published: 25 February 2020
Frontiers in Endocrinology | www.frontiersin.org 1February 2020 | Volume 11 | Article 80
John Even Schjenken,
University of Adelaide, Australia
Jinan University, China
This article was submitted to
a section of the journal
Frontiers in Endocrinology
Received: 10 October 2019
Accepted: 07 February 2020
Published: 25 February 2020
Shirasuna K, Karasawa T and
Takahashi M (2020) Role of the NLRP3
Inﬂammasome in Preeclampsia.
Front. Endocrinol. 11:80.
Role of the NLRP3 Inﬂammasome in
Koumei Shirasuna 1
*, Tadayoshi Karasawa 2and Masafumi Takahashi 2
1Department of Animal Science, Tokyo University of Agriculture, Atsugi, Japan, 2Division of Inﬂammation Research, Center
for Molecular Medicine, Jichi Medical University, Shimotsuke, Japan
Reproduction involves tightly regulated series of events and the immune system
is involved in an array of reproductive processes. Disruption of well-controlled
immune functions leads to infertility, placental inﬂammation, and numerous pregnancy
complications, including preeclampsia (PE). Inﬂammasomes are involved in the
process of pathogen clearance and sterile inﬂammation. They are large multi-protein
complexes that are located in the cytosol and play key roles in the production of
the pivotal inﬂammatory cytokines, interleukin (IL)-1βand IL-18, and pyroptosis. The
nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-
containing 3 (NLRP3) inﬂammasome is a key mediator of sterile inﬂammation induced
by various types of damage-associated molecular patterns (DAMPs). Recent evidence
indicates that the NLRP3 inﬂammasome is involved in pregnancy dysfunction, including
PE. Many DAMPs (uric acid, palmitic acid, high-mobility group box 1, advanced glycation
end products, extracellular vesicles, cell-free DNA, and free fatty acids) are increased and
associated with pregnancy complications, especially PE. This review focuses on the role
of the NLRP3 inﬂammasome in the pathophysiology of PE.
Keywords: NLRP3 inﬂammasome, pregnancy, preeclampsia, interleukin-1β, inﬂammation
Reproduction, including development of oocyte and sperm, ovulation, corpus luteum function,
fertilization, implantation, placentation, maintenance of pregnancy, and parturition, is essential
for species maintenance, and reproductive events for next generation are tightly regulated (1).
Pregnancy has been studied extensively over the years (2). From the perspective of the maternal
immune system, a conceptus is a semi-allogeneic tissue that must be rejected; however, that does
not generally happen. It was quickly ruled out that the fetus is shielded from the maternal immune
system via the placenta acting as a physical barrier because the fetal extravillous trophoblast cells
deeply penetrate the uterine mucosa and directly communicate with various maternal immune cells
to avoid rejection (3).
Inﬂammation is basically a complex protective immune response to harmful stimuli such as
pathogens, damaged or dead cells, and irritants (4). This response is tightly regulated by the
host, enabling survival after infection or injury and maintaining tissue homeostasis. However,
excessive inﬂammation may cause chronic or systemic inﬂammatory diseases. On the other hand,
the immune system also contributes to the regulation of reproductive function and pregnancy
(5). Immune-mediated processes such as tissue growth, remodeling, and diﬀerentiation are
crucial to maintain pregnancy (1,5). Disruption of well-controlled immune functions leads to
infertility, placental inﬂammation, and numerous pregnancy complications, such as preeclampsia
Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
(PE), obesity during pregnancy, gestational diabetes mellitus
(GDM), spontaneous abortion, and recurrent pregnancy
There is an increasing body of evidence to suggest that
inﬂammation and immune cells are involved in both physiology
and pathophysiology of pregnancy. Since infection is not
involved in the majority of the phenomena related to pregnancy
physiology and pathology, it remains unclear why inﬂammation
is involved. Recently, there have been numerous reports of
inﬂammasome mechanisms that control sterile inﬂammation
involved in pregnancy pathologies. Inﬂammasomes are large
multi-protein complexes found in the cytosol that play key
roles in the production of the pivotal inﬂammatory cytokines,
interleukin (IL)-1βand IL-18, and pyroptosis (inﬂammatory
cell death) [(9–11); Figure 1]. In particular, nucleotide-binding
oligomerization domain, leucine-rich repeat-, and pyrin domain-
containing 3 (NLRP3) inﬂammasome is a key mediator of sterile
inﬂammation. Excessive activation of the NLRP3 inﬂammasome
contributes to the pathogenesis of a wide variety of diseases,
such as diabetes, atherosclerosis, and obesity-induced insulin
resistance (12–17). The present review focuses on the role of the
NLRP3 inﬂammasome in placental inﬂammation and pregnancy
complications, especially PE.
IMMUNE CELLS INVOLVED IN
The most important immune cells that induce pregnancy
immune tolerance is CD4+regulatory T cells (Tregs) (18).
The transcription factor, forkhead boxP3 (Foxp3), is a master
regulator of the development and function of Tregs (19).
The frequency of Foxp3+Tregs increases during normal
pregnancy in the decidua and peripheral blood in humans
and mice (20–22). Shima et al. (23) used an animal model
to demonstrate that CD4+CD25+Foxp3+Tregs play a critical
role in regulating immune tolerance at the implantation site to
support implantation and successful pregnancy. The frequency
of Tregs is lower in human pregnancy complications such
as PE or miscarriage (24). In addition, seminal ﬂuid induces
and accumulates paternal-speciﬁc Tregs that are involved in
the preimplantation uterus, and insuﬃcient expansion of Tregs
against paternal antigens may trigger spontaneous abortion (25).
Natural killer (NK) cells, particularly decidual NK cells, are
also essential immune cells involved in establishing pregnancy;
they are the most abundant leukocyte population during the
ﬁrst trimester of human pregnancy (1,26). Decidual NK cells
directly communicate with extravillous trophoblast cells and
other immune cells in the fetal-maternal boundary area, and
promote fetal tolerance and pregnancy progression (26).
Monocytes also accumulate in the decidua, in a process
that involves communication with trophoblast cells (1,27).
They can diﬀerentiate into dendritic cells (DCs) in the decidua
during murine and human pregnancy (28,29). DCs regulate
immune tolerance by inducing eﬀector T cell apoptosis and
expansion of Tregs due to reduced antigen presentation, reduced
expression of co-stimulatory molecules, or enhanced production
of anti-inﬂammatory IL-10 (1,30). Monocytes also diﬀerentiate
into macrophages depending on the tissue, and polarization of
macrophages is well-understood (inﬂammatory M1 and anti-
inﬂammatory M2 type macrophages). It has been suggested
that dysfunction of decidual macrophages and dysregulation
of M1/M2 balance are critical events in the pathogenesis
of PE. Moreover, activation of NLRP3 inﬂammasome in the
reproductive organs including placenta is known to occur by
MECHANISMS OF NLRP3
Inﬂammasomes recognize various inﬂammation-inducing
stimuli, such as endogenous danger/damage-associated
molecular patterns (DAMPs) and exogenous pathogen-
associated molecular patterns (PAMPs). They tightly regulate
the production of proinﬂammatory cytokines such as IL-1βand
IL-18 (9,13,31). The NLRP3 inﬂammasome is the most widely
studied and is activated in response to a wide array of stimuli,
including exogenous and endogenous danger signals [(9,11);
Figure 1]. The NLRP3 inﬂammasome is typically composed
of NLRP3, apoptosis-associated speck-like protein containing
a caspase recruitment domain (ASC), and caspase-1 as an
IL-1β-converting enzyme (32). Activation of NLRP3 in response
to danger signals leads to nucleation of ASC into prion-like
ﬁlaments via pyrin domain (PYD)–PYD interactions (33).
ASC is then linearly ubiquitinated for NLRP3 inﬂammasome
assembly, followed by procaspase-1 interaction with ASC using
caspase recruitment domain (CARD)-CARD interactions,
forming its own prion-like ﬁlaments (34). Activated caspase-1
(a cysteine protease) cleaves the precursor cytokines, pro-IL-1β
and pro-IL-18, generating the biologically active cytokines, IL-1β
and IL-18, respectively (9–11). Moreover, active caspase-1 is
able to induce pyroptosis as an inﬂammatory form of cell death
due to cleaved gasdermin D (GSDMD) (35,36). Caspase-1
proteolytically cleaves GSDMD into a N-terminal domain and
C-terminal domain. Cleaved N-terminal domain of GSDMD
binds to phosphatidylinositol phosphates and phosphatidylserine
in the cell membrane, forming a 10–20 nm pore and induces
a lytic form of cell death, pyroptosis (36). Another feature of
gasdermin D-dependent pyroptosis is the release of IL-1βand
IL-18 via GSDMD-forming cell membrane pore.
The production and secretion of mature IL-1βare regulated
via two steps, including the transcription of pro-IL-1β
and proteolytic processing into a mature form IL-1βby
inﬂammasomes (9–11). Prior to its activation, NLRP3 must be
primed in most cell types. Nuclear factor κB (NF-κB)-activating
stimuli, such as lipopolysaccharide (LPS), upregulate mRNA
expression of NLRP3 and IL-1β, resulting in elevated expression
of NLRP3 and pro-IL-1βprotein (9–11). On the other hand,
another priming step facilitates the rapid induction of the NLRP3
inﬂammasome via deubiquitination of NLRP3 (37,38).
The upstream mechanisms of NLRP3 activation have
been elucidated by many studies, and include the release of
cathepsins into the cytosol after lysosomal destabilization,
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Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
FIGURE 1 | Schematic mechanisms of NLRP3 inﬂammasome activation. NLRP3 is activated by various endogenous DAMPs: uric acid crystals (MSU), cholesterol
crustal, cell-free DNA (cfDNA), high-mobility group box 1 (HMGB1), extracellular debris, extracellular vesicles (EVs), advanced glycation end-products (AGEs), and free
fatty acid. Various events such as intracellular ATP release, NLRP3 deubiqutination, relocalization, reactive oxygen species (ROS) generation, mitochondrial
dysfunction, lysosome rapture, and cathepsin release occur depending on the effects of damage-associated molecular patterns (DAMPs). Then, inﬂammasome
components, including NLRP3, ASC, and procaspase-1, form the NLRP3 inﬂammasome complexes. Finally, activated caspase-1 induces the inﬂammatory form of
cell death known as pyroptosis and cleaves the precursor cytokines pro-IL-1βand pro-IL-18, generating the biologically active cytokines IL-1βand IL-18.
potassium eﬄux, generation of mitochondrial reactive oxygen
species (ROS), and release of mitochondrial DNA (39,40).
Cytosolic leakage of cathepsin B via lysosomal rupture is
essential for NLRP3 inﬂammasome activation, especially by
endogenous DAMPs (41). Leakage of cathepsin B also leads to
potassium eﬄux and mitochondrial damage. Potassium eﬄux
and reduced potassium concentration within cells result in
NLRP3 inﬂammasome activation (10). In response to potassium
eﬄux, NEK7 (a member of the family of mammalian NIMA-
related kinases) directly interacts with NLRP3 inﬂammasome
(42,43). Cellular and mitochondrial ROS production also act as
NLRP3 inﬂammasome activators (44,45). Furthermore, recent
studies have demonstrated that the NLRP3 inﬂammasome
is tightly regulated by multiple mechanisms, including
ubiquitination, phosphorylation, nitrosylation, microRNAs, and
endogenous regulators (e.g., pyrin-only proteins and CARD-only
Following NLRP3 activation through the above mentioned
regulatory mechanisms, NLRP3 relocates from endoplasmic
reticulum to the mitochondria, where it forms complexes with
ASC (49). IL-1βand IL-18 secretion is regulated by caspase-1
activation by many NLRP3 inﬂammasome activators, including
monosodium urate (MSU) crystals, silica crystals, asbestos, and
cholesterol crystals (12,13,31,50). Additionally to the canonical
pathway of the NLRP3 inﬂammasome, the inﬂammasome
activation can also be indirectly triggered by caspase-11 in mice
(or the homologs caspase-4 and caspase-5 in humans), which
has been termed the non-canonical inﬂammasome pathway (51).
In this non-canonical pathway, caspase-11 directly recognized
and binds to intracellular LPS, resulting in its oligomerization
and activation by autoproteolytic cleavage (35). Then, caspase-
11 can directly induce the cleavage of GSDMD to induce
pyroptosis (35,36). Details of the structure and activation
mechanism of the NLRP3 inﬂammasome are refer to following
great reviews (10,17,39,40,52).
PREECLAMPSIA AND THE NLRP3
PE is a pregnancy-speciﬁc hypertensive syndrome that
complicates around 5–10% of all pregnancies worldwide
(53), and is a leading cause of maternal and fetal morbidity and
mortality. It is characterized by the onset of hypertension and
proteinuria in the third trimester of pregnancy, and is associated
with 12% of infants with fetal growth restriction (FGR) and
approximately 20% of preterm deliveries (54). The clinical
manifestations of PE reﬂect widespread systemic inﬂammation
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Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
and endothelial dysfunction, resulting in vasoconstriction,
end-organ ischemia and increased vascular permeability (55).
The placenta has been shown to play a central role in the
pathogenesis of PE due to the rapid disappearance of disease
symptoms after delivery. Thus, placenta-derived circulating
factor(s) may induce excessive inﬂammation and endothelial
defects, leading to PE (56).
During normal pregnancy, trophoblast cells invade, and
remodeling of maternal spiral arteries and the fetoplacental unit
produce angiogenic factors, such as vascular endothelial growth
factor (VEGF) and placental growth factor (PlGF), to support the
developing placenta (57,58). Inadequate trophoblast remodeling
of spiral arteries, which is a key feature of PE, is believed to result
of dysregulation in placental angiogenesis and maternal immune
response (55). Following that, various inﬂammatory factors are
produced by the diseased and hypoxic placenta, which activates
systemic inﬂammatory responses (27,59). It is widely recognized
that antiangiogenic factors, including soluble endoglin (sEng; a
coreceptor for transforming growth factor β) and soluble fms-
like tyrosine kinase (sFlt-1; a receptor for VEGF), induce PE-
like phenomena (57,60). Indeed, overexpression of sEng and
sFlt-1 in pregnant rats leads to severe PE symptoms including
hypertension, proteinuria, renal and endothelial dysfunction,
hemolysis, elevated liver enzymes, and FGR (60).
Pathophysiological changes of PE include inﬂammation and
immune cell activation (61–63). The main pathological features
of PE include a general inﬂammatory response by cytokines,
such as IL-1β, IL-6, IL-8, and tumor necrosis factor-α(TNFα)
(7,64,65). Siljee et al. (66) reported that IL-1βhas a potential to
improve prediction of PE during the ﬁrst trimester. A decreased
frequency of peripheral Tregs is characteristic immune cell
dynamics seen in PE patients (6). On the other hand, M2-like
immunomodulatory macrophages are abundantly present in the
decidua in healthy pregnant women and participate in spiral
artery remodeling via the angiogenic factors, VEGF and PlGF
(27). Increased numbers of M1-like inﬂammatory macrophages
are observed in PE patients and may be associated with increase
in inﬂammatory cytokines, decreased spiral artery remodeling,
and increased production of sFlt-1 and sEng (27).
In recent years, there has been a rapid increase in reports
that the NLRP3 inﬂammasome is involved in the pathogenesis of
PE (Figure 2). Higher expression of components of the NLRP3
inﬂammasome has been reported in peripheral blood mono-
nuclear cells and placental tissue from PE patients compared with
that of healthy normal pregnant women (67–69). In addition to
immune cells, human trophoblast cells express NLRP3, ASC and
caspase-1 that are components of the NLRP3 inﬂammasome (70–
72). IL-1βsecretion is induced in response to nigericin or nano-
silica crystals, typical activators of the NLRP3 inﬂammasome, in
human trophoblast cells (71,72).
HYPERTENSION AND THE NLRP3
INFLAMMASOME IN PE
Maternal hypertension is a characteristic of PE and the renin–
angiotensin system has been implicated in its pathogenesis of
PE (73,74) generated a mouse model of PE-like symptoms by
mating females expressing human angiotensinogen with males
expressing human renin, resulting in mice exhibiting maternal
hypertension, proteinuria, and FGR. Angiotensin II (AngII) is
a strong vasoconstrictor that contributes to hypertension and
stimulates sFlt-1 production and secretion from the placenta
in mice (75). Infusion of AngII in pregnant mice can lead to
high maternal blood pressure, proteinuria, and FGR (75,76).
Deﬁciency of NLRP3 inﬂammasome components attenuates the
development of AngII-induced hypertension, but does not aﬀect
FGR, proteinuria, or sFlt1 levels (76).
Furthermore, during non-pregnant conditions, infusion
of AngII induces hypertension with activation of NLRP3
inﬂammasome in the aorta, and NLRP3 deﬁciency attenuated
AngII-induced hypertension via inhibition of NLRP3
inﬂammasome activation in mice (77). A murine experimental
hypertension model (uninephrectomy and treatment with
deoxycorticosterone acetate and 0.9% NaCl in the drinking
water) induced activation of the NLRP3 inﬂammasome in kidney
and speciﬁc NLRP3 inhibitor, MCC950, inhibited the NLRP3
inﬂammasome and inﬂammation, resulting in improvement of
hypertension in mice (78). In rats, salt-induced hypertension
occurs partly due to the role of NLRP3 inﬂammasome activation
in the hypothalamic paraventricular nucleus, while blockade
of brain NLRP3 attenuates the hypertensive response (79).
An absence of ASC also reduces pulmonary hypertension
induced by hypoxia (80). These ﬁndings suggest that the NLRP3
inﬂammasome contributes to the development of hypertension
in both pregnant and non-pregnant situations. On the other
hand, NLRP3 inﬂammasome has been shown to contribute
to a wide range of acute and chronic kidney diseases (81);
the importance of NLRP3 inﬂammasome in renal pathologic
abnormalities in PE pathology is not well-understood.
ACTIVATION OF NLRP3 INFLAMMASOME
BY DAMPS IN PE
Release of DAMPs from various cells during stress has
been implicated in pregnancy complications. In PE patients,
many DAMPs, such as, cholesterol, uric acid crystals (MSU),
extracellular DNA, high-mobility group box 1 (HMGB1),
extracellular cell debris, advanced glycation end-products
(AGEs), and free fatty acids, have been detected in higher levels
in the peripheral blood and placenta (Figure 2) and act as NLRP3
CHOLESTEROL AND THE NLRP3
INFLAMMASOME IN PE
Cholesterol crystals activate inﬂammatory responses and
promote inﬂammatory cell inﬁltration, resulting in progression
of atherosclerosis and development of cardiovascular disease
(16,82). Cholesterol crystals also cause lysosome rupture,
resulting in the release of cathepsin B to the cytosol, and are a
candidate activator of the NLRP3 inﬂammasome (82,83).
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Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
FIGURE 2 | Concept of association of NLRP3 inﬂammasome in pathogenesis of pregnancy complications. Maternal risk such as hypertension and obesity are
associated with the elevation of damage-associated molecular patterns (DAMPs). DAMPs activate NLRP3 inﬂammasome, accumulate immune cells, and induce
inﬂammatory cytokine production and vascular dysfunction in placenta. These events result in placental inﬂammation and dysfunction, leading to pregnancy
complications, such as preeclampsia, spontaneous abortion, recurrent pregnancy loss, and fetal growth restriction.
Maternal cholesterol serum levels are elevated in PE and
cholesterol accumulates in placenta of PE patients, along with
increased levels of NLRP3 and IL-1βexpression (84,85). In
an in vitro human placental explant experiment, treatment
with cholesterol crystals signiﬁcantly increased the release
of IL-1β, and cholesterol crystal-induced IL-1βsecretion
was suppressed by treatment with MCC950, as a speciﬁc
inhibitor of the NLRP3 inﬂammasome (84). Cholesterol
crystals also strongly activated the NLRP3 inﬂammasome
in macrophages and induced IL-1βsecretion, dependent
on activation of the NLRP3 inﬂammasome (82,83,86). In
addition to macrophages, cholesterol crystals markedly increase
the formation and activation of NLRP3 inﬂammasome in
endothelial cells, as demonstrated by increased colocalization
of NLRP3 with ASC or caspase-1, enhanced caspase-
1 activity, and elevated IL-1βsecretion in mice (87).
These ﬁndings indicate that cholesterol induces placental
inﬂammation via the NLRP3 inﬂammasome pathway in human
placenta, suggesting the contribution of enhanced NLRP3
inﬂammasome activation to harmful placental inﬂammation
MSU AND THE NLRP3 INFLAMMASOME
Saturation of uric acid in body ﬂuids results in the formation of
MSU crystals. These are identiﬁed as danger signals from dying
cells, resulting in an acute and/or chronic inﬂammatory response
known as gout, which is associated with the deposition of MSU
crystals (41,88) demonstrated that MSU crystals activate the
NLRP3 inﬂammasome, resulting in the production of active IL-
1βand neutrophil accumulation in mice, suggesting a pivotal
role for inﬂammasomes in inﬂammatory diseases. In terms of the
mechanisms of NLRP3 inﬂammasome activation, MSU crystals
are taken up by phagocytosis and lysosomal damage is induced,
resulting in the release of cathepsin B and stimulation of ROS
production from mitochondria (89).
Elevated levels of MSU in the maternal circulation have
been shown in many pregnancy complications, especially PE
(69,84,90,91). In human ﬁrst trimester trophoblast cell lines,
IL-1βwas produced in response to MSU crystals via the NLRP3
inﬂammasome (91). Brien et al. (91) reported that MSU crystals
induce a proinﬂammatory proﬁle with predominant secretion of
IL-1βand IL-6 in human placental explants, and these eﬀects
were IL-1-dependent, as conﬁrmed using a caspase-1 inhibitor
and IL-1 receptor antagonist. In addition, administration of
MSU crystals to pregnant rats induced placental inﬂammation
(increase IL-1β, IL-6, and TNFαproduction, and macrophage
accumulation) and FGR. Indeed, MSU crystals elicit an increase
in the recruitment of macrophages and neutrophils with IL-
1βsecretion in the NLRP3 inﬂammasome-dependent manner
(41,92). These ﬁndings suggest that higher levels of MSU
in PE patients trigger placental inﬂammation via NLRP3
inﬂammasome activation, resulting in the pathogenesis of PE.
EXTRACELLULAR DNA AND THE NLRP3
INFLAMMASOME IN PE
Extracellular released cell-free DNA (cfDNA) circulating in
the blood, which is considered a product of apoptosis and/or
necrosis, acts as a DAMP and is related to many types of
inﬂammatory diseases (93,94). Toll-like receptor 9 (TLR9),
originally identiﬁed as a sensor of exogenous DNA fragments,
contributes to cfDNA-mediated inﬂammatory processes (95).
It is activated by bacterial DNA rich in unmethylated CpG
motifs, and can also be activated by DNA from mammalian
cells such as nucleic and mitochondrial DNA. Therefore, TLR9
signaling is of interest as a candidate molecule responsible for
the ﬁrst signal in sterile inﬂammation (96). It was previously
reported that cfDNA released from apoptotic hepatocytes
activates TLR9 systems, which in turn triggers a signaling cascade
that increases transcription of the genes encoding pro-IL-1β
and pro-IL-18. Furthermore, mice lacking components of the
NLRP3 inﬂammasome showed reduced amounts of cfDNA and
improved liver injury (96). Pan et al., reported that mitochondrial
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Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
DNA is directly recognized and binds with NLRP3, resulting
in the formation of NLRP3 inﬂammasome complex and its
During pregnancy, the amount of total cfDNA and cf-fetal
DNA (cﬀDNA) is signiﬁcantly increased in the maternal blood
depending on the stage of pregnancy (98). There are also
signiﬁcant associations between elevated cfDNA and cﬀDNA
with pregnancy complications such as PE and FGR (98–104).
We recently showed that expression levels of TLR9 and the
amount of cﬀDNA from the placenta were higher in PE patients
compared with that in women with normal placenta (NP), and
PE-derived cﬀDNA stimulated levels of inﬂammatory cytokine,
including IL-1βand sEng secretion depend on TLR9 signaling,
compared with NP-derived cﬀDNA (105). Moreover, a synthetic
TLR9 ligand activated inﬂammatory responses including IL-
6 secretion together with stimulation of sFlt1 secretion, while
inhibition of TLR9 reduced sFlt1 secretion in human trophoblast
cells (106). In mice, administration of a TLR9 ligand induces
PE-like symptoms, such as hypertension, proteinuria, placental
inﬂammation, and FGR. Moreover, injection of human fetal
DNA, but not adult DNA, induces placental inﬂammation, fetal
resorption, and preterm birth in pregnant mice, and notably,
these adverse eﬀects are improved in TLR9-knockout mice (107).
These ﬁndings suggest that excessive extracellular DNA acts as
a DAMP and causes pregnancy complications, especially PE, via
In trophoblast cells, cfDNA is also capable of detecting
danger signals via the intracellular DNA sensor, interferon-
inducible protein 16 (IFI16). Indeed, IFI16 agonist poly(dA:dT)
stimulates sFlt-1 and sEng production in human trophoblast
cells in an IFI16-dependent manner (108). Extracellular DNA
plays an essential role in the induction of inﬂammatory
responses; however, further research is required to clarify the
role of extracellular DNA in NLRP3 inﬂammasome activation in
HMGB1 AND THE NLRP3
INFLAMMASOME IN PE
HMGB1 is an important DAMP that acts as an architectural
chromatin-binding factor and is generally located in the nucleus
of most cell types under physiological conditions (109). When
cells are exposed to stress, HMGB1 is translocated into the
extracellular milieu and elicits inﬂammatory responses via the
production of proinﬂammatory mediators and accumulation
of inﬂammatory cells. HMGB1 interacts with TLR2, TLR4,
and receptor for AGE (RAGE), resulting in elevated levels of
HMGB1 in tissues and serum associated with the development of
inﬂammation during pathological conditions (110). It is reported
that HMGB1 induces the formation of the NLRP3 inﬂammasome
(111). HMGB1 also activates the NLRP3 inﬂammasome since
that stimulation with HMGB1 induces the release of IL-1β
with increase in NLRP3 inﬂammasome component, these eﬀects
can be attenuated by inhibition of the NLRP3 inﬂammasome
(112). In addition, Deng et al. (113) demonstrated that
HMGB1 directly binds LPS and targets its internalization into
the lysosomes of cells via the RAGE, resulting activation of
caspase-11-dependent non-canonical inﬂammasome signaling.
On the contrary, NLRP3 inﬂammasome activation accelerates
atherosclerosis induced by HMGB1 secretion, indicating that
HMGB1 is a key downstream signaling molecule of NLRP3
inﬂammasome activation (114). Therefore, the vicious cycle
of HMGB1 and the NLRP3 inﬂammasome may exacerbate
inﬂammation and pathological conditions.
In peripheral blood, HMGB1 concentrations are signiﬁcantly
elevated in PE patients compared with those of healthy pregnant
and non-pregnant women (115,116). Compared with healthy
placenta, protein and mRNA expression of HMGB1 and its
receptor RAGE, are increased in severe PE placentas (116).
In human trophoblast cells, HMGB1 stimulates inﬂammatory
cytokine production dependent on NF-κB activation and ROS
signaling via TLR4 (117). In human placenta, treatment with PE
serum increased the expression and release of HMGB1, which
induced endothelial cell activation (118). In addition, HMGB1
treatment increased NLRP3 protein expression and activation
of caspase-1, resulting in an increase of mature IL-1βsecretion
in human chorioamniotic membranes (119). These ﬁndings
indicated that HMGB1 contributes to placental inﬂammation
and NLRP3 inﬂammasome activation as endogenous DAMPs,
leading to PE. Interestingly, the expression levels of HMGB1 in
the uterus are lowest during the expected time of implantation,
and exogenous administration of HMGB1 leads to pregnancy
failure accompanied by induction of inﬂammatory responses in
rats, indicating a role of excessive extracellular HMGB1 in PE as
well as infertility (120).
PLACENTAL DEBRIS AND THE NLRP3
INFLAMMASOME IN PE
The outer layer of the placenta is covered by a single
syncytiotrophoblast that forms the maternal-fetal interface
(1). When portions of the syncytiotrophoblast become
damaged, cellular debris is extruded into the maternal blood
in membrane-enclosed vesicles (121). During normal healthy
pregnancy, trophoblastic debris is produced by programmed
cell death/apoptosis in the placenta. This extracellular debris is
believed to induce a tolerogenic response in maternal endothelial
and immune cells (122). On the other hand, extracellular debris
from PE placenta mainly originates from necrotic cell death, and
exposing endothelial cells to necrotic trophoblastic debris leads
to their activation (123). The amount of trophoblastic debris
shed into the maternal blood is greatly increased in PE patients
compared with that in healthy pregnant women (108).
It is likely that trophoblastic debris includes various types
of danger signals, such as DNA, RNA, adenosine, HMGB1,
and MSU (118,124). The degree of trophoblastic debris from
human placenta is increased by treatment with PE serum
and antiphospholipid antibodies, resulting in the activation of
endothelial cell activation and induction of immune cell adhesion
(118). Interestingly, necrotic, but not apoptotic, trophoblastic
debris contains IL-1βprotein, whereas much of the trophoblastic
debris is dead cell corpses that might not be able to produce new
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Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
proteins (124). On the other hand, adenosine in trophoblastic
debris and cell surface adenosine receptor A2B signaling also
contributes to the pathogenesis of PE (125). Iriyama et al. (125)
demonstrated that chronically elevated placental adenosine leads
to the hallmark features of PE (hypertension, proteinuria, and
FGR) in a mouse model. Moreover, elevated adenosine in PE
patients is correlated with Th1/Th2 imbalance, and adenosine
directly induces sFlt-1 production from placenta (126). Baron
et al. (127) showed that extracellular adenosine activates the
NLRP3 inﬂammasome and IL-1βsecretion by interaction with
adenosine receptors and through adenosine cellular uptake using
nucleotide transporters. These ﬁndings suggest that adenosine
signaling in debris activates NLRP3 inﬂammasome in placenta,
resulting in PE.
EXTRACELLULAR VESICLES AND THE
NLRP3 INFLAMMASOME IN PE
Extracellular vesicles (EVs) are also produced and released
by living cells and can be detected in all biological ﬂuids,
including blood. EVs are nanosized particles that are traditionally
classiﬁed into subtypes, such as exosomes, microvesicles, and
apoptotic/necrotic bodies (debris). EV cargo includes bioactive
molecules such as protein, lipids, and nucleic acid (DNA,
mRNA, microRNA, and non-coding RNA) (128). Signiﬁcantly
higher levels of syncytiotrophoblast-derived EVs are found in
the peripheral blood of women with PE compared with women
with normal pregnancies (129). EVs isolated from PE patients
diﬀer phenotypically and functionally from those isolated from
healthy pregnant women (130). Indeed, syncytiotrophoblast-
derived EVs (including exosomes) from patients with PE contain
higher levels of sFlt-1, sEng, and neprilysin, and treatment
with EVs from PE patients impairs angiogenesis of endothelial
cells and changes the characteristics of monocytes in vitro
(131,132). In addition, exosomes from PE patients cause
vascular dysfunction and directly result in adverse PE-like
birth outcomes in mice (131). Kohli et al. (133) demonstrated
that administration of EVs led to accumulation of activated
platelets and induced activation of NLRP3 inﬂammasome within
the placenta, resulting in a PE-like phenotype in pregnant
mice. Intriguingly, genetic deletion of NLRP3 inﬂammasome or
pharmacological inhibition of inﬂammasome abolished this PE-
like phenotype, indicating the pathogenesis of PE by EVs was
dependent the NLRP3 inﬂammasome.
FREE FATTY ACID AND THE NLRP3
INFLAMMASOME IN PE
Obesity is a major risk factor for PE and FGR (134,135). Obesity
represents low-grade chronic systemic inﬂammation (136), and
maternal obesity increases the risk of the oﬀspring developing
obesity and insulin resistance in the later stages of life (137–
141). The NLRP3 inﬂammasome is involved in the pathogenesis
of obesity-related inﬂammatory diseases, including metabolic
syndrome, type 2 diabetes, and cardiovascular diseases (12,13,
31,50). There are many common mechanisms between PE and
obesity-related pregnancy complications, and obesity accelerates
the systemic features of PE.
Free fatty acids levels are elevated in the plasma of obese
humans (142), and it has been proposed that they act to
promote inﬂammatory responses by directly engaging TLRs
and inducing the NF-κB-dependent production of inﬂammatory
cytokines (143,144). In particular, one of the major saturated
fatty acids, palmitic acid (PA), causes intracellular crystallization,
which in turn activates the NLRP3 inﬂammasome via lysosomal
dysfunction in macrophages (145). PA also induces NLRP3
inﬂammasome activation by generating ROS and inducing
autophagy dysfunction, resulting in secretion of mature IL-
1β(144,146,147). Similar to other crystalline molecules,
intraperitoneal administration of PA crystal induces neutrophil
recruitment in an IL-1β-dependent manner (145).
Serum PA levels are increased in women with PE and FGR
(148–150). Treatment with free fatty acid solution to mimic
the plasma of PE patients induces lipid droplet accumulation,
mitochondrial dysfunction, and apoptosis in human umbilical
vein endothelial cells (149). In addition, PA induces activation
of the NLRP3 inﬂammasome, resulting in the secretion of
mature IL-1βby human trophoblast cells (147). NF-κB activation
and IL-6 production are associated with higher levels of lipid
accumulation in the placenta of obese women compared with
those of lean women (151). These ﬁndings suggest that saturated
fatty acids directly induce placental inﬂammation, resulting
AGEs AND THE NLRP3 INFLAMMASOME
AGEs are heterogeneous, reactive, and irreversibly crosslinked
molecules formed from the non-enzymatic glycation of proteins,
lipids, and nucleic acids (152,153). They interact with RAGE
and/or TLR4 to induce inﬂammatory responses (154,155).
AGE-RAGE interactions may increase and perpetuate the
inﬂammatory condition, leading to obesity, diabetes mellitus,
and cardiovascular and kidney diseases. Both in vivo and
in vitro experiments have demonstrated that AGEs stimulate
NLRP3 inﬂammasome activation and IL-1βsecretion in human
umbilical vein endothelial cells, kidney, and pancreatic islets
(117,156,157). Ablation of the NLRP3 inﬂammasome improved
AGE-induced abnormal insulin sensitivity, pancreatic islet
damage, and inﬂammatory responses (158). These ﬁndings
suggest that consumption of AGEs increases obesity-related
dysfunction via NLRP3 inﬂammasome activation.
Increasing evidence indicates that AGEs and IL-1βare
associated with PE and obesity in pregnant women (134,135,
159–161). In human placenta, AGEs increase in vitro release
of IL-1β, IL-6, IL-8, and TNFαdepend on NF-κB activation
(162). We also demonstrated that in human placental tissues,
AGEs directly increase both the transcription and secretion of
IL-1β(117). In addition, AGEs stimulate pro-IL-1βproduction,
resulting in the acceleration of mature IL-1βsecretion by NLRP3
inﬂammasome activation in human trophoblast cells. AGEs also
induce sFlt-1 production through RAGE signaling, suggesting a
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Shirasuna et al. NLRP3 Inﬂammasome in Preeclampsia
direct link with the pathology of PE (163). Antoniotti et al. (164)
reported that AGEs led to activated inﬂammatory responses
in endometrial cells, impaired decidualization, compromised
implantation of blastocyst, and suppressed trophoblast invasion.
Therefore, AGEs adversely may impact not only PE but also
endometrial function and embryo implantation.
OTHER PREGNANCY COMPLICATIONS
ASSOCIATED WITH THE NLRP3
GDM is also classed as an obesity-related pregnancy
complication. In GDM, high levels of serum glucose are
associated with increased inﬂammation in blood as well
as placenta (165). Excess glucose induces IL-1βsecretion
from human trophoblast cells depending on the NLRP3
inﬂammasome (166). In addition to the placenta, caspase-
1 activation and mature IL-1βsecretion are higher in the
adipose tissue of pregnant patients with GDM compared
with healthy pregnant women (167), and treatment with
caspase-1 inhibitor suppresses IL-1βsecretion, suggesting
the contribution of NLRP3 inﬂammasome activation
Inﬂammation of the maternal-fetal interface such as
intra-amniotic inﬂammation or chorioamnionitis, which
can be induced by intra-amniotic infection or DAMPs, is a
causal link to spontaneous preterm birth, which is a leading
cause of perinatal mortality and morbidity (168). In a non-
primate rhesus macaques chorioamnionitis model induced
by intra-amniotic injection of LPS, the amnion upregulated
neutrophil accumulation via the chemoattractant IL-8 in an IL-
1-dependent manner (169). In a mouse model of intra-amniotic
inﬂammation-induced preterm birth, the NLRP3 inﬂammasome
was activated following IL-1βsecretion in the fetal membranes
and decidua basalis (170). In addition, IL-1βblockade decreased
inﬂammation-induced preterm labor in mice (171). These
ﬁndings suggest that the NLRP3 inﬂammasome plays a pivotal
role in inﬂammation of the maternal-fetal interface associated
with preterm birth, and IL-1 is a potential therapeutic target for
To understand the role of the NLRP3 inﬂammasome in
normal pregnancy and pregnancy complications, please refer the
essential review (172).
Accumulating evidence suggests that the NLRP3 inﬂammasome
plays an essential role in the pathogenesis of pregnancy
inﬂammatory complications. Various types of DAMPs act
as danger signals to activate the NLRP3 inﬂammasome in
reproductive organs, resulting in pregnancy inﬂammatory
complications (Figure 2). Once activated, the NLRP3
inﬂammasome drives the robust release of mature IL-1β,
initiating a positive feedback loop that results in the accumulation
of other immune cells (neutrophils and macrophages) and an
increase in the “danger” cytokines and chemokines. Considering
the potential for excessive NLRP3 inﬂammasome and IL-1β
production, it is not unexpected that several negative regulatory
mechanisms exist in nature to control inﬂammasome function.
Understanding how the NLRP3 inﬂammasome regulates
pregnancy complications and how to control excessive NLRP3
inﬂammasome activation is essential for the identiﬁcation of new
targets for the treatment of reproductive dysfunction.
KS and TK wrote the manuscript. MT critically revised the
manuscript. All authors read and approved the ﬁnal manuscript.
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Conﬂict of Interest: The authors declare that the research was conducted in the
absence of any commercial or ﬁnancial relationships that could be construed as a
potential conﬂict of interest.
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