Review Article
Natural Compounds as Regulators of NLRP3
Inflammasome-Mediated IL-1𝛽 Production
József Tyzsér
1
and Szilvia Benky
2
1
Department of Biochemistry and Molecular Biology , Faculty of Medicine, University of Debrecen, Debrecen, Hu n ga ry
2
Department of Physiology, Faculty of Medicine, Un iversity of Debrecen, Debrecen, Hungary
Correspondence should be addressed to Szilvia Benk
˝
o; benkosz@med.unideb.hu
Received May ; Revised July ; Accepted August
Academic Editor: Alex Kleinjan
Copyright © J. T
˝
ozs
´
er and S. Benk
˝
o. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
IL-𝛽 is one of the main proinammatory cytokines that regulates a broad range of immune responses and also participates in
several physiological processes. e canonical production of IL-𝛽 requires multiprotein complexes called inammasomes. One of
the most intensively studied inammasome complexes is the NLRP inammasome. Its activation requires two signals: one signal
“primes” the cells and induces the expression of NLRP and pro-IL-𝛽, while the other signal leads to the assembly and activation
of the complex. Several stimuli were reported to function as the second signal including react ive oxygen sp ecies, lysosomal rupture,
or cytosolic ion perturbation. Despite very intensive studies, the precise f u nction and regulation of the NLRP inammasome are
still not clear. However, many chronic inammatory diseases are related to the overproduction of IL-𝛽 that is mediated via the
NLRP inammasome. In this review, we aimed to provide an overview of studies that demonstrated the eect of plant-derived
natural compounds on NLRP inammasome-mediated IL-𝛽 production. Although many of these studies lack the mechanistic
explanation of their action, these compounds may be considered as complementary supplements in the treatment of chronic
inammatory diseases, consumed as preventive agents, and may also be considered as molecular to ols to study NLRP function.
1. Introduction
Inammation is an important host response triggered by
invading pathogens or damaged tissues, a response that is
aimed at diluting or destroying t he pathogens or isolating the
involved site. Moderate inammatory response contributes
to the host defense by removing pathogens or aiding in
therepairofdamagedtissue.However,uncontrolledor
prolonged inammation may promote further tissue damage
and could lead to serious disorders due to the overproduction
of inammatory cytokines.
Among inammatory mediators, IL-𝛽 is a master reg-
ulatory cytokine, functioning at several levels of immune
responses, such as activation of cells to produce other inam-
matory cytokines and chemokines, induction of endothe-
lial cells to express cell membrane adhesion molecules, or
assisting in the polarization of human cells [–].
Furthermore, it also participates in a variety of physiological
processes, such as the regulation of synaptic plasticity and
memory processes, in addition to participating in pain
development [–].
e production of IL-𝛽 requires a multiprotein complex
called inammasome. One of the most intensively studied
inammasomes is the NLRP inammasome that contains
NLRP sensor, ASC adaptor, and caspase- protease [].
e presence of NLRP inammasome has been shown not
only in immunocom petent cells b ut also in cells respon-
sible for various physiological functions, such as muscle
cells, neurons, or endocrine cells. Upon stimulation, NLRP
inammasome components assemble into large cytoplasmic
complexes, and the activat ion of caspase- eventually leads
to the maturation and secretion of IL-𝛽. Besides cytokine
production, NLRP inammasome activation may als o b e
accomp anied by caspase--mediated rapid cell death, which
is known as pyroptosis [].
Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2016, Article ID 5460302, 16 pages
http://dx.doi.org/10.1155/2016/5460302
Mediators of Inammation
ERK
p38
NLRP3
ASC
NLRP3
Caspase-1
ROS
ER stress
P2X7R
ATP
Cathepsin B
TLR
ROS
mtDNA
Sirt2
Ac
Ac
Ac
Ac
AMPK
Cardio-lipin
Acetylated 𝛼-tubulin
Ca
2+
influx
IL-1𝛽
Pro-IL-1𝛽
K
+
eux
𝛼-Tubulin
Pro-IL-1𝛽
NF𝜅B
F : Basic mechanisms of NLRP inammasome activation.
2. Mechanism of NLRP3 Inflammasome
Activation
NLRP inammasome can be activated by a broad range of
stimulithatbelongeithertopathogen-associatedmolecular
patter ns (PAMPs) released during viral, bacterial, fungal, or
protozoa infection [–] or to danger-associate d molecular
patterns (DAMPs) of endogenous or exogenous origin, like
extracellular ATP, reactive oxygen species (ROS), choles-
terol, monosodium urate (MSU) crystals, amyloid beta (A𝛽)
plaques, silica, or asbestos [–] (Figure ).
Due to the multiple functions of IL-𝛽,productionof
this cytokine is tightly regulated, requiring two signals. e
rst signal, called “priming,” is mediated through PAMPs
recognized by specic receptors like TLRs, activating signal
transduction pathways that induce the expression of the
inammasome components as well as that of the inactive
cytokine precursor pro-IL-𝛽. Signaling pathways through
NF𝜅B,p,andERKhaveallbeenassociatedwiththe
expression of NLRP and pro-IL-𝛽 [–]. e second
signal, provided by DAMPs or PAMPs, leads to the assembly
of the inammasome complex that is followed by activation
of caspase- and the cleavage and secretion of active IL-
𝛽 [, ]. Considering the diversity of the second signals
sensed by NLRP, it is highly unlikely that NLRP is capable
of interacting directly with chemically dierent activators.
It is more likely that NLRP senses a general signal that
induces the sequential events of inammasome activation.
Several cellular mechanisms were reported as requirements
for activation, including intracellular release of oxidized
mitochondrial DNA (mtDNA), increased intracellular Ca
2+
concentration, decreased intracellular cAMP level, or pore
formation by bacterial toxins [–]. C ommon cellular
eventsareassumedtobecriticalfortheinammasome
activation, including ROS formation, p otassium (K
+
)eux,
and cathepsin B leakage from lysosomes []. However,
due to the controversial results published in the eld, the
precise mechanism that mediates NLRP inammasome
activation is still not known. It is conceivable that more
than one single requirement needs to be fullled, and t hese
requirements may depend on the activat ing stimuli, also the
per se characteristics of the stimulated cell.
2.1. Reactive Oxygen Species. Several NLRP inammasome
activating stimu li like nigericin or ATP induce oxidative
stress and the production of intracellular ROS []. e
source of ROS is still unclear, as some of the studies
reportedtheroleofNADPHoxidaseinROSgeneration[],
whileotherstudiesprovedthatmitochondrialdysfunction
or ER stress leads to ROS production. As a consequence
of mitochondrial dysfunction, oxidized mtDNA is released
which can activate NLRP inammasome [], eventually
leading to IL-𝛽 production. It was also shown that, in
case of increased cytoplasmic ROS, reduced thioredoxin
(TRX) becomes oxidized and dissociates from thioredoxin
binding protein (TXNIP) that in turn binds to and acti-
vates NLRP. It was also shown that the expression of
TXNIP is upregulated during ER stress, which also induces
NLRP activation []. During oxidative stress, misfolded
proteins acc u mulate, which are sensed by the ER membrane-
bound inositol-requiring enzyme (IRE𝛼)andprotein
kinase RNA-like endoplasmic reticulum kinase (PERK).
In turn, they initiate ER stress response, activate NF𝜅B
pathways, and induce the upregulation of TXNIP expres-
sion[].eroleofnuclearfactor-(rythroid-derived)
Mediators of Inammation
like (Nrf) transcription factor-mediated antioxidant sig-
naling pathway has also been reported as a negative regulator
of NLRP inammasome, as disruption of Nrf leads to
increased production of IL-𝛽 []. Recent works highlight
the importance of metabolic changes such as in glycolysis and
oxidative phosphorylation that accompany cell activation and
inuence ROS production [, ]. Moreover, ROS inuences
signal transduction pathways such as NF𝜅B; thus, besides
NLRP inammasome activation, it is also thought to play
aroleinthe“priming”step[].
2.2. Ion Flux and Perturbation. Perturbation of cytosolic ion
concentration, such as K
+
and Ca
2+
,isacommonresult
of several NLRP inammasome activating stimuli, as many
activators were shown to directly induce ion uxes. Nigericin
or the activation of ATP-sensitive PX receptor induces
rapideuxofK
+
[–], and K
+
eux has been proven
to act as a common signal to trigger NLRP inammasome
activation []. Furthermore, several pore forming toxins
result in an increased intracellular Ca
2+
that triggers NLRP
activation []. Never theless, changes in intracellular ion
concentration would also stimulate other inammasome
complexes, which argues against ion ux as the exclusive
cause for NLRP inammasome activation.
2.3. Lysosomal Rupture and Cathepsin B Release. Destabi-
lization of the acidic lysosomal compartment and release of
lysosomal protease cathepsin B into the cytoplasm during
phagocytosis of large particles or crystals, such as silica,
asbestos, uric acid, or beta-amyloids, have also been impli-
cated in the activation of the NLRP inammasome [].
Furthermore,phagocytosiscouldalsoinduceK
+
eux.
Additionally, lysosomes contain high concentration of Ca
2+
,
and lysosomal rupture results in t he release of Ca
2+
into
the cytosol, triggering further Ca
2+
release from the ER.
However, the molecular details and connections of these
events are not yet claried. In conclusion, although NLRP
inammasome is the most intensively studied inammasome
complex, it is still unclear which mechanisms are responsible
for its activation.
2.4. Mitochondrial Dysfunction. In response to infection or
endogenous stimuli, the per turbation of intracellular ROS,
K
+
, or lysosomal stability leads to mitochondrial dysfunction
and to the release of mitochondrial D AMPs such as mtDNA,
cardiolipin, or dynamin-related protein (Drp) []. I t
was shown that due to increased mtROS level, released
mtDNA is oxidized and activates NLRP inammasome [].
Cardiolipin is a non-bilayer-forming phospholipid found
in bacteria and in the inner mitochondrial membrane of
eukaryotes, and its translocation from the inner membrane
to the outer membrane was shown to activate NLRP inam-
masome downstream of mtROS []. It was also reported that
GTPase Drp, that is needed for the fragmentation and aggre-
gation of mitochondria, also induces NLRP inammasome
activation and IL-𝛽 secretion in response to VSV infection
but not to ATP or nigericin []. Moreover, mitofusin, an
outer mitochondrial GTPase that regulates mitochondrial
fusion, was also described as an NLRP activator during
viral infection []. Recently, it was described that the
mitochondrial antiviral signaling protein (MAVS) located in
the mitochondrial membrane mediating interferon responses
also induces NLRP inammasome activation []. ese
reports demonstrate that the mitochondrion is a complex
regulator of cytosolic homeostasis and a central platform for
NLRP inammasome activation.
2.5. Autophagy. A u tophagy is an evolutionarily conserved
mechanism to main tain cellular homeostasis by selectively
eliminating damaged or aging organelles, microbes, and
ubiquitinated proteins, through the formation of autophago-
somes and using lysosomal degradation. Lines of evi-
dence show that aut ophagy suppresses organelle stress-
induced activation of NLRP inammasome. Furthermore,
it was reported that inducers of autophagy also induce
the autophagosomal engulfment of IL-𝛽 and its lysosomal
degradation []. It was also shown that damaged mitochon-
dria accumulate in autophagy-decient macrophages that in
turn lead to increased intracellular ROS and to the release of
mtDN A [].
2.6. Spatial Location of NLRP3. Originally, it was shown
that activated NLRP inammasome localizes to the mito-
chondria. However, recently, it was described that during
inammasome activation ER-associated NLRP colocalizes
with the mitochondria-associated ASC to the perinuclear
space via a microtubu le-mediated organelle transport. It was
shown that, upon cell activation, the decreased intracellular
NAD
+
level leads to the inhibition of sirtuin (SIRT)
deacetylase, resulting in the accumulation of acetylated alfa-
tubulin triggering a dynein-dependent transport of mito-
chondria to the perinuclear region, into the close proximity
to the ER [].
3. NLRP3 Inflammasome and Related Diseases
Understanding the steps and mechanism of NLRP inam-
masome activation would be of crucial importance for
the treatment of several diseases in which the inamma-
some complex has been implicated. By producing IL-𝛽
inammatory cytokine and inducing pyroptosis, the primary
function of NLRP inammasome is to protect the host
from invading microorganisms []. However, nonmicrobial
compoundsofeitherendogenous(self-derived)orexogenous
(environmental) origin are also eective inducers of NLRP
inammasome activation and lead to sterile inammation,
allergic responses, or other forms of inammation []. For
example, studies have shown that NLRP may be implicated
in Alzheimer’s disease, suggesting that misfolded A𝛽 pro-
teins form aggregates that lead to the activation of NLRP
inammasome [, ]. In gout, MSU crystals from purine
degradation form deposits in dierent tissues and specif-
ically activate NLRP inammasome []. Environmental
particulates such as inhaled asbestos and silica also act ivate
NLRP inammasome, and the high concentration of IL-
𝛽 is involved in the development of asbestosis and silico-
sis, two progressive pulmonary diseases leading to brosis
Mediators of Inammation
[, ]. NLRP inammasome has also been related to
allergic responses to a variety of allergens, such as aluminum,
dust mite, or ragweed pollen; however, the molecular details
of these responses are still not clear [–].
Other studies associated NLRP inammasome function
with metabolic syndrome and type diabetes, as NLRP de-
cient mice were protected from high fat diet- (HFT-) induced
inammation, glucose intolerance, insulin resistance, and
obesity [, ].
e dysregulated production of IL-𝛽 by the NLRP
inammasome is the main reason for the development of
Cryopyrin-Associated Periodic Syndromes (CAPS) which is
caused by a mutation in NLRP gene. CAPS is an autoin-
ammatory disorder rather than an autoimmune one, as
symptoms are media ted by the innate immune system, mainly
by monocytes and macrophages that produce huge amount
of IL-𝛽. Behcet’s disease (BD) is another chronic autoin-
ammatory disorder of unknown etiology, and increased
IL-𝛽 production has been noted as a central player in the
pathogenesis of this disease. Studies demonstrated increased
ROS production and, consequently, increased NLRP func-
tion in these patients, suggesting an important role for NLRP
inammasome in mediating cytokine production []. In
addition to autoinammatory diseases, NLRP inamma-
somefunctionhasalsobeenimplicatedinthedevelopment
of autoimmune diseases like rheumatoid arthritis (RA) [].
RA is a common inammatory disease aecting small joints,
and our knowledge about its pathogenesis is still incomplete.
Nevertheless, elevated levels of IL-𝛽 and high expression of
NLRP were detected from the serum and synovial tissue as
well as in macrophages of RA patients [].
At present, the role of NLRP inammasome and IL-
𝛽 in cancer is highly controversial. e microenvironment
of tumors is characterized by an inammatory milieu that
helps tumor survival with gr owth hormones, endothelial
activation, and angiogenesis that leads to metastasis accom-
panied by immune suppression. IL-𝛽 participates in each of
these mechanisms by stimulating the expression of TNF𝛼,
MMPs, VEGF, ICAM-, VCAM, and so forth. Cancer cells,
like melanoma or myeloma, as well as tumor-associated
macrop hages and dendritic cells, were shown to contribute to
the IL-𝛽 production that helps survival and growth of tumor
cells; furthermore, excessive production of IL-𝛽 can recruit
immunosuppressive cells like myeloid-derived suppressors
cells (MDSCs) []. On the other hand, it was also shown that
NLRP-induced IL-𝛽 production boosts T cell function in
patients receiving chemotherapy [].
e classical medical approach to treating the above-
mentioned diseases involves the use of synthetic drugs devel-
oped against individual elements of the signaling pathway,
the IL-𝛽 cytokine, or the IL-𝛽 receptor, as reviewed in
Ozakietal.,[].However,today,inthehealth-cautious
life era, the use of botanical and natural compounds has
gained popularity. While a few decades ago the use of natural
compounds was based exclusively on empirical experiences,
today, with the highly developed molecular biological, high
throughput methods, many molecular elements of their
action have already been identied. However, fundamental
questions remain to be answered, including bioavailability,
eective doses, body concentrations, cross-reactivity, half-
life and degradation, and synergistic eect of compounds
as well as carefully designed clinical trials. e aim of the
present review is to provide an overview of these plant-
derived natural compounds that would support the use of
medicinalplants(Figure).Asavarietyofcompoundscan
inuence inammasome activation and function, we have
focusedonnaturalcompoundsthathavebeeninlongterm
use in traditional medicine; therefore, their safety and overall
eect are mostly established. However, these compounds
typically inuence a multitude of pathways, and the exact
molecular mechanisms of their benecial actions are not
completely claried (Table ).
4. Natural Compounds Affecting NLRP3
Inflammasome Activation
4.1. Aloe vera. Aloe vera is a medical plant used traditionally
in diverse therapeutic applications. e gel of Aloe vera
has been reported to stimulate wound-healing and skin
hydration, induce hematopoiesis, and possess antidiabetic,
anticarcinogenic, antimicrobial, and antioxidant as well as
anti-inammatory activities. Over active components have
already been identied in Aloe vera leaf gels [], and
some of them have been implicated as immunomodulatory
compounds based on animal studies. In a mouse sepsis model
andinahumancolorectalmucosamodel,treatmentwith
Aloe vera signicantly inhibited the elevation of TNF𝛼,IL-
, and IL-𝛽 levels [, ]. Studying human THP- cells
and human monocyte-derived macrophages, it was found
that Aloe vera treatment signicantly reduced LPS-induced
IL-𝛽 production []. Aloe vera inhibited the expression
of pro-IL-𝛽,NLRP,andcaspase-aswellasthatofthe
PX receptor in the LPS-induced primary macrophages.
Furthermore, LPS-induced activation of signaling pathways,
such as NF-𝜅B, p, JNK, and ERK, were inhibited by Aloe
vera in these cells [].
Aloe emodin is an anthraquinone, present naturally in
Aloe leaves. It has been shown to promote natural killer cell
activity and macrophage phagocytosis in tumor []. Emodin
itself appears to have some protective eect in the inamma-
tory response. Recently , it was shown that emodin attenuated
nigericin-, ATP-, and silica-induced IL-𝛽 secretion from
LPS-activated murine bone marrow-derived macrophages
(BMDMs). It was also shown that mice treated intraperi-
toneally with emodin showed higher survival rates than con-
trol mice injected with LPS alone, indicating that emodin can
ameliorate the severity of NLRP inammasome-mediated
disease symptoms in vivo []. However, further molecular
details of the inhibitory eect are yet to be discovered.
4.2. Curcumin. Curcumin is a lipid soluble polyphenol, a
yellow pigment isolated f rom the rhizomes of Curcuma longa
(turmeric), but also found in other plants like ginger. It is
widely used in food coloring and avoring and it is also added
to cosmetics. It has gained attention in recent years for its
multiple pharmacological properties, being antioxidant, anti-
inammatory, and antimicrobial, as well as for its therapeutic
Mediators of Inammation
O
O
OH
OH
OH
Emodin
O
O
OH
HO
Curcumin
O
O
HO
OH
OH
OH
OH
Quercetin
O
S
N
C
S
Sulforaphane
O
OH
O
H
H
HO
Genipin
O
O
O
OH
OH
OH
HO
OH
OH
OH
EGCG
O
O
O
OH
OH
OH
OH
HO
HO
HO
Mangiferin
OH
OH
OH
OH
OGlc
OGlc
Ginsenoside
(basic)
OH
OH
OH
HO
OH
HO
Resveratrol
trans- cis-
H
3
C
H
3
CO
OCH
3
OCH
3
CH
3
F : Chemical structure of natural compounds inuencing NLRP inammasome activation.
potential in cancer, autoimmune, metabolic, pulmonary,
cardiovascular, and neurological diseases (reviewed in []).
Brain ischemia is known to induce ER stress and inam-
matory responses leading to neuronal damage [, ]. In
a recent study, glucose deprivation or hypoxia was reported
to strongly induce the production of glutamate and IL-𝛽 in
mouse hippocam pus []. It was shown that pretreatment
of the mouse hippocampus with curcumin reduced IL-
𝛽 production, and this eect was attenuated by
-AMP-
activated protein kinase (AMPK) inhibitor, suggesting the
possible involvement of AMPK. e authors also found
that curcumin attenuated glutamate-induced phosphoryla-
tion of PERK and IRE𝛼; the transmembrane sensors of
ER stress that mediate inammatory signals. Moreov er,
using a neuroblastoma cell line, they showed that curcumin
inhibited glutama te-induced ROS generation, as well as
reduced glutamate-induced TXNIP expression. As a possible
molecular mechanism, they found that, in mice hippocam-
pus, glutamate st i mulation increased NLRP expression and
the cleaved form of caspase- enzyme, while curcumin
attenuated NLRP and cleaved caspase- expression. e
production of IL-, a downstream target of IL-𝛽,wasalso
inhibitedbycurcumintreatment.Inaddition,curcumin
eectively attenuated mitochondrial function and prevented
caspase- activation in hippocampus exposed to glutamate
stimulation, eectively preventing glutamate-induced cell
apoptosis. Furthermore, in rats, curcumin administration
attenuated ischemic brain injury resulting from middle
cerebral artery occlusion (MCAO). Based on these ndings,
curcumin activates AMPK that inhibits glutamate-induced
ER stress and ROS production, thus inhibiting TXNIP-
induced NLRP inammasome activation and ultimately
reducing IL-𝛽 production in mouse h ippocampus limiting
brain injury.
Macrophages are popular models for the study of
NLRP inammasome activation. In another study per-
formedonmousemacrophagecelllineJA.andperi-
toneal macrophages, curcumin was shown to strongly inhibit
IL-𝛽 secretion triggered by LPS plus nigericin, aluminum,
ATP , or MSU []. Preincubation with curcumin prevented
nigericin-induced intracellular potassium level decrease,
attenuated lysosome damage and cathepsin B leakage, and
blocked high mobility group box (HMGB-) release; need-
less to say, all of these are components that may induce
NLRP inammasome activation. In BMDMs, curcumin
inhibited nigericin- or aluminum crystal-induced ROS pro-
duction. e authors did not nd obvious inhibitory eect
of curcumin on JNK and p phosphorylation enhanced
bynigericintreatmentintheLPS-primedmacrophages;
however, they found that ERK/ phosphorylation was