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J Cell Physiol. 2019;1–15. wileyonlinelibrary.com/journal/jcp © 2019 Wiley Periodicals, Inc.
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Received: 19 June 2019
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Accepted: 27 September 2019
DOI: 10.1002/jcp.29268
REVIEW ARTICLE
Emerging insights into molecular mechanisms underlying
pyroptosis and functions of inflammasomes in diseases
Fangfang Lu
1,2
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Zhixin Lan
2
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Zhaoqi Xin
2
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Chunrong He
2
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Zimeng Guo
2
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Xiaobo Xia
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Tu Hu
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1
Department of Ophthalmology, Xiangya
Hospital, Central South University, Changsha,
Hunan Province, China
2
Xiangya School of Medicine, Central South
University, Changsha, Hunan Province, China
Correspondence
Tu Hu, Department of Ophthalmology,
Xiangya Hospital, Central South University,
Changsha, 410013 Hunan Province, China.
Email: hutu1986@csu.edu.cn
Funding information
National Natural Science Foundation of China,
Grant/Award Number: 81900890; College
students’innovative project of CSU, Grant/
Award Number: ZY2016698; China
Postdoctoral Science Foundation, Grant/
Award Number: 2018M643004
Abstract
Pyroptosis is a form of necrotic and inflammatory programmed cell death, which
could be characterized by cell swelling, pore formation on plasma membranes, and
release of proinflammatory cytokines (IL‐1βand IL‐18). The process of pyroptosis
presents as dual effects: protecting multicellular organisms from microbial infection
and endogenous dangers; leading to pathological inflammation if overactivated. Two
pathways have been found to trigger pyroptosis: caspase‐1 mediated inflammasome
pathway with the involvement of NLRP1‐, NLRP3‐, NLRC4‐, AIM2‐, pyrin‐inflamma-
some (canonical inflammasome pathway) and caspase‐4/5/11‐mediated inflamma-
some pathway (noncanonical inflammasome pathway). Gasdermin D (GSDMD) has
been proved to be a substrate of inflammatory caspases (caspase‐1/4/5/11), and the
cleaved N‐terminal domain of GSDMD oligomerizes to form cytotoxic pores on the
plasma membrane. Here, we mainly reviewed the up to date mechanisms of
pyroptosis, and began with the inflammasomes as the activator of caspase‐1/caspase‐
11, 4, and 5. We further discussed these inflammasomes functions in diseases,
including infectious diseases, sepsis, inflammatory autoimmune diseases, and
neuroinflammatory diseases.
KEYWORDS
caspases, GSDMD, inflammasomes, pyroptosis
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INTRODUCTION
Pyroptosis was first described as lysis occurring following infection of
Shigella Flexner in macrophages in 1992, which was classified as
apoptosis mistakenly since initial recognition of programmed cell
death was limited to apoptosis (Zychlinsky, Prevost, & Sansonetti,
1992). In 2001, pyroptosis was first proposed by Cookson and
Brennan (2001), and was originally defined to describe the
nonapoptotic, caspase‐1 dependent cell death of Salmonella‐infected
macrophages.The subsequent research works identified that
pyroptosis was characterized by pore formation in the plasma
membranes, cell swelling and release of inflammatory intracellular
contents, which was substantially different from apoptosis (Table 1;
Aachoui, Sagulenko, Miao, & Stacey, 2013; Bergsbaken, Fink, &
Cookson, 2009). Pyroptosis is emerging as a form of inflammatory
programmed cell death mediated by inflammatory caspases (Man,
Karki, & Kanneganti, 2017). As the discovery of gasdermin D
(GSDMD), pyroptosis was redefined as gasdermin‐mediated pro-
grammed necrosis (Figure 1; Shi, Gao, & Shao, 2017).
Pyroptosis is substantially classified as innate immune response
which can be triggered by the process of recognizing structures
damage‐associated molecular patterns (DAMPs) and conserved patho-
gen‐associated molecular patterns (PAMPs) by host pattern recognition
receptors (PRRs; Takeuchi & Akira, 2010). Previous studies revealed
that PRRs presented as two types: the transmembrane and the
cytoplasmic. And the latter one facilitated the secretion of proin-
flammatory cytokines and removed the intracellular replicative niche of
the bacteria through mediating a series of signal cascades in cells at the
first line of defense against infection. These were considered as the key
events of pyroptosis (Lamkanfi & Dixit, 2014). In case of sensing
PAMPs/DAMPs, the cytoplasmic PRRs, including nucleotide‐binding
domain (NBD)‐like receptors (NLRs), absent in melanoma 2‐like
receptors (ALRs), and Pyrin, assembled into high‐molecular caspase‐
activating molecular platforms—“inflammasomes,”which served for
recruitment of the inflammatory caspases (Martinon, Burns, & Tschopp,
2002). Canonical inflammasomes are composed of cytoplasmic PRRs,
adapter protein apoptosis‐associated speck‐like protein containing a
CARD (ASC), and procaspase‐1 (Proell, Gerlic, Mace, Reed, & Riedl,
2013). Several canonical inflammasomes have been identified to
mediate pyroptosis in vivo, denoted by different cytoplasmic PRRs,
including NLRP1 inflammasome (Chavarria‐Smith & Vance, 2015),
NLRP3 inflammasome (Agostini et al., 2004), NLRC4 inflammasome
(Poyet et al., 2001), AIM2 inflammasome (Burckstummer et al., 2009),
and Pyrin inflammasome (de Zoete & Flavell, 2014). The activated
canonical inflammasomes could process zymogen procaspase‐1intothe
active noncovalently linked subunits p10 and p20 (active caspase‐1),
and the latter could facilitate the maturation of proinflammatory
cytokines (pro‐IL‐18 and pro‐IL‐1β) and induce the formation of pore on
plasma membranes (Elliott, Rouge, Wiesmann, & Scheer, 2009).
Previous studies demonstrated that the pores on plasma membranes
permitted ions to permeate and dissipated the cellular ionic gradient,
which led to a multifaceted cascade of events: cell swelling, release of
cytosolic pathogens, and inflammatory cytokines (IL‐1βand IL‐18; Miao
et al., 2010). Due to these pores, the immune defense function of
pyroptosis is realized by the released mature inflammatory cytokines
for recruitment of neutrophils and expelled bacteria for neutrophil‐
mediated killing in situ (Xia, Wang, Zheng, Jiang, & Hu, 2019). Recently,
Feng et al. reported GSDMD as a generic substrate for caspase‐1and
highlighted the crucial role of gasdermin D‐N terminus (GSDMD‐NT)in
the formation of these pores (Ding et al., 2016; Shi et al., 2015). Except
the caspase‐1 meditated canonical inflammasome pathway of pyropto-
sis, a series of subsequent studies identified murine caspase‐11 and
human caspase‐4/5 as cytosolic sensors in response to cytosolic
lipopolysaccharide (LPS) to meditate noncanonical inflammasome
pathway of pyroptosis (Baker et al., 2015; Kayagaki et al., 2011). In‐
depth studies demonstrated that noncanonical inflammasome could
also cleave GSDMD, triggering caspase‐1 independent pyroptosis, and
proinflammatory cytokines secretion (Figure 2; Kayagaki et al., 2015;
Yang, Zhao, & Shao, 2015).
In this review, we summarized the updated molecular mechan-
isms implicated in the regulation of inflammasomes which present as
the activators of canonical/noncanonical inflammasome pathways of
pyroptosis, and these inflammasomes function in relevant infectious,
autoinflammatory, and neuroinflammatory diseases.
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NLR FAMILY RELEVENT
INFLAMMASOME
NLRs structurally characterized by a C‐terminal LRR domain that
could recognize cytosolic ligands and retain the NLRs in an
TABLE 1 Characteristic comparisons between pyroptosis and apoptosis
Mechanism Morphology Function
Caspases Cellular contents DNA
Plasma‐
membrane Nucleus
Cyto-
plasm Reaction Result
Pyroptosis Caspase‐1, 4, 5,
and 11
Released through pores
on plasma membrane
DNA fragmentation Pores formation Intact nucleus Swelling Inflammatory response Expulsion of cytoplasmic
pathogens
Apoptosis Caspase‐3, 6, 7,
and 8
Packaged in apoptotic
body
Characteristic oligonucleosomal
DNA fragments
Intact plasma
membrane
Damaged
nucleus
Shrinkage Noninflammatory
response
Clearance of damaged
cells
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LU ET AL.
FIGURE 1 Milestones in the history of
pyroptosis exploration
FIGURE 2 Caspase‐1 mediated canonical inflammasome pathway and caspase‐4/5/11 mediated noncanonical inflammasome pathway of
pyroptosis. (Domain positions aren’t drawn to scale). In caspase‐1 mediated canonical inflammasome pathway of pyroptosis, different PAMPs or
DAMPs activate corresponding cytoplasm inflammasome sensors respectively, including NLRP1, NLRP3, NLRC4, AIM2, and Pyrin, and then
recruit caspase‐1 with or without adapter protein ASC to form inflammasome. The inflammasome subsequently triggers its dimerization and
autoproteolytic converting of procaspase‐1 into the active subunits p10 and p20. Activated caspase‐1 subsequently cleaves its downstream
effectors: the executioner GSDMD and pro‐IL‐1β/pro‐IL‐18. In noncanonical inflammasome pathway, cytosolic LPS from Gram‐negative bacteria
promotes activation of murine caspase‐11 or human caspase‐4/5 directly. The activated caspase‐11/4/5 also cleaves GSDMD, but it stimulates the
cleavage of pro‐IL‐1βand pro‐IL‐18 by activating the Nlrp3‐Asc‐caspase‐1 noncanonical inflammasome. The cleaved GSDMD via canonical or
noncanonical inflammasome pathway release the functional GSDMD‐NT, which forms pores on the plasma membrane and subsequently induces
pyroptosis and release of mature IL‐1β/IL‐18. ASC, apoptosis‐associated speck‐like protein containing a CARD; CARD, caspase activation and
recruitment domain; FIIND, function‐to‐find domain; GSDMD, gasdermin D; GSDMD‐NT, gasdermin D N‐terminal; GSDMD‐CT, gasdermin D
C‐terminal; LRR, leucine‐rich repeat; NATCH/ NOD, nucleotide‐binding oligomerization domain; PYD, Pyrin domain
LU ET AL.
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nonactivated status, a central highly conserved nucleotide‐binding
oligomerization domain (NACHT) that functions in process of
oligomerization, and a variable N‐terminal that mediates homotypic
interactions with downstream proteins (Lechtenberg, Mace, & Riedl,
2014). Different N‐terminal domains have been used as a structural
sub classification, like NLRP with a PYD (Pyrin domain), NLRB with
BIR domain (baculovirus inhibitory repeat domain), and NLRC with a
CARD (caspase activation and recruitment domain; Sharma &
Kanneganti, 2016). The most well‐recognized inflammasome‐forming
members in NLR family include NLRP3, NLRP1, and NLRC4 (Guo,
Callaway, & Ting, 2015). Upon recognizing specific stimuli, the
corresponding NLRs (NLRP3, NLRP1, and NLRC4) nucleates an
adapter protein ASC (composed of PYD‐CARD), forming an ASC
speck structure around nucleus for the subsequently clustering of
procaspase‐1 (Lu et al., 2014). Previous studies showed that the PYD‐
containing NLRP3 exclusively requires ASC for inflammasome
assembly via NLRP3
PYD
–ASC
PYD
and ASC
CARD
procaspase‐1
CARD
interaction, whereas CARD‐containing NLRP1 and NLRC4 could
recruit procaspase‐1 directly through CARD–CARD interaction
(Figure 2; Lechtenberg et al., 2014). Howbeit, some other studies
also claimed that NLRC4/NLRP1 induced procaspase‐1 activation
and cytokine processing could be significantly enhanced in the
presence of ASC, since its bipartite PYD‐CARD structure might
stabilize the CARD–CARD interaction (Van Opdenbosch et al., 2014).
Thus, there remains a dispute on whether ASC is unnecessary for the
activation of NLRP1/NLRC4 inflammasome.
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NLRP3 INFLAMMASOME
NLRP3 is the most extensively investigated sensor proteins in NLR
family and features a PYD in the N‐terminal. To date, a two‐step
model has been well‐established in NLRP3 inflammasome activation:
a priming signal requested to upregulate NLRP3 and pro‐IL‐1β, and a
second signal required for creating a functional inflammasome (He,
Hara, & Núñez, 2016). The priming signal could be simulated by
pattern recognition receptors, like TLR4, NOD2, TNFR, and IL‐1R. All
these signaling could cause activation of nuclear factor kappa B (NF‐
κB), which led to increased synthesis of pro‐IL‐1βand NLRP3
(Bauernfeind et al., 2009). Juliana et al. (2012) found an additional
rapid priming event: pattern recognition receptor TLR4 mediated
NLRP3 posttranslational deubiquitylation signaling by MyD88. Py,
Kim, Vakifahmetoglu‐Norberg, & Yuan (2013) further discovered that
BRCC3 functioned as the deubiquitinase in mouse cells that
specifically stimulated the deubiquitylation of the LRR domain of
NLRP3.Notably, posttranslational modification of protein is not
unique to NLRP3 deubiquitylation, subsequent research works
discovered SYK‐and JNK‐dependent ASC phosphorylation and
LUBAC‐mediated ASC ubiquitination to be novel priming processes
(Elliott & Sutterwala, 2015). In general, PRR signal‐dependent
priming in transcriptional or posttranslational modification level
enables NLRP3 to be responsive to agonists. The second signal of
activation of the NLRP3 inflammasome was triggered by extensive
structurally dissimilar agonists, including environmental crystalline
pollutants like silica, pathogen‐derived ligands like pore‐forming
toxins and nucleic acids as well as endogenous danger signals like
serum amyloid A and ATP (Elliott & Sutterwala, 2015).Due to the
diversity of its agonists, it was suggested that these unrelated
upstream activators might trigger cognate cellular downstream
events that directly activate NLRP3. Through further exploration,
researchers found that K
+
efflux, lysosomal disruption, calcium
signaling, mitochondria‐derived factors like oxidized mitochondrial
DNA (mt DNA), reactive oxygen species (ROS) and cardiolipin, and
microtubule‐driven spatial arrangement of mitochondria were all
direct upstream signals required for NLRP3 inflammasome activation
(Tang et al., 2019; Wang, Yuan, Chen, & Wang, 2019; Yabal, Calleja,
Simpson, & Lawlor, 2019). Despite intensive scrutiny on this topic, a
unified mechanism has not emerged yet. He et al. (2016) proposed
that the activation of the NLRP3 inflammasome is the integration of
above cellular signaling events. However, how these multiple signals
are interacted to activate the NLRP3 inflammasome remains elusive.
The negative regulation of NLRP3 equals the importance of the
priming and activation events described above (Sutterwala, Haasken,
& Cassel, 2014).In the priming level, the myeloid‐specific microRNA‐
223 (miR‐223) could specifically interact with the 3′‐untranslated
region of NLRP3 mRNA and target it for its degradation, thereby
interfering with the translation of NLRP3. Besides, type I interferons
(IFNs) could also suppress NLRP3 inflammasome priming by inducing
the upregulation of the anti‐inflammatory cytokine IL‐10 which
signaled through STAT3 to lessen expression levels of pro‐IL‐1βand
pro‐IL‐1α(Guarda et al., 2011). In the activating process, it was
suggested that nitric oxide (NO) inhibited the assembly of NLRP3
inflammasome by stabilizing mitochondria, thereby, obstructing IL‐1β
maturation and caspase‐1 activation (Mishra et al., 2013). In addition,
immunity‐related GTPase M (IRGM) was recently found to bind to
the conserved NACHT domain of NLRP3 and the PYD of ASC,
thereby limiting NLRP3/ASC oligomerization. On top of that, IRGM
could also target NLRP3 and ASC for their autophagic degradation
(Mehto et al., 2019; Nabar & Kehrl, 2019). To conclude, the
components both contributing to and negatively modulating the
activation of NLRP3 inflammasome ensure the validity of a perceived
threat with an appropriate response.
The activation of NLRP3 inflammasome exerts protective effects
against infection, including bacterium, virus, and fungi, through
caspase‐1 mediated pyroptosis (Xia et al., 2019). Nevertheless,
dysregulated NLRP3 inflammasome activation causes chronic in-
flammation, leading to neuroinflammatory diseases, and metabolic
diseases (Guo et al., 2015; Yin et al., 2019). For example, both spinal
cord injury (SCI) and traumatic brain injury (TBI) follow a biphasic
time course: after the initial primary injury resulting in neuronal
death, a complicated secondary phase of central nervous system
(CNS) injury cascade involving neuroinflammation follows, which are
mediated by proinflammatory cytokines. This secondary injury
causes further neurological deterioration and complicates the healing
process (Borgens & Liu‐Snyder, 2012). Previous studies identified the
expression of NLRP3 in neurons, astrocytes and microglia in rat
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LU ET AL.
model of SCI and TBI (Liu et al., 2013). More recently research works
provided evidence in a SCI or TBI model that during 24 hr–72 hr
postinjury, most of NLRP3 inflammasome components including
NLRP3, ASC, and caspase‐1were increased (Zendedel et al., 2018).
Besides, NLRP3 could be activated by α‐synuclein (α‐syn) and
amyloid‐β(Aβ) via microglial endocytosis and succeeding lysosomal
release of cathepsin B. The activated NLRP3 inflammasome then
processed IL‐18 and IL‐1β, and ultimately induce Parkinson disease
(PD) and Alzheimer disease (AD) pathology and tissue damage
respectively (Zhou et al., 2016). Moreover, NLRP3 inflammasome
functioned to promote multiple sclerosis (MS) through IL‐18 and IL‐
1β, which induced Th17 and Th1 cell responses and migration (Inoue,
Williams, Gunn, & Shinohara, 2012). These evidence suggested the
crucial role of NLRP3 inflammasome in neuronal disorders. In
addition, it has been well‐established in the past decade that chronic
inflammation contributes the development of metabolic disorders,
such as atherosclerosis obesity and type 2 diabetes mellitus (T2DM;
Donath & Shoelson, 2011). Recent evidence suggested that choles-
terol crystals could activate the NLRP3 inflammasome through
phagolysosomal damage and generate mature IL‐1βin both mouse
and human cells (Donath & Shoelson, 2011).Furthermore, elevated
glucose and islet amyloid polypeptide (IAPP) during T2DM could also
trigger the activation of NLRP3 inflammasome and result in
succeeding release of IL‐1β(Vandanmagsar et al., 2011; Zheng
et al., 2013).Based on these evidence, NLRP3 inflammasome induced
cellular pyroptosis may promote the progress of neurodegenerative
and metabolic diseases. Therefore, further explorations could be
focused on the inventions which compete against the formation of
NLRP3 inflammasomes to improve the treatment for these diseases
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NLRP1 INFLAMMASOME
The structure of NLRP1 proteins shows species‐specific differences
(Figure 3). It differs from the other NLRs in two respects. First, most
primates including human encode a single NLRP1, which contains
two annotated recruitment domains: A C‐terminal CARD and a N‐
terminal PYD. In other NLRs, the PYD functions to recruit caspase‐1
via ASC, while in human NLRP1 (hNLRP) it was C‐terminal CARD of
NLRP1 rather than PYD that mediated homotypic interactions with
downstream ASC and procaspase‐1 (Faustin et al., 2007).Notably, N‐
terminal PYD has recently been shown to be essential in sustaining
NLRP1 in its inactive state based on the observation that destabiliz-
ing the PYD of human NLRP1 increased self‐oligomerization (Zhong
et al., 2016). NLRP1 in mice has undergone three paralogs (Nlrp1a, b,
and c) that contain an NR100 (N‐terminal domain of rodent Nlrp1
proteins, approximately 100 amino acids) domain preceding the
NACHT domain (Moayeri, Sastalla, & Leppla, 2012). It was discovered
that proteolytic cleavage in the NR100 domain was necessary for
NLRP1B inflammasome formation and caspase activation (Chavarria‐
Smith & Vance, 2013). Second, between the LRR and the C‐terminal
CARD of NLRP1 proteins, there is a unique stretch of 30 kDa domain
called “function‐to‐find”domain (FIIND) (Finger et al., 2012). It was
reported that this domain possessed autoproteolytic activity and the
generated associated C‐terminal and N‐terminal fragments of NLRP1
was required for activating NLRP1 inflammasome. This autoproteo-
lytic activity is also identified as a maturation event before the
NLRP1 becoming responsive to activators (Chavarría‐Smith, Mitchell,
Ho, Daugherty, & Vance, 2016). To date, activators of NLRP1 remain
partially elucidated. Some researchers identified that Nlrp1b in mice
and rats could be activated by reduced intracellular levels of ATP and
Toxoplasma gondii infections (Ewald, Chavarria‐Smith, & Boothroyd,
2014; Liao & Mogridge, 2013). However, there are deficient evidence
to convincingly ascertain the role of ATP binding for NLRP1
activation and how T.gondii activates NLRP1 remains to be
discovered. For human NLRP1, there are no defined activators yet.
Although previous study showed that bacterial ligand muramyl‐
dipeptide (MDP) could induce NLRP1 oligomerization (Faustin et al.,
2007), growing evidence suggested that MDP is actually sensed by
other PRR pathway (Martino et al., 2016). Until now, the only well
FIGURE 3 Domain structure of NLRP1 proteins in human, mouse and rats. (Domain positions aren’t drawn to scale). All members of NLRP1
proteins contain NATCH, LRR, and CARD domains. Human NLRP1 also consists of a pyrin domain (PYD) in the N‐terminal. Compared with
human NLRP1, mouse NLRP1 has undergone three paralogs, termed Nlrp1a, ‐b, and ‐c, that contain an NR100 domain preceding the NACHT
domain instead of a PYD and the linker connecting this two units is shorter. Autolytic cleavage in the NR100 domain and FIIND is indicated
respectively by the split in the structure of corresponding domains. The FIIND and CARD domains are missing in the structure of Nlrp1c, which
is not expected to form a functional inflammasome
LU ET AL.
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identified activator of NLRP1 is anthrax lethal toxin (LT) which is
produced and secreted by Bacillus anthracis. It was show that LT
directly cleaved mouse Nlrp1b and rat Nlrp1at N‐terminal and such
cleavage is needed for activation of Nlrp1 inflammasome and
following macrophage pyroptosis (Chavarria‐Smith & Vance, 2013).
More recent studies indicate that the activation of human NLRP1 can
also be triggered by proteolysis in a specific N‐terminal linker region
between the PYD and NBD domains, which also confirms the auto‐
inhibitory role of N‐terminal PYD (Chavarria‐Smith et al., 2016).
Based on these evidence, we could conclude that the prerequisites of
NLRP1 activation are as follows: autoproteolytic cleavage of FIIND,
the sensation of stimulus and the N‐terminal proteolytic cleavage.
Anthrax disease is characterized by lung tissue necrosis,
circulatory collapse and pulmonary edema, which is resulted from
infection of B. anthracis whose major virulence determinant is lethal
toxin (LT; Quintiliani & Quintiliani, 2003). Studies indicated LT could
cleave the N‐terminus of NLRP1b, and thereby activating the
inflammasome and inducing pyroptosis in macrophage after infection
with B. anthracis in susceptible mouse strains (Nour et al., 2009). LT
induced pyroptosis in murine dendritic cells and macrophages was
originally believed to contribute to pathology of anthrax disease due
to the reduced bacterial clearance by these immunocytes (Hanna,
Acosta, & Collier, 1993). Further exploration illustrated that
activation of NLRP1 could defend against B. anthracis infection by
inducing NLRP1‐caspase‐1 mediated pyroptosis, inflammatory signal-
ing, and neutrophil recruitment in situ (Moayeri et al., 2012). In
addition, the previous study indicated that infection with Toxoplasma
triggers an NLRP1 inflammasome mediated inflammatory responses,
limiting parasite load and dissemination, which is protective to the
host (Ewald et al., 2014). These evidence suggest that NLRC4
inflammasome mediated pyroptosis and inflammatory responses
contribute to host‐defense against infection. However, aberrant
inflammatory signal could have deleterious effects. Recent multiple
studies demonstrated that activating mutations of NLRP1 in patients
were associated with hereditary autoinflammatory diseases, like
Crohn’s disease, autoinflammatory skin disorder, systemic lupus
erythematosus, and rheumatoid arthritis (Cummings et al., 2010;
Grandemange et al., 2017; Kitamura, Sasaki, Abe, Kano, & Yasutomo,
2014; Pontillo et al., 2012). Furthermore, similar to NLRP3, NLRP1 is
also implicated in neuroinflammatory process in neurons (Mortezaee,
Khanlarkhani, Beyer, & Zendedel, 2018). A study using immunoblot
analysis of cerebrospinal fluid (CSF) samples from both adult TBI
patients and nontrauma normal control revealed significantly higher
levels of NLRP1 and caspase‐1 in TBI patients (Adamczak et al.,
2012). Likewise, co‐immunoprecipitations of post‐SCI lysates in the
SCI rat model showed upregulated association between NLRP1 and
caspase‐1, suggesting NLRP1 contributing to the pathology of CNS
injury (Lin et al., 2016). Besides, growing evidence suggest NLRP1
also have a pathogenic role in AD (Saresella et al., 2016). Recently, a
study using APPswe/PS1dE9 transgenic mice (transgenic mice
developing chronic deposition of amyloid‐β) showed that cerebral
NLRP1 levels were upregulated and when NLRP1 or caspase‐1 was
knock downed, considerably decreased neuronal pyroptosis and
reversed cognitive impairments emerged. In cultured cortical
neurons, it was found that NLRP1‐mediated pyroptosis increased in
response to Aβ, which further confirmed the pathogenic role of
NLRP1 inflammasome in AD progression (Guo et al., 2015). Of note,
evidence indicate that composition of neuronal NLRP1 inflamma-
some differs from that described in peripheral macrophages. The
NLRP1 inflammasome in neuron is a preassembled multiprotein
complex, comprising of NLRP1, ASC, caspase‐1, caspase‐11, and the
inhibitor of apoptosis protein X‐linked inhibitor of apoptosis protein
(XIAP). The existence of XIAP in this preassembled complex in
neurons is not fully understood, although the cleavage of XIAP might
reduce its capability of inhibiting caspases. And whether caspase‐11
in the structure of NLRP1 inflammasome in neuron has the same
function of caspase‐1 to induce pyroptosis requires further con-
firmation as the current reports on caspase‐11 mediated pyroptosis
are limited in infectious diseases (de Rivero Vaccari, Dietrich, &
Keane, 2014). In contrast to the promotional function of NLRP3
inflammasome in metabolic diseases, NLRP1 has been recently
discovered to counteract metabolic syndrome through inflamma-
some activation and IL‐18 production (Murphy et al., 2016). Soares
et al. (2018) elucidated that diabetes metabolites, like high glucose
and glycated albumin, could modulate NLRP1 expression and further
suggested that NLRP1 functioned as a homeostatic factor protecting
host against diabetic kidney disease (DKD) in type‐1 diabetes.
Regardless of well‐defined NLRP3 inflammasome on neuroinflamma-
tory diseases and metabolic diseases, it should be a worthy research
path to explore new drug via targeting NLRP1.
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NLRC4 INFLAMMASOME
The NLRC4 inflammasome, insisting of a N‐terminal CARD, a central
NACHT and a C‐terminal LRR, was initially named ICE‐protease‐
activating factor (IPAF) for its function in activating caspase‐1 via
direct NLRC4
CARD
–caspase‐1
CARD
interaction (Poyet et al., 2001). In
contrast to the diverse stimuli for the activation of NLRP3, the
NLRC4 has been currently believed to be activated to only two
bacterial components: flagellin and type III secretion systems (T3SS)
apparatus (rod protein and needle protein; Miao et al., 2010). Unlike
other inflammasomes, NLRC4 activation required another protein,
neuronal apoptosis inhibitory proteins (NAIPs), to recognize the
cytosolic presence of these bacterial components (Kofoed & Vance,
2011). In the absence of these bacterial agonists, LRR domain in
NAIP and NLRC4 curves back to occlude the NBD, preventing NBD‐
mediated oligomerization (Hu et al., 2013). And in the presence of
bacterial ligands, NAIP directly recognizes and binds to specific
ligands with an internal region containing several NBD‐associated
α‐helical domains and thus breaks its autoinhibitory conformational
states, allowing its NBD‐mediated co‐oligomerization with NLRC4
(Tenthorey, Kofoed, Daugherty, Malik, & Vance, 2014). Such co‐
oligomerization induces formation of a large NAIP‐NLRC4 inflamma-
some for activating caspase‐1 and eventually leading to release of
IL‐1β/IL‐18 and pyroptosis (Zhao & Shao, 2015). Howbeit how
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LU ET AL.
NLRC4 autoinhibition is relieved to allow oligomerization with NAIP
proteins is elusive. NAIP protein, one of the NLR family member, is
featured by three tandem BIR domains at its N‐terminus (Kofoed &
Vance, 2012). Mice have four NAIP paralogues: NAIP5/NAIP6
recognize flagellin; NAIP1 and NAIP2 specifically recognize T3SS
needle protein and rod protein respectively. By contrast, human only
encodes one single NAIP gene (hNAIP) and is able to detect both
bacterial T3SS rod/needle protein and flagellin (Reyes Ruiz et al.,
2017). The discovery of NAIPs in mice and human NAIP that act
functionally upstream of NLRC4 maximizes the capacity of innate
immune detection in coping with bacterial infections. It remains
unknown whether the formation of hetero‐oligomeric structure is a
common feature of all NLRs or just unique to NLRC4. In addition to
the essential role of NAIPs functioning upstream of NLRC4
activation, recent several studies proposed that Ser533 phosphor-
ylation of NLRC4 prepared NLRC4 for subsequent activation by
NAIP detection of ligands (Matusiak et al., 2015). However, there are
also contradictory reports indicating that such phosphorylation event
also occurs in the inactive state of NLRC4 (Hu et al., 2013).
Therefore, it needs further investigation to determine the necessity
of the phosphorylation event.
Flagella and T3SS are molecular transport machineries employed
by bacteria to enhance the ability to reach and invade host cells
(Galán, Lara‐Tejero, Marlovits, & Wagner, 2014). It has been well‐
established that NAIP‐NLRC4 inflammasome in host immune cells,
like dendritic cells and macrophages, could detect bacterial flagellin,
T3SS rod/needle protein during infection (Zhao & Shao, 2015). And
the NAIP‐NLRC4 inflammasome mediated pyroptosis promotes host
innate antibacterial responses by expelling bacteria from their
protected intracellular niche and by released IL‐1β/IL‐18 caused
inflammation (Bergsbaken et al., 2009; Zhao & Shao, 2015). However,
in vivo,some pathogenic bacteria develop strategies to escape from
NAIP‐NLRC4 inflammasome mediated innate immune defense. For
instance, S. typhimurium normally represses flagellin and SPI1 T3SS to
evade the detection by NAIP‐NLRC4 inflammasome and conversely
activates SPI2 T3SS at the late phase of infection, which lacks
inflammasome‐stimulating activity, to promote its replication within
macrophages (Miao et al., 2010). To identify the exact pathway of
host‐defense against pathogens, Miao et al. use engineered S.
typhimurium constitutively expressing flagellin to infect mice and
the results showed that NLRC4 inflammasome activation efficiently
cleared the infected S. typhimurium in macrophage. Importantly, this
clearance was mediated by caspase‐1‐mediated pyroptosis while IL‐
1β/IL‐18 induced inflammatory responses only played a minor role,
based on the evidence that mice devoid of both IL‐18 and IL‐1βalso
cleared S. typhimurium, whereas pyroptosis released bacteria from
macrophages and exposed them to be cleared by the neutrophil
(Miao et al., 2010). Except the functions of NAIP‐NLRC4 inflammma-
some in macrophages and dendritic cells, recent studies reported
protection role of NAIP‐NLRC4 inflammasome against enteric
pathogens, like Salmonella and Citrobacter rodentium, in intestinal
epithelial cells (Nordlander, Pott, & Maloy, 2014). Still, it required
further study to determine whether this represents a general defense
mechanism protecting the intestinal mucosa. Despite the essential
role of NLRP4 inflammasome in controlling bacterial infections,
hyperactivation of the NLRC4 inflammasome could cause autoin-
flammation in human and mice. A spectrum of studies identified a
gain‐of‐function mutations in the NBD of NLRC4 to be related with
familial cold autoinflammatory syndrome (FCAS), neonatal onset
multisystem inflammatory disease (NOMID), and autoinflammation
with infantile enterocolitis (AIFEC) (Romberg, Vogel, & Canna, 2017).
It is expected that mutations of NLRP4 may destabilize the
interaction between the NBD and winged‐helix domain (WHD), or
directly affect ADP binding, either of which may be crucial for
maintaining NLRC4 in an auto‐inhibited state (Hu et al., 2013). Thus,
these evidence underscore the essential roles of the NLRC4
inflammasome in autoinflammatory diseases and suggest novel
targets for therapy.
6
|
AIM2 AND IFI16 INFLAMMASOME
AIM2 and IFI16 belong to the IFN‐inducible p200‐protein family,
which is also called AIM2‐like receptors (ALRs). Eight ALRs (like
p202a, p202b, p204, and Aim2) were found in mouse, while four
ALRs have been previously annotated in humans (IFI16, MNDA, IFIX,
and AIM2; Brunette et al., 2012). It was found that AIM2 could
interact with cytosolic double stranded DNA (dsDNA) and assembles
ASC and procaspase‐1 to form a functional inflammasome complex
(Fernandes‐Alnemri, Yu, Datta, Wu, & Alnemri, 2009). Recent studies
identified that IFI16 could also specifically recognize ASC and
procaspase‐1 in response to nuclear and cytosolic dsDNA, forming
a functional inflammasome for activating caspase‐1 (Kerur et al.,
2011). AIM2 and IFI16 are typical proteins of the ALRs, character-
ized by a PYD in the N‐terminus and one or two partially conserved
repeat of 200‐amino acid residues (HIN‐200 domain) in the C‐
terminus. The HIN‐200 domain contains two adjacent OB domains
(oligonucleotide/oligosaccharide‐binding domain), which could po-
tentially combine with dsDNA (Fernandes‐Alnemri et al., 2009). PYD
in AIM2 partly binds its HIN domain by electrostatic attraction so
that AIM2 persistently exists in the autoinhibition state in resting
cells. The dsDNA has higher affinity for OB domain of HIN domain
compared with PYD domain. Therefore, after intracellular dsDNA
appears, PYD will be replaced by dsDNA and thereby the auto‐
inhibited state of AIM2 is liberated (Jin, Perry, Smith, Jiang, & Xiao,
2013). The PYD therefore is exposed to ASC, recruiting ASC through
homotypic PYD–PYD interactions. Subsequently, An functional AIM2
inflammasome is assembled via combination of CARD of ASC and
CARD of procaspase‐1 which could induce pyroptosis and inflam-
matory responses (Choubey et al., 2010). Notably, both AIM2 and
IFI16 recognize dsDNA from a wide range of sources regardless of
their structure or sequences but the dsDNA strand is required to be
80 base‐pairs in length for optimum inflammasome activation. This
specific DNA recognition pattern is based on electrostatic charge
neutralization between the dsDNA sugar‐phosphate backbone and
the positively charged HIN domain residues (Jin et al., 2012).
LU ET AL.
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7
Moreover, despite there is no oligomerization domain in AIM2 or
IFI16, oligomerization could be accomplished by the multivalent
ligand dsDNA, which offered platform for AIM2 to cluster on (Jin
et al., 2012). Recognition of cytosolic DNA by cells is vital to the
innate immune system to initiate immunological responses and also
an essential contributor to autoimmune diseases when implicating
self‐DNA. Except the secretion of type I IFNs such as IFN‐β,an
effective immune response to the occurrence of cytosolic DNA is
dependent on proinflammatory cytokines (Medzhitov, 2007). Pre-
vious studies have illustrated that AIM2 and IFI16 could recognize
dsDNA and then form functional inflammasome to trigger pyroptosis
and IL‐18 and IL‐1βmediated inflammatory responses (Man, Karki, &
Kanneganti, 2016b). AIM2 detects dsDNA in a DNA sequence‐
independent manner, allowing its response to various microbial
threats and cellular stress. Indeed, AIM2 was shown to contributed
to provide immunosurveillance to microbial pathogens from bacteria
(like Francisella, Listeria, and Mycobacterium), DNA viruses (like
cytomegalovirus, vaccinia virus, and human papillomaviruses), fungi (like
Aspergillus fumigatus), and protozoa (like Plasmodium berghei; Man,
Karki, & Kanneganti, 2016a). These evidence highlighted the crucial
role of AIM2 inflammasome for host‐defense against cytosolic
pathogenic DNA. However, it occurs under certain circumstances
where host cell fails to distinguish host self‐DNA from pathogenic
nucleic acids, resulting in upregulated AIM2‐dependent release of IL‐
1βthat contributes to the pathogenesis of inflammatory diseases.
Studies have found abundant cytosolic self‐DNA and increased AIM2
expression in patients with chronic and acute skin conditions,
including contact dermatitis, psoriasis, and atopic dermatitis (de
Koning et al., 2012). Except the pivotal function of NLRP1 and NLRP3
inflammasome in TBI, recent studies suggested that AIM2 might
contribute to the pathogenesis of TBI. It was indicated that AIM2 was
associated with the pyroptotic cell death induced caspase‐1in
response to aberrant dsDNA in cortical neurons. It was also observed
that CSF samples from TBI patients contained considerably higher
levels of nucleic acids than CSF from control patients, and when
neurons were cocultured with CSF from TBI patients, AIM2
inflammasome activation emerged (Adamczak et al., 2014). However,
studies focusing on the function of AIM2 in CNS injury are limited. In
addition, IFI16 inflammasome has been described in HIV‐1 infected
CD4
+
T cells and DNA virus Kaposi's sarcoma‐associated herpesvirus
(KSHV) infected endothelial cells (Monroe et al., 2014). KSHV causes
an asymptomatic latent infection in normal individual, whereas it can
lead to Kaposi's sarcoma in immunocompromised patients, which
features an inflammatory microenvironment including IL‐1β(Ganem,
2006). Kerur et al. (2011) demonstrated that IFI16 acted as a nuclear
KSHV dsDNA sensor to induce the formation of inflammasome which
then translocates from nucleus to cytosol to process pro‐IL‐1β.Of
note, studies recently have made a breakthrough on the mechanism
underlying CD4 T‐cell death in HIV‐infected hosts. Doitsh et al.
(2014) suggested that the progressive depletion of more than 95% of
quiescent lymphoid CD4 T‐cells, abortively infected with HIV, die by
pyroptosis instead of apoptosis. Further studies identified that IFI16
was responsible for the detection of HIV‐1 dsDNA and consequently
resulted in pyroptosis of CD4
+
T cells and secretion of inflammatory
cell signaling molecule IL‐1β, which recruits more cells to die of
pyroptosis. This creates a vicious pathogenic cycle, contributing to
HIV pathogenesis (Monroe et al., 2014). To sum up, these evidence
indicate a novel and potential therapy for AIDS targeting the
pathway of host CD4
+
T cells pyroptosis rather than the virus.
7
|
PYRIN INFLAMMASOME
Pyrin (Marenostrin, TRIM 20), encoded by the human MEFV gene or
mouse analog Mefv, expresses mainly in neutrophils, monocytes and
dendritic (Seshadri, Duncan, Hart, Gavrilin, & Wewers, 2007). There
are four functional domains in human Pyrin, a N‐terminal pyrin
domain (PYD), two central zinc finger domain (bBox), a coiled coil
(CC) domain and a C‐terminal B30.2/SPRY domain. And mouse pyrin
lacks a B30.2/SPRY domain (Yu et al., 2006). The B‐box domain
interacts with the PYD to sustain Pyrin in an auto‐inhibited state and
the CC domain mediates self‐oligomerization of Pyrin into a trimer.
Thus, Pyrin exists as an inactive homotrimer in resting cells (Yu et al.,
2007). The PYD in Pyrin, which is also found in other known sensors
like NLRP1, NLRP3, and AIM2, mediates homotypic interaction with
ASC to assemble procaspase‐1 and form functional inflammasome
(Vajjhala et al., 2014). Despite the well‐established Pyrin inflamma-
some in vitro, the triggers and function of Pyrin in innate immunity
response remained obscure until a recent study elucidated that Pyrin
mediated inflammasome activation in response to pathogen‐
mediated modifications of Rho GTPases. And the modifications were
caused by effector proteins or pathogenic toxins, like FIC‐domain
adenylylation by VopS of Vibrio parahemolyticus, FIC‐domain adeny-
lylation by IbpA of Histophilus somni, Rho‐glycosylation by cytotoxin
TcdB of Clostridium difficile, deamination by Burkholderia cenocepacia,
and ADP‐ribosylation by C. botulinum C3 toxin and.These modifica-
tions all resided in Switch‐I region of Rho GTPases, and could lead to
the inactivation of the Rho GTPase. Pyrin functioned to recognize the
inactivation of Rho GTPases and thereby triggered Pyrin inflamma-
some activation (Xu et al., 2014). Interestingly, Pyrin seems not to
bind modified Rho GTPases directly, indicating a potential down-
stream signaling event. On topic of this, Park, Wood, Kastner, and
Chae (2016) recently showed that in the resting state, Rho GTPases
activated the serine‐threonine kinases PKN1 and PKN2 that bound
and phosphorylate human Pyrin at S208 and S242. Phosphorylated
Pyrin was combined with14‐3‐3 proteins, which maintained Pyrin
inflammasome in an inactive state (Park et al., 2016). When Rho
GTPases was modified by bacterial toxins or effector proteins, Pyrin
dephosphorylated and 14‐3‐3 proteins dissociated, resulting in a
conformational change that allowed Pyrin inflammasome activation.
Yet, it remains obscure how Pyrin is dephosphorylated and how 14‐
3‐3 proteins are released (Gao, Yang, Liu, Wang, & Shao, 2016).
Familial Mediteranean fever (FMF) is an autosomal recessive
autoinflammatory disease featured by attacks of fever and neutro-
phil‐mediated serosal inflammation, which was caused by mutations
of MEFV gene that encodes Pyrin (Centola, Aksentijevich, & Kastner,
8
|
LU ET AL.
1998). In particular, two thirds of such mutations are located in the
B30.2/SPRY domain (Chae et al., 2011). Such missense mutations
effect in FMF was due to gain‐of‐function of Pyrin, which caused
increased caspase‐1 activation and inflammatory responses (Chae
et al., 2011). In addition, A newly identified disease, Pyrin‐associated
autoinflammation with neutrophilic dermatosis (PAAND), is also
caused by mutations of MEFV gene, but it is distinct from FMF on
molecular mechanism. The mutation results in serine‐to‐arginine
replacement at position 242 (S242R) in pyrin and thereby eliminates
14‐3‐3 binding motif (Masters et al., 2016). Further studies
conducted by Moghaddas et al. (2017) reported a novel E224K
MEFV mutation that was located in the +2 position of the 14‐3‐3
binding motif in pyrin. Consistent with S242R mutation in Pyrin,
E224K in Pyrin could also induce spontaneous inflammasome
formation and occurrence of pyroptosis (Moghaddas et al., 2017).
In addition, a recent study identified that hyperimmunoglobulinemia
D syndrome (HIDS) was dependent on a dysregulation of pyrin
activation, which was caused by mutations of mevalonate kinase
(MVK). Loss‐of‐function mutations of MVK reduced prenylation of
proteins such as Rho‐GTPase, causing inactivation of Rho‐GTPase
and consequent activation of Pyrin inflammasome (Park et al., 2016).
Furthermore, Pyrin protein was also involved in syndrome of
pyogenic arthritis, pyoderma gangrenosum and acne (PAPA), an
inherited autoinflammatory disorder caused by proline‐serine‐threo-
nine phosphatase‐interacting protein1 (PSTPIP1) mutations (Wise
et al., 2002). The PAPA‐associated mutation was shown to increase
the binding of PSTPIP1 to B‐box of pyrin.Such binding unmasked
PYD of Pyrin protein, which allowed Pyrin to interact with ASC and
recruit procaspase‐1 to form Pyrin inflammasome (Yu et al., 2007).
Intriguingly, these evidence indicated a possibility that PSTPIP1
might be a downstream protein of RhoGTPase modification and more
studies are required to focus on this topic.
8
|
NONCANONICAL INFLAMMASOME
The functional significance of caspase‐11 in the process of pyroptosis
was masked for a long time, until Kayagaki et al. (2011) discovered
that proinflammatory caspase‐11 could trigger pyroptosis and IL‐1β
maturation in a Nlrp3‐Asc‐caspase‐1 inflammasome dependent
pathway in LPS‐primed mouse macrophages. Shi et al. (2014) found
that human caspase‐4/5 (homologs to murine caspase‐11) could also
be activated by direct binding to intracellular LPS in human
monocytes and non‐monocytes, like certain epithelial cells and
keratinocytes, which led to pyroptosis. This caspase‐4/5/11‐LPS
complex was classified as the noncanonical inflammasome (Ding &
Shao, 2017).
Mounting studies emphasized the crucial role of Gram‐negative
bacterium derived LPS in caspase‐4/5/11‐dependent pyroptosis (Yi,
2017). LPS of extracellular Gram‐negative bacterium was sensed by
transmembrane Toll‐like receptor 4 (TLR4). TLR4 activated MyD88/
TRIF/NF‐kB pathway and IFNR/JAK/STAT pathway to regulate the
transcription of caspase‐4/5/11, which was essential for initiating the
noncanonical inflammasome pathway (Yang et al., 2015). Caspase‐
11/4/5 acts as LPS sensors and only recognizes LPS that has entered
the cytoplasm (Hagar, Powell, Aachoui, Ernst, & Miao, 2013).
Howbeit extracellular Gram‐negative bacteria or internalized bacter-
ia within membrane‐bound vacuoles are believed to seldom get
access into cytoplasm, so exposure of bacterial LPS to cytoplasm is a
vital event for pathogen recognition. Vanaja et al. (2016) showed that
Gram‐negative bacteria produced outer membrane vesicles (OMVs)
that entered cells by endocytosis and the early endosomes could
release LPS into the cytoplasm. Recently, it was further identified
that hemolysin could combine with OMVs and promoted rupture of
OMV‐containing vacuoles in the cytoplasm, enabling OMV‐derived
LPS to access the cytoplasm (Chen et al., 2018). And for vacuole‐
residing Gram‐negative bacteria, studies suggested that guanylate‐
binding proteins (GBP) lysed vacuoles and thus exposed bacteria to
caspase‐11 in the cytoplasm (Meunier et al., 2014). The CARD in the
caspase‐4/5/11 could directly interact with the lipid A of cytosolic
LPS, leading to activation of the caspases (Hagar et al., 2013). The
active caspase‐4/5/11 then induced pyroptosis by cleaving GSDMD
into GSDMD‐NT (Kayagaki et al., 2015; Shi et al., 2015). Interestingly,
active caspase‐4/5/11 promotes the maturation of pro‐IL‐18 or pro‐
IL‐1βthrough stimulating the Nlrp3‐Asc‐caspase‐1 noncanonical
inflammasome pathway instead of direct interaction (Baker et al.,
2015). However, the underlying mechanisms by which caspase‐11
induces Nlrp3‐Asc‐caspase‐1 activation have not been well‐defined.
Several studies proposed that potassium outflow probably could
bridge caspase‐11 and NLRP3 activation based on the discovery that
activated LPS‐caspase‐11 could cleave the pannexin‐1 channel to
induce release of ATP, which activated purinergic receptor P2X7 to
open a larger reversible pore that allowed the efflux of potassium.
And the efflux of potassium was considered as one of the agonists of
NLRP3 inflammasome in the canonical inflammasome pathway (Ruhl
& Broz, 2015). However, the functional crosstalk between non-
canonical and canonical inflammasome signaling cascades still needs
further study. Furthermore, saturated fatty acids and nuclear DAMPs
(nDAMPs) induce lipid peroxidation were reported to trigger
caspase‐4/5 and caspase‐11 mediated pyroptosis respectively in
vitro (Chen et al., 2019; Pillon et al., 2016). Whether the
noncanonical inflammasome of cell pyroptosis could be activated
by other molecules except above agonists also remains to be
investigated.
Pyroptosis mediated by caspase‐11 is believed to contribute to
the clearance of cytosolic bacterial pathogens in vivo, such as
Salmonella and Burkholderia.Salmonella normally resides in the
vacuole. Specific mutant strains of S. typhimurium escaped from the
vacuole and were recognized by caspase‐11, and caspase‐11 induced
downstream signaling was shown to enhance clearance of the S.
typhimurium in vivo (Aachoui et al., 2013). Besides, caspase‐11
−/−
mice
were susceptible to Burkholderia thailandensis and Burkholderia
pseudomallei, which naturally invade the cytosol, whereas wild‐type
mice were resistant to the infection (Jorgensen & Miao, 2015).
Despite that caspase‐11 mediated pyroptosis provides protection
against pathogen by releasing potentially harmful bacteria from the
LU ET AL.
|
9
nutrient‐rich host cell cytosol and through secreted inflammatory
cytokine induced inflammatory responses, dysregulated caspase‐11
mediated pyroptosis during overwhelming infection contributes to
sepsis. Sepsis is defined as life‐threatening organ dysfunction caused
by a dysregulated host inflammatory response to infection (Singer
et al., 2016). LPS has been known as an essential stimulus of sepsis
(Sweet & Hume, 1996). Studies recently proposed caspase‐4/5/11 as
a sensor of cytoplasmic LPS, suggesting that caspase‐4/5/11
mediated noncanonical inflammasome pathway of pyroptosis might
play a pivotal role in regulating sepsis (Shi et al., 2014). Further
exploration confirmed this idea with the evidence that wild‐type mice
were more vulnerable to LPS than caspase‐11
−/−
mice when
challenged with the Gram‐negative bacteria (Hagar et al., 2013). In
addition, Kayagaki et al. (2015) reported that Gsdmd
−/−
mice were
protected from a lethal dose of LPS and highlighted that key role of
GSDMD in LPS‐induced septic shock. The resistance of mice lacking
GSDMD or caspase‐11 to LPS‐induced lethality inspires the
researchers to attempt to develop antagonists of human caspase‐4/
5 or GSDMD for the benefit patients suffering from Gram‐negative
bacteria‐induced sepsis. Moreover, based on the beneficial and
detrimental effects of caspase‐4/5/11 mediated pyroptosis, how to
weigh the delicate balance between combating infection and sepsis is
a question to be resolved in the future.
9
|
Gasdermin D (GSDMD)
The underlying molecular mechanism downstream of caspase‐1/4/5/
11 to mediate pyroptosis had remained undefined, until recently two
separated research groups, Vishva M Dixit and Feng Shao,
independently identified GSDMD (gasdermin D) as an essential
pyroptosis substrate of inflammatory caspases (Kayagaki et al., 2015;
Shi et al., 2015). Both group further generated mouse strains with a
genomic deletion of gasdermin D (Gsdmd
−/−
) to confirm its crucial
role in pyroptosis and found that Gsdmd
−/−
iBMDMs did not undergo
pyroptosis or secrete mature inflammatory cytokines (Kayagaki et al.,
2015; Shi et al., 2015). Shi et al. (2015) also found GSDMD deficiency
inhabit IL‐1βsecretion without influencing its cleavage by caspase‐1,
suggesting GSDMD functioning downstream of caspase. Moreover, it
was showed that after the assembly of canonical or noncanonical
inflammasome, the activated inflammatory caspases (caspase‐1/4/5/
11) cleaved the 53‐kDa GSDMD after Asp276 (mouse)/275 (human),
generating the gasdermin‐C terminus (GSDMD‐CT,22 kDa) and
gasdermin‐N terminus (GSDMD‐NT, 31 kDa). Of note, it was
GSDMD‐NT rather than full‐length GSDMD or GSDMD‐CT that
bore intrinsic pyroptosis‐inducing activity, based on the evidence
that when GSDMD‐CT, GSDMD‐NT, and full‐length GSDMD were
individually expressed in 293T cells, pyroptosis only occurred in
GSDMD‐NT group. Likewise, ectopic overexpression of GSDMD‐NT,
but not GSDMD‐CT or full‐length GSDMD, rapidly triggered
extensive cell death with pyroptosis morphology. As for the function
of GSDMD‐CT, it was indicated that GSDMD‐CT bound to GSDMD‐
NT in resting cells to maintain GSDMD protein in an auto‐inhibited
state (Kayagaki et al., 2015; Shi et al., 2015). Recently, Kuang et al.
(2017) reported the crystal structure of the GSDMD‐CT and
revealed that the first loop on GSDMD‐CT inserted into the N‐
terminal domain to help stabilize the full‐length GSDMD conforma-
tion. Such auto‐inhibited conformation could be broken upon
interdomain cleavage by inflammatory caspases. Once the auto‐
inhibition state is released, the positive potential surface of GSDMD‐
NT covered by GSDMD‐CT is exposed and then forms high‐order
oligomers via a charge‐charge interaction to trigger the pyroptosis
(Kuang et al., 2017; Shi et al., 2015). Thus, it further confirmed that
GSDMD‐NT was the key mediator of pyroptosis, which was
characterized by pore formation in morphology. This raised a
prediction that GSDMD‐NT either possessed pore‐forming activity
or promoted the formation of this pore (Broz, 2015). Through further
exploration on this topic, studies showed that the GSDMD‐NT
possessed lipid‐binding preferences and could specifically bind to
phosphatidylserine and phosphatidylinositol phosphates, which were
constrained to the inner leaflet of cell membrane, as well as
cardiolipin in the inner and outer leaflets of cell membranes (Ding
et al., 2016; Liu et al., 2016). Indeed, GSDMD‐NT formed 16‐mer
pore complex on the plasma membrane, which disrupted cell
membrane integrity. Then, cell pyroptosis and secretion of cytosolic
contents emerged (Ding et al., 2016). In addition, researchers
visualized GSDMD‐NT oligomers on PS‐containing liposomes by
using negative staining electron microscopy and observed that pores
in the plasma membrane were approximately 15 nm inner and 32 nm
outer diameters, which were larger than processed IL‐1βwith a
diameter of approximately 4.5 nm. These pores precisely explain the
release of IL‐18 and IL‐1βduring pyroptosis as these inflammatory
cytokines lack an N‐terminal signal peptide for endoplasmic
reticulum to Golgi secretion pathway (Liu et al., 2016). Interestingly,
the results from vitro experiments observed that GSDMD‐NT could
directly interact with cardiolipin on bacterial membranes and killed
them, suggesting a possible antibacterial role of GSDMD‐NT.
However, more experiments are required to distinguish such direct
killing of bacterium by GSDMD‐NT from killing of the GSDMD
induced host pyroptosis, and explain how GSDMD‐NT protein passes
through the thick cell wall to access inner membrane (Liu et al.,
2016).
GSDMD belongs to a gasdermin family that also includes
GSDMA, GSDMB, GSDMC, GSDME/DFNA5, and DFNB59. All
gasdermins (except for DFNB59) adopt GSDMD‐like auto‐inhibited
two‐domain architecture: gasdermin‐N and ‐C domains. Recently,
studies showed gasdermin‐N domains of all gasdermins could induce
mammalian cell pyroptosis and kill bacteria. However, except
GSDMD, other gasdermins are insensitive to inflammatory caspases
(Ding et al., 2016; Shi et al., 2017; Shi et al., 2015). Notably, there are
some recent evidence suggesting a role of GSDME in pyroptosis. It
was showed that GSDME could switch caspase‐3 dependent
chemotherapy drug‐induced apoptosis into pyroptosis. The shift
from apoptosis to pyroptosis relied on the expression or expression
level of GSDME. Upon processing chemotherapy drug, GSDME high
expression cells undergo pyroptosis; otherwise, undergo apoptosis
10
|
LU ET AL.
(Wang et al., 2017; Wang et al., 2018). In general, GSDME was
silenced in most cancer cells and expressed in many normal tissues.
Studies revealed that GSDME mediated pyroptosis in normal tissues
contributed to the chemotherapy drug‐induced toxicity, like tissue
damage and weight loss, while in GSDME‐expressed cancer cells, like
gastric cancer cells, GSDME mediated pyroptosis contributed to the
chemotherapy (Wang et al., 2017, 2018). These results break the
traditional concept that activation of caspase‐3 inevitably induce
apoptosis. More important, in addition to exploration of inflamma-
some inhibitors for future treatment of cancer (Xu et al., 2019), these
data highlighted an innovative and promising therapeutic approach
against cancer by targeting GSDME.
10
|
CONCLUTION AND PERSPECTIVES
Recent advances have now established the key events during process of
pyroptosis, which is inflammasome activation, inflammatory caspases
maturation, and GSDMD cleavage. A wide range of host and microbial
factors trigger inflammasome activation and induce a series of caspase
dependent signaling events, including proinflammatory cytokine ma-
turation, pyroptosis, and inflammatory responses. This caspase‐1
mediated canonical pathway of pyroptosis is paramount in host‐defense
against intracellular pathogens. Pyroptosis of infected cells could
remove intracellular replicative niche of the bacteria to extracellular
matrix and the released cytokine could recruit more immune cells to
amplify inflammation in situ. The discovery of LPS triggered caspase‐11/
4/5 mediated pyroptosis has expanded the understanding of signaling
pathway leading to pyroptosis. This newly discovered noncanonical
pathway is essential in host‐defense against pathogenic infection and is
responsible for the dysregulated inflammatory response, sepsis.
Importantly, GSDMD has been identified as substrate of inflammatory
caspases and pore‐forming activity of GSDMD‐N T is responsible for
pyroptosis execution.
Recent studies have clarified a number of mysteries that were
prevalent in the activators of canonical inflammasomes and noncanonical
inflammasome. However, whether these inflammasomes can also be
activated by other PAMPs/DAMPs is still uncertain, and the detailed
mechanisms governing the activation process above are still controver-
sial. Newly uncovered GSDMD opens up new avenues to specifically
suppress pyroptosis in health and disease. However, researchers have not
reached a consensus on the downstream molecular mechanisms involved
in GSDMD‐mediated cytotoxic pores, and the functions of other GSDMs
in pyroptotic process are still required further investigation. As reviewed
above, pyroptosis appears to be a double‐edged sword. Based on the
pathogenic and protective effects of pyroptosis, further exploration on its
therapeutic inhibition to control the excessive inflammation has to be
balanced against its beneficial contribution to infectious diseases.
ACKNOWLEGMENTS
This study is supported by National Natural Science Foundation of
China (no. 81900890), China Postdoctoral Science Foundation (no.
2018M643004), and the College Students’Innovative Project of CSU
(no. ZY2016698).
AUTHOR CONTRIBUTIONS
F. L. wrote the article. C. H. and Z. G. edited the article and Z. X. and
Z. L. draw the graphical illustrations. T. H. designed the work,
developed the idea, and supervised the writing process.
ETHICAL STATEMENT
Fangfang Lu, Zhixin Lan, Zhaoqi Xin, Chunrong He, Zimeng Guo, and
Tu Hu declare that they have no conflict of interests. This article does
not contain any studies with human or animal subjects performed by
the any of the author
ORCID
Fangfang Lu http://orcid.org/0000-0002-9214-3046
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How to cite this article: Lu F, Lan Z, Xin Z, et al. Emerging
insights into molecular mechanisms underlying pyroptosis and
functions of inflammasomes in diseases. J Cell Physiol.
2019;1–15. https://doi.org/10.1002/jcp.29268
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