STATE-OF-THE-ART REVIEW

NOD-like receptors interfacing the immune and

reproductive systems
Hanne Van Gorp1,2*, Anna Kuchmiy1,2*, Filip Van Hauwermeiren1,2 and Mohamed Lamkanfi1,2
1 Department of Medical Protein Research, VIB, Ghent, Belgium
2 Department of Biochemistry, Ghent University, Ghent, Belgium

Keywords Nucleotide-binding oligomerization domain receptors (NOD-like receptors,

disease, embryogenesis, imprinting,
inflammasomes, maternal effect gene,
mole, NOD-like receptors, oocyte,
reproduction, subcortical maternal complex
Correspondence
M. Lamkanfi, Department of Medical
Protein Research, VIB, Albert Baertsoenkaai
3, B-9000 Ghent, Belgium
Fax: +32 9 264 9490
Tel: +32 9 264 9341
E-mail: mohamed.lamkanfi@vib-ugent.be
*These authors contributed equally
(Received 30 June 2014, revised 13 August
2014, accepted 19 August 2014)
doi:10.1111/febs.13014
NLRs) are intracellular proteins that are chiefly known for their critical
functions in inflammatory responses and host defense against microbial
pathogens. Several NLRs have been demonstrated to assemble inflamma-
somes or to engage transcriptional signaling cascades that result in the pro-
duction of pro-inflammatory cytokines and bactericidal factors. In recent
years, NLRs have also emerged as key regulators of early mammalian
embryogenesis and reproduction. A subset of phylogenetically related
NLRs represents a new class of maternal effect genes that are highly
expressed in maturing oocytes and pre-implantation embryos. Mutations in
several of these NLRs have been linked to hereditary reproductive defects
and imprinting diseases. In this review, we discuss the expression profiles,
the emerging functions and molecular mode of action of these NLRs with
newly recognized roles at the interfaces of the immune and reproductive
systems. In addition, we provide an overview of coding mutations in NLRs
that have been associated with human reproductive diseases, and outline
crucial outstanding questions in this emerging research field.

Introduction
Nucleotide-binding oligomerization domain receptors
(NOD-like receptors, NLRs) are intracellular proteins
that have mainly been characterized in the context of
mammalian immunity. The vertebrate immune system
relies on the combined action of germline-encoded
(innate) and gene-rearrangement-based (adaptive) anti-
gen receptors to recognize microbial pathogens and
environmental challenges that threaten homeostasis.
Antigen receptor gene recombination in single T and B
cell clones empowers them to selectively recognize and
eliminate the threat. But clonal expansion of individual
antigen-specific T and B cells is temporally delayed
and often guided by innate immune cells [1,2].
Macrophages, dendritic cells, epithelial cells and innate
lymphoid cells that contribute to innate immunity are
equipped with a defined set of germline-encoded pat-
tern recognition receptors (PRRs). PRRs together with
the complement system enable innate effector cells to
mount fast and broadly effective protection against
external and internal threats. This is accomplished
through engagement of signaling cascades that result
in the production of inflammatory cytokines, microbi-
cidal agents, the activation and recruitment of addi-
tional immune cells to the affected tissue, and by

Abbreviations
BWS, BeckwithWiedemann syndrome; CARD, caspase-recruitment domain; IL, interleukin; LPS, lipopolysaccharide; NF-jB, nuclear factor
jB; NLR, NOD-like receptor; NOD-like receptor, nucleotide-binding oligomerization domain receptor; PRR, pattern recognition receptor; PYD,
pyrin domain; SCMC, subcortical maternal complex; siRNA, small interfering RNA; SRS, SilverRussell syndrome.

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H. Van Gorp et al.
tagging the foreign agents for degradation by profes-
sional phagocytes [3].
Surface-bound PRRs of the Toll-like receptor and
C-type lectin receptor family continuously monitor the
extracellular environment, while different classes of
more recently discovered intracellular PRRs safeguard
the intracellular space. The latter group comprises
members of the RIG-I-like receptor, AIM2-like recep-
tor, NLR protein families, along with a growing
collection of cytosolic nucleic acid sensors [3,4]. In the
last decade, NLRs have emerged as key components
of the innate immune system where they steer host
defense by recognizing pathogen-associated molecular
patterns (PAMPs) and danger-associated molecular
patterns (DAMPs) [5]. PAMPs constitute essential
microbial components, while DAMPs are generally
regarded as host-derived agents that accumulate under
conditions of (sterile) stress and tissue damage [4].
NLRs are subdivided according to their amino-ter-
minal effector domain, which generally comprises a
caspase-recruitment (CARD), a pyrin (PYD) or a
baculovirus inhibitor repeat motif (Fig. 1). As we learn
more about their biological roles, NLRs can also be
grouped according to their known physiological func-
tions in the immune system. NLRP1b, NLRC4 and
NLRP3 are NLRs with well-established roles in the
assembly of inflammasomes [6]. These cytosolic multi-
protein complexes recruit and activate the pro-inflam-
matory cysteine proteases caspase-1 and -11, which in
their turn promote secretion of the inflammatory cyto-
kines interleukin (IL) 1b and IL-18, and induce a pro-
grammed cell death mode (pyroptosis) that restricts
pathogen replication [6,7]. On the other hand, the
NLRs NOD1 and NOD2 are chiefly known for their
roles in promoting activation of nuclear factor jB
(NF-jB), MAPK and TRAF3 signaling cascades spe-
cifically upon cytosolic detection of bacterial peptido-
glycan fragments. Notably, mutations in the latter
NLRs are closely linked to inflammatory syndromes of
the intestinal tract [8]. Finally, CIITA is a transcrip-
tional co-activator that is critical for major histocom-
patibility complex II expression in antigen-presenting
cells, and mutations in CIITA cause bare lymphocyte
syndrome in humans [9]. Unlike the former NLRs, the
immune roles of other family members are less clear.
NLRP6, NLRP7 and NLRP12 have been implicated
in inflammasome signaling and are proposed to dam-
pen NF-jB-mediated cytokine production [1012], but
further analysis is required to clarify their molecular
modes of action.
While new and diverse roles exerted by NLRs in
immunity are rapidly emerging, many questions
remain regarding their potential involvement in other
FEBS Journal 281 (2014) 45684582 2014 FEBS
NLRs in immunity and reproduction
Fig. 1. Domain structure of human NLR subfamilies. NOD-like
receptors are composed of several protein domains, each fulfilling
a specific function. The different NLR subfamilies share a central
nucleotide-binding and oligomerization (NACHT) domain, and
carboxy-terminal leucine-rich repeats (LRRs). The NACHT domain is
an oligomerization domain that possesses NTPase activity, while
the LRRs are commonly involved in proteinprotein interactions.
Different subfamilies can be defined based on the presence of a
particular amino-terminal proteinprotein interaction domain. The
NLRA subfamily is equipped with a CARD supplemented with an
acid transactivation domain (AD), while the NLRB subfamily can be
identified based on the presence of a baculovirus inhibitory repeat
domain (BIR). The NLRC/X subfamily groups the proteins
containing either a CARD domain or a still unidentified domain, and
finally the NLRP subfamily is supplied with a PYD.
organ systems and signaling pathways. A significant
body of recent work has provided compelling evidence
that links a subset of NLRs to key functions in repro-
duction and during early stages of embryonic develop-
ment. In this paper, we review recently gained insight,
based on in vitro as well as in vivo models, into the
roles and mechanisms by which these NLRs regulate
early embryonic development. We argue for the exis-
tence of a clearly defined subset of NLRs that operates
at the interfaces of the immune and reproductive sys-
tems and that can be segregated from other NLRs
based on their distinctive phylogeny, expression pro-
files in gametes and the clustered genomic localization
of their genes. Finally, we also reflect on the functional
evidence linking mutations in these NLRs to reproduc-
tion-related syndromes in patients.
The ABC of reproduction: on maternal
effect genes and imprinting disorders
Humans like nearly all multi-cellular organisms in the
animal and plant kingdoms generate offspring
through sexual reproduction. At the first stage, this
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NLRs in immunity and reproduction
process requires meiosis to produce gametes, reproduc-
tive cells that bear half the number of chromosomes of
somatic cells. The 23 chromosomes of each haploid
human oocyte and sperm cell thus carry a unique com-
bination of genes obtained after homologous recombi-
nation of parental DNA strands. During fertilization,
the two gametes fuse to generate a unique diploid cell
(zygote) that develops into a new organism. Apart from
their own unique genetic material, the sperm cell and
oocyte also supply cytoplasmic factors such as mRNA
or proteins that are required for initial, vital stages of
embryogenesis. In particular, the products of maternal
effect genes are critical to guide the complex processes
that are needed for the fertilized oocyte to correctly
transition into a developing embryo by full transcrip-
tional activation of the zygotic genome [1315].
In vertebrates, maternal effect genes may be
involved in properly combining the maternal and
paternal haploid genomes in zygotes, in facilitating
embryonic genome activation shortly thereafter, and in
ensuring correct pre-implantation development [15].
Given their roles in these key pre- and post-fertiliza-
tion processes, it is not surprising that mutations in
maternal effect genes almost invariably lead to early
embryonic arrest [15,16]. Maternal effect gene products
are produced by the oocyte prior to fertilization, and
embryonic genome activation coincides with a steep
decline in the amount of maternal effect gene tran-
scripts. The kinetics of this distinctive expression pat-
tern is species-specific, since the precise moment in the
developmental plan at which embryonic genome acti-
vation occurs varies greatly among animal species.
Murine embryos undergo genome activation at the
two-cell stage, cattle at the 816-cell stage and humans
when the embryo comprises foureight cells [13,17].
Several maternal effect proteins are of critical
importance for fetal growth because they orchestrate
epigenetic reprogramming in primordial germ cells and
zygotes. After gametogenesis, terminally differentiated
male and female haploid genomes acquire a con-
densed, transcriptionally quiescent structure. This is
achieved by targeted DNA methylation and histone
modifications, which are reversible epigenetic mecha-
nisms that play key roles in switching transcriptional
activity on and off [18]. At fertilization, the embryonic
genome is globally demethylated until the developing
embryo reaches the morula stage. However, a few
genomic loci, called imprinted regions, escape this gen-
ome-wide epigenetic reprogramming and maintain the
methylation patterns that were set in the oocyte and
sperm cell [19]. As a consequence, transcription of
imprinted genes a subset of an estimated few hun-
dred autosomal genes in mammals occurs from a
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H. Van Gorp et al.
single allele. Which of the two alleles is expressed in
the developing embryo is determined by the parent-of-
origin of the considered allele [20]. For maternally
imprinted genes, the maternal allele is repressed to
ensure that transcription occurs uniquely from the
paternally inherited allele. In contrast, transcription
from the paternal allele is silenced in the case of pater-
nally imprinted genes. The methylation status of
imprinted regions is conserved in all somatic cells of
the adult organism, but primordial germ cells in the
gonads of the developing embryo remove the parental
imprinting patterns in order to allow oocytes and
sperm cells to subsequently re-establish proper gender-
specific imprinting patterns [21].
Other maternal effect proteins may ensure normal
early embryogenesis as components of intracellular
protein complexes in maturing oocytes. In this regard,
the subcortical maternal complex (SCMC) is a large
multiprotein complex that was recently discovered in
the subcortex of oocytes and pre-implantation
embryos. Zygotes originating from mutant oocytes
that are defective in SCMC assembly failed to progress
beyond the first embryonic cell divisions, suggesting an
essential role for the SCMC in early embryonic devel-
opment [14]. However, these mutant oocytes also
lacked oocyte cytoplasmic lattices, oocyte-specific cyto-
plasmic cytoskeletal elements that were suggested to
support maternal ribosome transport and organelle
(re)distribution during oocyte maturation [22]. Clarifi-
cation of the relative importance of the SCMC and
cytoplasmic lattice protein complexes in oocyte matu-
ration and early embryonic development requires addi-
tional analysis.
Although the molecular modes of action of mater-
nal effect genes are still vague, it has become clear in
recent years that mutations in some of these genes
and the resulting imprinting defects are associated
with multiple fetal growth disorders and reproductive
wastage in humans [21,23]. The BeckwithWiedemann
syndrome (BWS) is an imprinting disease that is
associated with congenital overgrowth, undescended
testes in male progeny, abdominal wall defects, sei-
zures and predisposition to embryonic tumorigenesis
[24]. SilverRussell syndrome (SRS) on the other
hand is characterized by growth retardation prior to
and after birth, and patients display colored spots on
the skin, excessive sweating and inward curving of
the fifth fingers. Hydatidiform moles are another
example of a human disorder caused by hypermethy-
lation or hypomethylation of imprinted control
regions. Moles constitute abnormal or molar preg-
nancies in which a non-viable fertilized egg implants
in the uterus, consequently leading to absent or
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H. Van Gorp et al.
abnormal embryonic development with abnormal
trophoblastic growth [25]. Abnormal molar tissues
have different genotypes (Fig. 2AG) [26,27]. Unlike
most types of moles, familial biparental hydatidiform
moles have a diploid genotype originating from both
parents (Fig. 2E). Notwithstanding the presence of a
paternal as well as a maternal genome, the embryo
does not develop properly because of multiple
imprinting defects. Familial cases of biparental hyda-
tidiform moles are typically caused by autosomal
recessive mutations in maternal effect genes that
affect oocyte sufficiency [28,29], while the paternal
genotype does not contribute to the pathogenesis
[30,31]. Molar pregnancies may also develop into an
invasive form, with persistent trophoblastic disease
(PTD) being strongly associated with familial bipa-
rental hydatidiform moles and developing in an esti-
mated 1015% of all hydatidiform mole cases
[32,33]. PTD can further lead to the development of
choriocarcinomas that necessitate chemotherapeutic
treatment for their eradication [34].
Each of the above developmental disorders has been
associated with mutations in a subset of NLR genes.
In the following, we highlight recent evidence suggest-
ing that this NLR subset may represent a novel group
of maternal effect genes, and review our current under-
standing of the molecular mechanisms by which they
exert their functions at the interfaces of the immune
and reproductive systems.
Fig. 2. Genetics of hydatidiform moles
(HM). Genetically, sporadic complete HM C
are androgenic diploid conceptions (AnHM)
hypothetically originating (A) from a
duplicated paternal haploid genome (46,
XX) or (B) after fertilization of an empty D
egg with two haploid sperm cells (46, XX
and 46, XY). (C) Sporadic partial HM
(PHM) are dispermic triploid conceptions E
and usually originate from dispermic
fertilization of an oocyte (69, XXX; 69,
XXY; 69, XYY). (D) Digynic triploid
conceptions originate from duplication of a F
maternal haploid set of chromosomes. (E)
Biparental HM are diploid and contain a
maternal and paternal set of genes. (F, G) G
Hypothetical schemes of conception
mosaicism.
FEBS Journal 281 (2014) 45684582 2014 FEBS
NLRs in immunity and reproduction
A phylogenetic NLR cluster implicated
in reproductive functions
The NLR gene family is evolutionarily ancient and
predates the emergence of mammals. Large sets of
NLR genes are found in several invertebrates with
sequenced genomes, including Hydra (simple freshwa-
ter animals with radial symmetry), Strongylocentrotus
(sea urchins) and Amphimedon (sponges) [3538]. The
apparent absence of NLR genes in the genomes of
Drosophila melanogaster (fruitfly) and Apis mellifera
(honey bee) form interesting exceptions that suggest
that a common insect ancestor has probably lost the
NLR repertoire during speciation. Also Caenorhabd-
itis elegans (roundworm) is devoid of NLR genes,
which is consistent with the particularly high levels
of gene loss observed in the model organisms D. mel-
anogaster and C. elegans [36,39]. The NLR gene sets
in mammals are highly plastic, with the human gen-
ome encoding 22 NLR genes and the mouse genome
34 NLR genes [40]. The evolutionary bifurcation of
rodents and primates about 80100 million years ago
is thought to have coincided with gene duplication
events that gave rise to novel and altered protein
functions [41,42]. In agreement, mice express three
orthologs of human NLRP1, four orthologs of
human NAIP, seven orthologs of human NLRP4
and three orthologs of human NLRP9. Gene duplica-
tion events are also suggested to account for the
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NLRs in immunity and reproduction
A C
D E
existence of primate-specific NLRP7. Apart from
being duplicated, genes might also get lost from the
genome during speciation. Indeed, rodents lack or-
thologs of human NLRP8, 11 and 13, most probably
reflecting loss of those genes during rodent evolution
[40,43,44].
4572
H. Van Gorp et al.
ys
da
Notably, phylogenetic analysis of the protein
sequences of the complete human NLR set along with
their murine orthologs reveals that NLRP2, 4, 5, 7, 8,
9, 11, 13 and 14 form a phylogenetic cluster that sepa-
rates from other NLR clusters with clearly established
immune roles (Fig. 3A) [43,44]. Interestingly, within
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H. Van Gorp et al. NLRs in immunity and reproduction

the PYD-containing NLR subfamily, the immune-
related NLRs NLRP1, 3, 6, 10 and 12 form a separate
cluster. In addition, the CARD-containing NLRC4
clusters together with the NAIPs with which it forms
an inflammasome complex in response to bacteria [45].
This suggests that NLRs may be divided in several
phylogenetic subgroups that might have distinct func-
tions [43,44,46]. In this regard, the cluster containing
NLRP2, 4, 5, 7, 8, 9, 11, 13 and 14 coincides remark-
ably with the emerging reproduction-associated NLR
subset. Meta-analysis of publicly available expression
data demonstrates that members of this reproduction-
associated human NLR gene cluster are expressed at
high levels in oocytes and ovaries (Fig. 3B) [47]. At
the protein level, human NLRP7, NLRP5 and NLRP9
are expressed at all stages of developing follicles
[31,48]. The murine orthologs belonging to this cluster,
namely Nlrp2, 4a-g, 5, 9a-c and 14, are similarly
expressed at high levels in oocytes and ovaries, while
immune-related NLRs are not (Fig. 3B) [49]. The fact
that expressed sequence tags corresponding to mouse
Nlrp4a, 4c, 4e, 4f, 9b and 9c were found in cDNA
libraries of oocytes, zygotes and two-cell stage
embryos supports this notion [50,51]. Several published
reports validated the high expression levels of mouse
Nlrp2, Nlrp4a-f [52], Nlrp5, Nlrp9a-c [53] and Nlrp14
in female and male gonads and maturing gametes
[50,54]. They also showed that mRNA expression lev-
els of these NLRs drop steeply shortly after fertiliza-
tion towards the blastocyst stage, a characterizing
feature of maternal effect genes [15,16]. Our meta-
analysis along 12 developmental stages in mouse
indeed nicely re-confirms the gradual decrease of
Nlrp4a, 4b, 4c, 4e, 4f, 4 g, 5, 9ac, and 14 levels dur-
ing early-stage embryogenesis (Fig. 3C). Immune-
related NLRs do not show similar kinetics of mRNA
decay during early developmental stages (Fig. 3C).
Another marked observation is that, apart from
NLRP14, all reproduction-related NLRP genes in the
human genome are located on chromosome 19, while
the murine syntenic region on chromosome 7 contains
all murine reproduction-related NLR genes with the
notable exceptions of Nlrp4f, Nlrp4g and Nlrp14
(Fig. 3D,E). Moreover, the genes coding for these
reproduction-associated NLRs are physically clustered
together in chromosomal loci that contain other genes
that are important for reproductive traits [55]. A good
example is the genomic localization of Nlrp4 and
Nlrp9 paralogs on mouse chromosome 7 in the vicinity
of the V1r genes which belong to a chemosensory
receptor family and are involved in pheromone detec-
tion (Fig. 3D) [44]. Also, NLRP9 in cattle and NLRP5
in pigs were mapped to a quantitative trait locus
region for reproductive traits [56,57]. In agreement,
mutations in some of these NLRs have been associated
with mice sterility [51] and human imprinting diseases
(see above) [58]. However, it should be mentioned
that possibly not all NLRs in these syntenic regions
are important for reproduction. The NLRP12 gene is
located in this region but is most probably not
involved in reproduction since Nlrp12-deficient mice
develop normally without apparent reproductive
defects [59,60]. In addition, the protein clusters phylo-
genetically with the immune-associated NLRs
(Fig. 3A) [44] and is known to play a role in immu-
nity based on hyper-responsive reactions to Toll-like

Fig. 3. Reproduction-related NLR cluster in humans and mice: phylogeny, chromosomal clustering and expression. (A) Full-length NLR
protein sequences from both human and mouse were retrieved from the Uniprot database and analyzed with the MEGA6 package hereby
using default settings [110]. Alignment was performed with MUSCLE, while MAXIMUM LIKELIHOOD was used for tree building. The resulting
phylogenetic tree file was uploaded to EVOLVIEW for visualization [111]. A reproduction-related NLR cluster (light blue) separates from the
immune-related NLRs. The whole NLRP subfamily is indicated in blue, with immune- and reproduction-related NLRPs in dark and light blue,
respectively. The NAIP subfamily forms a separate cluster together with NLRC4 (red), while most of the NLRC/X subfamily clusters
together with the NLRA subfamily (grey). (B) GENEVESTIGATOR [47] was used to explore NLR expression in human (left panel) and mouse (right
panel) revealing strong expression of reproduction-related NLRs in oocytes. In contrast, low expression levels were detected in
reproduction-related tissues for established immune-related NLRs. The darkest red color represents the maximum level of expression for a
given gene across all measurements available in the database for this gene. Adult but not embryonic or early postnatal tissue samples were
selected for analysis. The values for mouse Nod2, Naip4, Naip7, Nlrc4, Nlrp1a, Nlrp1b, Nlrp1c, Nlrp2, Nlrp4d are absent in the database. (C)
GENEVESTIGATOR [47] was used to explore expression of immune- and reproduction-related NLRs along different stages of mouse

embryogenesis. Reproduction-related NLRs are highly expressed during prenatal period day 01 (corresponding to two-cell stage embryos)
and their levels strongly decrease during prenatal period days 24 (corresponding to four-cell embryos and blastocysts). This particular
profile matches the expression pattern of maternal effect genes. (D) Reproduction-related NLRPs are clustered on chromosome 19 in
humans and on chromosome 7 in mice. (E) Based on Uniprot sequences, the domain structure of different reproduction-related NLRPs is
depicted: blue box, PYD; green box, nucleotide-binding and oligomerization (NACHT) domain; yellow circles, leucine-rich repeats. In humans,
nine different reproduction-related NLRPs could be identified, four of which are absent in mouse. In mouse, however, duplication events
occurred leading to an NLRP4 and NLRP9 cluster.

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NLRs in immunity and reproduction H. Van Gorp et al.

Fig. 4. Mutations in human NLRs associated with reproductive diseases. (A) Schematic view of the NLRP2 protein with indication of
harmful mutations (Uniprot Q9NX02-1; Unigene NP_001167552.1). (B) Schematic view of the NLRP7 protein with indication of harmful
mutations (Uniprot Q8WX94-3; Unigene NP_001120727.1). (C) Schematic view of the NLRP14 protein with indication of harmful mutations
(Uniprot Q86W24-1; Unigene NP_789792.1). NLR domains are represented as follows: blue box, PYD; green box, nucleotide-binding and
oligomerization (NACHT) domain; yellow circles, leucine-rich repeats. Mutations are grouped to a certain level corresponding to particular
diseases. Different colors indicate zygosity of the mutation, and cases with live birth are underlined. Mutations are presented based on the
nomenclature recommendations of the Human Genome Variation Society.

receptor stimulation in Nlrp12-deficient immune cells
[59,61].
In the following sections, we review the evidence
linking NLRP2, NLRP5 and NLRP7 to reproductive
functions and immunity. For most other reproduction-
related NLRs, however, much less is known and the
molecular mechanisms employed still need to be
unraveled. Small interfering RNA (siRNA) mediated
knockdown of Nlrp4e and Nlrp14 in fertilized eggs led
to early embryonic arrest indicating that they may be
maternal effect genes [50,62,63]. In addition, NLRP14
has been implicated in spermatogenesis (Fig. 4C)
[64,65], while human NLRP4 was shown to play a role
in immunity [6669]. Further research is clearly needed
to fully appreciate the role of all under-investigated
NLRs in reproduction and immunity.
NLRP2: at the interface of immunity
and reproduction
Together with NLRP3 and NLRP1, NLRP2 was one
of the first NLR family members shown to assemble a
functional inflammasome upon its ectopic expression
with ASC and caspase-1 in 293T cells [70]. In addi-
tion, endogenous NLRP2 was found to co-immuno-
precipitate with the inflammasome adaptor ASC in
lipopolysaccharide (LPS) stimulated THP-1 cells, and
siRNA-mediated knockdown of NLRP2 reduced the
levels of secreted IL-1b from these cells [70,71]. Apart
from its suggested role in inflammasome signaling,
ectopically expressed NLRP2 was demonstrated to
inhibit activation of the pro-inflammatory transcrip-
tion factor NF-jB in 293T cells [7173]. Human
NLRP2 transcripts were readily detectable under
steady-state conditions in thymus and spleen. In addi-
tion, NLRP2 mRNA levels in THP-1 cells were upreg-
ulated by diverse pro-inflammatory stimuli, including
LPS and IL-1b, supporting a potential role for
NLRP2 in immune signaling [71,73]. Moreover,
NLRP2 mRNA levels were upregulated in CD34+
progenitor cells during differentiation in macrophages
and neutrophils, and in mesenchymal stem cells under-
going adipogenesis [72]. Interestingly, common genetic
variants in NLRP2 were suggested to correlate with

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H. Van Gorp et al.
the outcome of HLA-identical allogeneic stem cell
transplantation. Donor cells with homozygous NLRP2
variants rs1043673, corresponding to missense muta-
tion A1052Q, and rs1043684, corresponding to a
mutation in the non-coding 30 -UTR, were associated
with non-relapse mortality and overall survival [74].
Further work is needed to determine how these
NLRP2-associated single nucleotide polymorphisms
may alter susceptibility to severe acute graft-versus-
host disease and infections [74].
While NLRP2 is expressed in immune cells and
other somatic cells in humans, in mice the highest
expression levels were noted in developing oocytes and
associated granulosa cells [49]. siRNA-mediated down-
regulation of Nlrp2 expression in oocytes did not inhi-
bit oocyte fertilization [63], but these cells failed to
progress normally after fertilization and most zygotes
arrested at the first and second cleavage events [49].
A similar early blockade of embryonic development
has been shown to occur upon disruption of Nlrp5
(see later) [51] and other maternal effect genes such as
Floped [14], Filia [75] and Stella [76] among others.
The functional relevance of NLRP2 in early human
embryonic development was inferred from a limited
set of disease-association studies (Fig. 4A). One of
these mutations R493SerfsX32 was discovered in a
familial case of BWS and causes a frameshift in
NLRP2 mRNA that is predicted to result in a trun-
cated protein [24]. However, analysis of another 11
cases of non-familial BWS failed to reveal mutations
in NLRP2, suggesting that defective NLRP2 expres-
sion is not the principal cause of sporadic BWS [24]. A
homozygous missense (Q884R) mutation in NLRP2
was also described in a female patient with biparental
hydatidiform mole [77]. In addition to the homozygous
mutations described above, a heterozygous (I352S)
missense mutation in NLRP2 was found in an SRS
patient presenting with defective methylation at six
maternally imprinted regions [25]. Screening larger
cohorts of BWS, biparental hydatidiform mole and
SRS patients is recommended to confirm the associa-
tion of NLRP2 mutations with these reproductive dis-
orders, and to estimate the fraction of these patients
presenting with homozygous NLRP2 mutations. It is
tempting to speculate that some etiological forms of
reproductive syndromes may have a common underly-
ing mechanism that involves defective activation of
NLRP2 and/or its signaling pathways. This might
resemble the causal association of several hereditary
autoinflammatory syndromes with mutations in the
inflammasome adaptor NLRP3 (familial cold auto-
inflammatory syndrome, MuckleWells syndrome and
chronic infantile neurological cutaneous and articular
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NLRs in immunity and reproduction
syndrome/neonatal onset multisystem inflammatory
disease [78,79]) and related inflammasome components
such as pyrin (familial Mediterranean fever) [80].
Undoubtedly, clarification of the molecular functions
and signaling partners of NLRP2 in maturing oocytes
and developing zygotes will shed light on this intrigu-
ing possibility.
NLRP5: a pioneering maternal effect
gene
NLRP5 was one of the first maternal effect genes
reported in mammals [51]. It was originally cloned as
an oocyte-specific factor targeted by autoantibodies in
a mouse model of autoimmune oophoritis, a syndrome
characterized by premature ovarian failure that results
in menopausal symptoms and infertility in young
women [81,82]. Autoantibodies against NLRP5 were
also described as a marker for hypoparathyroidism
that is detected in about half the patients suffering
from autoimmune polyendocrine syndrome type 1, a
rare autosomal recessive disorder that develops in
early childhood and affects a number of endocrine
organs such as the adrenal cortex, ovaries and para-
thyroid glands [83]. The development of mice with a
targeted hypomorph allele of Nlrp5 that reduced Nlrp5
expression by 9095% (Nlrp5tm/tm mice) formally
established the role of Nlrp5 in early embryogenesis
[51,84]. Both male and female Nlrp5tm/tm mice devel-
oped normally, and fertility of male Nlrp5tm/tm mice
was unaffected. In contrast, female Nlrp5tm/tm mice
produced oocytes that could be fertilized but the
resulting zygotes arrested at the two-cell stage [51].
Consequently, female Nlrp5tm/tm mice were incapable
of generating offspring and presented with a sterile
phenotype.
While the precise molecular function of NLRP5 in
maturing oocytes and zygotes remains enigmatic, it
was shown to localize primarily to the subcortex in
human oocytes [48]. In mice, Nlrp5 was shown to
assemble the SCMC, a large multiprotein complex that
lines the subcortex of mature oocytes and pre-implan-
tation embryos (Fig. 5) [14]. In the latter, the subcellu-
lar localization of the SCMC appears to result from its
active exclusion from regions of cellcell contact, thus
confining its localization to the cytocortex of the outer
morula cells and to the trophectoderm of blastocysts,
while the inner cell mass of the pre-implantation
embryos are devoid of the SCMC [14,84]. The molecu-
lar mechanism guiding this peculiar intracellular distri-
bution at the subcortex remains to be addressed.
However, it is clear that Nlrp5 is crucial for assembly
of the SCMC because Nlrp5tm/tm oocytes are devoid of
4575
NLRs in immunity and reproduction
Fig. 5. Subcellular localization of Nlrp5 in germinal vesicle oocytes.
Nlrp5 is part of the SCMC, which is a high molecular weight
multiprotein complex localized at the subcortex of oocytes and pre-
implantation embryos. The complex assembles during oocyte
growth and is essential for zygotes to progress beyond the first
embryonic cell divisions. Apart from Nlrp5, four additional
maternally encoded proteins have been identified to be part of the
SCMC: Floped, Tle6, Filia and Padi6. Nlrp5, Tle6 and Floped
interact with each other, while Filia binds to Nlrp5 independently.
The precise interaction partners of Padi6 in the SCMC remain to
be identified. In addition, Nlrp5, Padi6 and Floped co-localize at the
oocyte cytoplasmic lattices, a unique oocyte cytoskeletal element
involved in maternal ribosomal storage and transport, as well as in
organelle positioning and (re)distribution.
these ooplasm structures [14]. In addition to Nlrp5,
the maternal effect gene products Floped, Tle6 and
Filia also localized to the SCMC (Fig. 5) [14]. Floped
and Nlrp5 also co-localized with the oocyte-specific
peptidylarginine deiminase Padi6 in oocyte cytoplasmic
lattices, suggesting that these structures visible in elec-
tron micrographs may correspond to the SCMC
[22,8588]. Indeed, electron micrographs of Padi6/,
Nlrp5tm/tm and Floped/ oocytes contained markedly
reduced cytoplasmic lattice structures [85,87,88]. Of
note, Floped was also reported to be diffusely
expressed in the cytosol of agarose-embedded mature
oocytes and pre-implantation embryos, suggesting that
staining procedures may differentially affect the locali-
zation of SCMC components [88,89].
Importantly, both Floped/ and Padi6/ mice phe-
nocopied Nlrp5tm/tm mice because oocytes lacking
either Floped or Padi6 also failed to progress beyond
the initial cleavage events after fertilization [14,16,87].
Defective expression of Floped or Nlrp5 disrupted
assembly of the SCMC complex and led to virtual
depletion of SCMC components such as Tle6, Filia
and each others cellular pools in Floped/ and
Nlrp5tm/tm ovaries, respectively [14]. This was probably
4576
H. Van Gorp et al.
due to protein degradation because mRNA expression
of SCMC components was not affected in Floped/
and Nlrp5tm/tm oocytes. In contrast, Nlrp5tm/tm oocytes
expressed normal levels of Padi6 [85], suggesting that
Padi6 stability is not critically dependent on the
SCMC complex or that the protein has a significantly
longer half-life. Whether Padi6 deletion disrupts
assembly of the SCMC remains to be shown. How-
ever, Filia deficiency did not reduce expression of
other SCMC proteins in mature oocytes, nor was
assembly of the SCMC affected [75]. These observa-
tions suggest that Filia is not essential for SCMC
assembly. In agreement, Filia/ female mice were not
sterile, although they produced significantly fewer off-
spring than Filia-sufficient controls [75].
The role of NLRP5 in early embryogenesis is
likely to be conserved in primates because RNAi-
based downregulation of NLRP5 expression led to
embryonic arrest of in vitro fertilized rhesus macaque
monkey oocytes between the eight- and 16-cell stages
[90]. It would thus be of major interest to establish
the functional roles of the SCMC complex, and to
determine whether mutations in the human homologs
of Nlrp5, Floped, Filia and other genes encoding
other SCMC components are clinically associated
with fetal wastage in humans. It is noteworthy in
this regard that mutations in the human ortholog of
mouse Figla (factor in germline alpha) have been
identified in two patients with premature ovarian
failure [91]. Figla is a basic helixloophelix tran-
scription factor that drives expression of Floped,
Nlrp5, Tle6, Padi6 and possibly other SCMC compo-
nents in mice [14,92], suggesting that defective
SCMC function may be clinically associated with
female sterility in at least a subset of patients pre-
senting with infertility or recurrent spontaneous
abortion.
NLRP7: a mystery of molar
pregnancies
Unlike primates, rodents lack NLRP7. The human
gene is thought to have emerged from the duplication
of an ancestral NLRP2/7 gene after the split of the
rodent and primate lineages [40,43,44]. In agreement,
our phylogenetic analysis clustered mouse Nlrp2
together with human NLRP2 and NLRP7, with the
human paralogs being more closely related to each
other than to mouse Nlrp2 (Fig. 3A). While many
questions remain regarding the functions of this pri-
mate-specific NLR, recent work has illuminated some
aspects of the mechanisms by which it regulates
immune and reproductive functions.
FEBS Journal 281 (2014) 45684582 2014 FEBS
H. Van Gorp et al.
Human NLRP7 mRNA is detected in the spleen,
thymus and in peripheral blood leukocytes (PBMCs),
suggesting that NLRP7 contributes to immune signal-
ing [73,93]. In agreement, NLRP7 mRNA levels were
further upregulated in LPS- and IL-1b-stimulated
PBMCs and macrophages [73]. Initial studies with
ectopically expressed NLRP7 suggested it negatively
regulates extracellular IL-1b levels by inhibiting
inflammasome-mediated maturation of IL-1b or by
reducing cellular proIL-1b levels or by affecting its
secretion [73,94]. However, another report failed to
observe defective inflammasome-driven IL-1b secretion
upon transient expression of NLRP7 [69]. More
recently, siRNA-mediated knockdown of endogenous
NLRP7 in human primary macrophages and THP-1
cells suggested that NLRP7 assembles a canonical
inflammasome that in contrast drives IL-1b secretion
upon cytosolic detection of bacterial lipopeptides [12].
NLRP7 downregulation failed to affect secretion of
the inflammasome-independent cytokines IL-6 and
tumor necrosis factor, suggesting that NLRP7 specifi-
cally regulated inflammasome-driven IL-1b secretion
[12]. Notably, lipopeptide-induced NLRP7 activation
did not trigger pyroptosis, a lytic cell death mode that
usually accompanies IL-1b secretion by other canoni-
cal inflammasomes [7]. Determining how NLRP7 spe-
cifically engages inflammasome-dependent cytokine
secretion may reveal novel mechanisms that suppress
pyroptosis induction.
Apart from its expression in hematopoietic cells,
NLRP7 transcripts were found to be abundantly
expressed in testes and ovaries, suggesting that it exerts
reproductive functions (Fig. 3B) [73,95]. Notably,
NLRP7 transcript levels were shown to decrease pro-
gressively during oocyte maturation, and to rise signifi-
cantly 35 days after fertilization, when the embryo
has reached the blastocyst stage [96]. In contrast, tran-
script levels of the reproduction-associated NLRs dis-
cussed above decline irreversibly starting from the
zygote stage [50]. In addition, NLRP7 expression was
detected in endometrial cancer tissues and testicular
seminomas, showing the trend to be associated with
cancer pathogenesis [95,97].
NLRP7 was predicted to function as a maternal
effect gene based on the causal association of NLRP7
mutations with familial biparental hydatidiform moles
(Figs 2E,D, and 4B) [29,31]. NLRP7 mutations are
found in as many as 60% of clinical cases of familial
biparental molar pregnancies [93]. It is currently
debated whether mutations in NLRP7 also contribute
to the pathogenesis of other forms of molar pregnan-
cies (Fig. 2AD,F,G) and reproductive wastage
syndromes [77,93,98102]. While familial diploid
FEBS Journal 281 (2014) 45684582 2014 FEBS
NLRs in immunity and reproduction
biparental hydatidiform moles appear to be causally
linked to homozygous and compound heterozygous
NLRP7 mutations, it is not clear whether single het-
erozygous variants of NLRP7 lead to familial biparen-
tal hydatidiform moles [27,30,93,103,104]. These
homozygous and compound heterozygous NLRP7
mutations are often are located in coding exons and
splice junctions, and are predicted to affect the protein
by introducing deleterious amino acid changes, inser-
tions, intragenic duplications and deletions that lead to
a mutated or truncated protein product (Fig. 4B)
[105]. The identification of homozygous or compound
heterozygous NLRP7 missense and non-sense muta-
tions in men with apparently normal reproductive
functions suggests that NLRP7 may specifically regu-
late female reproduction [30,31].
So far, the molecular mechanisms of NLRP7 in
imprinting defects underlying familial biparental
molar pregnancies are not clear [106108], and this
is complicated by the wide spectrum of disease phe-
notypes observed. NLRP7 does not contain any
DNA-binding domains and is localized in the cyto-
plasm. Seemingly, the immunological function of
NLRP7 to control IL-1b secretion is not involved
since IL-1b-deficient mice are fertile. Besides, mono-
cytes from some patients with familial biparental
moles with NLRP7 mutations show reduced IL-1b
secretion [94], whereas others show increased IL-1b
secretion [104]. It should be noted, however, that
even the general population shows varying IL-1b
secretion levels [109]. Additional work is clearly
needed to fully understand the pathophysiological
role of NLRP7 in familial biparental molar pregnan-
cies, in order to comprehend its contribution to nor-
mal embryonic development.
Conclusion and perspectives
In addition to their well-established roles in the
immune system, the existence of an NLR subset that
operates at the interface of the immune and reproduc-
tive systems has emerged during the past 15 years. The
formal demonstration that NLRP5 acts as a maternal
effect gene product in mammals [51] has paved the way
for recognizing the critical importance of several addi-
tional NLR family members in the reproductive sys-
tem. Members of the reproduction-associated NLR
subset have in common that their sequences cluster in a
separate phylogenetic branch, that they are highly
expressed in mature oocytes and pre-implantation
embryos, and that deletions and mutations in their
genes are associated with human wastage syndromes.
However, many important questions remain to be
4577
NLRs in immunity and reproduction
addressed in the coming years, including how pre-
cisely NLRP2, NLRP5 and NLRP7 control early
embryonic development, what roles NLRP4, NLRP9
and other less-characterized NLRs play in reproduc-
tion, and how mutations in these genes affect human
fertility. Another question is whether there is func-
tional redundancy between members of the reproduc-
tion-related NLR cluster. Additional insight in their
biological roles will undoubtedly also come from
addressing whether these NLRs too contribute to
immune and host defense responses against microbial
pathogens that infect reproductive organs. Answering
these and other questions will markedly increase our
understanding of the importance of NLRs in human
reproduction, and may lead to the development of
improved diagnostics advancing patient stratification
as well as to better therapeutic strategies for repro-
ductive ailments and assisted reproductive technolo-
gies.
Acknowledgements
The authors apologize to those whose citations were
omitted owing to space limitations. H.V.G. and
F.V.H. are postdoctoral fellows with the Research
Foundation Flanders (FWO grant 1.2.449.13N and
1.2.E99.14N). M.L. is supported in part by the Ghent
University Concerted Research Actions (grant BOF14/
GOA/013) and by grants from the European Research
Council (Grant 281600) and the Research Foundation
Flanders (FWO grant G030212N).
Author contributions
HVG, AK and ML wrote the paper. AK performed
meta-analysis of expression data, while FVH per-
formed the phylogenetic analysis.
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