Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome.
ABSTRACT A common denominator among the multiple damage-inducing agents that ultimately lead to activation of NLRP3 has not yet been identified. Recently, production of reactive oxygen species (ROS) has been suggested to act as a common event upstream of the NLRP3 inflammasome machinery. Because de novo translation of NLRP3 is an essential step in the activation of NLRP3, we investigated the role of substances that inhibit either ROS production or its oxidative activity. Although we observe that NLRP3 inflammasome activation is unique among other known inflammasomes in its sensitivity to ROS inhibition, we have found that this phenomenon is attributable to the fact that NLRP3 strictly requires priming by a proinflammatory signal, a step that is blocked by ROS inhibitors. Although these data do not exclude a general role for ROS production in the process of NLRP3-triggered inflammation, they would put ROS upstream of NLRP3 induction, but not activation.
- SourceAvailable from: Klaus DalhoffPneumologie 02/2014; 68(S 01).
- [Show abstract] [Hide abstract]
ABSTRACT: The inflammasome is a molecular platform formed by activation of an innate immune pattern recognition receptor seed, such as NLRP3. Once activated, NLRP3 recruits the adapter ASC (apoptosis-related speck-like protein containing a caspase recruitment domain), which in turn recruits procaspase-1. Procaspase-1 autocatalyzes its cleavage and activation, resulting in maturation of the precursor forms of interleukin (IL)-1β and IL-18 into active proinflammatory cytokines and initiation of pyroptotic cell death. The NLRP3 inflammasome has been implicated in the pathogenesis of a wide variety of diseases, including genetically inherited autoinflammatory conditions as well as chronic diseases in which NLRP3 is abnormally activated. The NLRP3 inflammasome has been linked to diseases such as Alzheimer's disease, atherosclerosis, metabolic syndrome, and age-related macular degeneration. In this review, we describe the NLRP3 inflammasome complex and its activation in disease, and detail the current therapies that modulate either the NLRP3 inflammasome complex itself or the two cytokines it is responsible for activating, ie, IL-1β and IL-18.Journal of Inflammation Research 01/2015; 8:15-27.
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ABSTRACT: The acute respiratory distress syndrome (ARDS) is a form of severe hypoxemic respiratory failure that is characterized by inflammatory injury to the alveolar capillary barrier, with extravasation of protein-rich edema fluid into the airspace. Although many modalities to treat ARDS have been investigated over the past several decades, supportive therapies remain the mainstay of treatment. In this article, we briefly review the definition, epidemiology, and pathophysiology of ARDS and present emerging aspects of ARDS pathophysiology that encompass modulators of the innate immune response, damage signals, and aberrant proteolysis that may serve as a foundation for future therapeutic targets. Copyright © 2015 by The American Association of Immunologists, Inc.Journal of immunology (Baltimore, Md. : 1950). 02/2015; 194(3):855-860.
Cutting Edge: Reactive Oxygen Species Inhibitors Block
Priming, but Not Activation, of the NLRP3 Inflammasome
Franz Bauernfeind,*,1Eva Bartok,*,1Anna Rieger,* Luigi Franchi,†,‡Gabriel Nu ´n ˜ez,†,‡
and Veit Hornung*
A common denominator among the multiple damage-
inducing agents that ultimately lead to activation of
NLRP3 has not yet been identified. Recently, produc-
tion of reactive oxygen species (ROS) has been sug-
gested to act as a common event upstream of the
NLRP3 inflammasome machinery. Because de novo
translation of NLRP3 is an essential step in the activa-
tion of NLRP3, we investigated the role of substances
that inhibit either ROS production or its oxidative ac-
tivity. Although we observe that NLRP3 inflammasome
activation is unique among other known inflamma-
somes in its sensitivity to ROS inhibition, we have
found that this phenomenon is attributable to the fact
tory signal, a step that is blocked by ROS inhibitors.
Although these data do not exclude a general role
for ROS production in the process of NLRP3-trig-
gered inflammation, they would put ROS upstream of
NLRP3 induction, but not activation.
Immunology, 2011, 187: 613–617.
matory potential of bioactive IL-1b, its release is a tightly
controlled process, in which caspase-1–mediated cleavage of
pro–IL-1b is a rate-limiting step (1). Inflammasome com-
plexes control the regulated cleavage of pro–IL-1b and also
other procytokines by assembling a multicomponent protein
platform that leads to the activation of procaspase-1. In ad-
dition, the activation of inflammasome pathways leads to
a special type of inflammatory cell death that is commonly
referred to as pyroptosis. So far, several proteins have been
described that can initiate the formation of inflammasome
complexes: the nucleotide-binding domain leucine-rich repeat
(NLR) proteins NLRP1, NLRP3, and NLRC4 and the pyrin
The Journal of
nterleukin-1b–driven inflammation plays a pivotal role
both in antimicrobial immunity and in many sterile in-
flammatory conditions. Owing to the highly proinflam-
and HIN200 domain-containing protein AIM2. Up to now,
only AIM2 has been shown to directly bind to its activating
stimulus (dsDNA) (2–4), whereas the NLR inflammasome
proteins have not been established as bona fide receptors. Of
all of the NLR proteins, NLRP3 has attracted particular at-
tention because it seems to sense a large variety of stimuli of
diverse physiochemical nature [e.g., ATP, pore-forming tox-
ins, or crystalline material (5–7)] and also because it plays
a pivotal role in many inflammatory diseases. Prior to the
discovery of NLRP3 as an upstream component of caspase-
1 activation, it was already known that ATP critically requires
a proinflammatory priming step (e.g., LPS) for caspase-1
activation (8, 9). Moreover, priming cells is also necessary
for caspase-1 cleavage after exposure to pore-forming toxins
and crystalline inflammasome activators. We have recently
shown that induction of NLRP3 expression is the only critical
factor that determines the necessity of this priming step (10,
11). In fact, this requirement for priming can be overcome
solely by constitutive NLRP3 expression, as macrophages ex-
pressing heterologous NLRP3 do not require proinflam-
matory priming for their responsiveness toward ATP or other
NLRP3 activators (10). As trivial as this necessity for priming
might appear, it is important to consider when studying
mechanisms of NLRP3 activation or when exploring strate-
gies to specifically inhibit NLRP3 activation.
Various models of activation have been proposed for
NLRP3, and, most recently, the concept of reactive oxygen
species (ROS) being upstream of NLRP3 activation has gained
particular attention. Previous studies using RNA interference
and pharmacological inhibitors suggested that NADPH oxi-
dase (NOX)-dependent ROS production, which is observed
upon phagocytosis of crystalline material, would be upstream
of NLRP3 inflammasome activation (12). However, we and
others (13–15) found that macrophages deficient in NOX
subunits p47phox, p91phox, or p22phox (essential for func-
tional NOX1-4) responded normally to NLRP3 stimulation
(Supplemental Fig. 1A–D). Nevertheless, inhibitors of ROS
production or scavengers of ROS exhibit a strong inhibition
*Unit for Clinical Biochemistry, Institute for Clinical Chemistry and Pharmacology,
University Hospital, University of Bonn, 53127 Bonn, Germany;
Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and
‡Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor,
1F.B. and E.B. contributed equally to this work.
Received for publication March 1, 2011. Accepted for publication May 20, 2011.
This work was supported by grants from the German Research Foundation (SFB704 and
SFB670), the European Research Council (ERC-2009-StG 243046) (to V.H.), and the
National Institutes of Health (R01AI063331) (to G.N.) and by a Career Development
award from the Crohn’s and Colitis Foundation of America (to L.F.).
Address correspondence and reprint requests to Prof. Veit Hornung, Institute for Clin-
ical Chemistry and Pharmacology, University Hospital, University of Bonn, Sigmund-
Freud-Strasse 25, 53127 Bonn, Germany. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: CHX, cycloheximide; DPI, diphenyliodonium;
NAC, N-acetyl-L-cysteine; NLR, nucleotide-binding domain leucine-rich repeat;
NOX, NADPH oxidase; poly(dA:dT), poly(deoxyadenylic-deoxythymidylic) acid;
ROS, reactive oxygen species.
of NLRP3 inflammasome activation (12, 16). Indeed, in line
with the notion that mitochondria constitute the biggest
source of cellular ROS, it was subsequently shown that mi-
tochondria are, in fact, the site of ROS production during
NLRP3 inflammasome activation (17, 18). To this effect, it
has also been demonstrated that inhibitors of mitochondrial
ROS production (17) and the knocking down of mitochondrial
respiration by targeting the expression of voltage-dependent
anion channels (18) downmodulate NLRP3-mediated inflam-
masome activation. Furthermore, independent evidence also
exists that ROS activate proinflammatory transcription fac-
tors (19, 20) and that ROS production positively regulates
proinflammatory gene expression in various innate immune
signaling pathways (14, 21). On the basis of these findings, we
hypothesized that ROS inhibition does not directly affect
activation of the NLRP3 inflammasome, but, instead, nega-
tively regulates the priming step of NLRP3 inflammasome
Materials and Methods
Wild-type C57BL6/J, Ncf1m1J/J, and Cybb mutant mice in C57BL6/J
background were purchased from The Jackson Laboratory, and bones from
Laboratory) and have been previously described (22). All animal studies were
approved by the University of Michigan Committee on Use and Care of
Cells and cell culture
C57BL/6 or NLRP3-deficient macrophage cell lines (Figs. 1, 2; Supplemental
Figs. 1E, 1F, 2) were cultured and stimulated as previously described (10).
Macrophages stably overexpressing NLRP3 were obtained through lentiviral
transduction, as explained earlier (10).
ATP, poly(deoxyadenylic-deoxythymidylic) [poly(dA:dT)], nigericin, cyclo-
heximide (CHX), and N-acetyl-L-cysteine (NAC) were from Sigma-Aldrich.
Diphenyliodonium (DPI) was from Alexis. Flagellin and ultrapure LPS from
Escherichia coli were from Invivogen. Silica (U.S. Silica) was used at a final
concentration of 500 mg/ml. Poly(dA:dT) and flagellin were transfected
using Lipofectamine 2000 (Invitrogen) and 1,2-dioleoyloxy-3-(trimethyl-
ammonium)propane (DOTAP; Roche Applied Science), respectively. If in-
dicated, 5 mM nigericin or 5 mM ATP was added 1 h before supernatants
Caspase-1 cleavage was detected by immunoblot, as previously described
[(Figs. 1, 2; Supplemental Figs. 1E, 2) (10) or (Supplemental Fig. 1C, 1D)
(11)]. NLRP3 expression was assessed using the Cryo-2 Ab from Axxora.
Quantitative real-time PCR analysis
was performed on a Roche LC480. All gene expression data are presented as
relative expression to HPRT1. Primer sequences are available upon request.
Results and Discussion
Only the NLRP3 inflammasome requires priming by a
NLRP3 inflammasome activation is tightly controlled by a
priming step that requires de novo translation (10, 11). To
address whether other inflammasome pathways also require
de novo translation, we carried out experiments in murine
macrophages treated with the translation inhibitor cyclohex-
imide. These cells were then treated with prototypical stimuli
type (C57BL/6), NLRP3-deficient, or NLRP3-deficient macrophages reconstituted with NLRP3 (NLRP3-KO + NLRP3) treated with 100 ng/ml CHX or left
untreated. Stimulation was performed as indicated. B, Immunoblotting of caspase-1 from supernatants of wild-type macrophages pretreated with CHX for 1 h
and stimulated as indicated. C, mRNA expression in LPS-primed (200 ng/ml) or untreated macrophages. Relative expression data per Hprt1 are shown. Readouts
were performed 6 h (A, B) or 3 h (C) after stimulation, and data are from one representative experiment of three (A, B) or of four (C) experiments.
The requirement of priming is a distinctive feature of the NLRP3 inflammasome. A, Immunoblot of cleaved caspase-1 from supernatants of wild-
614CUTTING EDGE: ROS INHIBITORS BLOCK NLRP3 PRIMING
of the NLRP3 inflammasome (LPS/nigericin), the AIM2
inflammasome [poly(dA:dT)], or the NLRC4 inflammasome
(flagellin) and then monitored for caspase-1 activation as
a direct readout for inflammasome activation. As previously
shown, NLRP3 inflammasome activation was critically de-
pendent on the presence of LPS and abrogated by cyclohexi-
mide treatment (Fig. 1A, upper panel). Macrophages transduced
to constitutively express NLRP3 at levels that equal those of
LPS-primed macrophages (Supplemental Fig. 1E) did not re-
quire LPS priming, and inhibition of de novo translation had
no impact on NLRP3 activation in these cells (Fig. 1A, lower
panel). Conversely, activation of the AIM2 or the NLRC4
inflammasome was independent of LPS priming. Moreover,
even though AIM2 and NLRC4 ligands can serve as proin-
flammatory priming signals themselves, complete blockage of
de novo translation by cycloheximide also did not inhibit their
activation of caspase-1. Similarly, IL-18, which is constitu-
tively expressed in macrophages, was released in cycloheximide-
treated macrophages when stimulated via NLRC4, but not
when stimulated via NLRP3 (Supplemental Fig. 1F). Careful
titration of cycloheximide indicated that the inhibitory effect
on NLRP3 activation was dose dependent (Fig. 1B) and that
AIM2 or NLRC4 could be activated even at high concen-
trations of cycloheximide. The specific role of priming for
NLRP3 inflammasome activation was reflected by the fact that
NLRP3 was highly inducible in response to proinflammatory
stimuli such as LPS. Of interest, LPS priming enhanced neither
the expression of NLRP1a nor that of NLRP1b, NLRC4,
AIM2, caspase-1, or ASC. As expected, IL-1b was highly in-
ducible upon LPS priming (Fig. 1C). Similar results were
1 h with DPI (20 mM), NAC (20 mM), cytochalasin D (5 mM), or bafilomycin (250 nM), then stimulated as indicated and subsequently assessed for cleavage of
caspase-1. B, Wild-type macrophages were pretreated for 1 h with 0, 10, or 20 mM DPI, then stimulated as indicated; subsequently, IL-18 release was measured
by ELISA. C, Wild-type macrophages were treated with ascending doses of DPI, subsequently primed with LPS, and then assessed for NLRP3 mRNA expression.
D and E, Wild-type macrophages were treated with DPI (1 h) and then primed with LPS (3 h); alternatively, macrophages were primed with LPS (3 h), then
treated with DPI (1 h) and subsequently stimulated as indicated. Six hours following stimulation, IL-1b and IL-18 release was assessed in the supernatant. F,
Cleaved caspase-1 of wild-type and NLRP3-deficient macrophages reconstituted with NLRP3 is depicted. Data from one representative experiment of two (A, B,
D, E) or three (C, F) are presented.
Inhibitors of the ROS system block NLRP3-mediated caspase-1 activation by inhibiting cell priming. A, Wild-type macrophages were pretreated for
The Journal of Immunology615
obtained for other TLR ligands such as Pam3Cys (TLR2) or
R848 (TLR7/8) (data not shown). Altogether, these data in-
dicated that NLRP3 is unique among the known inflamma-
some pathways in its specific requirement of a proinflammatory
ROS inhibitors block the priming step of NLRP3 inflammasome
DPI is a competitive inhibitor of flavin-containing cofactors
and is thus a potent inhibitor of NOX-dependent ROS
production (23). At the same time, DPI also blocks mito-
chondria-derived ROS production, although higher concen-
trations are required for this effect (21, 24). NAC, in contrast,
functions as a scavenger of ROS regardless of the source of
production. ROS inhibitors have been reported to potently
inhibit NLRP3 activation (12, 16) and, in line with this
finding, we observed that both DPI and NAC potently
inhibited caspase-1 activation in response to various NLRP3
stimuli (LPS/ATP, LPS/nigericin, or LPS/silica), yet not in
response to AIM2 activation [poly(dA:dT)]. NLRP3 stimuli
that require phagosomal uptake and acidification, such as sil-
ica, were also inhibited by cytochalasin D or bafilomycin A
(Fig. 2A). Moreover, IL-18 release in response to NLRP3
stimulation, but not AIM2 or NLRC4 stimulation, was in-
hibited by DPI as well (Fig. 2B). Measuring IL-1b release in
response to NLRP3 activation confirmed the caspase-1 and
IL-18 activation data. However, ROS inhibition blocked
AIM2-mediated IL-1b release equally potently (Supplemental
Fig. 2A). In fact, assessing IL-1b expression at the protein
level in cell lysates or at the mRNA level using real-time PCR
revealed that ROS inhibitors downmodulated LPS-mediated
IL-1b expression per se (Supplemental Fig. 2B). At the same
time, the expression of other proinflammatory genes such
as TNF was also blocked by ROS inhibitors (Supplemental
Fig.2C). In accordance with this observation, ROS inhibition
dose-dependently inhibited the expression of NLRP3, which
is also induced in response to proinflammatory signals (Fig.
2C). On the basis of these findings, we speculated that ROS
inhibitors block NLRP3 inflammasome activation because
NLRP3 upregulation is inhibited. To address this assump-
tion, we performed experiments in which we added DPI
before or after LPS priming. Given its constitutive and thus
priming-independent expression, only IL-18 as a readout of
inflammasome activation suggested an NLRP3-specific in-
hibitory effect of DPI pretreatment. IL-1b release was again
completely blocked in DPI-pretreated macrophages stimu-
lated with either nigericin or poly(dA:dT) (Fig. 2D). More-
over, we observed an inhibition of NLRP3 activation only
when macrophages were treated with DPI before, but not
after, prolonged LPS priming (Fig. 2E). To further address
the hypothesis that DPI blocks NLRP3 inflammasome acti-
vation by inhibiting its upregulation, we re-evaluated ROS
inhibitors in macrophages stably expressing NLRP3. Indeed,
although DPI blocked NLRP3 activation in wild-type mac-
rophages stimulated with LPS/silica or LPS/nigericin, no in-
hibition of caspase-1 activation was observed when NLRP3
expression was uncoupled from the priming signal by stable
overexpression (Fig. 2F). Analogous data were obtained when
NAC was used to block ROS (Supplemental Fig. 2D–G). As
a whole, these data indicate that ROS inhibitors block
NLRP3 inflammasome activation by interfering with the
priming step that is required to induce NLRP3 expression,
whereas direct NLRP3 activation is not affected. In this
regard, the specificity of ROS inhibitors for the NLRP3 in-
flammasome can be explained by the fact that the NLRP3
inflammasome is critically dependent on priming because
NLRP3 is expressed at limiting levels in unprimed macro-
phages. AIM2- or NLRC4-mediated caspase-1 activation, in
contrast, is not affected by ROS inhibition, given their con-
stitutive expression and thus independence of de novo trans-
lation. Not only are these findings important for our under-
standing of the mechanistics of NLRP3 activation, but they
also have critical implications for the development of drugs
that specifically block NLRP3-mediated inflammation with-
out affecting proinflammatory transcription in general. In this
regard, we would favor approaches that explore inhibitory
strategies in the setting of constitutive NLRP3 expression.
We thank Millennium Pharmaceuticals for providing NLRP3-deficient mice
and E. Latz for discussion.
The authors have no financial conflicts of interest.
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