Sterile inflammation and pregnancy complications: a review.

TLDR: The role of sterile inflammation in reproduction, including early implantation and pregnancy complications is discussed, and major alarmins vastly implicated in numerous sterile inflammatory processes, such as uric acid, HMGB1, IL-1α and cell-free DNA are focused on while giving an overview of the potential role of other candidate alarmins.

Abstract: Inflammation is essential for successful embryo implantation, pregnancy maintenance and delivery. In the last decade, important advances have been made in regard to endogenous, and therefore non-infectious, initiators of inflammation, which can act through the same receptors as pathogens. These molecules are referred to as damage-associated molecular patterns (DAMPs), and their involvement in reproduction has only recently been unraveled. Even though inflammation is necessary for successful reproduction, untimely activation of inflammatory processes can have devastating effect on pregnancy outcomes. Many DAMPs, such as uric acid, high-mobility group box 1 (HMGB1), interleukin (IL)-1 and cell-free fetal DNA, have been associated with pregnancy complications, such as miscarriages, preeclampsia and preterm birth in preclinical models and in humans. However, the specific contribution of alarmins to these conditions is still under debate, as currently there is lack of information on their mechanism of action. In this review, we discuss the role of sterile inflammation in reproduction, including early implantation and pregnancy complications. Particularly, we focus on major alarmins vastly implicated in numerous sterile inflammatory processes, such as uric acid, HMGB1, IL-1α and cell-free DNA (especially that of fetal origin) while giving an overview of the potential role of other candidate alarmins.

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REPRODUCTION
© 2016 Society for Reproduction and Fertility DOI: 10.1530/REP-16-0453
ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
REVIEW
Sterile inflammation and pregnancy complications: a review
MathieuNadeau-Vallée
1,2
, DimaObari
2
, JuliaPalacios
3
, Marie-ÈveBrien
3,4
, CyntiaDuval
3
,
SylvainChemtob
1,2
and Sylvie Girard
2,3,4
1
Departments of Pediatrics, Ophthalmology and Pharmacology, CHU Sainte-Justine Research Center, Montreal,
Quebec, Canada,
2
Department of Pharmacology, Université de Montréal, Montreal, Quebec, Canada,
3
Department
of Obstetrics & Gynecology, CHU Sainte-Justine Research Center, Montreal, Quebec, Canada and
4
Department of
Microbiology, Virology and Immunology, Université de Montréal, Montreal, Quebec, Canada
Correspondence should be addressed to S Girard; Email:
sylvie.girard@recherche-ste-justine.qc.ca
Abstract
Inflammation is essential for successful embryo implantation, pregnancy maintenance and delivery. In the last decade, important
advances have been made in regard to endogenous, and therefore non-infectious, initiators of inflammation, which can act through
the same receptors as pathogens. These molecules are referred to as damage-associated molecular patterns (DAMPs), and their
involvement in reproduction has only recently been unraveled. Even though inflammation is necessary for successful reproduction,
untimely activation of inflammatory processes can have devastating effect on pregnancy outcomes. Many DAMPs, such as uric acid,
high-mobility group box 1 (HMGB1), interleukin (IL)-1 and cell-free fetal DNA, have been associated with pregnancy complications,
such as miscarriages, preeclampsia and preterm birth in preclinical models and in humans. However, the specific contribution of
alarmins to these conditions is still under debate, as currently there is lack of information on their mechanism of action. In this
review, we discuss the role of sterile inflammation in reproduction, including early implantation and pregnancy complications.
Particularly, we focus on major alarmins vastly implicated in numerous sterile inflammatory processes, such as uric acid, HMGB1,
IL-1α and cell-free DNA (especially that of fetal origin) while giving an overview of the potential role of other candidate alarmins.
Reproduction (2016) 152 R277–R292
Introduction
Inflammation is essential for successful female
reproduction. Inflammatory processes are implicated
in every step of fertility from menstrual cycle (ovulation
and menses) to early pregnancy (implantation and
decidualization) and later during labor (myometrial
activation, cervical ripening and weakening of fetal
membrane), whereas quiescence of these mechanisms
is maintained by local immune cells during gestation
to allow maternal tolerance of fetal antigen allograph.
However, untimely inflammatory triggers shifting the
immunological balance toward activation can lead to
adverse pregnancy outcomes including preterm birth.
Inversely, failure to mount a local inflammatory response
in early or late gestation can also lead to adverse
conditions, including miscarriages. Evidence shows
that impaired inflammatory response is implicated
in numerous female reproductive tract pathologies
including menstrual disorders (
Sales & Jabbour 2003),
endometriosis-associated infertility (
Gupta etal. 2008),
recurrent miscarriage (
von Wolff et al. 2000, Laird
et al. 2003
), intrauterine growth restriction (Heyborne
et al. 1994), preeclampsia (Redman et al. 1999,
Rinehart et al. 1999) and preterm labor (Romero
et al. 2006
, Christiaens et al. 2008). Infertility has an
estimated global prevalence of 9% with >72 million
infertile women worldwide (
Boivin etal. 2007), whereas
preterm birth and preeclampsia, the two leading causes
of perinatal mortality and morbidity, have an estimated
prevalence of >11% (
Blencowe etal. 2013) and 3–5%
(
Ananth et al. 2013, Chaiworapongsa et al. 2014)
respectively. Therefore, understanding the mechanisms
by which inflammation is untimely triggered in the uterus
is fundamental to developing effective therapeutics to
improve fertility and decrease poor obstetrical outcomes.
Infection is not an essential component in
reproductive disorders linked to inflammation. In
animals, sterile inflammation is sufficient to recreate
major features of common reproductive diseases
(
Romero et al. 1991, Scharfe-Nugent et al. 2012,
Gomez-Lopez et al. 2016), whereas in humans, a
significant part of patients suffering from preeclampsia,
preterm labor or other inflammatory diseases during
pregnancy display no clinical signs of infection. This
has been extensively studied in the context of preterm
birth; although observational, correlational and causal
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data accumulated for >30years have linked infection to
preterm labor, preterm birth without infection is more
prevalent (
Romero etal. 2001). Furthermore, antibiotics
are inefficient to prevent preterm labor in women with
infection (Olson etal. 2008), suggesting that infection-
induced pro-labor effects arise from inflammation (self)
rather than infection (non-self).
Pro-inflammatory stimuli can be classified as ‘danger’
(or damage) and ‘stranger’ signals; both types of signals
are pharmacologically active via pattern recognition
receptors (PRRs), a class of phylogenetically conserved
receptors ubiquitously expressed by mammalian cells.
These receptors act as sensors of damage-associated
molecular patterns (DAMPs) or pathogen-associated
molecular patterns by operating a transduction cascade
of intracellular and intranuclear signals leading to the
mounting of cytokine-based inflammatory responses.
PRRs include Toll-like receptors (TLRs) 1–11, scavenger
receptors, C-type lectins, and NOD-like receptors
and are expressed abundantly in decidua, placenta,
membranes and myometrium throughout pregnancy,
in immune and non-immune cells (Koga & Mor 2010,
Lappas 2013, Zhang et al. 2014). Therefore, the
uteroplacental compartment is a sensor of ‘danger’
and ‘stranger’ inflammatory stressors. We suggest that
inflammatory processes implicated in physiological
human reproduction are triggered mainly through
sterile pathways (e.g. via tissue injury or cell death)
compared to exogenous signals such as pathogens
(bacteria or viruses, namely stranger signals), whereas
pathological inflammatory events implicated in
pregnancy complications can be triggered by both
sterile and infectious pathways. This review focuses on
the role of sterile inflammation in common pathologies
of pregnancy.
Major players in sterile inflammation and their
mechanism of action
Sterile inflammation is triggered when DAMPs activate
PRRs (or other receptors including RAGE and IL-1R) to
mount an acute immune response in order to solve the
adverse condition that initially led to DAMP release.
As DAMPs are endogenous intracellular molecules
primarily released as a result of non-programmed cell
death to convey danger cues in the first few hours of an
injury, they are also referred to as alarmins (Matzinger
1994). Candidate alarmins include, but are not limited
to, high-mobility group box 1 (HMGB1), uric acid,
interleukin-1α (IL-1α) and cell-free DNA. A detailed
description of these alarmins and their mechanism
of action can be found below and in Fig. 1, whereas
their roles in pathological conditions of pregnancy are
presented in the next section.
HMGB1
HMGB1 is a highly conserved non-histone protein
(25 kDa) with cytokine-like activity in the extracellular
space. HMGB1 is abundantly and ubiquitously expressed
in nucleus where it plays a role in DNA replication,
transcription and repair, and nucleosome stabilization
(Boonyaratanakornkit et al. 1998, Stros 2010, Celona
et al. 2011). HMGB1 is structured into two DNA-
binding domains, HMG box A and B and an aspartic and
glutamic acid-rich C-terminal tail. Although originally
discovered in the nucleus, HMGB1 is also found in
cytosol, mitochondria, and on membrane surface and
can be released to the extracellular milieu through active
(secretion) and passive pathways (Erlandsson Harris &
Andersson 2004). First, active pathways are triggered by
Figure1 Inflammatory mechanism of action of HMGB1, uric acid, IL-1α and cell-free DNA. The general mechanism of action of alarmins at the
maternal–fetal interface is shown, with alarmins (uric acid/MSU, HMGB1, cffDNA and IL-1α) being released from cells of the maternal–fetal
interface (i.e., placenta and fetal membranes) following a stimulus or necrosis. They act on placental cells (primarily trophoblasts) and maternal
myeloid cells to induce an inflammatory response. The inflammatory cascade leads to the secretion/release of cytokines/chemokines, which
stimulate the recruitment of immune cells from the maternal circulation.
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pathogenic products (e.g. bacteria and viruses) or other
stressors (e.g. oxidative stress and cytokines), which
have been shown in immune cells and non-immune
cells (
Tsung etal. 2007, Tang et al. 2011, Harris et al.
2012). Secondly, whereas (ii) passive release is observed
following tissue injury and cell death, especially
necrosis (Scaffidi et al. 2002) and in specific cases of
apoptosis (Qin etal. 2006) – including when triggered
by sterile injury events (e.g. hypoxia, senescence and
autoimmune disease). The latter happens immediately
(Scaffidi et al. 2002), whereas the former is a slower
mechanism mediated by cellular signal transduction
(Tsung et al. 2014). Once HMGB1 accumulates in
the extracellular milieu, it conveys danger signals by
triggering inflammatory pathways, including NF-κB, ERK
and p38, in neighboring cells via numerous cell surface
receptors such as TLRs 2, 4 and 9; RAGE; CD24; and
others (Venereau etal. 2013). This leads to the activation
of innate and adaptive immunity, cytokine, chemokine,
and metalloprotease release and ensued pro-migration
and pro-inflammatory outcomes (Andersson etal. 2000,
Scaffidi etal. 2002, Rouhiainen etal. 2004, Andersson &
Tracey 2011). Of interest, HMGB1 has also been shown to
form complexes with many pro-inflammatory mediators
and enhance their respective actions in a synergistic
manner (Hreggvidsdottir et al. 2009). Furthermore,
HMGB1 levels are elevated in multiple animal models of
sterile injurious events (Tsung etal. 2014) and in humans
with acute organ injury, autoimmune diseases or cancer
(Tong etal. 2011, Wang etal. 2014). In vitro and in vivo,
HMGB1 administration induces inflammation (Yang
etal. 2005), and more importantly, HMGB1 antagonism
protects against sepsis (Yang etal. 2004). This evidence
highlights a critical alarmin role of HMGB1 as an
endogenous sterile driver of inflammation.
Uric acid
Uric acid (160 Da) is a product of metabolic breakdown
of purine nucleotides by xanthine oxidase, with normal
blood concentration ranging between 40 and 60 µg/mL.
Upon achieving concentrations of >70 µg/mL, uric acid
forms needle-like, immunostimulatory monosodium
urate (MSU) crystals, which cause acute inflammation
of gout. In the last few years, uric acid has been vastly
regarded as an alarmin of sterile inflammation because
of the high cytosolic concentration (≈ 4 mg/mL) released
upon cell death, which reacts with extracellular sodium to
form MSU in the immediate vicinity of cellular injury (Shi
etal. 2003). Transport of MSU inside antigen-presenting
cells through phagocytosis promotes its interaction with
NALP3 inflammasome and induces IL-1β maturation
and release thereby triggering an inflammatory response
(Martinon et al. 2006, Shi 2010). This is an important
step in sterile inflammation that enables immune cells
to sense injuries. Concordantly, administration of MSU
causes acute inflammation (Faires & Mccarty 1962), and
in mice, blocking uric acid is sufficient to inhibit the
immunological and inflammatory responses associated
with cellular death or injury in numerous cell types and
tissues (Shi etal. 2003, Kono etal. 2010).
Interleukin-1
The interleukin-1 family comprises 11 cytokines
that regulate inflammatory response to injuries and
stressors. Two major members of the family are IL-1α
and IL-1β, which bind to ubiquitous IL-1R1 to activate
the translocation of transcription factors NF-κB and
AP-1, thereby triggering the expression of numerous
cytokines including itself and initiating or sustaining
an inflammatory response (Di Paolo et al. 2009,
Dinarello 2009). Although IL-1α and IL-1β bind to the
same receptor and convey similar biological effect,
the two cytokines are encoded by different genes and
have distinct mode of action. Unlike IL-1β, IL-1α is not
actively secreted but instead translocates to the nucleus
to participate in the regulation of gene transcription
(Werman et al. 2004). Furthermore, while IL-1β
precursor requires exogenous (or endogenous in rare
cases) signals to trigger its transcription and to initiate
its inflammasome-dependent cleavage into a functional
cytokine, IL-1α precursor is, on the other hand,
ubiquitously expressed in the cytoplasm of healthy
cells, in the form of a biologically active precursor.
Consequently, only IL-1α is released in a functional
form upon necrosis; therefore, IL-1α is regarded as an
alarmin, whereas IL-1β is not (Eigenbrod et al. 2008,
Lukens et al. 2012). Accordingly, sterile cell death-
induced neutrophil inflammatory response in mice
requires both IL-1α and IL-1R, but not IL-1β (Chen
etal. 2007), suggesting that IL-1β is not essential for the
mounting of a functional sterile inflammatory response
to cell death. However, evidence shows that both
IL-1α and IL-1β are implicated in sterile inflammation
but have distinct timing of effect and roles (Rider
etal. 2011), suggesting that IL-1β contributes to sterile
inflammation, not as an initiator but as a redundant
mechanism to amplify the initial trigger. Accordingly, it
is documented that IL-1β can be produced to contribute
to sterile inflammation in response to non-cytotoxic
sterile stressors as those released upon necrosis (Lukens
etal. 2012). This was also recently reported for IL-1α
(Idan etal. 2015). Interestingly, the release of IL-1α is
tightly regulated during programmed cell death via
chromatin sequestration, which significantly reduces
its pro-inflammatory effect during apoptosis; this is not
observed during necrosis (Cohen etal. 2010). These data
suggest a critical role of IL-1α in sterile inflammation,
and a contributive, albeit non-essential, role of IL-1β.
The major role of IL-1α in sterile inflammation has been
reviewed elsewhere (
Lukens etal. 2012).
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Cell-free DNA
Circulating cell-free DNA refers to double-stranded,
cell-unbound DNA fragments in the blood of humans.
Cell-free DNA originates from genomic or mitochondrial
DNA released subsequently to cell death. Cell-free
DNA is present in small amounts in the blood of
healthy individuals, but its concentration is increased
in patients suffering from chronic diseases. In this
context, studies suggest that it acts as a contributor to
chronic diseases by inducing inflammation via TLR9, a
PRR classically activated by unmethylated CpG motif-
containing bacterial and viral DNA fragments (
Chan
& Lo 2002, Breitbach et al. 2012, Nishimoto et al.
2016). Mitochondrial DNA also triggers TLR9 to induce
inflammation (Zhang etal. 2010).
Circulating blood of pregnant women contains
an additional type of cell-free DNA, referred to as
cell-free fetal DNA (cffDNA) that originates from the
placenta. Evidence for the placental origin of cffDNA
includes the following: (1) it is detected in anembryonic
gestation (Alberry etal. 2007); (2) it is still detected after
therapeutic abortion in which placenta is incompletely
removed, albeit undetectable after normal delivery (Lo
etal. 1999, Wataganara etal. 2005); (3) it is detected
in cases of invasive placenta, a postpartum pregnancy
complication in which trophoblasts invade myometrium
(Sekizawa et al. 2002); and (4) it carries the placental
genotype in patients with confined placental mosaicism
(Masuzaki et al. 2004). In contrast to maternal cell-
free DNA, of which 32% of the fragments are >356 bp,
cffDNA are short hypomethylated fragments (<313 bp)
and potent inducer of sterile inflammation (Chan etal.
2004, Scharfe-Nugent etal. 2012, Schroeder etal. 2013).
The release of cffDNA is a physiological process present
in all mammals, but its possible roles and implications
in normal pregnancy (and more importantly parturition)
remain poorly understood. Placental growth involves
proliferation, differentiation and syncytial fusion of
cytotrophoblasts, which is associated with significant
release (grams per day) of microvesicles-encapsulated,
cffDNA-containing apoptotic trophoblasts content into
maternal circulation (Nelson 1996, Huppertz et al.
1998, Huppertz & Kingdom 2004, Bischoff etal. 2005,
Taglauer etal. 2014). These microparticles, also referred
to as syncytiotrophoblast microvesicles (SCTMs),
were first described more than 100years ago in lung
capillaries of women who died from preeclampsia
(Schmorl 1893) and were later described as a feature of
normal pregnancy, although increased in preeclampsia
(Johansen etal. 1999). SCTMs as well as cffDNA alone
are pro-inflammatory (Redman et al. 1999, Redman
& Sargent 2000, Phillippe 2015). Interestingly, once
pregnancy is past 20 weeks, the levels of cffDNA in
maternal circulation consistently increase 1% per
additional week of gestation to abruptly rise (up to
13-folds) when gestation nears the end (
Arigaetal.2001,
Majer et al. 2007, Wang et al. 2013). This evidence,
along with the established pro-inflammatory effects
of cffDNA, underpins the theory that cffDNA may
represent a common trigger to parturition in mammals
(Phillippe 2014). Furthermore, elevated cffDNA in the
maternal circulation has been observed in pathological
pregnancies (Levine et al. 2004, Alberry et al. 2009,
Girard et al. 2014) in association with placental
dysfunction and inflammation. For these reasons and
others, cffDNA is increasingly used for diagnostic
purposes to decrease the use of invasive amniocentesis.
Mechanistically, cffDNA can bind to TLR9 to induce a
conformational change in the homodimers of the receptor
resulting in the close apposition of the TIR signaling
domains and downstream activation of NF-κB and
transcription of inflammatory cytokine genes (Latz etal.
2007). Importantly, this TLR9, NF-κB-dependent pro-
inflammatory effect of cffDNA was shown in pregnant
mice and is characterized by IL-6 production in human
peripheral blood mononuclear cells (Scharfe-Nugent
etal. 2012). Classically, TLR9 is localized intracellularly
in endoplasmic reticulum (ER), endosomes and
lysosomes (Latz etal. 2004). Therefore, cffDNA must be
transported by endocytosis inside immune cells in order
to convey inflammatory effects via TLR9; this is likely
occurring through phagocytosis of cffDNA-containing
SCTMs by placental or circulating granulocytes. Given
the half-life of cffDNA (16.3 min in humans) (Lo etal.
1999), this inflammatory stimulation is short-lived, but is
likely sustained by unabated trophoblast turnover.
Others
Different levels of evidence have been accumulated,
suggesting that many other intracellular factors can
induce acute inflammation once released in their
environment and therefore may represent potential
alarmins. These include S100 proteins (Hofmann etal.
1999, Ryckman et al. 2003), nucleosomes (Decker
etal. 2005), purines (Cronstein etal. 1990), heat-shock
proteins (Basu et al. 2000), saturated fatty acids (Lee
etal. 2001) and antimicrobial peptides (Yang etal. 1999,
De et al. 2000, Zanetti 2004). Interestingly, possible
alarmin activity has been reported for molecules of
mitochondrial provenance such as mitochondrial
DNA (Zhang etal. 2010), N-formylated mitochondrial
peptides (Carp 1982) and others (Raoof et al. 2010),
which could arise from their probable prokaryote
origin. Noteworthy, the role and mode of action of the
aforementioned candidates in vivo are mostly unknown.
Along these lines, the possible alarmin activity of heat-
shock proteins is still under debate. Early studies have
shown that purified HSPs activate DCs ex vivo (Basu
etal. 2000) and in vivo (Binder et al. 2000) to trigger
an inflammatory response. This pro-inflammatory effect
has latter been attributed to bacterial contaminant
(
Bausinger et al. 2002, Gao & Tsan 2003), and the
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enthusiasm of a possible alarmin role of HSPs was
consequently severely dampened.
By definition, any endogenous molecule
physiologically expressed in low concentrations in the
extracellular milieu, which is upregulated and released
during pathological events, could be considered as an
alarmin candidate. Therefore many other mediators
could potentially be included in this category, such
as glucose that has recently been shown to have pro-
inflammatory actions in human trophoblast (
Han etal.
2015).
A role for sterile inflammation in pathological
conditions of pregnancy
Alarmin release, caused by tissue injury, hypoxia/
ischemia, cellular senescence or other stressors, is
implicated in pathologies of pregnancy independently
of infection. In the next section, we will focus on the
role of alarmins in miscarriages, recurrent pregnancy
loss (RPL), intrauterine growth restriction (IUGR),
preeclampsia and preterm labor. The proposed sites
of release of alarmins in diseases of pregnancy are
shown in Fig.2.
Early pregnancy
Many inflammatory mediators (and immune cells) are
implicated in the early events of pregnancy, primarily
embryo implantation. A tight regulation of the immune
system is required for proper invasion and remodeling of
the spiral arteries by fetal trophoblasts, which has been
recently reviewed (Erlebacher 2013). The involvement
of DAMPs in RPL or miscarriages has only recently
been addressed. Elevated HMGB1 levels in uterine
fluids have been associated with pregnancy failure in
rats and lower abundance of HMGB1 observed in the
receptive phase of implantation in humans (Bhutada
et al. 2014). Additionally, a genetic polymorphism of
HMGB1 characterized by higher expression of HMGB1
in placenta has been associated with RPL (Jin et al.
2015). Furthermore, HMGB1 was shown to induce
inflammation, characterized by an NF-κB- and reactive
oxygen species-dependent increased secretion of IL-6,
IL-8 and CCL2 in first-trimester trophoblast (Shirasuna
et al. 2016). Other alarmins, including S100A8 (Nair
etal. 2015) and cell-derived microparticles (Martinez-
Zamora etal. 2016), were also found to be elevated in
early pregnancy loss. Likewise, increased inflammatory
cytokine levels (namely IL-18, LIF, MIF, IL-12, IFN-
γ and ICAM-1) in the blood and endometrium
were also associated with RPL (Comba et al. 2015).
Correspondingly, increased expression of the NALP3
inflammasome and its products IL-1β and IL-18 was
observed in the endometrium of patient with RPL
(D’Ippolito et al. 2016). This evidence points toward
a potential role of DAMPs in early pregnancy failure,
although there are still many unresolved issues.
Figure2 Principal sites of release and actions of alarmins in pathological pregnancy. Multiple causes of cellular stress will affect cell viability
and lead to the release of DAMPs (alarmins) by the fetal membranes and the placenta. These DAMPs will then act not only on the placenta itself
but also on the uterus, cervix and fetal membranes inducing inflammation and contributing to many complications of pregnancy.
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