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Loop Between NLRP3 Inflammasome
and Reactive Oxygen Species
Abishai Dominic,
1,2
Nhat-Tu Le,
2
and Masafumi Takahashi
3,i
Abstract
Significance: Inflammasomes are cytosolic multiprotein complexes that mediate innate immune pathways.
Inflammasomes activate inflammatory caspases and regulate inflammatory cytokines interleukin (IL)-1band
IL-18 as well as inflammatory cell death (pyroptosis). Among known inflammasomes, NLRP3 (NLR family
pyrin domain containing 3) inflammasome is unique and well studied owing to the fact that it senses a broad
range of stimuli and is implicated in the pathogenesis of both microbial and sterile inflammatory diseases.
Recent Advances: Reactive oxygen species (ROS), especially derived from the mitochondria, are one of the
critical mediators of NLRP3 inflammasome activation. Furthermore, NLRP3 inflammasome-driven inflamma-
tion recruits inflammatory cells, including macrophages and neutrophils, which in turn cause ROS production,
suggesting a feedback loop between ROS and NLRP3 inflammasome.
Critical Issues: The precise mechanism of how ROS affects NLRP3 inflammasome activation still need to be
addressed. This review will summarize the current knowledge on the molecular mechanisms underlying the
activation of NLRP3 inflammasome with particular emphasis on the intricate balance of feedback loop between
ROS and inflammasome activation.
Future Directions: Understanding that this relationship is loop rather than traditionally understood linear mech-
anism will enable to fine-tune inflammasome activation under varied pathological settings. Antioxid. Redox Signal.
00, 000–000.
Keywords: cytokines, inflammation, interleukins, macrophages, mitochondria, pyroptosis
Introduction
Inflammation is the response of the body’s innate im-
mune system to harmful stimuli derived from patho-
gens, damaged or dead cells, and irritants. The inflammatory
response is tightly regulated to control its duration and mag-
nitude while preserving host tissue structure and function.
Exposure to harmful stimuli activates multiple signaling cas-
cades to induce the expression of inflammatory cytokines
and chemokines by resident tissue cells. This leads to the
1
Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, College Station, Texas, USA.
2
Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, Houston,
Texas, USA.
3
Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan.
i
ORCID ID (https://orcid.org/0000-0003-2716-7532).
ANTIOXIDANTS & REDOX SIGNALING
Volume 00, Number 00, 2022
ªMary Ann Liebert, Inc.
DOI: 10.1089/ars.2020.8257
1
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recruitment of inflammatory cells, including macrophages
and neutrophils, inducing a cascade of further production
of substantial amounts of inflammatory cytokines, ultimately
leading to the clearance of the pathogen or stimuli. In addi-
tion, this process also leads to the resolution of inflammation
by clearance of necrotic cells and damaged tissue and initi-
ating the process of tissue repair.
In this manner, inflammatory cytokines are key contribu-
tors to mounting an effective inflammatory response. Inter-
leukins (ILs) are the major type of cytokines that regulate
crucial inflammatory responses. Although >40 cytokines are
designated as ILs, IL-1bis considered the prototypical in-
flammatory cytokine that evokes the secretion of other in-
flammatory cytokines and chemokines, and is implicated in
a wide variety of diseases (54); therefore, targeting of IL-1b-
mediated inflammation has received increasing attention.
One such example is the recent CANTOS (Canakinumab
Anti-inflammatory Thrombosis and Outcome Study) trial
revealed that IL-1binhibition with canakinumab could de-
crease systemic inflammation and cardiovascular events in-
dependent of lipid lowering (75).
A great body of evidence indicates that IL-1bsecretion
is predominantly regulated by intracellular multiprotein com-
plexes called inflammasomes, which create molecular plat-
forms for activating caspase-1 (11, 44, 88). Because caspase-1
is reported to be an IL-1bconverting enzyme (ICE) (47), its
activation drives the processing of pro-IL-1binto active
IL-1b, resulting in inflammation and injury to the surround-
ing tissue. Although there are many different types of in-
flammasomes, the NLRP3 (NLR family pyrin domain
containing 3, also known as NALP3) inflammasome is con-
sidered to be a promising target of various diseases because
its activation is triggered by a broad spectrum of stimuli
derived from pathogens, damaged or dead cells, and irri-
tants, which are known as pathogen-associated molecular
patterns (PAMPs) or damage/danger-associated molecular
patterns (DAMPs).
For this reason, NLRP3 inflammasome has been exten-
sively studied, and it has been revealed that multiple mech-
anisms can participate in NLRP3 inflammasome activation
(44, 53, 58). In particular, reactive oxygen species (ROS)
are one of the main mediators of this activation (44, 88).
In contrast, NLRP3 inflammasome-driven inflammation re-
cruits inflammatory cells, including macrophages and neu-
trophils, which in turn cause ROS production (22, 64). IL-1b
can also promote intracellular accumulation of ROS by dis-
turbing antioxidant enzymes, suggesting a positive feedback
loop between ROS and NLRP3 inflammasome. This review
will summarize the current knowledge on the molecular
mechanisms underlying the activation of NLRP3 inflamma-
some with particular emphasis on the role played by ROS.
NLRP3 Inflammasome
Inflammasomes
The term ‘‘inflammasome’’ was first coined by Martinon
et al. (55) to describe the assembly of an intracellular
multiprotein complex as a molecular platform that triggers
activation of inflammatory caspases (cysteine-dependent
aspartate-specific proteases). The assembly of inflamma-
somes occurs in response to PAMPs and DAMPs. Caspase-1
is a representative inflammatory caspase that regulates the
maturation and secretion of potent pro-inflammatory cyto-
kines such as IL-1b, leading to inflammatory responses (47).
Most inflammasomes typically consist of a sensor protein, the
adaptor protein ASC (apoptosis-associated speck-like protein
containing a caspase-recruitment domain), and caspase-1
(44, 88), although the exact constituents depend on the acti-
vating DAMPs.
Each inflammasome is generally named according to the
identity of the sensor protein. To date, many sensor proteins
that assemble the inflammasomes have been reported: these
include members of the NLR (nucleotide-binding oligo-
merization domain-like receptor) family and the PYHIN
(pyrin and HIN domain) family. Well-known sensor proteins
include NLRP3, NLRP1, NLRC4 (NLR family caspase-
recruitment domain [CARD] containing 4), and AIM2 (ab-
sent in melanoma 2). NLRP1 is activated by lethal toxin and
muramyl dipeptide, whereas NLRC4 is activated by NAIP
(NLR family of apoptosis inhibitory protein) that can rec-
ognize flagellin or components if the bacterial type II secre-
tion system. AIM2 is activated by cytosolic double-strand
DNA.
Among these different types of inflammasomes, NLRP3
is unique and activated by a wide variety of stimuli, including
PAMPs and DAMPs, and is implicated in various diseases
associated with microbial and sterile inflammation, includ-
ing infection, autoinflammatory disorders, cardiovascular
and metabolic diseases, neurological disorders, and cancer
(24, 41, 88, 91, 97). Therefore, NLRP3 inflammasome is the
most intensively studied to date and is considered to be a
promising target for treating a wide variety of diseases.
Regulation of NLRP3 inflammasome assembly
and downstream effects
The NLRP3 inflammasome consists of the sensor NLRP3,
the adaptor ASC, and the effector enzyme pro-caspase-1
(Fig. 1) (44, 88). NLRP3 consists of an N-terminal pyrin
domain (PYD), a central nucleotide-binding and oligomeriza-
tion(NACHT,alsoreferredtoasNOD)domain,anda
C-terminal leucine-rich repeat (LRR) domain. ASC consists of
an N-terminal PYD and a C-terminal CARD. Pro-caspase-1
consists of a CARD and catalytic domains (termed p20/p10
subunits). Although it was previously deemed that the LRR
domain of NLRP3 has autoinhibitory function via an intra-
molecular interaction, a recent study reported that the LRR
domain is dispensable for NLRP3 inflammasome activa-
tion (27).
Upon NLRP3 activation in response to DAMPs or PAMPs,
NLRP3 recruits ASC through PYD–PYD interactions, which
in turn recruits pro-caspase-1 through CARD–CARD inter-
actions, leading to autocleavage of pro-caspase-1 and the
release of active caspase-1 p10 and p20 tetramer, thereby
terminating inflammasome activity (8). The NLRP3 inflam-
masome is mainly expressed in innate immune cells, in-
cluding macrophages and neutrophils, but is also expressed in
nonimmune cells, including epithelial cells, endothelial cells,
and fibroblasts (87).
To assemble the NLRP3 inflammasome and secrete the
active form of IL-1b, a ‘‘two-step’’ signal, the priming signal
(Signal 1) and the activation signal (Signal 2), is required
(Fig. 2) (44, 88). Because the protein levels of NLRP3 and
pro-IL-1bare usually low or undetectable in resting cells,
2 DOMINIC ET AL.
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a priming signal to increase these protein levels is needed.
This priming signal is mediated at a transcriptional level by
nuclear factor-jB (NF-jB)-via Toll-like receptor (TLR)- or
cytokine receptors. The rapid priming of NLRP3 protein ex-
pression is also mediated by post-translational modification
(PTM), such as de-ubiquitination and phosphorylation (58).
Thereafter, the activation signal promotes the assembly of
NLRP3 inflammasome and induces the activation of caspase-
1 (initially known as ICE) (47). Activated caspase-1 pro-
cesses pro-IL-1b(31 kD) into its active form (17 kD), leading
to the secretion of active IL-1band causing inflammatory
responses. Thus, NLRP3 inflammasome-driven IL-1brelease
is regulated by a ‘‘two-step’’ process. Active caspase-1
also processes pro-IL-18 (inactive, 24 kD) into its active form
(18 kD). However, pro-IL-18 protein is expressed more
constitutively in many types of cells (98), suggesting that
priming for pro-IL-18 may not be necessary. NLRP3
inflammasome-driven activation of caspase-1 also induces
pyroptosis, a lytic form of regulated cell death (37, 63, 82).
Pyroptosis is morphologically distinct from apoptosis and
characterized by cell swelling, plasma membrane rupture,
and the subsequent loss of cytosolic contents. In 2015, gas-
dermin D (GSDMD) was discovered to be a substrate of
caspase-1 and identified as the executioner of pyroptosis (30,
43, 83). The gasdermin was named after the exclusive ex-
pression profile in the gastrointestinal tract and the epithe-
lium of the skin (77), and consists of a family of recently
identified pore-forming effector proteins (9). In addition
FIG. 2. Regulation of NLRP3 inflammasome activation. A two-step signal is required for NLRP3 inflammasome-
driven downstream events. The priming signal (Signal 1) to increase the protein levels of pro-IL-1band NLRP3 is mediated
by NF-jB-mediated signaling via a TLR or cytokine receptors. The rapid priming of NLRP3 protein expression is also
mediated by PTM, such as de-ubiquitination and phosphorylation. The activation signal (Signal 2) promotes the assembly of
NLRP3 inflammasome and induces the activation of caspase-1, leading to the processing of pro-IL-1band pro-IL-18 into
the active forms. Active caspase-1 also induces the processing of GSDMD and its cleaved N-terminus (GSDMD-N) forms
pores on the cell membrane, leading to pyroptotic cell death. Active IL-1band IL-18 are released outside of the cells via the
GSDMD-forming pores, and cause inflammation. GSDMD, gasdermin D; IL, interleukin; NF-jB, nuclear factor-jB; PTM,
post-translational modification; TLR, Toll-like receptor.
FIG. 1. Components and assembly of NLRP3 inflam-
masome. NLRP3 consists of a PYD, a NACHT domain,
and a C-terminal LRR domain. ASC consists of an
N-terminal PYD and a C-terminal CARD. Pro-caspase-1
consists of a CARD and catalytic p20/p10 domains. Upon
NLRP3 activation in response to DAMPs or PAMPs,
NLRP3 recruits ASC through PYD–PYD interactions,
which in turn recruits pro-caspase-1 through CARD–CARD
interactions, leading to autocleavage of pro-caspase-1 and
the formation of active caspase-1 p10 and p20 tetramer.
ASC, apoptosis-associated speck-like protein containing a
caspase-recruitment domain; CARD, caspase-recruitment
domain; DAMP, damage/danger-associated molecular pat-
tern; LRR, leucine-rich repeat; NLRP3, NLR family pyrin
domain containing 3; PAMP, pathogen-associated molecu-
lar pattern; PYD, pyrin domain.
NLRP3 INFLAMMASOME AND ROS 3
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to processing pro-IL-1band pro-IL-18, active caspase-1 can
process GSDMD into an N-terminal domain (GSDMD-N,
31 kD) and a C-terminal domain (GSDMD-C, 22 kD).
This processing separates autoinhibitory GSDMD-C from
the active GSDMD-N, which translocates to the cell mem-
brane, binds to phosphoinositides (or cardiolipin of bacterial
membrane) on the inner leaflet of the plasma membrane, and
oligomerizes to form membrane pores (inner diameter of 10–
20 nm) that disrupt membrane integrity, thereby leading to
pyroptosis. Concomitantly, the leakage of bioactive IL-1b
and IL-18 as well as intracellular DAMPs, such as high
mobility group box-1 (HMGB1) and S100 proteins, occurs
and this cause inflammatory responses. Because IL-1band
IL-18 lack an N-terminal signal peptide for secretion via the
endoplasmic reticulum (ER) to a Golgi exocytosis pathway,
the mechanism for their secretion was previously unclear.
It is now considered that these cytokines are secreted by at
least two mechanisms. One is secretory autophagy (21, 99)
and the other is gasdermin-mediated pore formation. IL-1b
and IL-18 (*5 nm) are small enough to pass through the
GSDMD-forming pores. Interestingly, a recent study re-
ported that the GSDMD-mediated pores are repaired by en-
dosomal sorting complexes required for transport machinery
and suggested that IL-1bcan be released in the absence of
pyroptotic cell death (76). GSDMD-mediated pyroptosis is
also induced by murine caspase-11 (caspase-4/5 in humans)
(43, 83). Caspase-11 directly detects cytosolic lipopoly-
saccharides (LPS) and promotes GSDMD-mediated pyr-
optosis in the absence of cytokine secretion.
This pathway is known as a noncanonical inflammasome.
Furthermore, Wang et al. (95) recently reported that pyr-
optosis can also be driven by caspase-3-mediated processing
of GSDME (also known as deafness autosomal dominant
nonsyndromic sensorineural 5 [DFNA5]). We also more re-
cently found that NLRP3 inflammasome induces pyroptosis
via an ASC/caspase-8/caspase-3 pathway independent of
caspase-1 (5). Because the pore-forming ability is conserved
throughout gasdermin family proteins (9), it is possible that
other members of the gasdermin family may contribute to
the process of pyroptosis. Accordingly, although pyroptosis
was initially defined as caspase-1-dependent necrotic cell
death, it is now redefined as gasdermin-dependent necrotic
cell death (82).
Activation of NLRP3 inflammasome
Considering that NLRP3 activation is induced by a wide
variety of diverse stimuli and that most NLRP3 activators
have not been shown to directly bind NLRP3, it seems likely
that NLRP3 can sense common cellular events triggered
by the activating stimuli. These cellular events include K
+
efflux (decrease in intracellular K
+
concentration), lyso-
somal dysfunction-induced release of cathepsin B, and
production of ROS, especially from the mitochondria
(Fig. 2) (44, 88). Of these, K
+
efflux is considered to be the
common upstream mediator of NLRP3 inflammasome acti-
vation (67).
However, recent studies have suggested that K
+
efflux is
not a prerequisite for NLRP3 inflammasome activation (26).
In general, lysosomal dysfunction-induced release of ca-
thepsin B is responsible for NLRP3 inflammasome activation
in response to crystals and particles, including monosodium
urate, cholesterol crystals, and silica. ROS are another critical
mediator of NLRP3 inflammasome activation, which is dis-
cussed in detail later in this review.
Several other regulators of NLRP3 inflammasome acti-
vation, including Nek7 (NIMA-related kinase 7) (31, 78, 81),
GBP5 (guanylate-binding protein 5) (80), PKR (double-
stranded RNA-dependent protein kinase) (52), macrophage
migration inhibitory factor (49), and MARK4 (microtubule-
affinity regulating kinase 4) (51), have been reported previ-
ously, but some of them are controversial (44). In addition
to K
+
efflux, other ionic fluxes, including Ca
2+
mobilization,
Na
+
influx, and Cl
-
efflux, can also be involved in NLRP3
inflammasome activation (44). Regarding Cl
-
efflux, chloride
intracellular channels trigger Cl
-
efflux as downstream of
the K
+
efflux-mtROS axis, which activates NLRP3 inflam-
masome via an NLRP3-Nek7 interaction (89).
Furthermore, Chen recently reported that PtdIns4P
(phosphatidylinositol-4-phosphate) on dispersed trans-Golgi
network mediates NLRP3 inflammasome activation (13). To
date, many investigations have shown that the priming and
activation of NLRP3 inflammasome is fine-tuned by nega-
tive and/or positive regulators, including endogenous mod-
ulators (e.g., CARD-only proteins and pyrin-only proteins),
PTMs (e.g., ubiquitination, phosphorylation, nitrosylation, and
sumoylation), and microRNAs (39, 42, 44, 58). The activation
mechanism underlying NLRP3 inflammasome has been re-
vealed to be much more complicated than previously thought
because it appears that the regulation of NLRP3 inflamma-
some varies depending on the types of cells and stimuli
(Table 1). For more information on NLRP3 inflammasome
regulation, see recent excellent reviews (44, 53, 58).
ROS and NLRP3 Inflammasome
ROS are defined as oxygen-containing highly reactive
chemical species with unpaired electron, and mainly include
hydrogen peroxide (H
2
O
2
), superoxide anion (O
2
-
), and
hydroxyl radical (OH
) (18, 64). ROS are produced as a
by-product of oxygen metabolism in the mitochondria,
or produced by cellular enzymes, such as nicotinamide
adenine dinucleotide phosphate (NADPH) oxidases (NOXs),
xanthine oxidase (XO), lipoxygenases (LOXs), and cyclo-
oxygenases (COXs) (12, 18).
Excessive production of ROS causes oxidative damage
to proteins, nucleic acids, and lipids in membrane, and is
generally thought to be harmful; however, ROS also play
an important role in pathogen elimination, especially in
neutrophils and macrophages, and pathophysiological
processes, including cell signaling, proliferation, and dif-
ferentiation (12, 18, 19). Numerous studies using antioxi-
dants (e.g., N-acetyl cysteine [NAC]) support the notion
that ROS are critical mediators of NLRP3 inflammasome
activation (3, 4).
Although previous studies have reported that O
2
-
,H
2
O
2
,
and peroxynitrite (ONOO
-
) play a role in NLRP3 in-
flammasome activation in some cell types, little informa-
tion is available on which species of reactive oxygen induce
the activation (2, 3, 33, 103). ROS also contribute to the
priming signal of NLRP3 inflammasome via ROS-dependent
transcriptional factor NF-jB and LPS-mediated NLRP3
deubiquitination (38). In contrast, NLRP3 inflammasome
activation induces inflammatory responses and recruits
4 DOMINIC ET AL.
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inflammatory cells, including neutrophils and macrophages,
which also produce ROS, leading to further inflammatory
responses.
Furthermore, IL-1bpromotes intracellular accumulation
of ROS by disturbing superoxide dismutase (SOD) and cat-
alase (29, 57). Thus, it is likely that there is some positive
loop regarding ROS-mediated NLRP3 inflammasome acti-
vation (Fig. 3). However, the role and mechanisms of ROS in
NLRP3 inflammasome activation are still a matter of debate
and ongoing investigation. Furthermore, since NLRP3 in-
flammasome is highly expressed in innate immune cells, in-
cluding macrophages and neutrophils, but is also expressed in
nonimmune cells, including endothelial cells, epithelial cells,
and fibroblasts, the mechanisms of ROS-mediated NLRP3
inflammasome activation may differ depending on cell type.
ROS derived from mitochondria
It is widely accepted that mitochondrial ROS (mtROS) is a
major mediator of NLRP3 inflammasome activation (Fig. 4).
FIG. 3. Proposed mechanisms of ROS-mediated NLRP3 inflammasome activation. Proposed mechanisms of ROS-
mediated NLRP3 inflammasome activation include mtROS, ox-mtDNA, and TXNIP. mtROS are released from damaged
mitochondria and induce NLRP3 inflammasome activation. mtDNA are also released from damaged mitochondria and
oxidized by mtROS or cytosolic ROS (derived from NADPH oxidase), and directly binds NLRP3, leading to NLRP3
inflammasome activation. The newly synthesized mtDNA via a TLR/IRF-1/CMPK2 pathway also contributes to NLRP3
inflammasome activation. Mitophagy-mediated clearance of damaged mitochondria inhibits its activation. We hypothesize
that ROS derived from mitochondria or NADPH oxidase may cause the dissociation of TXNIP from Trx, and induce the
binding of TXNIP with NLRP3, leading to NLRP3 inflammasome activation. IRF-1, interferon regulator factor-1; mtROS,
mitochondrial ROS; NADPH, nicotinamide adenine dinucleotide phosphate; ox-mtDNA, oxidized mtDNA; ROS, reactive
oxygen species; Trx, thioredoxin; TXNIP, thioredoxin-interacting protein.
Table 1. Investigations of Inflammasome Modulation in Preclinical Studies
of Various Pathological Disorders
Disorders investigated Pathway or functional consequence targeted References
Type 2 diabetes Islet mass and insulin resistance (46, 47)
Endothelial dysfunction Inflammation and oxidative stress, pyroptosis,
endothelial cell function and senescence
(48)
Traumatic brain injury and depression Neuroinflammation, chronic stress, microglial
activation, and neuronal cell death
(49, 50)
Cancer Inflammatory microenvironment, IL-1b
dysregulation, oxidative stress, and clonal
expansion
(51, 52)
Cardioprotective signaling IL-6-mediated caspase activation (47, 53)
Pathogenic infection and autoimmune disorders Cytokine production and phagocytosis (54)
IL, interleukin.
NLRP3 INFLAMMASOME AND ROS 5
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Zhou et al. (104) was the first to demonstrate the role of
mitochondria and mtROS in NLRP3 inflammasome activa-
tion by demonstrating that first, the mitochondrial respiratory
chain complex I inhibitor rotenone and the complex III in-
hibitor antimycin considerably increased mtROS produc-
tion and NLRP3 inflammasome activation. Conversely, the
downregulation of voltage-dependent anion channels by
shRNA decreased mtROS production and NLRP3 inflamma-
some activation.
Second, upon inflammasome stimulation, NLRP3 relo-
cates from ER to mitochondria-associated ER membranes,
and colocalizes with ASC. In this regard, a later study re-
vealed the mechanism of NLRP3 relocation mediated by the
mitochondrial outer membrane protein mitochondrial anti-
viral signaling protein (86). Third, inhibition of autophagy/
mitophagy by 3-MA (3-methyladenine) results in the accu-
mulation of damaged mitochondria and mtROS in parallel
with increased IL-1bproduction. Similarly, downregulation
of autophagy-associated proteins, Atg5 and beclin-1, en-
hances NLRP3 activator-induced IL-1bproduction.
Consistently, Nakahira et al. (69) showed that deficiency of
beclin-1 and another autophagy-associated protein LC3B
promotes mtROS production and inflammasome activation
in macrophages. Interestingly, upon NLRP3 inflammasome
activation, mtDNA is released into the cytosol through in-
creased mitochondrial membrane permeability, and cytosolic
mtDNA can activate AIM2 inflammasome and subsequent IL-
1bproduction. AIM2 is a member of the PRRs family that
recognizes dsDNA and assemblesAIM2 inflammasome (101).
Similarly, Shimada et al. (84) reported that macrophages
lacking mtDNA were unable to secrete IL-1bin response to
NLRP3 activators. As the responsible mechanism, they fur-
ther showed that mtROS lead to oxidized mtDNA that
directly binds NLRP3, leading to activation of NLRP3 in-
flammasome. Intriguingly, Zhong et al. (102) recently re-
ported that TLR signaling promotes the synthesis of mtDNA
by interferon regulator factor-1 (IRF-1) and mitochondrial
deoxyribonucleotide kinase UMP-CMPK2 (CMPK2), and
then the newly synthesized mtDNA is oxidized and drives
NLRP3 inflammasome activation.
Another potential mechanism of mtROS-induced NLRP3
inflammasome activation is thioredoxin-interacting pro-
tein (TXNIP; also known as thioredoxin-binding protein-2
[TBP2] or vitamin D3 upregulated protein 1 [VDUP1]).
TXNIP was originally characterized as a negative regulator
of the antioxidant protein thioredoxin-1 (Trx1) (70). Zhou
et al. (103) found that, under resting conditions, TXNIP binds
to Trx1 and suppresses its antioxidant and thiol-reducing
function. Once excessive ROS are produced, TXNIP is dis-
sociated from oxidized Trx1, which in turn directly binds the
LRR and NACHT domain of the NLRP3, leading to NLRP3
inflammasome activation.
They further confirmed that caspase-1 activation and IL-1b
processing in response to monosodium urate crystals, ATP, and
imiquimod (R-837) were inhibited in bone marrow-derived
macrophages (BMDMs) from TXNIP
-/-
mice. Although Trx1
is a cytosolic protein, it is possible that interaction between
mitochondrial Trx2 and TXNIP may play a similar role in
NLRP3 inflammasome activation. Supporting this idea, Zhou
et al. (104) observed that NLRP3 activators induce TXNIP to
redistribute to mitochondria in a ROS-dependent manner.
Thereafter, numerous studies have demonstrated that mtROS
can act as a critical mediator of NLRP3 inflammasome
activation.
Most of these studies showed that mitochondria-targeting
antioxidants (e.g., MitoTEMPO, SS-31, and MitoQ) had in-
hibitory effects on NLRP3 inflammasome activation and
IL-1bproduction (15, 91, 96). We have previously shown
using an angiotensin II (AII)-infused murine abdominal
aortic aneurysm (AAA) model that mtROS is robustly in-
creased in the adventitial macrophages in the early phase
of AAA formation (91). Moreover, MitoTEMPO or cyclos-
porin A (an inhibitor of mitochondrial permeability transi-
tion) significantly blocked AII-induced IL-1bproduction in
macrophages.
Similarly, the pathogenic role of the mtROS/NLRP3 in-
flammasome pathway has been demonstrated in various ex-
perimental disease models.
ROS derived from NADPH oxidase and other enzymes
The NADPH oxidases are another major source of ROS
production. Earlier studies reported that NLRP3 inflamma-
some activation in response to ATP, silica, and asbestos was
inhibited by diphenyleneiodonium (DPI) and other ROS in-
hibitors (14, 20, 33). In particular, the asbestos-induced
processing of pro-IL-1bwas inhibited in p22
phox
-knockdown
THP-1 cells (20). Based on these observations, it had been
thought that NADPH oxidase-induced ROS could be invol-
ved in NLRP3 inflammasome activation. However, subsequ-
ent studies showed that NADPH oxidases negatively impact
its activation.
Chronic granulomatous disease (CGD) is an inherited dis-
order that is caused by mutations in subunits of the NADPH
oxidase, and characterized by defective ROS production
in phagocytes, resulting in recurrent infections and dysre-
gulated inflammatory responses. Using peripheral blood
mononuclear cells (PBMCs) from CGD patients, several
groups have shown that NADPH oxidase-derived ROS
FIG. 4. Loop between NLRP3 inflammasome and ROS.
ROS activate NLRP3 inflammasome via the priming and
activation signals, and produces IL-1b, which induces in-
flammatory responses and recruits neutrophils and macro-
phages. The accumulated neutrophils and macrophages
produce ROS and lead to further inflammatory responses.
Furthermore, IL-1bpromotes intracellular accumulation of
ROS by disturbing antioxidant enzymes. Thus, our hypo-
thesis is that this phenomenon is due to the likely positive
loop regarding ROS-mediated NLRP3 inflammasome acti-
vation and not a linear activation.
6 DOMINIC ET AL.
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production did not affect caspase-1 activation or IL-1bse-
cretion in response to known NLRP3 activators (60, 92, 93).
Notably, one study reported that IL-1bsecretion in re-
sponse to uric acid crystals in PBMCs from CGD patients was
fourfold higher than that in PBMCs from control subjects
(93). Consistent with these clinical findings, stretch-induced
caspase-1 activation and IL-1bsecretion were not prevented
in alveolar macrophages deficient in Nox2 subunit gp91
phox
(96). In addition, it has been shown that DPI, commonly used
as a pharmacological inhibitor of NADPH oxidase-derived
ROS, can inhibit mtROS at higher doses (10). These findings
indicate that NADPH oxidases are dispensable for NLRP3
inflammasome activation.
However, many studies report that NADPH oxidase acti-
vation upon inflammasome activation (3). Although the
reason for this contradiction regarding the role of NADPH
oxidases is currently unclear, it might be due to species
variations, different types of cells, spatial-temporal locali-
zation of ROS, and the presence of functionally redundant
Nox proteins (29). In either case, NADPH oxidase is involved
in the inflammasome mediation, but whether it is a conse-
quence, or a trigger is still be delineated. Cross talk between
mtROS and NADPH oxidase (e.g., Nox1, Nox2, and Nox4)
has been demonstrated (16, 46).
In particular, Nox4, localized to the mitochondria, can
contribute to the production of mtROS by inactivation of
mitochondria respiratory chain complex I and uncoupling of
endothelial nitric oxide synthase (45, 50). Regarding Nox4,
Moon et al. (66) recently showed that Nox4 promotes NLRP3
inflammasome activation via fatty acid oxidation. ROS are
also generated by other cytosolic enzymes, including XO,
LOXs, and COXs, and these enzyme-derived ROS have been
shown to contribute to the activation of NLRP3 inflamma-
some (1, 36). In conclusion, there is an undisputed link be-
tween the role of mtROS and NLRP3 activation, whereas the
link between ROS produced by other cellular machinery and
NLRP3 inflammasome still requires clarification.
Antioxidant molecules and species of ROS
Endogenous antioxidant molecules have been reported
to play a role in NLRP3 inflammasome activation. An earlier
study reported that SOD1 deficiency markedly increases
ROS production, and subsequently inhibits caspase-1 acti-
vation and IL-1bsecretion in macrophages. Regarding the
mechanism, higher superoxide production specifically in-
hibits caspase-1 activation by glutathionylation of cysteine
residues of caspase-1 (59). Supporting this, an excessive
amount of ROS has been shown to inactivate caspase-3 (90).
Thus, ROS may act as double-edged sword in NLRP3 in-
flammasome activation depending on their amount. Inter-
estingly, a recent study showed that de-glutathionylation of
Nek7, a regulator of NLRP3 inflammasome, by glutathione
transferase mega 1-1promotes NLRP3 activation (34).
Nrf2 (nuclear factor E2-related factor or nuclear factor
[erythroid-derived 2]-like 2) is a transcription factor that
regulates antioxidant gene expression and acts as a ROS
detoxification factor (32). For this reason, it was previously
anticipated that Nrf2 might have inhibitory effects on NLRP3
inflammasome activation. However, a previous study showed
that Nrf2 deficiency reduces cholesterol crystal-induced in-
flammasome activation and attenuates the development of
atherosclerosis (23). In this regard, Nrf2 has been shown to
upregulate CD36, a scavenger receptor for oxidized low-
density lipoprotein (LDL), in macrophages (35), suggesting
that elevated CD36 levels by Nrf2 potentiate oxidized LDL
incorporation into macrophages, causing the formation of
cholesterol crystals and subsequent NLRP3 inflammasome
activation.
Furthermore, Nrf2 deficiency inhibits caspase-1 activation
and IL-1bsecretion as well as ASC speck formation in re-
sponse to activators of NLRP3 and AIM2, but not NLRC4, in
macrophages (100). Indeed, it has been increasingly reported
that many Chinese herbal medicines that activate Nrf2 can
inhibit NLRP3 inflammasome activation and attenuate dis-
ease severity in inflammation-associated disease models
(32). Although these herbal medicines are not specific to
NRF2, Garstkiewicz et al. (25) recently reported interesting
results that Nrf2 is a positive regulator of the NLRP3 in-
flammasome, whereas Nrf2-activating compounds inhibit
NLRP3 inflammasome activation.
In conclusion, although at first NRF2 was speculated to be
a negative regulator of inflammasome owing to its antioxi-
dant properties as discussed earlier, studies show that the
relationship is not as straightforward as predicted. One of
the major reasons to this is that the pleotropic nature of both
NRF2 and NLRP3. It can also be speculated that under
acute conditions ROS levels can be sustained by NRF2, but
under chronic conditions the effect of NRF2 might be di-
minished leading to be disengaged from inflammasome
pathways. Further studies directed on the acute and chronic
conditions will be able to establish the link between NRF2
and NLRP3.
As mentioned earlier, TXNIP has been shown to function
as a ROS-dependent regulator of NLRP3 inflammasome
activation. Several other studies have reported that TXNIP
plays a similar role in NLRP3 inflammasome activation (1,
65). In contrast, however, Masters et al. (56) showed that
TXNIP
-/-
BMDMs did not influence IL-1bsecretion in re-
sponse to NLRP3 activators, compared with wild-type mac-
rophages. Muri et al. (68) very recently reported that Trx1
promotes NLRP3 inflammasome-driven IL-1bprocessing
and secretion independent of TXNIP. Therefore, it is as-
sumed that ROS-mediated NLRP3 inflammasome activation
is regulated not only by TXNIP/NLRP3 interaction but also
by other unknown mechanisms.
Conclusions and Clinical Perspective
The NLRP3 inflammasome is a pivotal component of the
innate immune system and plays a significant role in the path-
ogenesis of a wide variety of diseases that include infection,
autoinflammatory disorders, cardiovascular and metabolic
diseases, neurological disorders, and cancer. In particular,
sustained activation of NLRP3 inflammasome contributes to
the development of chronic inflammatory cardiovascular
diseases, such as atherosclerosis and AAA.
Although the mechanism of the sustained NLRP3 activation
remains largely unclear, a previous study suggested that the
adenosine A
2A
receptor (A
2A
R) signaling amplifies the prim-
ing and activation signals and increases the duration of NLRP3
inflammasome activation. As the mechanism, the amplification
by A
2A
R signaling is mediated through a cAMP/PKA/CREB/
HIF-1apathway although the inflammasome regulation by
NLRP3 INFLAMMASOME AND ROS 7
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adenosine does not replace either the priming and activation
signal (72).
In addition, it seems likely that positive loop of NLRP3
inflammasome and ROS participates in the process of the
sustained activation. Moreover, several key emerging con-
cepts of precondition signaling and concepts of hormesis can
be used to explain the redox state and the activation and
inactivation of inflammasomes (85, 94). Specifically, the
dose–response for ROS and subsequent inflammasome acti-
vation is not clearly understood. This becomes crucial in
neurodegenerative diseases where it is believes that cellular
threshold for redox signaling is not adaptive (62, 74).
NLRP3 inflammasome has been extensively studied for
the past decade and substantial knowledge has been gained;
however, it is becoming clearer that the mechanisms regu-
lating NLRP3 inflammasome and how these influence the
pathogenesis of diseases are much more complicated.
Regarding the ROS and NLRP3 inflammasome, several un-
solved questions still need to be addressed. In particular, the
most important question of how ROS affects NLRP3 in-
flammasome activation is not yet fully understood. In gen-
eral, the redox state is involved in wide variety of diseases
and pathologies from neurological to metabolic disorders
(Table 1). As discussed in this review, the loop concept be-
tween ROS and NLRP3 becomes crucial under innate im-
mune activation settings.
In many settings, NLRP3 indirectly is activated by ROS
produced in response to danger signals. Understanding and
fine-tuning the inflammatory response under various condi-
tions will be key in regulating the several pathological con-
ditions. Inhibition of ROS can inhibit NLRP3 activation,
whereas chronic activation of ROS can lead to negative reg-
ulation of NLRP3 (73). Several recent studies have developed
small molecule inhibitors that can target inflammasome at
various stages of the pathway, whereas the specific drug se-
lection can be determined only by understanding the intricate
balance between redox state and inflammasome activation
(79). Thus, understanding the fine balance that governs this
feedback loop is essential in treating chronic conditions.
In contrast, the recent CANTOS trial using canakinumab
validated the concept that IL-1b-driven inflammation di-
rectly contributes to the pathogenesis of atherothrombotic
events and is a new therapeutic option for reducing cardio-
vascular diseases in clinical practice (75); therefore, both
researchers and clinicians should pay more attention to
NLRP3 inflammasome. Because canakinumab is a fully
human anti-IL-1 monoclonal antibody that inhibits IL-1band
has no cross-reactivity with IL-1a, clinical trials using ca-
nakinumab are rather focused on autoinflammatory disorders
such as cryopyrin-associated periodic syndromes with
NLRP3 mutation, rheumatoid arthritis, and gout (48).
However, the widespread use of canakinumab is limited
because of its high cost. In addition, chronic inhibition of
IL-1bmay cause adverse effects such as infection and
impairment of immune homeostasis. Thus, the develop-
ment of less expensive and direct NLRP3 inhibitors is
highly desirable. Hence, a deeper understanding of the
mechanism of NLRP3 inflammasome regulation and the
development of specific NLRP3 inhibitors should not only
offer new therapeutic modalities but also break new
ground for studying the role of NLRP3 inflammasome in
various disorders.
Authors’ Contributions
M.T. conceptualized the content of this study. A.D. and
M.T. reviewed the literature and drafted the article. N.-T.L.
discussed and edited the review.
Acknowledgments
The authors thank past and current members of the
Inflammation Research laboratory for their contributions to
the research.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported by grants from the Japan Society
for the Promotion of Science ( JSPS) through Grants-in-Aid
for Scientific Research (18K08112), the Private University
Research Branding Project, the Agency for Medical Research
and Development-Core Research for Evolutional Science
and Technology (AMED-CREST: 18gm0610012h0105).
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Address correspondence to:
Dr. Masafumi Takahashi
Division of Inflammation Research
Center for Molecular Medicine
Jichi Medical University
3311-1 Yakushiji, Shimotsuke
Tochigi 329-0498
Japan
E-mail: masafumi2@jichi.ac.jp
Date of first submission to ARS Central, December 24, 2020;
date of final revised submission, September 2, 2021; date of
acceptance, September 3, 2021.
Abbreviations Used
3-MA ¼3-methyladenine
A
2A
R¼adenosine A
2A
receptor
AAA ¼abdominal aortic aneurysm
AII ¼angiotensin II
AIM2 ¼absent in melanoma 2
ASC ¼apoptosis-associated speck-like protein
containing a caspase-recruitment domain
BMDM ¼bone marrow-derived macrophage
CANTOS ¼Canakinumab Anti-inflammatory
Thrombosis and Outcome Study
CARD ¼caspase-recruitment domain
CGD ¼chronic granulomatous disease
COX ¼cyclooxygenase
CREB ¼cAMP response element binding protein
DAMP ¼damage/danger-associated molecular
pattern
DFNA5 ¼deafness autosomal dominant
nonsyndromic sensorineural 5
DPI ¼diphenyleneiodonium
ER ¼endoplasmic reticulum
GBP5 ¼guanylate-binding protein 5
GSDMD ¼gasdermin D
H
2
O
2
¼hydrogen peroxide
HIF-1a¼hypoxia-inducible factor-1a
HMGB-1 ¼high mobility group box-1
ICE ¼IL-1bconverting enzyme
IL ¼interleukin
IRF-1 ¼interferon regulator factor-1
LDL ¼low-density lipoprotein
LOX ¼lipoxygenase
LPS ¼lipopolysaccharides
LRR ¼leucine-rich repeat
MARK4 ¼microtubule-affinity regulating kinase 4
mtROS ¼mitochondrial ROS
NAC ¼N-acetyl cysteine
NADPH ¼nicotinamide adenine dinucleotide phosphate
NAIP ¼NLR family of apoptosis inhibitory protein
Nek7 ¼NIMA-related kinase 7
NF-jB¼nuclear factor-jB
NLR ¼nucleotide-binding oligomerization
domain-like receptor
NLRC4 ¼NLR family caspase-recruitment
domain containing 4
NLRP3 ¼NLR family pyrin domain containing 3
NOX ¼NADPH oxidase
Nrf2 ¼nuclear factor E2-related factor or nuclear
factor [erythroid-derived 2]-like 2
O
2
-
¼superoxide anion
OH
¼hydroxyl radical
ONOO
-
¼peroxynitrite
PAMP ¼pathogen-associated molecular pattern
PBMC ¼peripheral blood mononuclear cell
PKR ¼double-stranded RNA-dependent protein
kinase
PtdIns4P ¼phosphatidylinositol-4-phosphate
PTM ¼post-translational modification
PYD ¼pyrin domain
12 DOMINIC ET AL.
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Abbreviations Used (Cont.)
PYHIN ¼pyrin and HIN domain
ROS ¼reactive oxygen species
SOD ¼superoxide dismutase
TBP2 ¼thioredoxin-binding protein-2
TLR ¼Toll-like receptor
Trx ¼thioredoxin
TXNIP ¼thioredoxin-interacting protein
VDUP1 ¼vitamin D3 upregulated protein 1
XO ¼xanthine oxidase
NLRP3 INFLAMMASOME AND ROS 13
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