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Oncology
NLRP7 is a member of a new family of proteins that contributes to innate immune processes. Depending on its level of expression, NLRP7 can function in an inflammasome-dependent or independent pathway.
- NLRP7
- inflammasome
- choriocarcinoma
- pregnancy
- maternal immune tolerance
- tumor microenvironment
1. NLRP7 Inflammasome: Generalities
NLRP7 Expression Pattern and Functions
Inflammasomes are cytosolic multi-protein complexes that link pathogen recognition by specific cytosolic pattern recognition receptors (PRRs) [22,23]. The nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) serves as intracellular guards that coordinate the innate immunity and inflammatory responses upon the perception of adverse signals within the cell [22,23]. The activation of these inflammasomes is mediated by two signals. The first signal activates the nuclear factor (NF-κB) pathway that induces the transcription of the pro-IL-1β and pro-IL-18 [24], while the second signal is the direct sensing of the stimulus by the inflammasome, mediating its assembly and later the maturation of the pro-IL-1β and pro-IL-18, in a caspase-1 dependent manner (Figure 1).
Figure 1. Illustration of the domains that comprise the NLRP7 inflammasome. NLRP7 inflammasome is composed of the NLRP7 receptor, the ASC adaptor protein and procaspase 1. PYD: pyrin domain; NATCH: nucleotide binding domain (allows the ATP-dependent oligomerization of the NLRs); LRR: leucine-rich repeats.
Under physiological conditions, NLRP7 mediates the maturation and secretion of IL-1β via its inflammasome’s activity [20]. However, its overexpression under pathological conditions, such as cancer, causes an inhibition of the procaspase-1 and pro-IL-1β maturation through direct physical interaction with these pro-proteins and without any interference with the NF-kB pathway.
2. NLRP7 and Normal Pregnancy
Beside its contribution to inflammatory responses in numerous physiological systems, NLRP7 exhibits an important role in the control of the female reproduction processes [20,28,29,31,32,33,34]. NLRP7 has been reported to be highly expressed in the oocyte and to interact with other maternal-effect genes to regulate ovarian reproductive activities [35]. Immunohistochemical localization of NLRP7 within adult ovary sections revealed that the protein was present in follicles regardless of their developmental stages [35]. In addition, NLRP7 knockdown is unfavorable for the pre-implantation embryo development, in vitro [36,37].
Because maintenance of human pregnancy is considered as an immunological paradox, it has been established that its normal outcome depends on finely tuned adaptations at the fetomaternal interface of numerous systems, including the innate and adaptive immune system. At this interface, two distinct genomes must interact in order to maintain tolerance of the allograft and to preserve the pregnancy. In this way, the placenta has to employ several mechanisms to regulate immune tolerance and modulate the way the maternal immune system adapts in the presence of potentially dangerous signals [38,39].
Importantly, placental trophoblasts, endothelial cells and fetal macrophages (Hofbauer cells) have been reported to be sensitive to infectious agents via the PRRs [40]. The PRRs are known to sense both pathogen-associated molecular patterns (PAMPs) and host-derived damage-associated molecular patterns (DAMPs). The latter include exosomes, reactive oxygen species (ROS), uric acid, cholesterol and microparticles [41,42]. During pregnancy, both maternal and fetal compartments have been reported to express mRNA and protein of the following NLRPs, 1 to 4 and NLRP7, as well as the adaptor protein ASC and the main caspase (caspase 1). These proteins have been reported in the syncytiotrophoblast (ST) and cytotrophoblast (CT), in the cells of the myometrium and in the amnion cells [43,44]. At term, the activity of the inflammasomes have been reported to be increased in the cervix and the decidua [45]. In addition, recent studies have demonstrated that pyroptosis can also occur upon the activation of the NLRP3 inflammasome during the process of labor [46].
3. NLRP7 in Pregnancy Pathologies
3.1. NLRP7 and Recurrent Hydatidiform Moles
Until 2014, studies on NLRP7 in relation to pregnancy pathologies have mainly been focused on its association with recurrent HM, as ample evidence has been collected to convincingly link HM to NLRP7 [9,31,49]. Several NLRP7 gene variants are clearly associated with reproduction and imprinting defects [9,31,49,50]. HM patients have been reported to carry nonsynonymous variants of NLRP7, and more than 200 sequence variants have thus far been reported in 48–80% of recurrent HM patients [50,51]. Mutations in the nlrp7 gene include insertions, substitutions, deletions and duplications. While the association between NLRP7 mutations and HM occurrence is convincing, the functional consequences and the underlying molecular mechanism are still unknown.
3.2. NLRP7 and Fetal Growth Restriction
In relation to pregnancy pathologies, we demonstrated that NLRP7 expression is elevated in the placentae of pregnancies complicated by fetal growth restriction (FGR), a pregnancy often characterized by increased inflammation [13,20,52,53]. These findings strongly suggest that the NLRP7 inflammasome could be involved in the etiology of FGR. In addition, we demonstrated that the expression of other NLRP7 inflammasome components, including ASC, cleaved caspase-1 and mature IL-1β, were also increased in FGR placentae and that circulating IL-1β, but not IL-18 levels, were significantly increased in the sera from FGR patients [20].
3.3. NLRP7 and Preeclampsia
While deregulation of NLRP7 expression in the placenta of preeclamptic patients is likely, assuming that inflammation is one of the main causes of PE development, no study has thus far reported NLRP7 status in relation to this significant and life-threatening pathology of pregnancy complications. Because the NLRP gene family has been reported to be associated with the etiology of imprinting defects and that PE has also been observed in disorders associated with aberrant methylation at genomically imprinted loci, it was hypothesized that the NLRP gene family may be implicated in PE. To verify this hypothesis, Soellner et al. analyzed a cohort of 47 PE patients for NLRP gene mutations using next generation sequencing [54]. The screening indicated that NLRP mutations are not a relevant cause of PE. Further studies are warranted to show the potential clinical and biological significance of NLRP7 in the etiology of PE.
4. NLRs and Cancer
Because elevated serum concentrations of IL-1β and IL-18 are often correlated to malignancies, it was suggested that all members of the NLR family are associated with pro-tumoral activities. Nevertheless, numerous studies, especially the comprehensive review by Terlizzi et al., reported that members of this family can exhibit both pro-and anti-tumoral activities, depending on the type of cancer and whether they function in inflammasome dependent or independent pathways [26].
While inflammasome activation in cancer is supposed to control its expansion, some stimuli of inflammasomes can behave as tumor promoters through the induction of chronic inflammation that rather facilitates tumor development. This sight is contrasted in animal models of colon cancer in which the activation of some inflammasome complexes is associated with tumor protection. For instance, NLRC4- and caspase-1-deficient mice have been reported to develop increased colonic inflammation, responsible for higher colon adenocarcinoma burden, in an azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. NLRC4 and caspase-1 were inferred to exert a protective function in that model via a direct effect on epithelial cell proliferation [26]. In addition, knockdown of NLRP6 in mice increased their risk of developing colorectal cancer, suggesting its significant role in the onco-suppressive activity [55]. Conversely, NLRP3, the most studied NLR has been reported to be associated with pro- and anti-carcinogenic roles. In the DSS/AOM cancer model, NLRP3 has been reported to play a protective role [56]. However, this member has been associated with poor survival rate of colorectal cancer [57] and to higher susceptibility to melanoma [58] and myeloma [59]. In addition, NLRP3 has been reported to suppress NK (natural killer) and T cell-mediated anti-tumor actions and immune-editing in a mouse model of carcinogen-induced sarcoma and metastatic melanoma [25]. This phenomenon was mediated by IL-1β-dependent recruitment of immune suppressive cells, such as myeloid-derived suppressor cells (MDSCs) and Treg cells [25]. Taken together, these findings strongly suggest that the roles of NLRs in human cancers are yet to be elucidated.
4.1. NLRP7 and Gestational Trophoblastic Diseases
Distinct from normal placental development, GTDs are a rare subgroup of placental pathologies, encompassing PHM or CHM and their non-molar counterpart such as CC, which constitutes the most aggressive form of placental cancer [62]. CC is a highly proliferative and invasive tumor as trophoblast cells forming the tumor metastasize into multiple organs, including the vagina, lungs and brain [18,62]. CC has an estimated incidence of 2 to 7 in 100,000 pregnancies in Europe and North America. This incidence is higher in Asia and Africa, with 5 to 202 in 100,000 pregnancies [62]. CHM is a morbid pathology that is associated with a high risk (20%) for patients to develop post-molar CC [62]. More often, CC may also develop after normal delivery. The incidence of this type of CC is 1 per 67,000 live births [19,63]. Recent studies have shown that 50% of patients with recurrent HM have mutations in the gene nlrp7 [62]. While the association of biallelic mutations in nlrp7 with recurrent HM is well established, its role in the development of GTDs, especially CC, is poorly understood and often controversial [28].
4.2. NLRP7 and Choriocarcinoma
Since the identification of nlrp7 as a highly mutated gene in recurrent HMs, no study has been conducted to determine whether deregulations in the expression of this gene may contribute to the change in the behavior of the tumor trophoblast cells and their metastasis. Recently, we investigated the role of NLRP7 in these processes [64]. We used three approaches to define the role of NLRP7; (i) a clinical study in which we used human sera and placentae that were collected from normal pregnant women and from patients with CHM or CC; (ii) an in vitro study in which we investigated the influence of NLRP7 knockdown on the tumorigenesis of the choriocarcinoma cell line, JEG3, which used both 2D and 3D culture systems; and (iii) an in vivo study in which we used an orthotopic model of CC and a metastatic model of this cancer [65]. This study demonstrated that NLRP7 was upregulated in tumor cells, and in CHM and CC placentae. In JEG3 cells, NLRP7 increased proliferation and 3D organization of malignant cells.
NLRP7 increased expression in JEG3 cells and in CHM and CC tissues strongly suggested that its inflammasome is highly activated. Nevertheless, no production or secretion of mature IL-1β have been observed in JEG3 cells. This finding strongly suggests that NLRP7 may function in an inflammasome-independent pathway in malignant trophoblast cells. This statement is in line with previous studies reporting that overexpression of NLRP7 exerts negative feedback on the production and maturation of IL-1β. [9,30]. Recent studies from the literature demonstrated that IL-1β might negatively control the proliferation of trophoblast cells through the deregulation of the cell cycle [66,67]. Importantly, Chow et al. demonstrated that another member of the NLR family, the NLRP3, promotes metastasis in an inflammasome independent manner and that knock-out mice for NLRP3 exhibit lower numbers of lung metastases upon intravenous inoculation of prostate or melanoma malignant cells [25]. It has also been reported that overexpression of NLRP12 is associated with the aggravation of prostate cancer without any increase in the levels of mature IL-18 or IL-1β by these cells [55]. Overall, these results roughly suggest that NLRP7, similar to NLRP12 and NLRP3, functions in an inflammasome independent manner in malignant cells [55,68].
Importantly, the in vivo study that used the orthotopic model of CC, which was injected within its placenta with NLRP7 invalidated-CC cells, showed higher maternal immune response and that the mice developed smaller tumors and displayed less metastases. Furthermore, we observed a strong increase in the levels of IL-1β, both locally in mouse placenta and in the maternal serum. This finding strongly suggests that the expression of NLRP7 by the trophoblast cells contributes to its camouflage by the maternal environment (Figure 2). In line with this assumption, we observed that malignant cells that were inactivated for NLRP7 exhibited significant decrease in the expression of proteins that contribute to maternal immune tolerance. These include PD-L1, HLA-G and hCG. The latter hormone has recently been reported to increase the activity of regulatory T cells (Treg) and to retain the tolerogenic activity of dendritic cells [69,70]. Importantly, these findings strongly support a local immune tolerance that is mediated by malignant cells-secreted hCG. This hormone is known to act as a strong chemoattractant for T-suppressors that are apoptotic actors for T-lymphocytes.
Figure 2. Proposed model for the role of NLRP7 protein in the development of gestational choriocarcinoma.
4.3. Proposed Mechanism of NLRP7 Control of Maternal Microenvironment
Because the fetus is considered as a semi-allograft by the maternal immune system, a slowdown in the function of the immune system is required [39,72]. Yet, the normal pregnancy progresses and leads to birth in most cases. This is actually due to a fine adaptation of the maternal immune system to tolerate the foreign body. Hence, one can speculate that a similar mechanism of tolerance may occur during tumor development [73]. Among proteins that contribute to trophoblast tolerance from the maternal system [39,72] are the HLA family members, as well as the PDL-1/PD-1 system.
4.4. HLA Family in Normal and Tumor Placenta
HLA (human leukocyte antigen) proteins are mainly expressed by the EVT. This family of proteins has been shown to be involved in the attenuation of the pool of immune cells present at the fetomaternal interface from implantation to delivery [74,75]. The HLA family is composed of numerous members that are differentially involved in the immune tolerance during pregnancy. Among all HLA members, HLA-G is exclusively expressed on EVT [76]. During pregnancy, HLA-G plays an immunosuppressive role rather than an antigen-presenting role [39,72]. In non-pathological conditions, it is expressed only at the surface of the EVT, thymic epithelial cells, the cornea and in the cells facing the amniotic fluid [74]. The main role of HLA-G is to inhibit cytotoxic T lymphocytes and Natural killer cells through an interaction with their ILT-2 and KIR receptors [77]. Recent studies also showed that HLA-G regulates trophoblast invasion, a key parameter of placental development in normal and tumor conditions [78]. In addition, soluble HLAG (sHLA-G) has been shown to impair the expression and function of different chemokines receptors in T, B, and NK cells through the ILT2 receptor [77]. HLA-G has also been shown to be upregulated by βhCG in JEG3, suggesting that this hormone also contributes to the mechanism by which choriocarcinoma cells develop immune tolerance [69,70].
4.5. PDL-1 in Normal and Tumor Trophoblast Cells
The survival of the trophoblast cells depend on their ability to evade the immune system through the inhibition of their anti-tumoral activity [39,72]. A common ligand found in several aggressive cells is the protein PD-L1 (programmed death ligand -1), which mediates immunosuppression upon binding to its receptor PD-1, commonly expressed by immune cells [79]. During normal pregnancy, the immunosuppressive role of PD-L1 is major, as it is expressed on the ST. PDL-1 interaction with these cells promotes an immune tolerance to the fetal tissues [80].
In CC, PD-L1 is expressed by the ST and CT. PD-L1/PD-1 interaction provides an immune tolerance through the activation of the paternal antigen-specific naïve helper T cells Tregs [81]. Importantly, we demonstrated that NLRP7 knockdown caused a decrease in PD-L1 expression, suggesting that this protein is directly involved in the NLRP7-mediated immunosuppression [64]. Altogether, these findings suggest that a tight relationship exists between the maternal immune system and the NLRP7 inflammasome.
Conclusion
It clearly appears that appropriate NLRP7 expression and NLRP7 inflammasome activity are essential during early pregnancy. However, further investigation is required to establish how HM-associated NLRP7 overexpression and variants might affect NLRP7 function and lead to reproductive wastage.
Overall, it appears that the NLRP7 mode of function will tightly depend on the cellular status. Under physiological conditions, NLRP7 will function in an inflammasome- dependent pathway to contribute to the maintenance of the required fine balance between pro-inflammatory and anti-inflammatory settings. This will be ensured through the processing of pro-IL-1β to IL-1β. This is what clearly has been reported in normal trophoblast cells and in placental explant model systems. The NLRP7 inflammasome activity can be exacerbated in the context of pregnancy pathologies such as FGR pregnancy, to overcome the stressful conditions of the trophoblast cells.
This entry is adapted from the peer-reviewed paper 10.3390/biomedicines10020252
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