FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2015903912, filed on 25 September, 2015, entitled "A method of treatment and prophylaxis", the entire contents of which, are incorporated herein by reference.

BACKGROUND FIELD

[0002] The present invention relates to the treatment or prevention of preeclampsia or symptoms thereof. The use of biomarkers indicative of preeclampsia or a risk of developing preeclampsia is also taught herein.

DESCRIPTION OF RELATED ART

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0005] Preeclampsia (PE) is a pregnancy-induced disorder unique to humans characterized by hypertension and proteinuria. It affects approximately 8% of pregnancies (Sibai et al. (2005) Lancet 3(55:785-799). The etiology is poorly understood and there are no early detection tests and a complete lack of a pharmacological treatment option (Kaufmann et al. (2003) Biol. Reprod. 69: 1-1). The only known cure is delivery of the placenta generally requiring a premature birth. Nevertheless, there is substantial evidence that abnormal placentation is the key underlying cause. During pregnancy, highly invasive extravillous trophoblasts (EVT) acquire vascular- like properties to remodel uterine spiral arterioles. This creates low-resistance, large-diameter vessels that promote utero -placental blood supply required to sustain fetal growth and development (Pijnenborg (1994) Reprod Med Rev 3:53-73; Brosens et al. (1972) Obstet. Gynecol. Annu. 1 : 177-191). It is widely accepted that inadequate trophoblast invasion and spiral artery remodeling is an initiating factor in the development of PE (Naicker et al. (2003) Acta obstetrician et gynecological Scandinavica 82(8):Ί22-Ί29). This leads to reduced utero -placental arterial flow, oxidative stress (Burton et al. (2009) Placenta 30 Suppl A:S43-48) and placental secretion of pro-inflammatory cytokines (Otun et al. (2011) Reprod Immunol 88(l): \-\ \) and angiogenic regulators such as soluble fms-like tyrosine kinase- 1 (sFlt-1) [Maynard et al. (2003) Journal of Clinical Investigation 111 :649-659]. This in turn triggers maternal endothelial dysfunction causing hypertension, proteinuria and peripheral and/or cerebral edema. Symptoms can differentially manifest during the second (early-onset, EO), or third (late-onset, LO) trimester (Vatten and Skaerven (2004) BJOG: an international journal of obstetrics and gynaecology 111(4 J:298-302). In addition to the maternal symptoms, PE is also frequently associated with prematurity (Sibai et al. (2005) supra) and intrauterine growth restriction (IUGR), related to impaired insulin-like growth factor (IGF-1) signaling (Liu et al. (1993) Cell 75(1 J:59-72).

[0006] It is well established that cytokines produced within the local uterine environment, can alter trophoblast action (Dimitriadis et al. (2005) Hum Reprod Update ll(6):6l3-630). Interleukin-(IL)-l l is a pleiotropic cytokine that regulates cell cycle, invasion and migration in numerous cell types (Paul et al. (1990) Proc Natl Acad Sci USA 87(19):! '512- 7516), all roles critical to placental development. IL-11 is a member of the IL-6-type cytokines and signals via the IL-11 receptor (R) chain and signal transducer gpl30 (Heinrich et al. (1998) Biochemical Journal 334:297-314) to activate the Janus kinase (JAK)/Signal transducers and activators of transcription (STAT)3 pathway in human endometrium (Dimitriadis et al. (2006) Endocrinology i47:3809-3817), primary human EVT (Paiva et al. (2009) Biology of reproduction 80:302-310; Paiva et al. (2007) Endocrinology 48:5566-5572). IL-11 and IL-11R are expressed by syncytio- and cyto- trophoblast cells, endovascular EVT and decidual cells during the first trimester (Paiva et al. (2007) supra). IL-11 is required for decidualization in humans (Dimitriadis et al. (2002) Mol Hum Reprod 8:636-643) and mice (Robb et al. (1998) Nature Medicine 4:303-308), though levels are elevated in PE decidual tissue (Basar et al. (2010) Reproduction 40:605-612). More recently, IL-11 has been shown to impede human EVT invasion in vitro (Paiva et al. (2009) supra; Sonderegger et al. (2011) Human Reproduction 2(5:2841- 2849), although its function in placentation in vivo has not been investigated.

[0007] There is a need to further understand and determine the casual mechanisms of PE. Early detection of PE or the identification of at risk individuals will better facilitate clinical intervention. There is also a need to identify therapeutic targets. An animal model of PE is also needed.

SUMMARY

[0008] The present invention is predicated in part on identifying IL-11 -mediated signaling as a casual factor in preeclampsia. This leads to the development of a protocol for the clinical management of this condition and its symptoms.

[0009] Hence, enabled herein is a method for preventing or ameliorating symptoms of PE in a pregnant human female subject, by the administration of an agent in an amount effective to normalize the level of signaling mediated by the IL-11 signaling pathway. Examples of agents include an antagonist of IL-11, IL-11 receptor a (IL-l lRa) and of IL- 11-IL-l lRa interaction. Alternatively, the agent antagonizes the activity or level of a component of the IL-11 signaling pathway such as but not limited to pregnancy-associated plasma protein A2 (PAPPA2). Furthermore, the agent may normalize the activity or level of a component regulated by IL-11 -mediated signaling such as IL-10 or placental growth factor (PLGF). Without limiting the present invention to any one theory or mode of action it is proposed that IL-11 or IL-11 signaling acts on the placenta to damage it and this results in an abnormal placenta which releases toxins into the maternal blood stream and this leads to symptoms of PE.

[0010] The method herein generally provides administering the agent for a time and under conditions sufficient to prevent or ameliorate the symptoms of PE. In an embodiment, the pregnant human female subject is first screened for the level of IL-11 in a blood or uterine fluid sample wherein an elevated level of IL- 11 compared to a normal control is indicative that the subject requires treatment with the agent. The normal control may be a sample from the same patient taken prior to PE developing or may be a statistically validated control cover. Reference herein to PE includes, in an embodiment, early onset PE. Early onset PE includes between about 10 weeks and 34 weeks gestation.

[0011] Taught herein is the use of an agent which normalizes the level of signaling mediated by the IL-11 signaling pathway in the manufacture of a medicament for the prevention or amelioration of symptoms PE in a pregnant human female subject. This extends to the treatment of a dysfunctional placenta leading to or with a potential to lead to PE.

[0012] Further taught is an agent which normalizes the level of signaling mediated by the IL-11 signaling pathway for use in preventing or ameliorating symptoms of PE in a pregnant human female subject.

[0013] The present specification also teaches a method for determining the level of risk that a pregnant human female subject will develop PE or symptoms of PE, by determining the level of IL-11 in the blood stream or uterine fluid of the subject and comparing the level to a control wherein an elevation of the level of IL-11 relative to the control is indicative of a high risk of developing PE and a control level or less than control level is indicative of a zero to low risk of developing PE. In an embodiment, the level of IL-11 is determined using an antibody specific for IL-11.

[0014] In relation to the latter embodiment, the antibody may be labeled with a reporter molecule capable of providing a detectable signal or the first mentioned antibody is detected bound to IL-11 by a second antibody specific for the first antibody wherein the second antibody is labeled with a reporter molecule capable of providing a detectable signal. The assay may also be in a multiplex format whereby multiple biomarkers are screened indicative of the state of a pregnancy wherein at least one marker is IL-11. Any number of ways may be employed to quantitate IL-11 levels or an associated component.

[0015] A therapeutic kit is also contemplated herein comprising an agent which normalizes the level of signaling of the IL-11 signaling pathway and reagents for use in determining the level of IL-11 in the blood stream or uterine fluid of a pregnant human female subject.

[0016] Yet another embodiment contemplated herein is a method of treating a pregnant human female subject, the method comprising adding a polyethylene glycol (PEG) group to a drug indicated for use in the subject to treat a disease or condition wherein the PEG group is selected to prevent the drug crossing the placenta and the position of the PEG group on the drug is selected to minimize any deleterious effect on the activity of the drug.

[0017] In an embodiment, the drug indicated is a drug to treat inter alia hypertension, diabetes, cancer, heart disease or liver or kidney dysfunction or microbial or viral infection.

[0018] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

[0019] A summary of sequence identifiers used throughout the subject specification is provided in Table 1.

Table 1

Summary of sequence identifiers

020] A list of abbreviations used in the subject specification is defined in Table 2. Table 2

Abbreviations

Abbreviation D iiiilion

CT Cytotrophoblast

EO Early onset

EVT Extravillious trophoblast

FSTL3 Follistatin-like-3 hCG Human chorionic gonadotropin

ML- 11 Human IL- 11

IGF-1 Insulin-like growth factor- 1

IL- 11 Interleukin-11

IL- l lRa Interleukin- 11 receptor alpha

IS Implantation sites

ISB4 Isolectin4

IUGR Intrauterine growth restriction

LO Late onset

PAPPA2 Pregnancy-associated plasma A2

PE Preeclampsia

PEG Polyethylene glycol

PEGIL- 11 PEGylated IL- 11

PLGF Placental growth factor rhIL- 11 Recombinant human IL- 11

SBP Systolic blood pressure

ST Syncytiotrophoblast

VSMCs Vascular smooth muscle cells

WT Wild type a-SMA a-Smooth muscle actin BRIEF DESCRIPTION OF THE FIGURES

[0021] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0022] Figures la through f are graphical and photographic al representations showing IL-11 and IL-l lR in wild-type mouse placenta and decidua throughout gestation. Wild type (WT) mouse implantation sites were collected from n=3 mice/timepoint. IL-11 and IL-l lR mRNA and protein expression were analyzed in E6, 8 and 10 embryos as whole implantation sites (IS) and E13, 15 and 17 were dissected into placenta and decidua. (a) IL-11 and (b) IL-l lR mRNA expression was determined by semi-quantitative PCR normalized to 2-microglobulin. Data are mean + SEM, ANOVA, Tukey's post-hoc test, **p<0.01. (c, d) IL-11 protein expression in placenta, IS and decidua were determined by Western blot normalized to GAPDH. Data are mean + SEM, ANOVA, Tukey's post-hoc test, ***p<0.001. Representative photomicrographs of mid-gestation (E13) implantation site sections immunostained for (e) IL-11 or (f) IL-l lR . (e; i) IL-11 localized to mononuclear trophoblasts (arrow) associated with maternal blood sinuses and (ii) endothelial cells lining fetal capillaries (asterix) in the placental labyrinth, (e; iii) IL-11 also localized to glycogen trophoblasts within the spongiotrophoblast layer at the maternal- fetal interface and (iv) trophoblast giant cells within the spongiotrophoblast layer at the maternal-fetal interface. Bars represent 100 m (i) and 20 m (ii-iv). (f; i) IL-l lR was produced abundantly throughout the decidua and placenta, (f; ii) IL-l lR localized to EVTs lining maternal spiral arteries in the decidua (arrow) and (iii) glycogen trophoblasts within the spongiotrophoblast layer at the maternal-fetal interface. Within the placental labyrinth, IL-l lR localized to all trophoblast subsets (iv) and endothelial cells lining fetal capillaries (asterix) (v). Bars represent 200 m (i) and 20 m (ii-iv). Insets are negative controls.

[0023] Figures 2a through h are graphical and photographical representations showing IL-11 promotes pSTAT3 in placental villous tissue from 1st trimester placentas and IL-11 and IL-11R are produced by the human placenta throughout gestation, (a) pSTAT3 immuno staining is localized in the syncytiotrophoblast (ST), cytotrophoblast (CT) and villous stroma of the placenta. Bar 200 m. Insets are negative controls, (b) IL-11 treatment increased pSTAT3 staining in CT and stroma of the villous core compared to control. Bar graph shows semi-quantitative analysis of staining intensity for pSTAT3 in ST, CT and stroma. Data are mean + SEM, students t-test, **p<0.01, (n=6/gp). (c) IL-11 and (d) IL- 11R localization and respective staining intensity scores (e, f) in human placenta and decidua throughout gestation. 1 ^st trimester <12 weeks; 2nd trimester 13-28 weeks; term delivery >38 weeks. Bars represent 200 m. Insets are negative controls. Staining intensity is reduced in 2nd trimester and term decidua compared to first trimester. Data are mean + SEM, ANOVA, Tukey's post-hoc test, *p<0.05, n=5/ gp. (g) IL-11 and (h) IL-11R mRNA expression in human placenta and decidua throughout gestation. 1st trimester <12 weeks; 2nd trimester 13-28 weeks; term delivery >38 weeks, n=5/gp.

[0024] Figures 3a through f are graphical and photographic al representations showing Administration of IL-11 (500 g/kg/day) to mice mimics IL-11 levels in PE women and activates STAT3. (a) Non-pregnant (NP) and pregnant (P) female mice at E13 were administered with recombinant human IL-11 and serum collected at 10 minutes (m), 30m, 1 hour (h), or 2h. (b) Representative photomicrographs of E13 saline or IL-l l-treated placental labyrinth (L) [top panel] and decidual regions (D) [lower panel] containing maternal spiral arteries immunostained for phosphorylated (p) STAT3. Bars represent 200 m. (c) IL-11 treatment increased numbers of positive pSTAT3 stained cells in the placenta and decidua. (d) Renal ischemia in pregnant IL-11 treated mice. Representative photomicrographs of E17 maternal kidneys, stained with H&E and Periodic acid Schiff (PAS) show enlarged glomerular morphology. Bars represent 50 m. (e) Narrow glomerular capillary lumen (L) and basement membrane thickening (asterix) in pregnant IL-11 treated mice at E17 shown by electron microscopy, low power magnification, xl500. (f) Circulating soluble (s)-Endoglin (Eng) levels in non-pregnant or pregnant serum at E13 were quantified by ELISA. Data are mean + SEM, 1-way ANOVA. [0025] Figures 4a through m are graphical and photographical representations showing IL-11 administration during pregnancy in mice alters placental development and contributes to maternal and fetal features of PE in mice. Non-pregnant or pregnant mice were treated with saline vehicle control or IL-11 (500 g/kg/day) from E8-13 (n=5) or E10- 17, or 8 days equivalent in non-pregnant mice (n=8). (a) E13 embryo sections were stained for trophoblasts (cytokeratin; red) or smooth muscle ( -SMA; green). Bars represent 50 m. (b) Decidual vessel area was measured in cross-sections from three placentas per mouse at E13. Data are mean + SEM, students t test, ***p<0.001; n=5. (c) Desmin immuno staining highlights decidual area at E13. (d) Staining intensity was quantified as pixel intensity/area (%). Data are mean + SEM; n=5. (e) Systolic blood pressure (SBP) was measured in IL-11 or saline-treated non-pregnant or pregnant mice at mid- (E14-15) and late-gestation (E16-17) by tail-cuff plethysmography. Data are mean + SEM, 1-way ANOVA, *p<0.05, ***p<0.001; n=8. (f) Circulating soluble (s)-Fltl levels were quantified by ELISA in mouse sera. Data are mean + SEM, 1-way ANOVA, **p<0.01; n=5. (g) Total urinary protein was quantified by Bradford colorimetric assay. Data are mean + SEM, students t test, **p<0.01; n=5. (h) Glomerular pathology of maternal kidneys from IL-11- treated pregnant mice at E17 including narrow glomerular capillary lumen (L) and basement membrane thickening (asterix) shown by electron microscopy at high power magnification, x8000. (i) Glomerular basement membrane thickening was quantified across all treatment groups. Data are mean + SEM, 1-way ANOVA, ***p<0.001; n=8. (j) Haematoxylin and eosin (H&E) staining shows E17 placental labyrinth structure is abnormal, with areas of necrosis (arrow head) and reduced vascular spaces following IL-11 administration. Masson's trichrome staining (MTC; middle panel) shows areas of collagen deposition (arrow heads) in the labyrinth following IL-11 treatment. Isolectin B4 (ISB4; lower panel) highlights extracellular matrix surrounding maternal sinusoid branches. Labyrinth vascular branching development is impaired (arrow head) in IL- 11 -treated mice. Inset is negative control. Bars represent 200 m. (k) Number of labyrinth vascular branches were counted, expressed as branches per placental area (mm2). Data are mean + SEM, students t test, **p<0.01, ****p<0.0001; n=8. (1, m) Fetal weight (E17) was reduced following IL-11 or PEGIL-11 treatment compared with saline or PEG control respectively. Data are mean + SEM, students t test, ****p<0.0001; n=8. (n) Representative photomicrographs of E17 PEG-treated implantation site immunostained for PEG, localized to mouse placental trophoblasts (arrow), but not fetal tissue. Bar represents 500 m (top panel) and 100 m (lower panel). Inset is negative control.

[0026] Figures 5a through e are graphical and photographical representations showing IL-11 administration during placental development in mice reduced invasive decidual trophoblast area and lead to spiral artery vascular smooth muscle cell retention, but did not alter decidual area. Representative photomicrographs of immunostained E13 implantation sites treated with saline or IL-11 from E8-13. (a) Cytokeratin highlights decidual trophoblasts (EVT). Bars represent 200 m (top panel) and 50 m (lower panel). (b)!Decidual and myometrial vessels from IL-11 treated pregnant mice have altered, narrow vessel morphology and thicker -smooth muscle actin ( SMA) lining compared to saline control. Bars represent 200 m (top panel) and 50 m (lower panel), (c) Staining intensity was analyzed in 3 mid-sagital implantation site sections per mouse and averaged using CellSens software, quantified as staining intensity (pixels) per decidual area (%). Data are mean + SEM, students t-test, *p<0.05; n=5. (d) F4/80 highlights decidual macrophages. Bars represent 50 m. Arrows denote F4/80-positive macrophages, (e) Macrophages were quantified expressed as number of positive cells per field (x20 magnification; 3 fields per tissue were analyzed from 3 placentas per mouse) Data are mean + SEM, students t test; n=5. Insets are negative controls.

[0027] Figures 6a and b are photographical representations showing In situ hybridization performed on implantation sites from mice treated with saline or IL-11 from E8-13 at E13 (a) or treated from El 0-17 at E17 (b). Markers include Syna -syncytiotrophoblast-II; Gmcl - syncytiotrophoblast-I; Mest - endothelial cells; Prlb - invasive extravillous trophoblast; Plf- trophoblast giant cells.

[0028] Figures 7a and b are photographical and graphical representations showing representative Western blot demonstrates PEGylated IL-11 [PEGIL-11] (lOOng/ml) and IL-11 (lOOng/ml) have similar activity, both activate pSTAT3 in HTR8 trophoblast cells after 30 minutes compared with PEG control. Data are mean + SEM; n=2. [0029] Figures 8a through k are graphical and photographical representations showing IL-11 impairs insulin growth factor-1 (IGF-1), up-regulates pappasylin-A2 (PAPPA2) in mouse and human placenta to alter extravillous trophoblast function and alters decidual uterine natural killer (uNK) cells, (a) Effects of single dose IL-11 treatment (500 g/kg) after 2h, on mouse placental gene expression normalized to control from highest to lowest abundance quantified by real-time PCR array; n=3. (b) Circulating IGF-1 levels were quantified by ELISA in mouse sera. Data are mean + SEM, 1-way ANOVA, *p<0.05, n=5. (c) PAPPA2 immuno staining shows increased placental and decidual levels following IL- 11 administration in E8-13 treated mouse embryo sections. Bars represent 500 m. Inset is negative control, (d) Data are mean + SEM, students t test, *p<0.05, **p<0.01, n=5. (e) First trimester human placental villous explants cultured with saline or IL-11 (lOOng/ml) for 48 hours were immunostained for PAPPA2 or negative control IgG (inset), (f) PAPPA2 levels were increased in the syncytiotrophoblast (arrows). Data are mean + SEM, students t test, ***p<0.001, n=5. (g) Representative images of first trimester human placental villous explants cultured on collagen drops to model villous outgrowth and invasion, (h) Treatment with recombinant human PAPPA2 (lOOng/ml) or IL-11 (lOOng/ml) similarly reduced trophoblast outgrowth compared to saline vehicle control. PAPPA2 knockdown by siRNA rescued IL-11 -impaired EVT outgrowth. Data are mean + SEM, ANOVA, Tukey's post-hoc, **p<0.01; n=5. (i) DBA lectin staining highlights decidual uNK cells in E13 implantation sites (upper panel) and high power images show double nuclei uNK cells in IL-11 treated implantation sites (lower panel), (j) Decidual uNK cells and (k) double nuclei uNK cells were quantified expressed as number of positive cells per field (x20 magnification; 3 fields per tissue were analyzed from 3 placentas per mouse) Data are mean + SEM, students t test, ***p<0.0001, n=5.

[0030] Figures 9a through g are graphical and photographical representations showing (a) Quantitative real-time PCR shows PAPPA2 mRNA levels in placenta and decidua of mice at E17, treated with saline or IL-11 from ElO-17. (b) Western blot shows protein levels of PAPPA2 up-regulated in placental tissue from IL-11 treated mice compared to control at E17, treated with saline or IL-11 from ElO-17. (c) Representative Western blot shows serum levels of PAPPA2 up-regulated in IL-11 treated mice compared to control at E13 (IL-11 treatment E8-13) and E17 (IL-11 treatment E10-17). (d) PAPPA2 is produced in the non-pregnant mouse uterus (arrows), (e) IL-11 does not alter circulating PAPPA2 in non-pregnant mice treated for 8 days (equivalent to E10-17 treatment) in pregnant mice, (f) Knockdown of PAPPA2 by siRNA resulted reduced PAPPA2 protein in human placental villous explants compared to scrambled sequence control (scr), shown by (g) Western blot and quantification of PAPPA2 by densitometry, normalized to GAPDH. Data are mean + SEM, students t test, *p<0.05, n=5.

[0031] Figures 10a through g are graphical and photographical representations showing IL-11 withdrawal at mid gestation rescues PE features in mice. Pregnant mice were treated with IL-11 (500 g/kg/day) from E10-14 (IL-11 withdrawal) n=8. (a) In the IL-11 withdrawal treatment group, systolic blood pressure was reduced at late-gestation (E16- 17); (b) total urinary protein was reduced and (c) fetal weight (E17) was significantly increased at late gestation compared to IL-l l-treated mice from E10-17. Data are mean + SEM, 1-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001; n=8. (d) E17 embryo sections were stained for trophoblasts (cytokeratin; red) or smooth muscle ( - SMA; green) [upper panel]; hematoxylin and eosin H&E (middle panel), highlighting increased red blood cells (arrows) in IL-11 withdrawal implantation sites compared to IL- 11 treated mice; and isolectin-B4 (lower panel). Bars represent 50 m, insets are negative controls, (e) Decidual vessel area was measured in cross-sections from three placentas per mouse at E17. Data are mean + SEM, 1-way ANOVA, **p<0.01; n=8. (f) IL-11 withdrawal rescues IL-11 -impaired vascular branching in the placental labyrinth, 1-way ANOVA, **p<0.01; n=8. (g) IL-11 withdrawal altered placental growth factor (PLGF) and follistatin-like-3 (FSTL3) placental gene expression when normalized to IL-11 treated placenta at E17, quantified by real-time PCR array; n=3.

[0032] Figures 11a through c are photographical and graphical representations showing IL-11 withdrawal from E14 of gestation alleviated STAT3 activation, (a) IL-11 withdrawal abolished IL-11 -induced pSTAT3 in the mouse placental labyrinth (L) and decidua (D) to control levels compared with IL-11. Bars represent 50 m. (b) Number of pSTAT3 positive cells were counted. Data are mean + SEM, 1-way ANOVA, ****p<0.0001. (c) Representative photomicrographs of E17 maternal kidneys, stained with H&E and rescue of glomerular hypertrophy in IL-11 withdrawal mice compared to IL-11 treated mice. Bars represent 50 m.

[0033] Figures 12a though c are graphical representations showing IL-11 does not affect syncytialization in human first trimester placental villous explants. (a) Cultures were grown under normoxia or hypoxia (2% v/v 0 ₂ ) conditions for 48h. Secreted human chorionic gonadotropin (hCG) was measured by ELISA in media. IL-11 did not alter hCG levels compared to control n=8/gp. First trimester placental villous explants were cultured for 72 hours with PBS vehicle control, IL-11 (lOOng/ml), STAT3 inhibitor (STAT3i, 30uM) or IL-11+STAT3L SYN1 (b) and SYN2 (c) mRNA expression was determined by quantitative real-time PCR, normalized to 18s and expressed as fold-change from PBS control. Data are as mean + SEM, ANOVA, n=8/gp.

[0034] Figures 13a through e are graphical and photographical representations showing IL-11 levels are elevated in the sera of women prior to onset of PE. (a) IL-11 protein was quantified in pregnant human serum using ELISA (a) during the first or second trimester, prior to the development of late onset (LO) or early onset (EO) preeclampsia, or (b) after preeclampsia onset. Data expressed as mean + SEM, Mann-Whitney U test, *p<0.05, **p<0.01, **p<0.001, n=15. (c) IL-11 and IL-11R mRNA levels in PE placental villous or preterm match control villous whole tissue were determined by quantitative real-time PCR, normalized to 18s and expressed as mean ddCT values + SEM, students t test; n=15. (d) IL-11 protein levels in PE placental villous or preterm match controls were determined by immunohistochemistry. Bars represent 50 m. Inset is negative control, (e) Staining intensity in the syncytiotrophoblast (ST) [arrow heads], cytotrophoblast (CT) and stroma were scored from 0-no staining to 3 -intense staining by two independent, blinded assessors. Data expressed as mean + SEM, students t test, **p<0.01; n=15. DETAILED DESCRIPTION

[0035] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method steps or group of elements or integers or method steps.

[0036] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a single biomarker, as well as two or more biomarkers; reference to "an agent" includes a single agent, as well as two or more agents; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention. All such aspects are enabled within the width of the present invention. Any variants and derivatives contemplated herein are encompassed by "forms" of the invention.

[0037] Preeclampsia (PE) is an insidious disease affecting approximately 8% of pregnancies in humans. Until the advent of the present invention, the only effective treatment was placental delivery by labor induction or cesarian. In accordance with the present invention, a direct casual relationship is identified between IL- 11 -mediated signaling and PE or at least predictive of a risk of developing PE. It is proposed herein to use IL-11 or a component in the IL-11 signaling pathway or a molecule whose regulation is effected by IL-11 signaling as a biomarker predictive of PE. It is further proposed to use IL-11 or a component in the IL-11 signaling pathway or a molecule whose regulation is effected by IL- 11 signaling as a target for therapeutic intervention to result in normalized IL-11 signaling. Without limiting the present invention to any one theory or mode of action it is proposed that IL-11 or IL-11 signaling acts on the placenta to damage it and this results in an abnormal placenta which releases toxins into the maternal blood stream and this leads to symptoms of PE. [0038] Accordingly, the present specification enables a method for preventing or ameliorating symptoms of preeclampsia (PE) in a pregnant human female subject, the method comprising administering to the subject an agent in an amount effective to normalize the level of signaling mediated by the IL-11 signaling pathway.

[0039] Further enabled herein is a use of an agent which normalizes the level of signaling mediated by the IL-11 signaling pathway in the manufacture of a medicament for the prevention or amelioration of symptoms of preeclampsia (PE) in a pregnant human female subject.

[0040] The present invention further extends to the treatment of a dysfunctional placenta by normalizing IL-11 signaling including antagonizing IL-11, IL-l lRa or IL-l lR-IL-l lR interaction.

[0041] The subject specification also teaches an agent which normalizes the level of signaling mediated by the IL-11 signaling pathway for use in preventing or ameliorating symptoms of preeclampsia (PE) in a pregnant human female subject.

[0042] Reference herein to "PE" or "preeclampsia" includes symptoms of PE indicative of a pregnant human female subject having PE or is in the stages of developing PE or is at risk of developing PE. This includes early to late onset PE. Early onset PE includes from 12 weeks to 34 weeks gestation. Such symptoms include but are not limited to one or more of hypertension, or a condition associated with elevated systolic blood pressure (SBP), proteinuria, abnormal extravillious trophoblast (EVT) invasion and intrauterine growth restriction. For the avoidance of doubt, reference may be made to either the treatment of PE or symptoms of PE without departing from the scope of the invention. In an embodiment, the symptoms of PE result from toxins emitted from a dysfunctional placenta.

[0043] By "pregnant human female subject" means a subject at any stage of pregnancy from first to third trimester including from early, mid or late stage second trimester to early, mid or late stage third trimester.

[0044] The terms "antagonist", "agonist", "medicament", "active", "drug", "medicine", "compound" and "therapeutic" may be used interchangeably herein to refer to a substance that induces a desired pharmacological and/or physiological effect including normalizing IL-11 signaling activity or the effects of IL-11 signaling activity. The pharmacological and/or physiological effect includes inhibiting IL-11 -dependent activation of STAT3. The terms also encompass pharmaceutically acceptable and pharmacologically active forms thereof, including salts. The desired effect is the inhibition or normalization of IL-11 activity or IL-11 receptor complex signaling or IL-11 signaling. This includes components effected by IL-11 signaling.

[0045] Hence, the agents contemplated herein include IL-11 signaling modulators which encompass molecules which can down-regulate or up-regulate a target component in or associated with IL-11 signaling.

[0046] The terms "treating" and "treatment" as used herein refer to therapeutic treatment. For example, treatment may result in a reduction in severity and/or the frequency of symptoms of PE, the elimination of symptoms and/or underlying cause of the PE, the prevention of the occurrence of symptoms of PE and/or its underlying cause and improvement or remediation or amelioration of damage following PE. Hence, the treatment may not result in a "cure" but rather an amelioration of symptoms including a reducing in proteinuria and/or hypertension in a pregnant human female subject. In addition, treatment may not commence until an exacerbated event occurs. In this context, the term "prophylaxis" also applies to the prevention or treatment of a likelihood of an exacerbated event occurring. The terms "treating" and "treatment" also refer to treating a dysfunctional placenta.

[0047] The terms "treating" and "treatment" as used herein also refer to the reduction of one or more symptoms or characteristics associated with PE. In an embodiment, the PE is early onset PE including from 12 to 34 weeks gestation.

[0048] The terms "condition" and "disease" are used interchangeably throughout the subject specification to refer to PE or symptoms of PE depending on when in progression of PE a patient presents for treatment.

[0049] The agent may be a proteinaceous or non-pro teinaceous (i.e. a non-protein chemical) molecule. The term "agent" may also be described as an antagonist, agonist, medicament, active drug, therapeutic, medicine, compound or other molecule having an ability to normalize IL-11 signaling. Examples include antibodies to IL-11 or IL-11 receptor a (IL-1 IRa) or to a component in, or affected by, IL-11 signaling, soluble IL- l lRa; a mutated form of soluble IL-1 IRa or IL-11 (each of which binds to IL-11 and IL- 11, respectively, to block IL-11 signaling); an oligonucleotide; or microRNA which down- regulates a gene or mRNA encoding IL-11, IL-1 IRa or a component in, or affected by, IL- 11 signaling. For example, International Patent Publication No. WO 03/099322 describes certain IL-11 antagonists and their antagonist activity.

[0050] In an embodiment, the agent is soluble IL-1 IRa or a mutated form thereof. The "mutated form" includes a portion of IL-1 IRa which contains one or more amino acid substitutions, additions or deletions. Such mutated forms may increase serum half-life of the mutated soluble IL-1 IRa or increase binding avidity to IL-11 thus reducing the amount of IL- 11 available to bind to an IL- 11 receptor.

[0051] In an embodiment, the agent is a non-functional form of IL-11 which can bind to IL-11 receptor without inducing IL-11 signaling. Such non-functional forms of IL-11 generally contain single or multiple amino acid substitutions, additions or deletions to a portion of IL-11 required for IL-11 signaling but which does not prevent it from binding to IL-11 receptor. Such a mutated IL-11 thus prevents active IL-11 from binding to its receptor to induce signaling.

[0052] In an embodiment, the agent is an antibody specific for IL-11 or IL-1 IRa or is a IL-l l/IL-l lRa-binding fragment thereof. The antibody may be a human antibody or a deimmunized or humanized form of a non-human antibody.

[0053] The terms "antibody" and "antibodies" include polyclonal and monoclonal antibodies and all the various forms derived from monoclonal antibodies, including but not limited to, full-length antibodies (e.g. having an intact Fc region); antigen-binding fragments, including for example, Fv, Fab, Fab' and (F(ab') ₂ fragments; and antibody- derived polypeptides produced using recombinant methods such as single chain antibodies. The terms "antibody" and "antibodies" as used herein also refer to human antibodies produced, for example, in transgenic animals or through phage display, as well as chimeric antibodies and humanized antibodies. It also includes other forms of antibodies that may be therapeutically acceptable and antigen-binding fragments thereof, for example, single domain antibodies derived from cartilage marine animals or Camelidae, or from libraries based on such antibodies. In relation to the latter embodiment, the antibody may be an immunoglobulin new antigen receptor (IgNAR) as defined in International Patent Publication No. WO 2005/118629 derived from a cartilaginous marine animal such as a shark. Reference may also be made to Nuttal et al. (2003) Eur. J. Biochem 270:3543- 3554.

[0054] The term "monoclonal antibody" is used herein to refer to an antibody obtained from a population of substantially homogenous antibodies. That is, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" as used herein therefore indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not used to indicate that the antibody was produced by a particular method. For example, monoclonal antibodies in accordance with the present invention may be made by the hybridoma method described by Kohler and Milstein (1975) Nature 25(5:495-499, or may be made by recombinant DNA methods (such as described in US Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628 or Marks et al. (1991) J. Mol. Biol. 222:581-597. [0055] Chimeric antibodies may include antibodies to IL- 11 or IL-1 IRa or a component in or affected by UIL-11 signaling comprising the heavy and light chain variable regions of mouse, rat or rabbit antibodies to IL-11 or IL-1 IRa and human heavy and light chain constant domains.

[0056] The agent may be a high level antagonist of the activity of IL-11, IL-1 IRa or a component of, or effected by, IL- 11 signaling or it may be a low level antagonist designed or selected to reduce to non-zero levels the activity of the targeted molecule. The aim is to "normalize" IL-11 signaling to reduce the adverse effects of IL-11 signaling in or around the placenta. Hence, some components may need to be upregulated. Consequently, the agent may be an agonist of the target component. The desired effect is to promote remodeling of uterine spiral arterioles by EVTs to create low -resistance, large-diameter vessels that promotes utero-placental blood supply required to sustain fetal growth and development.

[0057] In another embodiment, the agent is a nucleic acid molecule such as an antisense, sense, microRNA, hairpin nucleic acid, or other molecule which can prevent or reduce expression of a gene or translation of an mRNA into IL-11, IL-1 IRa or a component of, or effected by, IL-11 signaling. Again, the aim is to normalize the level of IL-11 signaling to the pregnant human female subject. Hence, the nucleic acid molecule may be required to up-regulate gene expression.

[0058] The agent is generally formulated in a pharmaceutical composition comprising the agent and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0059] The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils or alcohols. [0060] Pharmaceutical compositions of the present invention used for PE therapy suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil- in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients.

[0061] The components and/or extracts identified of the present invention may also be administered, parenterally (including subcutaneous, intravenous, intramuscular or intraperitoneal injection or by infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

[0062] When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

[0063] In an embodiment, the agents of the present invention are administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. When administered by injection, the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. [0064] The effective dosage of the agents employed in therapy may vary depending on the particular agent employed, the mode of administration, the stage of PE and/or the severity of the PE being treated. Thus, the dosage regimen utilizing the agents of the present invention is selected in accordance with a variety of factors including age, gestation stage, sex and medical condition of the patient; the severity of the PE to be treated; the route of administration; the renal and hepatic function of the patient; and the particular agent employed. A physician or clinician of ordinary skill can readily determine and prescribe the effective amount of the agent required to prevent or arrest the progress of PE. Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug. Importantly, the result of the treatment is to normalize IL-11 signaling.

[0065] Another aspect of the invention provides a method for the prophylaxis and/or treatment of PE in a pregnant human female subject comprising the administration to the subject of cells capable of expressing a genetic construct encoding an IL-11 signaling modulating component.

[0066] The present invention also includes pharmaceutical compositions and formulations comprising nucleic acid molecules which target and modulate the level of expression or translation of a target component associated with IL-11 signaling. Such nucleic acid pharmaceutical compositions of the present invention may be administered in a number of ways. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-0-methoxyethyl modification are useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Aptamers capable of targeting a particular gene or mRNA may also be employed.

[0067] The formulation of therapeutic compositions and their subsequent administration (dosing) is within the skill of those in the art. Dosing is dependent on severity and responsiveness of the PE to be treated, with the course of treatment lasting from several days to several months, or until the symptoms of the PE are ameliorated. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC ₅₀ S found to be effective in in vitro and in vivo animal models.

[0068] The present invention further contemplates the use of IL-11 or a component in the IL- 11 signaling pathway or a component affected by IL- 11 signaling as a biomarker for PE or a risk of developing PE or as a biomarker of a dysfunctional placenta. Conveniently, the assay is conducted on a blood sample such as whole blood, serum, plasma or blood fraction or on a uterine fluid sample. In an embodiment, the level of blood or fluid IL-11 is measured although, as indicated above, any component in or affected by IL- 11 signaling may be assayed.

[0069] For example, a blood or fluid sample is isolated from the pregnant human female subject and optionally fractionated or serially diluted. It is then contacted with an antibody specific for IL-11 (or other target). The antibody itself may be labeled with a reporter molecule capable of providing an identifiable signal (e.g. a fluorescent molecule). Alternatively, a second antibody specific for an immunoglobulin is employed which is the antibody labeled with the reporter molecule. This second antibody is used to bind to the IL- 11 -specific antibody bound to IL- 11.

[0070] There are many variations to the assay which is designed to quantitate the level of IL-11 in the sample. A statistically validated control level of IL-11 may be the reference level or the patient's own blood or fluid may be used to monitor for an elevation in IL-11. For example, a female subject may be monitored for fluid levels of IL-11 from the time the subject is pregnant and then throughout pregnancy.

[0071] Any elevation in IL-11 would then trigger the decision to administer an IL-11 antagonist or an agent which targets a component in or which is effected by IL-11 signaling.

[0072] Hence, contemplated herein is a method for determining the level of risk that a pregnant human female subject will develop preeclampsia (PE) or symptoms of PE, the method comprising determining the level of IL-11 in the blood stream or uterine fluid of the subject and comparing the level to a control wherein an elevation of the level of IL-11 relative to the control is indicative of a high risk of developing PE and a control level or less than control level is indicative of a zero to low risk of developing PE. In an embodiment, the control is the level of IL-11 (or other component) from the subject or is a statistically validated control.

[0073] In an embodiment, the level of IL- 11 is determined by an antibody specific for IL- 11. The IL- 11 specific antibody or an antibody specific for the first mentioned label with a reporter molecule capable of providing an identifiable signal. Any number of quantitative assays such as HPLC may be used to determine IL-11 (or other component) levels.

[0074] The assay may also be part of a multiplex assay where a number of parameters are screened to monitor the health of a pregnancy.

[0075] Further enabled herein is an animal model for PE. The animal model comprises a pregnant non-human animal to which IL-11 is administered. The amount of IL-11 administered is from 10(Vg/kg/day to lOmg/kg/day including 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000μg (lmg), 2, 3, 4, 5, 6, 7, 8, 9 and lOmg/kg/day. [0076] The half-life of IL-11 is approximately 12 hours in serum. The dosage regime is suitably adjusted so that at a certain time after IL-11 administration, the serum levels of IL-11 will equate to levels in a pregnant human female.

[0077] In an embodiment, the animal used in the animal model is a murine animal such as, but not limited to, a mouse. Other animals include a rat, rabbit, guinea pig, hamster, pig, sheep, dog and cat. Non-human primates may also be employed.

[0078] As indicated above, in an embodiment, the animal model is a mouse model to which recombinant IL-11 at 50( g/kg/day is administered. At 6 hours post administration, the serum levels of IL-11 equate to levels in pregnant human female subjects with PE.

[0079] Accordingly, contemplated herein is an animal model comprising a non-human pregnant animal manipulated to artificially elevated levels of IL-11 wherein the levels equate to levels observed in pregnant human female subjects with onset of PE. In an embodiment, the animal model is a mouse model.

[0080] In an embodiment, the artificially elevated level of IL- 11 is introduced recombinant IL-11. In an embodiment, 50( g/kg/day of recombinant IL-11 is administered. At approximately 6 hours post-administration, the level of serum IL-11 is similar to that observed in human female subjects with PE.

[0081] The animal model is useful in the study of the etiology of PE, the effect of IL-11 rescue by, for example, the administration of an IL-11 antagonist or an antagonist of a component of the IL-11 signaling pathway or a modulator of a molecule whose level is effected by the IL-11 signaling pathway.

[0082] Therapeutic and diagnostic kits are encompassed by the present invention. The kits may comprise agents for treatment or reagents for diagnostic applications such as serum or fluid levels of IL-11. [0083] During work towards the present invention, it was observed that PEGylated IL-11 (PEGIL-11) was unable to cross the placenta. This enables a treatment protocol for treating pregnant women. In particular, contemplated herein is a method of treating a pregnant woman with a drug while minimizing the risk of fetal toxicity. The method comprises selecting a drug required such as to treat hypertension, diabetes, cardiac disease, liver or kidney disease or failure or cancer or microbial or viral infection and subjecting it to PEGylation to thereby reduce passage of the drug across the placenta. A pharmaceutical composition comprising the PEGylated drug and one or more pharmaceutically acceptable carriers, excipients and/or diluents is also contemplated herein.

EXAMPLES

[0084] Aspects disclosed herein are further described by the following non-limiting Examples.

Material and Methods

Animals

[0085] Female (virgin 8 to 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 a 12L: 12D cycle. C57BL/6 mice were mated, and, the time of gestation is denoted by embryonic day Έ', where E0 represents the day of detection of a vaginal plug.

Recombinant IL-11 administration during pregnancy in mice

[0086] To determine the effect of elevated IL-11 on placentation, mated female mice were administered with 500 g/kg/day IL-11 (Genetics Institute, MA, US) or vehicle IP, twice daily from E8-13 (n=5), E10-17 (n=8) or from E10-14 for the withdrawal study (n=8). This treatment regime was based on extensive pilot data; tl/2 of recombinant IL-11 is 12 hours in mouse serum and levels at 6 hours after administration are equivalent to PE women (Figure 3). For PCR array, pregnant mice at E13 were administered with a single dose of IL-11 and killed after 2 hours (h). The placenta and decidua were dissected from implantation sites and snap frozen at -80°C. Human IL-11 was PEGylated as described previously (Takagi et al. (2007) Journal of controlled release : official journal of the Controlled Release Society 119(3):2Ί\-2Ί% White et al. (2007) PNAS 04: 19357-19362) with some modifications. Briefly, hIL-11 was reacted with poly(ethylene glycol) [40 kDa]- NHS Ester (Y-NHS-40K) [Jenkem Technology, Allen, TX] in 0.1 M MOPS, pH 8.0 at a protein/reagent molar ratio of about 1: 14 for 3 hours at room temperature. After the reaction, PEGylated ML- 11 (PEGIL-11) was diluted 1:3 in 25 mM MES, pH 6.0 and loaded onto an 1-mL HiTrap SP Sepharose column (GE Healthcare) equilibrated in 25 mM MES, pH 6.0. Bound PEGIL-11 was eluted and purified by size-exclusion chromatography (GE Healthcare). SDS-PAGE analysis shows that purified PEGIL-11 has molecular weight of approximately 120,000.

Animal tissue collection

[0087] All mice were euthanized by carbon dioxide gas followed by cardiac puncture to collect peripheral blood, which was immediately separated by centrifugation to obtain serum and stored at -80°C before use. Implantation sites (at least n=3/mouse) were dissected to obtain placenta, decidua and fetus and these tissues were weighed and imaged using a dissecting microscope, then snap frozen at -80°C or fixed in 4% v/v neutral buffered formalin solution for 24 hours and paraffin embedded.

Mouse sFLTl, sENG, IGF-1 and IL-11 ELISA

[0088] To analyze sFltl, sEng, IFG-1 and IL-11 in neat mouse serum, the established ELISA protocol was followed from the manufacturer using the commercial ELISAs: VEGF Rl/Flt-1 Quantikine ELISA Kit (MVR100: R&D Systems), Mouse Endoglin ELISA Kit (ELM-ENG, Ray Biotech), Mouse IGF-1 Quantikine ELISA Kit (MG100, R&D Systems) and Mouse IL-11 ELISA Kit (ELM-IL-11, Ray Biotech).

Systolic blood pressure measurement

[0089] SBP was measured in pre-trained, conscious pregnant mice on E10, 13, 15 or 17, or corresponding days in non-pregnant mice (n=8/group) by tail-cuff plethysmography, following a procedure adapted from the ITCC Life Science manual (ITCC Life Science, Woodland Hills, California, USA), detailed previously (Rickard et al. (2006) Endocrinology 147(12 J:5901-5906). Briefly, following 15mins of stabilization in the preheated (30-32°C) chamber, three consecutive manual inflation -deflation cycles were performed and SBP calculated from the tracing provided by the analyzer. The average reading was calculated from three accurate SBP tracings and reported as the SBP value. Urinary protein measurement

[0090] Urine samples were collected from non-pregnant mice and pregnant mice at mid (E10-13) and late (E16-17) gestational stages. Urinary protein levels were measured using a colorimetric assay based on a modified Bradford method (Bio-Rad).

Electron microscopy

[0091] Kidneys from non-pregnant or pregnant mice at E17 were fixed in 2.5% w/v glutaraldehyde, treated with 1% w/v osmium tetroxide and embedded in an Araldite-Epon mixture. Semi-thin sections (0.6 mm) were prepared and examined with a transmission electron microscope (Hitachi H7500) at Monash Micro Imaging, Monash University, Victoria, Australia. Data are from experiments using at least three mice per treatment group. Images captured by a blinded observer were later used to evaluate ultrastructural alterations of the GBM. Quantitative assessment of GBM thickness (GBM area/GBM length) was performed using Image-Pro Plus version 6.0 software.

Histology and immunohistochemistry

[0092] All tissues were sectioned at 5 um, placed onto SuperFrost slides, dried, deparaffinized, and rehydrated. Wild-type implantation sites were immunostained for IL- 11 and IL-11R as described previously (Dimitriadis et al. (2003) Reproductive Biology and Endocrinology i:34-44). PEG immunohistochemistry was performed as previously (Menkhorst et al. (2009) Biology of reproduction 80:920-927). Treated mouse placental sections were stained with Hematoxylin and eosin (H&E) and Masson's trichrome. H&E and periodic acid Schiff (PAS) staining were performed on maternal kidneys. For immunohistochemical analysis, antibodies against desmin (Dako, #D-33), pancytokeratin (Santa-Cruz, sc-H-240) -SMA (Dako, clone 1A4) and F4/80 (Serotec) were used to label decidual cells, trophoblasts, smooth muscle cells and macrophages respectively. Phospho- STAT3 (Tyr705) [Cell Signaling Technologies #9145] and PAPPA2 (Abeam #117743) immuno staining were also performed. Primary antibody or isotype negative control goat IgG in blocking solution were applied for 18 hours incubated at 4°C. After stringent washing with 0.6% v/v Tween-20 in TBS, antibody localization was detected by sequential application of biotinylated horse anti-goat IgG (1:200; Vector Laboratories) in blocking solution for 30 minutes and an avidin-biotin complex conjugated to HRP (Vector Laboratories). Protein was visualized as a brown precipitate using diaminobenzidine tetrahydrochloride substrate (Dako). Sections were counterstained with Harris hematoxylin (Sigma Chemicals), and mounted. For immunofluorescence, formalin-fixed sections were treated as described above except: non-immune serum diluted in and washes performed in phosphate buffered saline; primary antibody for pan-cytokeratin, -SMA or non-immune goat IgG (isotype negative control); secondary antibody incubation (Donkey -mouse alexa fluor 488 and Donkey -goat alexa fluor 594; both 1:200) in non-immune serum for 2 hours at room temperature; following further washes, sections were mounted using Vectastain containing DAPI (DAKO). Dolichos biflorus agglutinin (DBA lectin, Sigma) and isolectin-B4 (ISB4, Sigma) staining were performed to highlight decidual uNK cells and the extracellular matrix surrounding fetal blood vessels respectively.

Placental morphometry

[0093] Mid-sagital sections from at least 3 implantation sites per mouse were analyzed using CellSense software (Olympus). To assay vessel density, hemisected placentas were stained with ISB4. Six to twelve photographs at 20X magnification were taken from two different sections (middle region) representing more than 90% of the labyrinth and vessels counted using Image J software as previously described (Garcia-Gonzalez et al. (2010) PLoS One 5(9)). Digital photographs at IX were taken and CellSense software was used to quantify pixel density and expressed as intensity per area to give a percentage. For cell counts; 3 fields per tissue were analyzed from 3 placentas per mouse at x20 magnification. A blinded observer counted the number of positive macrophages, uNK cells and nuclear pSTAT3 positive cells expressed as number of cells per field. Decidual area was quantified by measuring the cross- sectional area and expressed per total implantation site area as a percentage.

In situ hybridization

[0094] In situ hybridization was performed on implantation sites as previously described (Simmons et al. (2007) Dev Biol 304(2 J:567-578). Briefly, for in situ hybridization, sections were re-hydrated in PBS, post-fixed in 4% v/v PFA for 10 min, treated with proteinase K (15 g/ml for 5 minutes at room temperature for E9.0 implantation sites and 30 g/ml for 10 minutes at room temperature), acetylated for 10 minutes (acetic anhydride, 0.25% v/v; Sigma), and hybridized with digoxigeninlabeled probes overnight at 65°C. Digoxigenin labeling was done according to the manufacturers instructions (Roche). Hybridization buffer contained lxsalts (200mM sodium chloride, 13 mM Tris, 5 mM sodium phosphate monobasic, 5mM sodium phosphate dibasic, 5mM EDTA), 50% v/v formamide, 10% w/v dextran sulfate, lmg/ml yeast tRNA (Roche), lxDenhardt's (1% w/v bovine serum albumin, 1% w/v Ficoll, 1% w/v polyvinylpyrrolidone), and DIG-labeled probe (final dilution of 1:2000 from reaction with 1 g template DNA). Two 65°C post- hybridization washes were carried out (lxSSC, 50% formamide, 0.1% v/v Tween-20) followed by two RT washes in lxMABT (150mM sodium chloride, lOOmM maleic acid, 0.1% v/v Tween-20, pH 7.5), and 30 minutes RNAse treatment (400mM sodium chloride, 10 mM Tris pH7.5, 5mM EDTA, 20 g/ml RNAse A). Sections were blocked in lxMABT, 2% blocking reagent (Roche), 20% v/v heat inactivated goat serum for lh, and incubated overnight in block with anti- DIG antibody (Roche) at a 1:2500 dilution. After four 20 minutes washes in lx MABT, slides were rinsed in lxNTMT (100 mM NaCl, 50 mM MgC12, lOOmM Tris pH 9.5, 0.1% v/v Tween-20) and incubated in NBT/BCIP in NTMT according to the manufacturer's instructions (Promega). Slides were counterstained with nuclear fast red, dehydrated and cleared in xylene, and mounted in cytoseal mounting medium (VWR).

SDS-PAGE and Western blotting

[0095] Whole tissues were lyzed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM -glycerolphosphate, protease inhibitor cocktail (Calbiochem) and the protein was quantified by the Bradford assay. Equal protein per sample was resolved on 8-10% w/v sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels, transferred to polyvinyldifluoride (PVDF) membranes (GE Healthcare Bio-Sciences), blocked with 5% w/v non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% v/v Tween-20 (Bio-Rad) and probed with polyclonal antibodies against IL-11 (1:500; Santa Cruz, sc-H169,), IL-11R (1:500; Abeam, ab 109697), pSTAT3 (as above), or PAPPA2 (as above) overnight at 4°C, followed by three wash steps. Membranes were incubated for 1 hour at room temperature with secondary anti-rabbit Ig-horseradish peroxidase (HRP) linked, (1:5000; DakoCytomation) and signals were developed with enhanced chemiluminescence detection system reagent (Pierce). Membranes were stripped and incubated with anti-GAPDH as a protein loading control.

Participants and collection of human sera samples

[0096] Blood samples were drawn from women with moderate or severe preeclampsia (n=9) and from pregnant women with normal pregnancies (controls) throughout the gestational weeks (n=l l-15/trimester). Patients with preeclampsia (PE) did not have any prior history of hypertension or renal disease and all women in control group did not show any clinical or pathological signs of preeclampsia, infections or any other maternal or placental disease. Blood samples were taken from all pregnant women in each trimester and samples from women that developed preeclampsia were retrospectively analyzed. Sera collected were immediately separated by centrifugation and stored at -70°C before use.

Human IL-11 Enzyme-linked immunoassays (ELISA)

[0097] Total IL-11 was measured from human serum using the established ELISA protocol from the manufacturer (Human IL-11 Quantikine ELISA Kit Dl lOO, R&D Systems).

Collection of placental tissues

[0098] Placental samples were collected from healthy women undergoing first-trimester termination of pregnancy (6-10 weeks) for psychosocial reasons. Tissues were washed in 0.9% w/v saline before transfer to medium Dulbecco's modified Eagle's medium (DMEM)/F12 1: 1 (Invitrogen).

First-trimester villous explant culture

[0099] Small pieces of first-trimester placental villous tissue (n=6) were dissected and cultured in DMEM/Ham's F12 with IL-11 (lOOng/ml), or PBS control. Explants were collected and snap frozen at 12 hours and fixed and processed at 72 hours. Human chorionic gonadotroph (hCG) ELISA

[0100] Total human chorionic gonadotrophin (hCG) [Diagnostic Systems Laboratories] was measured in conditioned media from the first trimester placenta explants, diluted 1: 10 using the established ELISA protocol from the manufacturer. The sensitivity for the hCG assay was 3mIU/ml.

Primary human first trimester placenta villous (EVT) outgrowth assay

[0101] Small pieces (Ixlmm) of villous tissue of first-trimester placentas (n=5) were dissected under the microscope and cultured overnight in serum-free medium. (DMEM/F12). To analyze the effects of IL-11 on trophoblast outgrowth, villous explants were seeded into 48-well plates for 3 hours on collagen I (serum-free medium), allowing anchorage, and then stimulated with recombinant human (rh) IL-11 (lOOng/ml) [R&D Systems], rhPAPPA2 (lOOng/ml) [R&D Systems] or PBS vehicle control. After 48 hours, anchoring villi (3 per treatment group) were photographed. The area of outgrowth was measured and quantified using the imaging software Adobe Photoshop (Adobe).

Small interfering RNA (siRNA) transfection of Primary human first trimester placenta villous tissue

[0102] Small pieces (Ixlmm) of villous tissue of first-trimester placentas (n=5) as above were transfected with commercially generated and validated ON-TARGETplus SMARTpool siRNA (Dharmacon) that targeted either PAPPA2 or no specific sequences (scr) as a scrambled control. siRNA delivery was performed using the LipofectamineRNAiMAX (Invitrogen, Life Technologies) according to manufacturer's instructions. Tissue was transfected for 72 hours prior to RNA and protein collection to test for transfection efficiency or prior beginning the functional experiments.

RNA preparation and quantitative real time RT-PCR

[0103] Placental tissues were lyzed, RNA extracted using TriReagent and analyzed using the Nanodrop spectrophotometer (Thermo Scientific). Mouse Preeclampsia RT2 Profiler PCR Arrays were performed in accordance with manufacturer instruction (QIAGEN). For qPCR, 500ng RNA was converted to cDNA using Superscript III RNA polymerase (Life Technologies). Realtime RT-PCR analyzes were performed on the ABI 7500HT fast block real-time PCR system (Applied Biosystems). Primer sequences are detailed in Table 3. The PCR protocol was as follows: 95°C for 10 minutes and 40 cycles of 95°C for 15 s followed by 60°C for 1 min. Relative expression levels were calculated by the comparative cycle threshold method ( Ct) as outlined in the manufacturer's user manual, with 18s ribosomal RNA serving as the endogenous control for normalization.

Statistical analysis

[0104] Statistical analysis was carried out using GraphPad Prism (GraphPad Software), and the data were assessed by Student's t-test assuming normal distribution or Kolmogorov-Smirnov test. Multiple groups were compared using one-way ANOVA, with Tukey's post-hoc test. Results of P<0.05 were considered statistically significant.

Table 3

Primer sequences for (a) human; and (b) mouse genes

A.

IL-11 F 5'-GTGGCCAGATACAGCTGTCGC3' (SEQ I D NO:l)

R 5'-GGTAGGACAGTAGGTCCGCTC3' (SEQ I D NO:2)

IL-llRa F 5'-CAGGGCCTGCGGGTAGAGTCAG3' (SEQ I D NO:3)

R 5'-CTCCTTGGTATGGTCCCAGTG3' (SEQ I D NO:4)

VEGFA F 5'-TCTACCTCCACCATGCCAAGT3' (SEQ I D NO:5)

R 5'-GCCGTCCGAGTACATTTGAT3' (SEQ ID NO:6)

PAPPA2 F 5 ' - AG G G G ATAGTCCTATTG G G C A3 ' (SEQ I D NO:7)

R 5'-CCTCACCTAGAGACTCCTTGG3' (SEQ ID NO:8)

SYN1 F 5'-CCCCATCGTATAGGAGTCTT3' (SEQ ID NO:9)

R 5'-CCCCATCAGACATACCAGTT3' (SEQ ID NO:10)

SYN2 F 5'-AGCCTTAACGACCATGCAAGA3' (SEQ ID NO:ll)

R 5'-CTGTGCTGCCGTTAACATGTCTA3' (SEQ I D NO:12)

18s F 5'-GATCCATTGGAGGGCAAGTCT3' (SEQ I D NO:13)

R 5'-CCAAGATCCAACTACGAGCTT3' (SEQ ID NO:14)

B.

IL-11 F 5'-GTTTACAGCTCTTGATGTCTC3' (SEQ I D NO:15)

R 5'-GAGTCTTTAACAACAGCAGG3' (SEQ ID NO:16)

IL-llRa F 5'-GTCCCCTGCAGGATGAGATA3' (SEQ ID NO:17)

R 5'-AGGCCAAGGCAAGAGAAGAT3' (SEQ ID NO:18)

PAPPA2 F 5'-GTTGGAAGGGGAACGCTGTT3' (SEQ ID NO:19)

R S'-ACC 1 1 G 1 AG 1 1 1 1 CGAGGC3' (SEQ I D NO:2U)

VEGF F 5'-GCTGCGCTGATAGACATCCA3' (SEQ I D NO:21)

R 5'-GCTGCGCTGATAGACATCCA3' (SEQ I D NO:22) β2- microglobulin F S'-GG I U 1 I U GG I GU I G I U CA3' (SEQ ID Νϋ:23)

R 5'-GTTCGGCTTCCCATTCTCC3' (SEQ I D NO:24) EXAMPLE 1

IL-11 administration impairs EVT invasion and spiral artery remodeling in vivo

[0105] In vivo evidence for a functional role of IL-11 in placentation is lacking. In this example, IL-11 and IL-11R were localized in the mouse placental endovascular trophoblast and endothelial cells in implantation sites throughout gestation (Figure 1). This reflects expression patterns in women (Figure 2), implying a role in placentation in vivo. To model elevated levels of IL-11 seen in women with PE (Basar et al. (2010) supra), mice were administered with physiologically relevant doses of IL-11 (Figure 3) or control twice daily from embryonic day (E)8-13 to determine effects on spiral artery remodeling and placentation (Rossant and Cross (2003) Nature Reviews Genetics 2:538-548), or E10- 17 to determine effects on PE features. At E13, IL-11 enhanced phosphorylated (p) STAT3 in the placental labyrinth and maternal spiral arterioles, suggesting that IL-11 acts at these sites via STAT3 (Figure 3). Implantation sites at E13 showed decidual trophoblast invasion and displacement of -smooth muscle actin ( -SMA) positive vascular smooth muscle cells (VSMCs), indicating normal spiral artery remodeling (Figure 4A and B; Figure 5). In IL-11 -treated mice, trophoblast invasion and spiral artery remodeling was impaired, decidual vessel area was significantly reduced and VSMCs lining decidual spiral arterioles retained (Figure 4A and B; Figure 5), supporting in vitro findings in humans (Paiva et al. (2009) supra; Sonderegger et al. (2011) supra). Since IL-11 plays a crucial role in decidualization from E3-6 (Robb et al. (1998) supra), the potential role of elevated IL-11 was investigated during mid-late gestation on the decidua. In mice treated with IL-11 from E8-13, desmin immuno staining (decidual marker) was unchanged between groups at mid- gestation (Figure 4C and D). EXAMPLE 2

IL-11 administration recapitulates the features ofPE in mice

[0106] Since IL-11 impaired trophoblast invasion in vivo, the effect of elevated IL-11 was determined on hallmark features of PE was determined in mice. In pregnant mice treated with IL-11 from E10-17, SBP increased by 20% at E15 (116.20mm/Hg + 2.37 versus control 92.96mm/Hg + 1.85, p<0.001) [Figure 4E]. IL-11 had no effect on SBP in nonpregnant mice. The anti-angiogenic factor s-Fltl, associated with endothelial dysfunction and PE in mice and women (Levine et al. (2004) N Engl J Med 350:672-683) was increased in a pregnancy specific manner in response to IL-11 at E13 (46455pg/ml + 10836 versus control 18750pg/ml + 2723, p<0.01) [Figure 4F]. IL-11 treatment from E10- 17 increased urinary protein in pregnant mice at mid- and late-gestation (117.4 g/ 1 ± 5.86 versus control 65.64 g/ 1 ± 12.38, p<0.001) [Figure 4G]. Kidney glomeruli from IL- 11 -treated pregnant mice were enlarged and ischaemic, indicated by fewer red blood cells compared to pregnant controls (Figure 3). Glomeruli had narrow capillary lumen and thickened basement membrane that facilitates filtration (Figure 4H and I), suggesting endotheliosis and extracellular matrix deposition in the maternal kidneys.

EXAMPLE 3

IL-11 impairs placental labyrinth vasculature development and promotes IUGR via placental insufficiency

[0107] Elevated IL-11 dramatically altered placental labyrinth structure and morphology at mid and late-gestation compared to control (Figure 4J; 6), which may alter maternal-fetal exchange (Rossant and Cross (2003) supra). It was demonstrated that there was impaired labyrinth endothelial cell differentiation (Mest) and reduced invasive trophoblasts (Prll7bl) at the maternal-fetal interface (Figure 6). Labyrinth fetal vascular branching (Isolectin-B4) was significantly reduced (Figure 4J and K) and labyrinth morphology was abnormal with necrotic/fibrotic areas and collagen deposition (Figure 4J) in IL-11 -treated placenta compared to control at E17. IL-11 reduced fetal weight at E17 (0.63g + 0.02 versus control 0.87g + 0.01, p<0.01) [Figure 4L and M]. To confirm that IL-11 likely acts via the placenta and not directly on the fetus to cause IUGR, polyethylene glycol (PEG) was ligated to IL-11. Very little PEGIL-11 crossed the placenta (Figure 4N; Figure 6). PEGIL-11 resulted in a similar reduction in fetal weight as IL-11 alone, compared to respective controls (Figure 4M). IGF-1, an important regulator of placental and fetal growth (Liu et al. (1993) supra) was identified as an IL-11 regulated target in the mouse placenta. IL-11 down-regulated IGF1 mRNA by 2.05-fold (p<0.01) [Figure 8A]. Indeed circulating maternal serum levels showed a trend in reduced levels at E13 and a significant reduction at E17 (Figure 8B).

EXAMPLE 4

IL-11 impedes human EVT invasion via the pregnancy-associated plasma protein A2

(PAPPA2) protease

[0108] IL-11 targets were identified in the placenta by quantitative PCR array. IL-11 up- regulated 3 genes, and down-regulated 4 genes (>2-fold) [Figure 8A]. Pregnancy- associated plasma protein A2 (PAPPA2) was up-regulated 2.6-fold (p<0.001). PAPPA2 protein is elevated in EO PE placenta (Macintire et al. (2014) Reproduction, Fertility and Development 2(5:351-357) and maternal serum prior to PE onset (Crosley et al. (2014) Reprod Sci 21(6):Ί '54-Ί '60) and expressed by first trimester EVT in women and invasive trophoblasts in mice (Wang et al. (2009) Endocrinol 202(3 ):331-3A5). IL-11 regulated PAPPA2 rriRNA and protein in human and mouse EVTs and decidual cells and in IL-11- treated mouse serum, in a pregnancy-specific manner (Figure 8C-F; Figure 9). In first trimester placental explants, PAPPA2 reduced trophoblast outgrowth by 55%+8 compared to control (p<0.05), to a similar extent as IL-11 (Figure 8G and H). Knockdown of endogenous PAPPA2 (Figure 9) did not significantly alter outgrowth (p>0.05) [Figure 8H], suggesting that only abnormally elevated PAPPA2 levels alter EVT invasion. PAPPA2- knockdown rescued IL-11 -impaired EVT outgrowth (79%+6) compared to IL-11 (50%+4, p<0.05) [Figure 8H)]

EXAMPLE 5

IL-11 alters decidual immune cells required for normal spiral artery remodeling

[0109] Immune cells function at the maternal-fetal interface to mediate maternal tolerance and facilitate decidual and arterial tissue remodeling during EVT invasion (Cartwright et al. (2010) Reproduction i40(<5):8O3-813). IL-11 reduced placental gene expression of IL10, IL15 and IL18 (Figure 8A), which play important roles in regulating decidual immune cell functions. As indicated by DBA lectin staining (Figure 81), IL-11 dramatically reduced the number of decidual uNK cells at mid-gestation (17 + 2 versus control 50 + 3, p<0.0001) [Figure 8 J] and also promoted a significant increase in the number of abnormal double nuclei decidual uNK cells (p<0.05) [Figure 8K]. A trend in reduced decidual macrophages was found (Figure 5), although it was not significant.

EXAMPLE 6

IL-11 withdrawal

[0110] The data indicate that IL-11 contributed to the pathogenesis of PE features in mice via placental alterations. Normalizing elevated IL-11 levels during pregnancy in mice was investigated, in particular, after the onset of placental dysfunction/PE features. Mice received IL-11 from E10-14 so that administration was ceased at the onset of IL-11- induced PE features. By late-gestation, IL-11 withdrawal restored IL-l l-induced elevated SBP in pregnant mice to control levels (withdrawal 100.21mm/Hg + 1.32 versus IL-11 117.65mm/Hg + 1.89, p<0.0001) [Figure 10A]. Similarly, proteinuria was alleviated (withdrawal 50.22 g/ 1+10.75 versus IL-11 121.20 g/ 1 + 2.53, p<0.001) [Figure 10B] and kidney glomerular hypertrophy seen in pregnant IL-11 -treated mice subsided (Figure 11). Fetal weight (E17) was significantly increased by 18% (withdrawal 0.72g + 0.04 versus IL-11 0.61g + 0.04, p<0.05) [Figure IOC]. To determine how quenching mid-late gestation elevations in IL-11 may ameliorate PE features after diagnosis, implantation sites from IL-11 treated and IL-11 withdrawal mice were examined at E17. IL-11 withdrawal did not rescue IL-11 -impaired spiral artery remodeling (Figure 10D, E). Differences in placental labyrinth morphology were evident in the IL-11 withdrawal group compared to IL-l l-treated mice at E17 (Figure 10D). In the IL-11 -withdrawal labyrinth, more red blood cells were apparent in the circulations (Figure 10D). Isolectin-B4 staining highlighted impaired labyrinth branching and structure in IL- 11 treated placenta, but normal branching in the IL-11 withdrawal placenta (Figure 10D, F). The potential for IL-11 to modify syncytialization was investigated, since the syncytium is the barrier between the maternal and fetal circulations in the human and mouse placenta. However, IL-11 had no affect on Syna in the mouse placenta (Figure 6) or SYN1, SYN2, or E-CADHERIN gene transcription, or hCG protein secretion in primary human first-trimester placental explants (Figure 12). IL-11 withdrawal had no effect on the decidua, although IL-11 withdrawal did alleviate IL-l l-induced STAT3 activation in the mouse decidua and also the placenta (Figure 11). Finally, to gain some insight into the molecular changes between the IL-11 treated and IL-11 withdrawal placenta, quantitative array analysis was performed. Placental growth factor (PLGF) showed a 2.2-fold increase follistatin-like-3 (FSTL3) a 2- fold decrease in the IL- 11 withdrawal mouse placenta compared to IL-11-treated at E17 (p<0.05) [Figure 10F].

EXAMPLE 7

IL-11 in PE in women and the potential to target IL-11

[0111] Circulating IL-11 was significantly increased in women with EO PE (35.48pg/ml + 6.29) compared to LO PE (0.77pg/ml + 0.58) or normal pregnant gestation-matched controls (4.83pg/ml + 1.88) prior to PE development (p<0.001) [Figure. 13A] and also following diagnosis (p<0.01) [Figure. 13B]. In PE placenta, IL-11 and IL-11R mRNA showed an up regulation trend (Figure 4C) and IL-11 protein was significantly increased (Figure 13D and E). IL-11 immunostaining was increased in syncytiotrophoblast, villous stroma, endovascular EVTs and decidual cells (Figure 13D).

EXAMPLE 8

Role ofIL-11

[0112] The data herein answer a long standing questions regarding the role of IL-11 in trophoblast invasion and placental development in vivo. Heretofore, in vivo evidence for a functional role of IL-11 in placentation was lacking. Female IL-11R -/- mice are infertile ((Robb et al. (1998) supra) attributed to defective decidualization between E3-E6, leading to mid-gestation pregnancy loss (Menkhorst et al. (2009) Biology of reproduction 80:920- 927), thus not a useful model for studying placentation. This is the first study to demonstrate that IL-11 is causal of PE features in a mouse model. Defective decidual trophoblast invasion and spiral artery remodeling were observed and placental labyrinth defects associated with IL-11 elevation in mice were also evident during mid-gestation. Moreover, IL-11 -administered pregnant mice exhibit diagnostic criteria for PE, including elevated SBP, sFlt-1 dysregulation and proteinuria with kidney pathology. The data herein indicate that elevated SBP and proteinuria can be reversed after damage to the placenta, vasculature and the kidney has ensued, while totally reversing abnormal EVT invasion is not necessary in this model. Furthermore, IL-11 overexpression is connected to early manifestation of PE in vivo, characteristic of the more severe cases of this syndrome (EO PE) and evidence obtained that IL-11 is causal of PE in women. IL-11 is elevated in the serum of women with EO PE prior to diagnosis and disease onset, showing that IL-11 is up-regulated early during placentation prior to PE. Similar to IL-l l-treated mice that exhibit impaired embryo growth, EO PE in women is almost always associated with IUGR.

[0113] It is determined herein that non-pregnant female mice administered with IL-11 did not develop PE features. These data demonstrate a pregnancy- specific effect of IL-11 in eliciting PE features in mice, showing that this occurs via placental alterations. Administration of PEGIL-11 confirmed at least that the IUGR phenotype seen in IL-11 treated mice was attributed to placental insufficiency and not direct IL-11 -signaling in fetal tissues. Likely due to the large hydrodynamic volume displayed by the PEG moiety, PEGIL-11 did not cross the placenta. This finding indicates that PEGylation of potential pharmacological therapeutics may reduce potential embryotoxic effects in pregnancy. In accordance with IUGR, IL-11 treated mice also exhibited placental fibrosis, associated with placental insufficiency and IUGR in women (Chen and Aplin (2003) Placenta 24(4):316-325). It is determined herein that IL-11 reduced placental gene expression and circulating protein levels of IGF-1, an important regulator of placental and fetal growth (Liu et al. (1993) supra), suggesting a mechanism by which IL-11 contributes to IUGR via placental alterations. Interestingly, IGF-1 itself can also alter trophoblast migration (Lacey et al. (2002) BMC developmental biology 2:5).

[0114] The examples herein highlighted a role for exogenous IL-11 in activating STAT3 in the human and mouse placenta. Other pathways activated by IL-11 include the mitogen- activated protein kinase (MAPK) [Yin and Yang (1994) The Journal of Biological Chemistry 269(5):3131-3738], Src-family kinases (Fuhrer and Yang (1996) Experimental hematology 24(2 : 195-203), and phosphatidylinositol 3-kinase (PI3K) signaling pathways (Fuhrer and Yang (1996) Biochemical and biophysical research communications 224(2 J:289-296). The relative importance of each signaling pathway is tissue specific (Du and Williams (1994) Blood S3(SJ:2023-2030).

[0115] Although it is established that IL-11 signals through STAT3 to regulate EVT function, previously, the mechanism of IL-11 -impaired trophoblast invasion and a potential causative role of IL-11 in the development of PE were unknown. IL-11 did not regulate common proteases know to be associated with human EVT invasion and PE, including matrix metallopeptidases (MMPs), tissue inhibitors of MMP (TEVIPs), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and serpin peptidase inhibitors (SERPINEs) [Paiva et al. (2009) supra]. It is shown herein that IL-11 significantly up-regulates PAPPA2 in the human and mouse placenta and also circulating levels in pregnant mouse serum. While it is known that IL-11 itself impedes primary human EVT invasion (Paiva et al. (2009) supra; Sondewregger et al. (2011) supra), the functional role of PAPPA2 in EVT invasion has not previously been investigated. It is demonstrated herein that PAPPA2 ligand impaired primary human EVT outgrowth, while PAPPA2-knockdown was able to rescue IL-11 -impaired EVT outgrowth, implying that IL-11 impedes trophoblast invasion, at least partly via PAPPA2. This demonstrates a novel mechanism by which IL- 11 impairs trophoblast invasion and spiral artery remodeling.

[0116] Additionally, it is demonstrated herein that high levels of IL-11 alter decidual immune cells required for normal spiral artery remodeling. IL10, among other factors, can induce M2 macrophage polarization, which promotes tissue remodeling and immune tolerance (Brown et al. (2014) Frontiers in immunology 5:606). In the IL-11 treated mouse placenta, IL10 was significantly down regulated, suggesting that IL-11 may promote M2 macrophage polarization. Total macrophages only showed a trend in reduced numbers in the IL-11 treated mouse decidua. Interestingly, IL10 is also reduced in women with PE (Makris et al. (2006) Placenta 27(4-5):AA5-A5l). Uterine natural killer cells are the most abundant immune cell type at the maternal-fetal interface (Cartwright et al. (2010) supra). In addition to IL10, IL-11 also significantly reduced placental gene expression of IL15, required for uNK cell maturation and differentiation (Ye et al. (1996) The Journal of Experimental Medicine iS4((5J:2405-2410) and IL18, secreted by uNKs to mediate normal tissue remodeling (Zhang et al. (2003) Biology of reproduction 69(2 J:404-411). Decidual uNKs were dramatically reduced in IL-11 treated pregnant mice at mid-gestation. This finding somewhat contradicts a previous report in IL-11R ^_/~ mice, in which IL-15 production was compromised and uNK cell localization within the decidual compartment was virtually absent (Ain et al. (2004) Developmental dynamics : an official publication of the American Association of Anatomists 23 Ί(4):Ί '00-708). Together, these findings indicate that IL-11 signaling is required for normal uNK cell recruitment, but elevated levels also affect recruitment and/or differentiation and normal function. The present findings highlight a complex role for IL-11 in impairing trophoblast invasion and spiral artery remodeling in vivo, possibly attributed to impaired immune cell recruitment and/or differentiation.

[0117] IL-11 withdrawal after the development of PE features in mice alleviated elevations in blood pressure, proteinuria and also reduced fetal weight. Differences in placental labyrinth morphology were evident in the IL-11 withdrawal group compared to IL-11- treated mice at E17, proposing that this layer is dynamic during late-gestation. In the IL- 11 -withdrawal labyrinth, more red blood cells were apparent in the circulations, advocating improved or restored blood flow. IL-11 did not alter the expression of syncytialization genes or hCG in the human or mouse placenta, suggesting that IL-11 does not affect syncytialization. Impaired labyrinth branching and structure in IL-11 -treated placenta was restored in the IL-11 withdrawal placenta, implying that rescue of the labyrinth may mediate the reduced blood pressure and proteinuria. Labyrinth defects alone have been shown to induce hypertension in mice (Bainbridge et al. (2012) Hypertension 59(3):T32- 739). This finding could potentially be attributed to a significant increase in placental growth factor (PLGF) in the IL-11 withdrawal mouse placenta compared to IL-11 -treated at E17. Inducing PLGF has been shown to ameliorate PE symptoms in mice (Kumasawa et al. (2011) Proc Natl Acad Sci USA i0S(4J: 1451-1455), indicating a potential mechanism by which alleviating high levels of IL- 11 could rescue PE features in vivo in the present model.

[0118] Clinically diagnosed most often in the late second or third trimester, the only current available treatment for PE is placental delivery by labor induction or Cesarian- section. Therefore, the identification of biomarkers in early stages of the PE syndrome will help to target women at elevated preeclampsia risk for closer follow-up, optimizing delivery timing, and avoiding unnecessary premature deliveries. According to the present invention, circulating IL-11 is significantly increased in women with EO PE. IL-11 protein was also significantly increased in human PE villous and EVT.

[0119] Despite its well-characterized role in decidualization in humans and mice, IL-11 withdrawal had no effect on the decidua. In women and mice, decidual IL-11R protein levels are significantly reduced during the second trimester or mid gestation respectively, highlighting the ability to target IL-11 to ameliorate PE in women, without affecting the decidua or pregnancy viability.

[0120] In summary, the present invention is predicated in part on the demonstration that IL-11 is causal of PE features in a mouse model and in women. These findings highlight the potential of IL- 11 inhibition to rescue PE symptoms in women. An appropriate model is validated to test potential therapeutics for the treatment of PE.

[0121] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure contemplates all such variations and modifications. The disclosure also enables all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features or compositions or compounds.

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