolecular complexes of
varying complexity to mediate the cleavage of proIL1
(FIG.3). On recognition of certain fungal and myco
bacterial infections in human dendritic cells (DCs),
dectin1 (also known as CLEC7A) and its adaptor SYK
promote the formation of a CARD9BCL10MALT1
(mucosa-associated lymphoid tissue lymphoma trans-
location protein 1) complex that drives the transcrip-
tion of the gene encoding IL1. This complex further
recruits apoptosis-associated speck-like protein con-
taining a CARD (ASC; also known as PYCARD) and
caspase 8 to form a socalled non-canonical caspase8
inflammasome, whereby caspase 8 directly mediates
the processing of proIL1 in the absence of caspase1
(REF.104). By definition, this complex cannot be clas-
sified as an inflammasome as it does not contain an
inflammatorycaspase.
REVIEWS

Direct cleavage of proIL1 by caspase 8 in macro cellular IAP1 (cIAP1; also known as BIRC2) and
phages has been observed under conditions in which cIAP2 (also known as BIRC3) or inhibition of these
inhibitor of apoptosis proteins (IAPs) are blocked. Genetic IAPs by mimetics of second mitochondrial-derived acti-
deletion of X-linked IAP (XIAP; also known as BIRC4), vator of caspases (SMAC; also known as DIABLO) in

A. fumigatis Candida spp. Yersinia spp.

Ligands M. leprae LPS FASL

Dectin 1 FAS
TLR TLR4

Classical Endosome
MDP inammasome Stress-induced Pro-apoptotic
MYD88 Poly(I:C) drugs
activators stimuli
TLR3 TRIF ER
SYK
NOD2
TRIF Mitochondrion
ER stress
LRR CARD9 ?
SMAC

Caspase 8 NLRP3 MALT1 BCL-10

RIPK3 IAPs
PYD
FADD
Caspase 8 RIPK1 FADD
DED
ASC
FADD
FADD CARD Yersinia
Caspase 1 Caspase 8 Caspase 8 spp. Caspase 1
p50 p65
NF-B Caspase 1 Non-canonical Caspase 8 Caspase 1
inammasome caspase 8 inammasome processing unit processing unit

NLRP3

FAS Pro-IL-1 IL-1

Pro-inammatory
cytokines
TNF
Nucleus and IL-6

Figure 3 | Caspase 8 mediates initiation of NFB signalling and
maturation of IL1. Ligand recognition by Toll-like receptors (TLRs) leads
to signalling via the adaptor MYD88 and subsequent nuclear factor-B (NFB)
activation. Caspase 8 and its adaptor FAS-associated death domain (FADD)
contribute to this pathway by inducing the transcription of genes encoding
pro-inflammatory cytokines, some of which such as prointerleukin1
(pro-IL1) and proIL18 require further proteolytic processing93100. Caspase8 kinases receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and
or FADD is also recruited to the nucleotide-binding domain (NOD)-,
leucine-rich repeat (LRR)- and caspase activation and recruitment domain
(CARD)-containing protein 4 (NLRC4), NOD-, LRR- and pyrin domain(PYD)-
containing protein 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasome
complexes activated by classical inflammasome activators (Salmonella
enterica subsp. enterica serovar Typhimurium, lipopolysaccharide (LPS) plus
ATP or nigericin, Citrobacter rodentium, Escherichia coli, Francisella novicida,
double-stranded DNA and Aspergillus fumigatus), in which caspase8 either
activates caspase 1, leading to caspase 1dependent processing of proIL1
and proIL18, and/or modulates cell death95,96,111,112,117. However, in the
absence of caspase 1, caspase 8 may induce apoptosis in the inflammasome
speck112,113. Caspase 8 also assembles a range of structurally diverse complexes
to directly mediate processing of proIL1 independently of caspase 1.
Candida spp., A.fumigatus and Mycobacterium leprae infection trigger dectin1
and its adaptor SYK to engage a socalled non-canonical caspase 8
inflammasome complex that is composed of CARD9, BCL10,
mucosa-associated lymphoid tissue lymphoma translocation protein 1
(MALT1), apoptosis-associated speck-like protein containing a CARD (ASC)
and caspase 8. Caspase 8 directly processes Nature Reviews
proIL1 | Immunology
in the absence of
caspase 1 and this process does not require internalization of the pathogen104.
In response to TLR3 or TLR4 activators, the adaptor TIR domain-containing
adaptor protein inducing IFN (TRIF) promotes assembly of a caspase 8
processing unit for direct cleavage of IL1 in a manner that may require the
RIPK3, and the adaptor FAS-associated death domain (FADD)101,105108. For
example, LPS signalling via TLR4TRIF and an as-yet-unidentified signal
released as a result of stress in the endoplasmic reticulum (ER) engage
caspase8dependent processing of IL1 with or without RIPK3 (REF.108).
LPS-induced TLR4TRIF signalling and release of second mitochondrial-
derived activator of caspases (SMAC), owing to exposure to pro-apoptotic
signals delivered by doxorubicin, staurosporine or cycloheximide, activate
caspase 8dependent processing of IL1106,107. Here, SMAC or SMAC mimetics
suppress inhibitor of apoptosis proteins (IAPs) from blocking RIPK3. During
Yersinia pestis infection, RIPK1 and FADD are essential in mediating caspase8
activation via TRIF- and RIPK3dependent and -independent mechanisms,
whereby activation of caspase 8 ultimately drives caspase 1mediated
processing of IL1115,116. Signalling via TLRs induces upregulation of FAS.
Interaction between FAS and FASL licenses caspase 8dependent IL1
release via FADD, independently of RIPK3 or caspase 1 (REFS109,110).
DED, death effector domain; MDP, muramyl dipeptide; poly(I:C),
polyinosinicpolycytidylic acid; TNF, tumour necrosisfactor.

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2015 Macmillan Publishers Limited. All rights reserved


Inhibitor of apoptosis
proteins
(IAPs). A family of proteins
thathave multiple functions,
including inhibition of
apoptosis by binding to
apoptotic caspases, E3
ubiquitin ligase activity,
regulation of MAPK and
NFBsignalling pathways
andregulation of signal
transduction by
pattern-recognition receptors.
Second mitochondrial-
derived activator of caspases
(SMAC). Endogenous
antagonist of inhibitor of
apoptosis proteins (IAPs)
released from mitochondria
inresponse to death stimuli.
SMAC binds and degrades
XIAP, cIAP1 and cIAP2,
resulting in increased
caspaseactivation.
NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 9
macrophages results in LPS-induced secretion of IL1
through a mechanism that depends on both caspase8
and the caspase 1NLRP3 inflammasome102. When
XIAP, cIAP1 and cIAP2 are inhibited, both caspase 1-
and caspase 8dependent pathways of IL1 secretion
are licensed by the common upstream kinase RIPK3,
which is supressed by IAPs102. The complex interplay
between IAPs was further revealed by studies showing
that bone marrow-derived macrophages (BMDMs)
ormice with a deletion of the gene encoding cIAP1 or
cIAP2 have an impaired ability to activate the NLRP3
inflammasome133, whereas deletion of the gene encod-
ing XIAP promotes LPS-induced secretion of IL1 in
a RIPK3dependent manner 134. A later study further
confirmed that RIPK3 is required for LPS-induced
caspase1- or caspase8dependent secretion of IL1
in mouse bone marrow-derived DCs (BMDCs) 105.
The caspase 8containing proIL1 processing com-
plex is reported to also contain RIPK1, RIPK3 and
FADD, and requires signalling via the TLR adaptor
TIR domain-containing adaptor protein inducing
IFN (TRIF; also known as TICAM1)105. This find-
ing is consistent with a previous study showing that
the Cterminal receptor-interacting protein homo-
typic interaction motif (RHIM) of TRIF is essential for
driving caspase 8dependent IL1 processing 101.
A role for caspase 1 and caspase 8 in the release
of IL1 has also been described in the context of ER
stress106108,135137. The NLRP3 inflammasome responds
to ER stress and induces caspase 1dependent release
of IL1 in human and mouse cells135,137. A further
study reported that ER stress induced by an attenuated
strainof Brucella abortus leads to NLRP3dependent
activation of caspase 2 (REF. 136) . Caspase 2 then
mediates activation of BH3interacting domain death
agonist (BID) and release of mitochondrial DNA,
which leads to the activation of caspase 1 and secre-
tion of IL1136. Additional studies found that pro-
apoptotic chemot herap eutic agents and drugs that
induce stress in the ER promote caspase 8dependent,
caspase1independent secretion of IL1 in LPS-primed
BMDCs or BMDMs106108. Furthermore, TRIF is required
to mediate IL1 release in this process106,108. The amount
of cIAP1 is decreased in cells treated with LPS and the
pro-apoptotic chemotherapeutic agent doxorubicin106,
which may imply that cIAP1 has a role as a suppressor
of the caspase8dependent IL1processing unit 102.
Ithas, therefore, been proposed that pro-apoptotic stim-
uli dysregulate mitochondrial functions and induce the
release of SMAC from the mitochondria. SMAC then
binds to and inhibits IAPs, and thus prevents both
IAP-mediated degradation of RIPK3 and inhibition of
caspase 8dependent processing of IL1102,106 (FIG.3). Itis
not known whether ER stress directly causes reduced
levels of IAPs leading to the secretion of IL1. However,
RIPK3 has been shown to be partially involved in this
TRIFcaspase 8dependent pathway 108.
Engagement of FAS by FAS ligand (FASL; also
known as TNFSF6 and CD95L) is another pathway
by which caspase 8 is activated to cleave IL1 and
IL18 independently of caspase 1 (REFS109,110). In
2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
this context, TLR-induced FAS expression drives
caspase8- and FADD-mediated release of IL1 and
IL18 (REF.109). The observations that RIPK3 is dis-
pensable for this pathway and that priming by TLR7
which uses the adaptor MYD88 rather than TRIF
sufficiently induces FAS expression and FASL-mediated
IL1 production109 indicate that the FAS-regulated
caspase 8 complex probably differs in composition
to the aforementioned TRIF-dependent caspase 8
RIPK3RIPK1 complex. The precise architecture of
these caspase 8containing units remains to be defined
(FIG.3). Inhibition of inflammation and of the inflam-
masome by caspase 8 have also been observed and are
further discussedbelow.
Caspase 1dependent processing of IL1. More recent
studies have revealed a requirement for caspase 8 in
activating caspase 1 within the inflammasome complex
to generate bioactive IL1138,139. Confocal microscopy
studies have identified the presence of caspase 8 within
the ASC or caspase 1 inflammasome speck in macro
phages or DCs infected with S. Typhimurium 95,111,
F.novicida 112, C.rodentium 96 and Aspergillusfumiga
tus 117 (BOX1). Super resolution and confocal microscopy
studies have further identified bioactive caspase 1 and
caspase 8 molecules being bound by a concentric ring of
endogenous NLRC4 or NLRP3 and ASC in macrophages
infected with S. Typhimurium111.
In response to canonical inflammasome activators,
caspase 1 is required for the processing of proIL1
and proIL18 (REFS95,96,117,140156). The role of
caspase8 in this context is to activate caspase 1 or modu
late pyroptosis, rather than to participate directly in the
processing of proIL1 and proIL18 (FIG.3). Indeed,
recombinant caspase 8 cleaves caspase 1 invitro 96,
allowing caspase 1 to be fully activated to induce the
cleavage of pro-IL1 and pro-IL18 in response to
inflammasome activators LPS and ATP, C.rodentium,
Yersinia pestis, Yersinia pseudotuberculosis and A.fumi
gatus96,115117. During Yersinia spp. infection, activation of
caspase 1 by caspase 8 also requires RIPK1 and FADD,
with a partial contribution from TRIF and RIPK3
(REFS115,116). In response to glucans and heat-killed
C.albicans, both caspase 1 and caspase 8 regulate IL1
release independently of one another 118. Consistent
with this concept, prolonged stimulation with LPS
and nigericin or infection with S. Typhimurium in the
absence of caspase 1 activates a delayed pathway lead-
ing to caspase8dependent processing of IL195,114.
Caspase 1 and caspase 8 also synergistically contribute to
the production of IL1 that drives the development of
osteomyelitis119. Mouse neutrophils carrying a missense
mutation in the gene encoding proline-serine-threonine
phosphatase-interacting protein 2 (PSTPIP2) induce
more robust cleavage of IL1 and release large amounts
of IL1 relative to wild-type neutrophils, which is the
basis for the development of spontaneous bone inflam-
mation and destruction. However, genomic deletion of
the genes encoding caspase 1 and caspase 8 prevents
excessive production of IL1 and the development of
PSTPIP2mediatedosteomyelitis119.
REVIEWS
Precisely how caspase 1 and caspase 8 operate syner-
gistically and independently of one another and the nature
of the activators that determine this relationship remain
to be fully elucidated. Interestingly, caspase 8dependent
activation of the NLRP3 inflammasome is regulated by
RIPK3 and the necroptosis effector mixed lineage kinase
domain-like (MLKL) only after prolonged stimulation157.
This would, in part, reflect the compositional flexibility
and dynamics of the caspase 8containing complex, which
is observed when different contextual cues are sensed by
the cell at different times. Moreover, the growing evidence
supporting a role for caspase 8 in the initiation of inflam-
mation reinforces the perspective that the function of this
caspase is clearly not confined to apoptosis.
Inhibition of inflammation and the inflammasome. In
addition to its pro-inflammatory function, caspase8
has anti-inflammatory roles. Mice lacking caspase 8 in
hepatocytes or DCs develop chronic inflammatory or
autoimmune diseases158,159. Increased inflammation in
mice lacking caspase 8 in DCs could be prevented by dele-
tion of the TLR adaptor MYD88, which suggests a role
for caspase 8 in supressing TLR signalling 159. In the skin,
loss of caspase 8 expression promotes IL1 secretion and
the transcriptional activation of NFBresponsive genes
during wound healing 160.
One of the abilities of caspase 8 to modulate inflam-
mation includes inhibition of inflammasome activity
(FIG.4). The deficiency of caspase 8 in mouse DCs results
in assembly of the NLRP3 inflammasome and release of
IL1 in response to LPS or Pam3CSK4 even without an
inflammasome activator 120. LPS-induced IL1 produc-
tion in DCs lacking caspase 8 requires RIPK1, RIPK3,
the effector MLKL and the serine/threonine protein
phosphatase PGAM5. This study suggested that LPS
stimulation of DCs promotes an association of caspase8
with FADD to inhibit the assembly of a RIPK1RIPK3
complex and its downstream effector functions, leading
to activation of the NLRP3 inflammasome120. It has also
been proposed that caspase 8 and FADD bind NLRP3
to directly inhibit the inflammasome120. It remains
unclear in what context caspase 8 functions as a negative
regulator of inflammation and whether this function is
cell-typespecific.
Inhibition of necroptosis. An indirect way in which
caspase 8 inhibits inflammatory responses is through its
ability to inhibit necroptosis an inflammatory form of
programmed cell death mediated by RIPK1 and RIPK3
independently of caspases20,123,124. In addition to its ability
to bind and inhibit RIPK1RIPK3mediated inflamma
some activation120, caspase 8 cleaves RIPK1, RIPK3 and
the deubiquitylating enzyme CYLD, which is respon-
sible for removing ubiquitin chains from RIPK1 and
directing its function towards necroptosis161163 (FIG.4).
Indeed, spontaneous inflammation that develops in the
absence of caspase 8 is prevented by the inhibition of
necroptosis164,165. Mice with a conditional deletion of
the gene encoding caspase 8 in the intestinal epithelium
exhibit spontaneous ileitis, loss of Paneth cells and gob-
let cells, increased infiltration of granulocytes and CD4+
10 | ADVANCE ONLINE PUBLICATION www.nature.com/nri
2015 Macmillan Publishers Limited. All rights reserved
Tcells into the lamina propria, and increased expres-
sion of inflammatory markers164. Importantly, inhibition
of necroptosis by necrostatin1 largely prevents TNF-
induced lethality and small intestinal tissue destruction
in these mice164. Another report also found that caspase8
prevents RIPK3mediated necroptosis, death of entero-
cytes and immune cell infiltration of the colon165. These
findings are further supported by a study showing that
mice lacking FADD in the intestinal epithelium are
hypersusceptible to necrosis of epithelial cells, loss of
Paneth cells and the development of colitis defects
that are largely prevented by genomic deletion of the
gene encoding RIPK3 (REF.166). The ability of caspase 8
to provide a checkpoint to suppress necroptosis-induced
inflammation adds another layer of functional complex-
ity to this enigmatic protease.
Apoptotic caspases inhibit typeI IFNs
Apoptotic caspases have a major role in suppressing
typeI IFN responses. Defective caspase 8 functions have
been shown to lead to inflammatory skin disease owing
to increased IFN activity 122. In this study, a mouse strain
was generated to carry a set of wild-type caspase 8 alleles
and a set of enzymatically defective caspase 8 alleles122.
This was achieved by microinjecting fertilized oocytes
from wild-type mice with a bacterial artificial chromo-
some carrying a set of alleles encoding caspase 8 lacking
enzymatic activity. Mice with a set of wild-type caspase8
alleles develop normally in the presence of a second set
of alleles encoding caspase 8 lacking enzymatic activ-
ity 122. However, deletion of one of the two wild-type
alleles encoding caspase 8 results in the development of
a chronic inflammatory skin disorder as well as infiltra-
tion of leukocytes into the lungs and pancreas122. This
same study also found that mice lacking caspase 8 spe-
cifically in keratinocytes develop cutaneous inflamma-
tion. The disease pathogenesis was not a result of TNF,
IL1 or TLR signalling but of increased activation of
the transcription factor IFN-regulatory factor 3 (IRF3)
and TANK-binding kinase 1 (TBK1) in the epidermis122.
Furthermore, keratinocytes lacking caspase 8 express
increased levels of IFN and IFN-inducible proteins fol-
lowing exposure to transfected double-stranded DNA
compared with transfected wild-type keratinocytes122.
Increased expression of IRF3 and IRF7 in BMDCs
lacking caspase 8 has also been observed159.
More recent studies further highlight a relationship
between caspase 8 and other apoptotic caspases and
typeI IFNs. Recognition of RNA viruses by the cyto-
solic sensor RIGI triggers signalling through the adap-
tor mitochondrial antiviral signalling protein (MAVS;
also known as IPS1, VISA and CARDIF) at the mito-
chondria. RIPK1 is recruited to the mitochondria where
it is polyubiquitylated to drive IRF3dependent pro-
duction of typeI IFNs121. Importantly, caspase 8 cleaves
polyubiquitylated RIPK1, converting RIPK1 into an
inhibitor of RIGI121 (FIG.4). In the absence of caspase8,
fibroblastoid cells or hepatocytes infected with the RNA
viruses Sendai virus and vesicular stomatitis virus or
stimulated with the synthetic double-stranded RNA
poly(I:C) have enhanced activation of IRF3 (REF.121).
 REVIEWS

The intrinsic pathway of apoptosis regulated by
caspase 9 must also maintain immunological silence.
Released mitochondrial DNA owing to mitochondrial
outer membrane permeabilization activates the DNA
sensor cyclic GMPAMP synthase (cGAS) and its
adaptor stimulator of IFN genes (STING), which induce
typeI IFNs and IFN-stimulated genes for antiviral
defence167,168. However, caspase 3, caspase 7 and caspase9
inhibit the induction of typeI IFN responses167,168 (FIG.4).
Although the molecular mechanism remains to be
defined, it has been suggested that apoptotic caspases
might cleave cGAS, a regulator of cGAS or mitochon-
drial DNA itself 167,168. Release of mitochondrial DNA
could engage inflammasome sensors to potentiate the

Apoptotic Sendai virus,

stimuli VSV or poly(I:C)
TLR

Caspase 8
Intrinsic ssRNA or
Mitochondrion dsRNA FADD
apoptotic RIG-I DED
pathway
BAX BAK RIPK3 RIPK3
CYLD
MAVS Caspase 8 RIPK1 RIPK1
RIPK1 MLKL
Ub PGAM5
Ub
Ub
Cytochrome c mtDNA Ub

APAF1 Caspase 9 Cleaved RIPK3,

cGAS LRR RIPK1 and CLYD
WD40 Cleaved
RIPK1
NLRP3
PYD Necroptosis
Apoptosome

ASC

TBK1
CARD Pro-caspase 1
NLRP3 inammasome Pyroptosis
P P
IRF3 IRF3

STING
STING Pro-IL-1 IL-1

Caspase 3
Caspase 7 ER
P P Type I IFNs
IRF3 IRF3 and ISGs

Apoptosis
Nucleus

Figure 4 | Inhibition of inflammation by caspase 8 and other apoptotic
caspases. In the intrinsic pathway of apoptosis, mitochondrial DNA (mtDNA)
is released owing to mitochondrial outer membrane permeabilization
mediated by the pro-apoptotic effector proteins BCL2associated X protein
(BAX) and BCL2 antagonist/killer (BAK). Released mtDNA binds to the DNA
sensor cGAMP synthase (cGAS), which signals through its adaptor stimulator
of interferon (IFN) genes (STING), TANK-binding kinase 1 (TBK1) and
IFN-regulatory factor 3 (IRF3) to induce typeI IFNs and activate IFN-stimulated
genes (ISGs)167,168. Cytochromec is also released by mitochondria, which is
sensed by apoptotic protease-activating factor 1 (APAF1). APAF1
andcaspase9 assemble the apoptosome to activate effector caspase 3 and
caspase 7 (REFS1216). During apoptosis, caspase 3, caspase 7 and caspase9
prevent signal transduction by the cGASSTING pathway leading to typeI IFN
production through an asyet-undefined mechanism167,168. The RNA sensor
retinoic acid-inducible gene I (RIGI) recognizes the double-stranded RNA
(dsRNA) viruses Sendai virus and vesicular stomatitis virus (VSV) or the
synthetic dsRNA ligand polyinosinicpolycytidylic acid (poly(I:C)). RIGI
interacts with the adaptor mitochondrial antiviral signalling protein (MAVS)
at the mitochondria, where the kinase receptor-interacting serine/
threonine-protein kinase 1 (RIPK1) is recruited and undergoes Lys63linked
polyubiquitylation at site Lys377. Mitochondria-associated
Nature Reviewspolyubiquitylated
| Immunology
RIPK1 induces activation of antiviral responses via TBK1 and IRF3. Following
activation of IRF3, polyubiquitylated RIPK1 is cleaved by caspase 8, whereby
cleaved RIPK1 inhibits RIGI signalling121. Caspase 8 also suppresses activation
of the nucleotide-binding domain (NOD)-, leucine-rich repeat (LRR)- and
pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome121. Upon
stimulation of Toll-like receptor 2 (TLR2) or TLR4 in dendritic cells, caspase 8
and FAS-associated death domain (FADD) inhibit the ripoptosome composed
of the kinases RIPK3 and RIPK1. In the absence of caspase 8 or FADD, the
ripoptosome signals through the effector mixed lineage kinase domain-like
(MLKL) and the mitochondrial serine/threonine-protein phosphatase PGAM5
to activate the NLRP3 inflammasome121. Caspase 8 and FADD have also been
proposed to directly interact with and inhibit NLRP3. There is also evidence
to suggest that caspase 8 mediates cleavage of RIPK3, RIPK1 and the
deubiquitylating enzyme CYLD as a mechanism to inhibit necroptosis and
inflammation161163. ASC, apoptosis-associated speck-like protein containing
a caspase activation and recruitment domain (CARD); DED, death effector
domain; IL1, interleukin1; ssRNA, single-stranded RNA.

NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 11

2015 Macmillan Publishers Limited. All rights reserved

REVIEWS

Cell-autonomous immunity
A defence mechanism used by
the cell to control infection that
is not traditionally considered
as part of the immune system.
Examples include compart-
mentalization to prevent
inappropriate entry of bacteria
into the cytoplasm within a
eukaryotic cell and production
of nitric oxide synthases to
mediate killing of an invading
microorganism.
activation of caspase 1 (REFS146149,169,170). Indeed,
oxidized mitochondrial DNA released during apopto-
sis has been shown to bind directly to NLRP3 and acti-
vate the inflammasome in LPS-primed BMDMs169; this
example illustrates that immunological silence during
apoptosis is not always maintained171.
Caspases and cell-autonomous immunity
The induction of cell-autonomous immunity is essential
for host survival in response to infection. The functional
roles of caspases and the cytoskeleton are in many ways
inextricably linked to cell-autonomous host defence172.
Caspase 1 activation via the NLRC4 inflammasome
controls replication of L.pneumophila by inducing mat-
uration of the L.pneumophila-containing vacuole150.
Importantly, caspase 1 cleaves caspase 7 to promote
fusion between phagosomes and pathogen-containing
vacuoles173,174. A further study has shown that caspase4,
caspase 5 and caspase 11 are essential for the control
of L.pneumophila replication in macrophages175. The
mechanism by which caspase 11 contributes to the inhib
itionof L.pneumophila replication depends on the abil-
ity of caspase 11 to license actin polymerization through
phosphorylation of the actin-binding protein cofilin,
ultimately enabling fusion of the L.pneumophila vacuole
with lysosomes175. In addition, the CARD of caspase 11
has been shown to physically interact with the Cterminal
WD40 propeller domain of actin-interacting protein 1
(AIP1; also known as DAB2IP), whereby this interaction
potentiates cofilin-mediated actin depolymerization176.
The relationship between caspases and cell-
autonomous immunity is further demonstrated during
S.Typhimurium infection. In infected macrophages,
the NLRC4caspase 1 axis is required to induce cel-
lular stiffness, leading to impaired movement and
uptake of additional bacteria, as well as increased reac-
tive oxygen species production for bacterial killing 177.
Earlier studies have validated a role for caspase 1 in
cleaving actin and actin, rendering them unable
to undergo polymerization173,178180. It is possible that
caspase1dependent restriction of cytoskeletal functions
prevents overwhelming infection at the single cell level,
which enables the cell to direct resources to killing resid-
ing bacteria before phagocytosing more bacteria. This
hypothesis would align with the idea that commitment
to pyroptosis is made only when the cell no longer has
the capacity to kill the residing bacteria177.
In the absence of caspase 1, macrophages infected
with S. Typhimurium and F. novicida or trans-
fectedwith DNA undergo apoptosis and induce robust
cleavage of caspase 3, caspase 7, caspase 8 and caspase9
or the apoptotic substrate PARPs112,113,181. It is likely that
pyroptosis driven by caspase 1 overrides apoptotic cell
death during infection by certain bacteria. However,
if caspase 1mediated cell death is somehow bypassed
or compromised by bacterial or viral virulence fac-
tors that strategically block or mediate evasion of the
inflammasome, apoptosis eventually ensues to remove
the replicative niche for these pathogens. The synergy
between caspases in the context of cell-autonomous
immunity further blurs the line between inflammatory
and apoptotic caspases. Importantly, these findings pro-
vide elegant examples of how caspases control facets of
immunity beyond cell death and inflammation.
Conclusions
The caspase family members are architects of the body.
They maintain homeostasis by dismantling unwanted or
damaged cells in a systematic manner. Although caspases
are broadly classified into apoptotic and inflammatory
caspases, new and exciting studies are continuously
unveiling the multifaceted nature of individual caspases
in the immune system. Caspases function as pro-inflam-
matory and anti-inflammatory molecules, as well as hav-
ing cell-intrinsic roles to orchestrate cell-autonomous
immunity. Converging roles of caspases in the immune
system are further exemplified by their ability to com-
municate with one another to maintain immunological
silence or to promote inflammatory responses to elim-
inate invading pathogens. Furthermore, the capacity
for inflammatory caspases to mediate sensing of LPS
signifies the importance of these proteases in pattern
recognition. These elegant and surprising findings
challenge the existing functional classification of the
caspase family members. The intertwining relationship
between caspases will be further defined in the future.
Understanding the expanding and interconnected
roles of caspases will contribute to the development of
therapies that target disease processes associated with
dysregulated caspaseactivity.

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Acknowledgements
Research studies from the authors laboratory are supported
CA163507 and AI101935 to T.-D.K.), the American
Lebanese Syrian Associated Charities (to T.-D.K.) and the
National Health and Medical Research Council of Australia
(NHMRC/R.G. Menzies Early Career Fellowship to S.M.M.).
The authors apologize to their colleagues whose work was
not cited owing to space limitations.
Competing interests statement
The authors declare no competing interests.