Leukemia Inhibitory Factor (LIF) Inhibition during Mid-Gestation Impairs Trophoblast Invasion and Spiral Artery Remodelling during Pregnancy in Mice

The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Trophoblast cell proliferation, migration and invasion into the endometrium are fundamental events in the initiation of placentation. Leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion in vitro, however its precise role in trophoblast invasion in vivo is unknown. We hypothesized that LIF would be required for normal trophoblast invasion and spiral artery remodeling in mice. Both LIF and its receptor (LIFRα) co-localized with cytokeratin-positive invasive endovascular extravillous trophoblasts (EVT) in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via administration of our unique LIFRα antagonist, PEGLA, resulted in abnormal trophoblast invasion and impaired spiral artery remodeling compared to PEG control. PEGLA-treated mouse decidual vessels were characterized by retention of α-smooth muscle actin (αSMA)-positive vascular smooth muscle cells (VSMCs), while PEG control decidual vessels were remodelled by cytokeratin-positive trophoblasts. LIF blockade did not alter F4/80-positive decidual macrophage numbers between treatment groups, but resulted in down-regulation of decidual transcript levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10), which are important immune cell activation factors that promote spiral artery remodeling during pregnancy. Our data suggest that LIF plays an important role in trophoblast invasion in vivo and may facilitate trophoblast-decidual-immune cell cross talk to enable adequate spiral artery remodeling.


Introduction
Trophoblast cell proliferation, migration and invasion into the endometrium are critical events in the initiation of placental development [1].Specialized extravillous trophoblast (EVT) cells migrate and invade into the decidua and remodel the maternal spiral arterioles to create widebore arteries and reduce utero-placental resistance [2].Inadequate or inappropriate spiral artery remodeling and placentation is thought to be a leading cause of pregnancy complications [3].
Placental development is highly regulated spatially and temporally by numerous factors, such as cytokines produced within the local uterine environment [4].Such factors can ultimately determine the success or failure of pregnancy.Leukemia inhibitory factor (LIF) is one of the most important cytokines in the female reproductive tract [5].LIF is a member of the Interleukin-6 (IL-6) family of cytokines and is a secreted glycoprotein that signals via the LIF receptor α-chain (LIFRα)/gp130 heterodimeric complex to activate the downstream signalling cascade including the JAK-STAT [1,[6][7][8][9] and the extracellular signal regulated kinase (ERK) pathways [10].
LIF is required for uterine blastocyst implantation in mice [11,12] and is thought to play a critical role in implantation in primates and women [13][14][15].LIF-null female mice are infertile due to defects in embryo implantation [14,16].Since implantation fails, these mice are not useful in investigating the role of LIF in placentation or trophoblast function.We have shown that blocking endogenous LIF action during the peri-implantation period using a novel polyethylene glycol (PEG) conjugated LIF-antagonist (PEGLA) mimicked this phenotype [12,17].Conversely, LIFR-knockout mice are perinatal lethal and die within 24 hours of birth [18].In these mice, placental morphology is dramatically altered, which likely contributes to the perinatal loss.However the fetus is also LIFR-deficient, which results in impaired skeletal and neuronal development [18], preventing the generation of adult LIFR -/-mice to investigate the role of LIF in placentation.These studies do highlight however, the critical importance of LIF action during pregnancy.
Well-characterised animal models can provide insight into the mechanisms of abnormal placentation.The mouse and human placenta share a high degree of proteomic and molecular genetic homology [19] and overall physiological similarities [20].EVT remodeling of maternal spiral arteries occurs in both humans and mice, although this remodeling is shallower in mice [21].LIF and LIFR have never been localized in the mouse placenta, so it is unclear in which cell types LIF signalling is most important.In women, LIF and LIFR mRNA and LIFR protein are expressed in the chorionic villi, decidua and EVTs of first trimester and term placenta/ decidua [22,23].Functionally, LIF activates STAT3 in human primary extravillous trophoblast (EVT) cells and stimulates their adhesion to primary endometrial epithelial cells [24].LIF also promotes primary human first trimester placental villous trophoblast migration and invasion [25], suggesting a potential role in EVT invasion and spiral artery remodeling, however, this has never been investigated in vivo.
Strong expression of LIF mRNA has been also detected in decidual leukocytes in women [22], suggesting that LIF may mediate interactions between maternal decidual leukocytes and invading trophoblasts.During optimal spiral artery remodeling, there is a transient influx of decidual leukocytes, including activated macrophages, thought to initiate the disruption of the organized VSMC layer and endothelium required for trophoblast penetration of the vessel [26].Impaired macrophage recruitment or activation can prevent complete spiral artery remodeling [27].However, it is unknown whether LIF alters vessel remodeling and whether this occurs via a macrophage-dependent mechanism.
Due to the pattern of localisation of LIF in human EVTs and its functional in primary human first trimester EVT in vitro, we hypothesised that LIF is required for normal trophoblast invasion and spiral artery remodelling at the fetal-maternal interface in vivo.Given the difficulties of studying first trimester placental development in women, we employed a mouse model and co-localized LIF and LIFR with cytokeratin-positive trophoblasts in wild-type mouse decidua.In order to circumvent the lack of available knockout mouse models, we utilized our unique LIF inhibitor, PEGLA to determine the effect of transient LIF blockade during trophoblast invasion and spiral artery remodeling in mice.

Ethics statement
All procedures were approved by the Monash Medical Centre Animal Ethics Committee (#MMCB/2012/17) and followed the NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (S1 Table ).

Wild-type mouse implantation site tissue collection
Female (virgin 8-12 weeks old) and male C57BL/ 6J mice (Monash Animal Services, Clayton, Australia) were housed under conventional conditions, with food and water available ad libitum and held in a 12h light and dark cycle.Implantation sites were collected from wild-type mated female mice on embryonic days (E) 6-17 of gestation (E0 = day of plug detection) (n = 3 mice/time point).Whole, intact implantation sites (IS) containing the placenta, maternal decidua and fetus were dissected out and fixed in 10% neutral buffered formalin overnight, or snap frozen.

LIF inhibition during pregnancy in mice
We produced polyethylene glycol (PEG) conjugated LIF-antagonist (PEGLA) as previously described [12].The LIF antagonist binds to the LIF receptor α-chain with high affinity but no longer binds to the co-signal transducing subunit, gp130, preventing the initiation of downstream signalling.LA was covalently conjugated to PEG (PEGLA) to increase the period of serum retention [28].To inhibit LIF action, mated female mice were administered by intraperitoneal (IP) injection with 600μg/dose PEGLA (20mg/kg/dose) or equal molarity PEG as a control (as optimized previously [17,29,30]), twice daily at E8-10 or E10-13 (n = 4 mice/group).Mice were killed at E10 or E13 of pregnancy respectively, on the final day of PEGLA treatment.Whole, intact implantation sites (IS) containing the placenta, maternal decidua and fetus were dissected out and fixed in 10% neutral buffered formalin overnight, or snap frozen.

RNA extraction and polymerase chain reaction
Total RNA was isolated from snap frozen wild-type or PEG/PEGLA treated mouse decidual tissue using TriReagent.Genomic DNA was digested using the DNAfree kit (Ambion) according to the manufacturer's instructions.RNA samples were analysed by spectrophotometry (Nanodrop) at an absorbance ratio of A260/280nm to determine RNA concentration, yield and purity.cDNA was synthesized from total RNA (250ng) using Superscript III reverse transcriptase (Invitrogen) and analyzed by spectrophotometry at an absorbance ratio of A260/ 280nm to determine concentration and purity.PCR reactions were performed using PCR express machine (Thermo Fisher Scientific) and GoTaq master mix (Promega) according to the manufacturer's instructions.cDNA was analysed for LIF, LIFR, gp130, MCP-1, IL-10, IFNγ and β2-microglobulin (Table 1) using reaction conditions of an initial denaturation at 95°C for 3 mins, followed by 30 cycles of: denaturation, 95°C for 30 secs; annealing, 60°C for 30 secs; extension, 72°C for 1 min; with a final extension at 72°C for 10 min.The PCR products were run on a 1.5% agarose gel with 1000bp DNA ladder (Invitrogen).To semi-quantify gene expression, the gel was scanned using Bio-Rad Gel Doc EZ Imager and band intensity (optical density) of the gene of interest was normalized to β2-microglobulin, using Image lab Software (version 4.1, Bio-Rad).

Primer
Sequence: forward (F) and reverse (R) containing DAPI (DAKO).Digital photographs at 1X were taken and CellSense software was used to quantify pixel density and expressed as intensity per area to give a percentage.Decidual area was quantified by measuring the cross-sectional area and expressed per total implantation site area as a percentage.

Statistical analysis
GraphPad Prism 6.0 (GraphPad Software) was used for all statistical analyses.Data were analysed by students t-test when comparing two groups, or ANOVA followed by Tukey's post-hoc test when comparing more than two groups.Data were expressed as the mean ± SEM.Differences were considered significant at p<0.05.

Results
LIF, LIFR and gp130 are expressed in the mouse decidua throughout gestation LIF mRNA was produced in the mouse decidua throughout gestation and levels were significantly increased in E17 late-gestation decidua compared to E6 whole implantation sites (Fig 1).Levels of LIFR and gp130 mRNA were unchanged in whole mouse implantation sites at E6 and E8, or decidua alone from E10-17 of gestation in mice (Fig 1).

LIF and LIFR co-localize with endovascular EVTs in mouse implantation sites
To determine whether EVTs in mice produce LIF and LIFR, we co-localized these with cytokeratin-positive trophoblasts in serial sections from wild-type implantation sites at mid-gestation (E13).Both LIF and LIFR localized to endovascular EVTs in mouse implantation sites (Fig 2).

LIF blockade during placentation impairs trophoblast invasion and spiral artery remodeling in mice
In order to determine the role of LIF on trophoblast invasion in vivo, which occurs maximally during mid-gestation from E8-E13 [31], mice were treated with PEG vehicle control or PEGLA from either E8-10 or E10-13 to block LIF action.Mice were sacrificed on the final day of PEGLA

LIF inhibition reduced decidual transcript levels of immune cell activation factors that promote spiral artery remodeling during pregnancy
To determine the effect of LIF inhibition during trophoblast invasion and placentation in mice, we immunolocalized macrophages in PEG or PEGLA treated mouse implantation sites at E10

Discussion
This study is the first to determine the role of LIF in mediating trophoblast invasion during placental development in vivo.We found that LIF and its signalling components, LIFR and gp130 are expressed in the mouse decidua at the transcript level during the time of trophoblast invasion and spiral artery remodeling.Furthermore, LIF and LIFR both co-localized to invasive endovascular EVTs within the mid-gestation mouse decidua, supporting a potential functional role in EVT invasion that has previously been reported in humans [24,25].By temporally blocking LIF action during the initiation of trophoblast invasion and placentation in mice  using a unique antagonist, PEGLA, we demonstrated that LIF is required for normal trophoblast invasion in vivo.
In women the high levels of LIF expression in first trimester decidua and the localization of its receptor on trophoblast cells suggests paracrine actions during placentation [22,23].Previously, we localized LIF to the mouse decidua post-implantation at E5 [32], however this is before the time of EVT invasion and spiral artery remodeling during mouse pregnancy, which occurs predominantly between E8-13 [33].In the present study, we confirmed LIF and LIFR mRNA production in the mouse decidua during this time and also EVT production of LIF and LIFR.However, we observed no decidual alterations following LIF inhibition, likely due to the fact that mice were administered with PEGLA from E8 of gestation onwards, after the time of critical establishment of the maternal decidua in mice [31].
As hypothesized, LIF inhibition resulted in abnormalities in EVT invasion and spiral artery remodeling in mice.These in vivo findings strongly support previous in vitro findings in humans where LIF has been shown to promote JEG-3 choriocarcinoma cell line invasion [34] and also primary trophoblast invasion [24,25].Meanwhile, PEGLA has been demonstrated to inhibit this effect [25].In women, abnormal trophoblast invasion and impaired spiral artery remodeling is associated with pregnancy complications such as placental insufficiency, intrauterine growth restriction (IUGR) and preeclampsia [35].At term, placental LIF is elevated in women with preeclampsia [32].However, it is unclear whether LIF may be a causal factor or a byproduct of abnormal placentation.To our knowledge, LIF levels in maternal serum have not been investigated during the first trimester of pregnancy, when abnormal trophoblast invasion occurs in women, prior to the onset of preeclamptic symptoms.This may give some indication of placental LIF levels and offer some insight as to whether reduced LIF levels are present in women with likely impaired spiral artery remodeling as seen in the PEGLA-treated mice.
In vitro studies suggest that LIF may exert direct effects on trophoblasts and/or decidual cells to facilitate EVT invasion.Previously, we reported that LIF alters the balance between matrix metalloproteinase (MMPs) and tissue inhibitors matrix metalloproteinase (TIMPs) required for decidual extracellular matrix (ECM) breakdown [36], in primary human EVTs [24].Strong expression of LIF mRNA has been detected in decidual leukocytes in women [22], suggesting that LIF may also act to facilitate immune-cell mediated trophoblast invasion and spiral artery remodeling.LIF is a macrophage-derived cytokine [37] and furthermore, macrophages numbers are reduced by more than half in implantation sites during early pregnancy in LIF-null mice [38], suggesting that LIF acts as a chemo-attractant for these cells.
We investigated this mechanism of LIF-mediated trophoblast invasion in the present study.However, there were no differences in the number of macrophages in between PEGLA or PEG-treated mouse implantation sites at E10 or E13.Macrophages were detected by immunohistochemistry in multiple decidual cross-sections per mouse, although a preferable method from complete quantitation of these cells could be by flow cytometry.Our findings could also suggest that LIF is required for macrophage recruitment very early during pregnancy in mice, prior to E8 when PEGLA was administered.Indeed, changes in macrophage recruitment in LIF -/-mice were found as early as post-implantation D3 [38].Despite this finding, interestingly, we did see a reduction in transcript factors required for macrophage recruitment and activation, suggesting that LIF may contribute to macrophage activation status.
Decidual macrophages are the second most abundant immune cell population at the implantation site in women during the first trimester, comprising 20-30% of immune cells in the uterine decidua [39,40].Dysregulated macrophage activation in the decidua has been implicated in recurrent miscarriages [41].Thus, understanding the signalling pathways that regulate decidual macrophages may elucidate causes of early pregnancy failures.Macrophages are broadly classified into the classically activated M1 phenotype or the alternatively activated M2 phenotype.Macrophages can be polarized by IFN-γ into M1, which are 'proinflammatory', present antigens, produce reactive oxygen species and skew cell mediated immune responses [26].Alternatively, IL-10, among other factors can induce M2 polarization, which promote tissue remodeling and immune tolerance [26].During pregnancy, MCP-1 (CCL2) produced by decidual cells attracts new macrophages into the decidua, leading to protection against rejection of the fetus and appropriate vessel remodeling by balancing the M1/M2 ratio [26].MCP-1 transcript levels were significantly reduced in PEGLAtreated decidual tissue compared to control.Although there were no statistical differences in the numbers of decidual F4/80-positive macrophages between treatment groups, there was a trend in reduced macrophage numbers in PEGLA-treated decidua at both E10 and E13, which supports the reduced MCP-1 transcript levels, suggesting that reduced LIF signalling could impair macrophage recruitment.It would be interesting to determine the effect of impaired LIF signalling earlier in pregnancy during decidualization, when macrophage recruitment is maximal [42].Furthermore, IL-10 transcript levels were significantly down regulated following LIF inhibition compared to control, suggesting that LIF may alter macrophage polarization.However, this would need to be investigated using specific macrophage markers to differentiate the subtypes.

Conclusion
In this study we have demonstrated that targeted inhibition of LIF signalling during mid gestation leads to abnormal trophoblast invasion and maternal decidual spiral artery remodeling in vivo in mice.These data warrant future studies investigating the broader relationship between LIF and immune cells during pregnancy and strengthen the rationale that LIF is a critical player in the establishment of a healthy pregnancy.

Fig 2 .
Fig 2. LIF and LIFRα co-localization with cytokeratin to detect invasive EVTs in wild-type mouse decidua.Wild type (WT) mouse implantation sites were collected from n = 3 mice/time point and 2μm serial sections were immunostained for cytokeratin, LIF and LIFRα.Representative photomicrographs of mid-gestation (E10 and E13) implantation site sections are shown here.Both LIF and LIFRα co-localized with cytokeratin in maternal decidual vessels.Bars represent 20μm.Insets are negative controls.doi:10.1371/journal.pone.0129110.g002