NOD-like receptors interfacing the immune and reproductive systems

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1 STATE-OF-THE-ART REVIEW NOD-like receptors interfacing the immune and reproductive systems Hanne Van Gorp 1,2 *, Anna Kuchmiy 1,2 *, Filip Van Hauwermeiren 1,2 and Mohamed Lamkanfi 1,2 1 Department of Medical Protein Research, VIB, Ghent, Belgium 2 Department of Biochemistry, Ghent University, Ghent, Belgium Keywords 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: Tel: mohamed.lamkanfi@vib-ugent.be *These authors contributed equally (Received 30 June 2014, revised 13 August 2014, accepted 19 August 2014) Nucleotide-binding oligomerization domain receptors (NOD-like receptors, 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 inflammasomes or to engage transcriptional signaling cascades that result in the production 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. doi: /febs 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) antigen 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 pattern 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, microbicidal agents, the activation and recruitment of additional immune cells to the affected tissue, and by Abbreviations BWS, Beckwith Wiedemann 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, Silver Russell syndrome FEBS Journal 281 (2014) ª 2014 FEBS

2 H. Van Gorp et al. NLRs in immunity and reproduction tagging the foreign agents for degradation by professional 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 receptor, 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-terminal 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 functions in the immune system. NLRP1b, NLRC4 and NLRP3 are NLRs with well-established roles in the assembly of inflammasomes [6]. These cytosolic multiprotein complexes recruit and activate the pro-inflammatory cysteine proteases caspase-1 and -11, which in their turn promote secretion of the inflammatory cytokines interleukin (IL) 1b and IL-18, and induce a programmed 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 specifically upon cytosolic detection of bacterial peptidoglycan fragments. Notably, mutations in the latter NLRs are closely linked to inflammatory syndromes of the intestinal tract [8]. Finally, CIITA is a transcriptional co-activator that is critical for major histocompatibility 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 dampen NF-jB-mediated cytokine production [10 12], 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 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 protein protein interactions. Different subfamilies can be defined based on the presence of a particular amino-terminal protein protein 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 reproduction and during early stages of embryonic development. 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 existence of a clearly defined subset of NLRs that operates at the interfaces of the immune and reproductive systems and that can be segregated from other NLRs based on their distinctive phylogeny, expression profiles in gametes and the clustered genomic localization of their genes. Finally, we also reflect on the functional evidence linking mutations in these NLRs to reproduction-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 FEBS Journal 281 (2014) ª 2014 FEBS 4569

3 NLRs in immunity and reproduction H. Van Gorp et al. process requires meiosis to produce gametes, reproductive 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 combination of genes obtained after homologous recombination 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 transcriptional activation of the zygotic genome [13 15]. 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-fertilization 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 transcripts. The kinetics of this distinctive expression pattern is species-specific, since the precise moment in the developmental plan at which embryonic genome activation occurs varies greatly among animal species. Murine embryos undergo genome activation at the two-cell stage, cattle at the 8 16-cell stage and humans when the embryo comprises four eight 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 condensed, transcriptionally quiescent structure. This is achieved by targeted DNA methylation and histone modifications, which are reversible epigenetic mechanisms 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 genome-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 hundred autosomal genes in mammals occurs from a single allele. Which of the two alleles is expressed in the developing embryo is determined by the parent-oforigin 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 paternally 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 genderspecific 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 development [14]. However, these mutant oocytes also lacked oocyte cytoplasmic lattices, oocyte-specific cytoplasmic cytoskeletal elements that were suggested to support maternal ribosome transport and organelle (re)distribution during oocyte maturation [22]. Clarification of the relative importance of the SCMC and cytoplasmic lattice protein complexes in oocyte maturation and early embryonic development requires additional analysis. Although the molecular modes of action of maternal 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 Beckwith Wiedemann syndrome (BWS) is an imprinting disease that is associated with congenital overgrowth, undescended testes in male progeny, abdominal wall defects, seizures and predisposition to embryonic tumorigenesis [24]. Silver Russell 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 hypermethylation or hypomethylation of imprinted control regions. Moles constitute abnormal or molar pregnancies in which a non-viable fertilized egg implants in the uterus, consequently leading to absent or 4570 FEBS Journal 281 (2014) ª 2014 FEBS

4 H. Van Gorp et al. NLRs in immunity and reproduction abnormal embryonic development with abnormal trophoblastic growth [25]. Abnormal molar tissues have different genotypes (Fig. 2A G) [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 hydatidiform 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 biparental hydatidiform moles and developing in an estimated 10 15% 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 suggesting that this NLR subset may represent a novel group of maternal effect genes, and review our current understanding of the molecular mechanisms by which they exert their functions at the interfaces of the immune and reproductive systems. 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 freshwater animals with radial symmetry), Strongylocentrotus (sea urchins) and Amphimedon (sponges) [35 38]. 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 Caenorhabditis elegans (roundworm) is devoid of NLR genes, which is consistent with the particularly high levels of gene loss observed in the model organisms D. melanogaster and C. elegans [36,39]. The NLR gene sets in mammals are highly plastic, with the human genome encoding 22 NLR genes and the mouse genome 34 NLR genes [40]. The evolutionary bifurcation of rodents and primates about 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 duplication events are also suggested to account for the A B Fig. 2. Genetics of hydatidiform moles (HM). Genetically, sporadic complete HM are androgenic diploid conceptions (AnHM) hypothetically originating (A) from a duplicated paternal haploid genome (46, XX) or (B) after fertilization of an empty egg with two haploid sperm cells (46, XX and 46, XY). (C) Sporadic partial HM (PHM) are dispermic triploid conceptions and usually originate from dispermic fertilization of an oocyte (69, XXX; 69, XXY; 69, XYY). (D) Digynic triploid conceptions originate from duplication of a maternal haploid set of chromosomes. (E) Biparental HM are diploid and contain a maternal and paternal set of genes. (F, G) Hypothetical schemes of conception mosaicism. C D E F G FEBS Journal 281 (2014) ª 2014 FEBS 4571

5 NLRs in immunity and reproduction H. Van Gorp et al. days A C B D E existence of primate-specific NLRP7. Apart from being duplicated, genes might also get lost from the genome during speciation. Indeed, rodents lack orthologs of human NLRP8, 11 and 13, most probably reflecting loss of those genes during rodent evolution [40,43,44]. 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 separates from other NLR clusters with clearly established immune roles (Fig. 3A) [43,44]. Interestingly, within 4572 FEBS Journal 281 (2014) ª 2014 FEBS

6 H. Van Gorp et al. NLRs in immunity and reproduction the PYD-containing NLR subfamily, the immunerelated 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 functions [43,44,46]. In this regard, the cluster containing NLRP2, 4, 5, 7, 8, 9, 11, 13 and 14 coincides remarkably with the emerging reproduction-associated NLR subset. Meta-analysis of publicly available expression data demonstrates that members of this reproductionassociated 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 levels of these NLRs drop steeply shortly after fertilization towards the blastocyst stage, a characterizing feature of maternal effect genes [15,16]. Our metaanalysis along 12 developmental stages in mouse indeed nicely re-confirms the gradual decrease of Nlrp4a, 4b, 4c, 4e, 4f, 4 g, 5, 9a c, and 14 levels during early-stage embryogenesis (Fig. 3C). Immunerelated 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 detection (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 phylogenetically with the immune-associated NLRs (Fig. 3A) [44] and is known to play a role in immunity 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 0 1 (corresponding to two-cell stage embryos) and their levels strongly decrease during prenatal period days 2 4 (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. FEBS Journal 281 (2014) ª 2014 FEBS 4573

7 NLRs in immunity and reproduction H. Van Gorp et al. A B C 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_ ). (B) Schematic view of the NLRP7 protein with indication of harmful mutations (Uniprot Q8WX94-3; Unigene NP_ ). (C) Schematic view of the NLRP14 protein with indication of harmful mutations (Uniprot Q86W24-1; Unigene NP_ ). 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 reproductionrelated 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 [66 69]. 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 addition, endogenous NLRP2 was found to co-immunoprecipitate 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 transcription factor NF-jB in 293T cells [71 73]. Human NLRP2 transcripts were readily detectable under steady-state conditions in thymus and spleen. In addition, NLRP2 mrna levels in THP-1 cells were upregulated 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 undergoing adipogenesis [72]. Interestingly, common genetic variants in NLRP2 were suggested to correlate with 4574 FEBS Journal 281 (2014) ª 2014 FEBS

8 H. Van Gorp et al. NLRs in immunity and reproduction the outcome of HLA-identical allogeneic stem cell transplantation. Donor cells with homozygous NLRP2 variants rs , corresponding to missense mutation A1052Q, and rs , corresponding to a mutation in the non-coding 3 0 -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-versushost 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 downregulation of Nlrp2 expression in oocytes did not inhibit 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 truncated protein [24]. However, analysis of another 11 cases of non-familial BWS failed to reveal mutations in NLRP2, suggesting that defective NLRP2 expression 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 association of NLRP2 mutations with these reproductive disorders, 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 underlying 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 autoinflammatory syndrome, Muckle Wells syndrome and chronic infantile neurological cutaneous and articular 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 intriguing 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 parathyroid glands [83]. The development of mice with a targeted hypomorph allele of Nlrp5 that reduced Nlrp5 expression by 90 95% (Nlrp5 tm/tm mice) formally established the role of Nlrp5 in early embryogenesis [51,84]. Both male and female Nlrp5 tm/tm mice developed normally, and fertility of male Nlrp5 tm/tm mice was unaffected. In contrast, female Nlrp5 tm/tm mice produced oocytes that could be fertilized but the resulting zygotes arrested at the two-cell stage [51]. Consequently, female Nlrp5 tm/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-implantation embryos (Fig. 5) [14]. In the latter, the subcellular localization of the SCMC appears to result from its active exclusion from regions of cell cell 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 molecular mechanism guiding this peculiar intracellular distribution at the subcortex remains to be addressed. However, it is clear that Nlrp5 is crucial for assembly of the SCMC because Nlrp5 tm/tm oocytes are devoid of FEBS Journal 281 (2014) ª 2014 FEBS 4575

9 NLRs in immunity and reproduction H. Van Gorp et al. 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 preimplantation 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 electron micrographs may correspond to the SCMC [22,85 88]. Indeed, electron micrographs of Padi6 /, Nlrp5 tm/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 localization of SCMC components [88,89]. Importantly, both Floped / and Padi6 / mice phenocopied Nlrp5 tm/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 other s cellular pools in Floped / and Nlrp5 tm/tm ovaries, respectively [14]. This was probably due to protein degradation because mrna expression of SCMC components was not affected in Floped / and Nlrp5 tm/tm oocytes. In contrast, Nlrp5 tm/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. However, Filia deficiency did not reduce expression of other SCMC proteins in mature oocytes, nor was assembly of the SCMC affected [75]. These observations suggest that Filia is not essential for SCMC assembly. In agreement, Filia / female mice were not sterile, although they produced significantly fewer offspring than Filia-sufficient controls [75]. The role of NLRP5 in early embryogenesis is likely to be conserved in primates because RNAibased 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 helix loop helix transcription factor that drives expression of Floped, Nlrp5, Tle6, Padi6 and possibly other SCMC components in mice [14,92], suggesting that defective SCMC function may be clinically associated with female sterility in at least a subset of patients presenting 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 primate-specific NLR, recent work has illuminated some aspects of the mechanisms by which it regulates immune and reproductive functions FEBS Journal 281 (2014) ª 2014 FEBS

10 H. Van Gorp et al. NLRs in immunity and reproduction Human NLRP7 mrna is detected in the spleen, thymus and in peripheral blood leukocytes (PBMCs), suggesting that NLRP7 contributes to immune signaling [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 specifically 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 canonical inflammasomes [7]. Determining how NLRP7 specifically 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 progressively during oocyte maturation, and to rise significantly 3 5 days after fertilization, when the embryo has reached the blastocyst stage [96]. In contrast, transcript levels of the reproduction-associated NLRs discussed 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 pregnancies (Fig. 2A D,F,G) and reproductive wastage syndromes [77,93,98 102]. While familial diploid biparental hydatidiform moles appear to be causally linked to homozygous and compound heterozygous NLRP7 mutations, it is not clear whether single heterozygous variants of NLRP7 lead to familial biparental 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, insertions, 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 mutations in men with apparently normal reproductive functions suggests that NLRP7 may specifically regulate female reproduction [30,31]. So far, the molecular mechanisms of NLRP7 in imprinting defects underlying familial biparental molar pregnancies are not clear [ ], and this is complicated by the wide spectrum of disease phenotypes observed. NLRP7 does not contain any DNA-binding domains and is localized in the cytoplasm. Seemingly, the immunological function of NLRP7 to control IL-1b secretion is not involved since IL-1b-deficient mice are fertile. Besides, monocytes 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 pregnancies, in order to comprehend its contribution to normal 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 reproductive 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 additional NLR family members in the reproductive system. 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 FEBS Journal 281 (2014) ª 2014 FEBS 4577

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