The Residual Innate Lymphoid Cells in NFIL3-Deficient Mice Support Suboptimal Maternal Adaptations to Pregnancy

Uterine NK cells are innate lymphoid cells (ILC) that populate the uterus and expand during pregnancy, regulating placental development and fetal growth in humans and mice. We have recently characterized the composition of uterine ILCs (uILCs), some of which require the transcription factor NFIL3, but the extent to which NFIL3-dependent cells support successful reproduction in mice is unknown. By mating Nfil3−/− females with wild-type males, here we show the effects of NFIL3 deficiency in maternal cells on both the changes in uILCs during pregnancy and the downstream consequences on reproduction. Despite the presence of CD49a+Eomes− uILC1s and the considerable expansion of residual CD49a+Eomes+ tissue-resident NK cells and uILC3s in pregnant Nfil3−/− mice, we found incomplete remodeling of uterine arteries and decidua, placental defects, and fetal growth restriction in litters of normal size. These results show that maternal NFIL3 mediates non-redundant functions in mouse reproduction.

pregnancy and reproduction, e.g., IL-5 produced by uILC2s and IL-17 and IL-22 produced by uILC3s (9,10). Indeed, mice lacking lymphocytes, including uILCs, show impaired uterine arterial modifications (11) and fetal growth restriction (13). Defective uterine vascular remodeling and shallow trophoblast invasion are associated with great obstetrical syndromes, including preeclampsia and fetal growth restriction (14), and unbalanced cytokine production may lead to preterm labor (15). Mice lacking the basic leucine zipper transcription factor NFIL3/E4BP4 display immune abnormalities (16), including severely reduced numbers of cNK cells (17) as well as reduced ILCs in several tissues (18)(19)(20)(21). NFIL3 also regulates the emergence of common ILC precursors (22). However, the requirement for NFIL3 during development, homeostasis, and function of different ILCs is lineage and tissue specific. Indeed, Nfil3 −/− mice retain trNK cells in uterus, liver, salivary glands, and skin, as well as thymically derived NK cells (23)(24)(25), and we have shown that they also have uILC3s (9). Moreover, the few residual cNK cells in Nfil3 −/− mice exhibit normal effector functions in response to mouse cytomegalovirus infection (26). How the absence of NFIL3 impacts maternal responses to the endocrine and immunological changes occurring during gestation is unknown. We have begun to answer these questions by describing the changes in uILCs during pregnancy in Nfil3 −/− mice mated with wild-type (WT) males. Using these mice, here we show that absence of maternal NFIL3 lead to a dramatic reduction of cNK cells and uILC2s during pregnancy. Despite the expansion of residual CD49 + Eomes + trNK cells and uILC3s and the presence of abundant CD49 + Eomes − uILC1s at midgestation, the uterine vasculature in Nfil3 −/− females failed to complete the vascular remodeling process necessary to ensure ample and steady blood supply to the fetoplacental unit. This was associated with placental abnormalities and reduced fetal growth, although litter sizes were normal. The results demonstrate that NFIL3 regulates important functions for reproduction in mice, including key aspects of uILCs. resUlTs anD DiscUssiOn curtailed expansion of nfil3 −/− unK cells in response to Pregnancy We asked if the residual uNK in virgin Nfil3 −/− females require NFIL3 to expand at midgestation. To do this, we quantified CD3 − NK1.1 + NKp46 + uNK numbers in both virgin and pregnant Nfil3 −/− females and compared them with numbers in WT females. Both Nfil3 −/− and WT females were mated with WT males to restrict NFIL3 deficiency to maternal tissues. Pregnant uteri were dissected into decidua -which is the transformed mucosa that forms in response to blastocyst implantation in mice -and the muscular outer layer, the myometrium, which includes a transient lymphoid structure that forms in rodents during pregnancy, the mesometrial lymphoid aggregate of pregnancy (MLAp). CD3 − NK1.1 + NKp46 + uNK expanded from 2.3 × 10 4 cells in virgin to 13.7 × 10 4 cells in pregnant WT mice (5.9-fold increase). The expansion in Nfil3 −/− mice was less pronounced, from 0.9 × 10 4 cells in virgin to 2.4 × 10 4 cells in pregnant mice (2.7-fold increase). Most of this expansion in both WT and Nfil3 −/− dams was due to accumulation of uNK cells in the myometrium ( Table 1).
Based on the expression of the two integrins CD49a (ITGA1, VLA-1) and DX5 (CD49b, ITGA-2, VLA-2), uterine CD3 − NK1.1 + NKp46 + cells can be divided in two subsets: CD49a + DX5 − and CD49a − DX5 + cells, which resemble similar subsets in liver trNK and cNK cells, respectively (25). Figure 1A shows a representative flow cytometry analysis, Figure 1B a diagrammatic representation, and Table 1 summarizes the quantification of the expansion of the two subsets during pregnancy. The expression of CD49a and DX5 has been described as mutually exclusive on the two subsets both in liver and uterus (25); however, we found that DX5 was expressed on most uterine CD49a + cells. In Nfil3 −/− mice, the expression of DX5 on many CD49a + cells increased at midgestation, albeit less than on cNK cells in WT mice ( Figure 1A). NFIL3 may regulate expression of integrins (27), and we found that in Nfil3 −/− dams, expression of DX5 on some of the CD49a + cells became even higher than that on cNK cells ( Figure 1A).
Ex vivo, both CD49a + cells and cNK cells stained positive for Ki-67, a marker strictly associated with cell proliferation ( Figure 1C). Given the considerable expansion and the lower rate of proliferation in decidual cNK cells in WT mice (Figures 1A,C), it is conceivable that some of decidual cNK cells are cNK cells that came from the periphery. Another possible explanation is the in situ generation from progenitors, and both scenarios remain to be tested. Like the hepatic CD49a + cells, also uterine CD49a + cells were CD69 + and CD62L − (Figure 1C). We analyzed Eomes expression, granularity, and DBA reactivity on the two subsets of CD49a + and CD49a + uterine cells gated on CD45 + CD3 − CD19 − NK1.1 + NKp46 + cells. All CD49a − cNK and most CD49a + cells expressed Eomes in WT mice (Figure 2A), thus marking them as "bona fide" NK cells. The minority of the CD49a + cells that did not express Eomes are Eomes − uILC1s (9). uNK have also been known as "granulated" cells in several species (28). The granularity of CD49a + cells was more pronounced than that of cNK cells (Figure 2A). Perhaps the most distinctive feature of mouse uNK cells is the positivity for DBA staining (29), although more recently, it has become apparent that some uNK cells are not reactive for DBA (30,31), may be the largest producer of IFN-γ (32) and, if inhibited, contribute to insufficient uterine  adaptations to pregnancy (13). DBA staining mostly colocalized with Eomes staining in tissue sections of both WT and Nfil3 −/− dams ( Figure 2B). Although both CD49a + cells and cNK cells stained positive for DBA, CD49a + cells showed a brighter staining than cNK cells (Figure 2A), suggesting that these cells may relate more closely to what traditionally has been referred to as a major subset of uNK cells. In line with this, CD49a + cells produced less IFN-γ than cNK cells after stimulation with PMA and ionomycin or with IL-12 and IL-15 (not shown).

expansion of group 1 Uterine ilc1 subsets in Pregnancy
The data here and those in the accompanying manuscript (33) corroborate our recently published data, which show that Eomes expression defines two populations of group 1 ILCs within the CD49a + subsets, which, along with cNK cells make up the three subsets of group 1 uILC1, defined collectively as CD45 + CD3 − CD19 − NK1.1 + NKp46 + cells (9). The three subsets are CD49a + Eomes + trNKs, CD49a + Eomes − ILC1s, and CD49a − DX5 + cNK cells (9). We next set out to define how pregnancy and the lack of NFIL3 affect the changes in these three subsets of group 1 ILC1s. Eomes + trNK cells expanded about sevenfold in Nfil3 −/− mice ( Figure 2C). This expansion was nearly as extensive as in pregnant WT mice; however, trNK cells were still over fivefold less numerous in Nfil3 −/− dams than in WT dams, presumably because they were already decreased in virgin mice. Eomes − uILC1s did not seem to expand in Nfil3 −/− dams and expanded very little in pregnant WT dams too. CD49 − DX5 + cNK cells did not expand at all in Nfil3 −/− dams, whereas they expanded 3.7-fold in WT dams (Figures 1A and  2C; Table 1). These results show that the population of uterine group 1 ILC that expand the most during pregnancy in WT mice is trNK cells, with some expansion of cNK cells and little or no expansion of Eomes − uILC1 cells ( Figure 2C; Table 1). NFIL3 is required for development and expansion of uterine cNK cells and for development of trNK cells, but is dispensable for the expansion of trNK cells and the development of Eomes − uILC1s.

expansion of Uterine ilc2 and ilc3 in Pregnancy
NFIL3 is important also for the development of other ILCs. Nfil3 −/− mice are severely deficient in ILCs in several tissues, such as the intestine or the lungs (19)(20)(21). Recently, we have shown that only uILC2s strictly require NFIL3 to develop in the uterus of virgin mice (9). Indeed, uILC2s were not detectable in virgin Nfil3 −/− mice and barely detectable in pregnant Nfil3 −/− mice (Figure 2D), and uILC3s were present in virgin Nfil3 −/− mice but they did not expand as in WT mice during pregnancy ( Figure 2C). It will be interesting to study the potential role of uILC2s and uILC3s during murine pregnancy, for example, in MLAp formation. Moreover, ILC3s are the most abundant non-NK ILCs in the human uterus (9, 10), and they may play roles in human pregnancy.
Altogether, the data show that NFIL3 may serve different roles in different uILC lineages. Homeostatic development of trNK in virgin mice, but not expansion in response to pregnancy, requires NFIL3, whereas the opposite seems to be true for uILC3s. On the other hand, cNK and uILC2s require NFIL3 for both development and expansion.
Maternal, Placental, and Fetal abnormalities in nfil3 −/− Dams Our data show that Nfil3 −/− mice exhibit defects in the development and/or the expansion of uterine cNK cells and other uILCs during pregnancy. The residual trNK cells, as well as the Eomes − uILC1s and the uILC3s found in Nfil3 −/− dams, may however sustain the uterine adaptations necessary for placentation and fetal growth. In order to test this, we compared uterine arterial changes, litter sizes, and placental and fetal growth in WT and Nfil3 −/− dams mated with syngeneic WT mice. In contrast to what was found in allogeneic pregnancies (34), we found normal litter sizes. Therefore, the lack of NFIL3 does not impact on the number of live births in our crosses. Previous work with mice that do not develop uNK cells or have hypofunctional uNK cells showed that these mice too have normal litter sizes. However, analysis of uterine tissues and fetoplacental growth revealed clinically relevant abnormalities in these mice (11,13).
To assess the role of residual ILCs in Nfil3 −/− mice during pregnancy, we used stereology and immunohistochemistry to analyze morphological aspects of implantation sites in Nfil3 −/− females mated with WT males. DBA + uNK cells were readily detected in uterine sections of both WT and Nfil3 −/− mice, including in the decidua and in the MLAp (Figures 3A,B). The MLAp is reduced in size in NK-deficient mice (11) or in mice engineered to have hypofunctional uNK cells (13), and we found here that it was also the case in Nfil3 −/− dams (Figures 3A,B). Vascular walls were thicker with incomplete loss of smooth muscle actin (SMA) in Nfil3 −/− dams (Figures 3B,C), a phenotype typical of NK-deficient mice (11,13). Impaired arterial remodeling in uNK-deficient mice results in placental abnormalities and fetal growth restriction. Nfil3 −/− dams and their fetuses displayed phenotypes similar to those of NK-deficient mice ( Figure 4A). Conversely, WT females mated with Nfil3 −/− males had normalsized fetuses and placentas ( Figure 4B). The comparison of Nfil3 −/+ with Nfil3 +/− conceptuses showed that the reduction in fetal weight and the increase in placental weight is a phenotype of maternal origin (Figure 4C).
The labyrinth zone of mouse placenta is analogous to the human placental villi, where nutrient and gaseous exchange take place. The volume of the labyrinth zone was larger in placentas of Nfil3 −/− dams (Figures 4D,E). Trophoblast and maternal blood spaces, as well as the thickness of the barrier separating the fetal and maternal blood spaces, were increased in placentas of Nfil3 −/− dams ( Figure 4E). These abnormalities, with the exception of the surface area of placental exchange, mirror those in NK-deficient Rag2 −/− Il2rg −/− dams ( Figure 4F) and are consistent with altered placental hemodynamics secondary to deficient spiral artery remodeling (35,36). The reason and the significance of the divergent phenotype of the surface area of placental exchange in Nfil3 −/− and Rag2 −/− Il2rg −/− dams are unclear; however, it may reflect the difference in cellularity in the two strains, with residual ILCs and other lymphocytes in Nfil3 −/− dams and total lack of all lymphoid cells in In a mouse model of allogeneic pregnancy, Fu et al. found increased Th17 cells in the decidua of Nfil3 −/− dams, which was associated with fetal demise, and they suggested that the absence of uNK cells in Nfil3 −/− dams might have caused Th17 expansion and breakdown of tolerance (34). We find here that some NK are instead present in Nfil3 −/− dams at midgestation, alongside Eomes − uILC1s, and uILC3s. In conclusion, our results show that the residual uILCs in Nfil3 −/− dams are not sufficient to mediate normal uterine adaptations to pregnancy. Placentation and fetal growth is compromised in the absence of NFIL3, which emerges as an essential mediator of non-redundant functions for reproduction in mice.

MaTerials anD MeThODs
Mice C57BL/6J mice were purchased from Charles River UK and were used as WT mice in all experiments. Nfil3 −/− and Rag2 −/− IL2rg −/− mice were in C57BL/6J background as previously described (17,37). All mating experiments used virgin females mated either with C57BL/6J males or with Nfil3 −/− males as indicated. Gestation day (gd) 0.5 was counted at noon of day of the appearance of a copulation plug. Virgin and pregnant females were age matched (7-12 weeks). All procedures were conducted in accordance with the University of Cambridge Animal Welfare and Ethical Review Body and United Kingdom Home Office Regulations.

cell Preparation
Ovaries, mesometrium, and cervix were removed from all uteri in ice-cold PBS without Ca 2+ /Mg 2+ . Virgin uteri were entirely minced with scissors. For gd9.5 and gd10.5 mid-gestated uteri, myometrium tissue (including MLAp) were first dissected from the decidua and pooled. The decidua basalis (containing placenta) tissues were also separately minced and pooled. Embryos, yolk sacs, and decidua parietalis were discarded.

stereology analysis of Mlap Volume and Decidual arteries
Tissue volumes were quantified from serial sections using the Cavalieri method (40). Briefly, MLAp cross-sectional areas were calculated on serial sections and volumes were calculated by accounting for the spacing between the sections as previously described (41). Arterial remodeling was evaluated in the central decidua basalis on sagittal sections and veins localized at the periphery were excluded. Three to five implantation sites from randomly chosen uterine horn were analyzed per litter. In every implantation site, three to five largest vessels were measured in triplicate (three sections with distance of 49 μm apart). Lumen area and vessel-to-lumen ratios were quantitated by using outlines tools from NDP viewer (Hamamatsu) and Aperio ePathology (Leica Biosystems) softwares. SMA IHC staining was used for further qualitative assessment of arterial remodeling (41).

stereology analysis of Placentas
Midsagittal cross-sections of placental tissue (gd18.5) were stained with H&E using standard protocols. From each of nine WT and five Nfil3 −/− mice, the placenta closest in weight to the litter mean was selected for stereology analysis. The fractional volumes of the labyrinth zone (Lz) were assessed by point counting (10× objective lens, the newCAST Computer Assisted Stereological Toolbox system Visopharm, Hoersholm, Denmark) with systematic random sampling (42). Assuming a density of 1 mg mm −3 , fractional volumes were converted to absolute volumes in cubic millimeter by multiplying by total placental volume (43). The other placental halves were fixed in glutaraldehyde and embedded in Spurr's epoxy resin and a single mid-line section taken (1 μm thickness) then stained with toluidine blue. Volumes of maternal blood space and trophoblast in the Lz, surface area, and thickness of the barrier were calculated at 100× magnification using stereological methods that were described previously (42).
statistics Data were analyzed using paired or unpaired parametric Student's t-test or non-parametric Mann-Whitney test. Fetal and placental weights were analyzed using a mixed model approach to test the effect of maternal genotype and account for both gestational age and clustering of observations by litter (44). P < 0.05 was taken as statistically significant for all tests. Analyses were performed using GraphPad Prism and IBM SPSS.