SB273005

Integrin αvβ3 Is Essential for Maintenance of Decidua Tissue Homeostasis and of Natural Killer Cell Immune Tolerance During Pregnancy

Shaojuan Wang, MD1, Xiaoli Zhou, MM2, and Jing Yang, MD3
Reproductive Sciences 1-7
ª The Author(s) 2018
Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719117746766
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Abstract
Physiological functions of villous trophoblasts are essential for normal implantation and pregnancy, which are under fine reg- ulations. Integrin avb3 has been shown to mediate cellular attachment of villous trophoblasts; however, the physiological functions of integrin avb3 in pregnancy have not been revealed. Here, we found that suppression of integrin avb3 in vivo by a small molecule inhibitor, SB-273005, resulted in abnormal pregnancy in mice. Mechanistically, suppression of integrin avb3 in vivo broke down the tissue homeostasis of the decidua, as revealed by disorganized histology and compromised cellular proliferation in the decidua. Compromised cellular proliferation was also observed in integrin av knocked down human villous trophoblast cell lines, suggesting that compromised proliferation of trophoblast might contribute to the abnormal pregnancy after SB-273005 injection. Moreover, increased NK cells, as well as elevated serum levels of IFN-r in pregnant mice after SB- 273005 injection, further linked aggravated pathogenic NK cell responses with the abnormal pregnancy in mice with integrin avb3 inhibition. Together, these data demonstrate a role of integrin avb3 in maintenance of decidua tissue homeostasis and of NK cell immune tolerance during pregnancy.

Keywords
pregnancy, trophoblast, integrin, natural killer cells

Introduction
Villous trophoblasts represent the maternal–fetal interfaceplaying critical roles in maintaining both the homeostasis of placental tissues and maternal–fetal immune tolerance.1 Prolif-uterine receptivity.8 Also, aberrant integrin b3 expression in the endometrium is associated with endometriosis and defects in uterine receptivity.9 In addition, integrin-binding galectins in the endometrium were proposed to contribute to normal
10implantation. Functional studies using neutralizing antibodyeration and invasion of the villous trophoblasts into the deciduaare essential for implantation and normal pregnancy.2 Physio-against integrin avb3 indicate its critical role in normal implan-11,12 logical functions of villous trophoblasts are under fine regula-tions, whose underlying mechanisms are not fully understood. Integrins, the major adhesion molecules, are a family of heterodimeric transmembrane glycoproteins with subunits a and b.3 Integrins mediate cell–cell recognitions and cellular interactions with extracellular matrix (ECM). During implanta- tion, recognition of ECM components (eg, vitronectin and fibronectin) by integrin avb3 is required for the cell–cell inter- actions to attach the trophoblast to the uterine epithelium.4 Upon ligand recognitions, downstream protein kinases are acti- vated, such as focal adhesion kinase (FAK) and MAP kinases p42 and p44.5 Integrin avb3 expression was detected in the epithelial compartment of the endometrium, as well as in the decidua of pregnancy.6,7 The expression pattern of integrin avb3 in the endometrium was shown to be related to the win- dow of implantation, suggesting its role in establishment of integrin avb3 in pregnancy, the physiological role of integrin avb3 in pregnancy has not been fully investigated.

1 Department of Gynaecology and Obstetrics, People’s Hospital of Longgang Distract, Shenzhen, Guangdong, China
2 Department of Gynaecology and Obstetrics, Women and Children’s Hos- pital of Longgang Distract, Shenzhen, Guangdong, China
3 Department of Gynaecology and Obstetrics, People’s Hospital of Wuhan University, Wuhan, Hubei, China.

Corresponding Author:
Shaojuan Wang, Department of Gynaecology and Obstetrics, People’s Hospital of Longgang Distract, No.53, Ailong Road, Centre of Longgang Distract, Shenzhen, Guangdong 518174, China.
Email: [email protected]

NK cells are innate immune cells that play important roles in both host defense and tissue homeostasis.13 Through IFN-g production, NK cells regulate adaptive immune responses,14 immune pathology,15 tissue regeneration,16 and pregnancy.17,18 During early pregnancy, NK cells extraordinarily accumulated in the decidua in humans.19-22 In the deciduae, interactions between NK cells and trophoblasts maintain homeostasis of placental tissues, which are essential to pregnancy.23 However, the molecular mechanisms, as well as the physiological impact, of NK–trophoblast interaction have not been revealed.Here in this study, we aim to investigate the role of integrin avb3 in maintaining decidua tissue homeostasis, as well as in preserving immune tolerance during pregnancy.

Materials and Methods
Animal Model
Six- to eight-week-old female KM mice were purchased from the Animal Experiment Facility of Sun Yat-sen University. Mice were iv injected with 100 mL of dimethylsulfoxide (DMSO) or 30 mg/kg of small molecule inhibitor SB-273005 (SmithKline Beecham, London, UK) dissolved in 100 mL of DMSO every day from days 3 to 5 since gestation day and were then sacrificed on day 8 for analyses. The animal experiments were approved by the Animal Experiments Committee of the Wuhan University.Histological Analysis and Histoimmunochemistry Endometrium tissues from pregnant mice were fixed with 4% paraformaldehyde and embedded in paraffin, and 5-mm sec- tions were used for HE staining or for histoimmunochemistry staining of integrin avb3 expression using anti-integrin avb3 antibody from Abcam (Cambridge, UK).

Antibodies and Reagents
We purchased anti-phospho-PDK1 and anti-PDK1 from Pro- spec (Rehovot, Israel), anti-phospho-mTOR, anti-mTOR, anti-integrin a 5, anti-phospho-P38, anti-phospho-P70S6K, anti-phospho-RPS6, anti-RPS6, and anti-integrin 5 b 3 from Abcam (Cambridge, UK); anti-SPP1, anti-AKT, anti-P38, anti-ERK, and anti-P70S6K from Santa Cruz (San Diego, California, USA); SPP1 protein from Millipore (Burlington, New Jersey, USA); anti-SPP1 from Pierce (Appleton, Wisconsin, USA); and anti-phospho-AKT from Cell Signal (Danvers, Massachusetts, USA).

Immunoprecipitation
Protein G agarose beads (GE) were incubated with cell lysate overnight, together with specific antibody or control IgG. Beads were then washed thoroughly before Western blot (WB) analysis.
Flow Cytometry
We purchased fluorescein isothiocyanate (FITC) anti-CD19, Percp-CD3, and APC anti-NK1.1 from BD Biosciences. Gating of T, B, and NK cells was based on surface expression of CD3, CD19, and NK1.1 (Supplementary Figure 1).

Proliferation Assay
Cells treated with the indicated concentrations of SPP1 were allowed to proliferate for 1 week. Culture medium was replaced with fresh medium every 3 to 4 days. After 1 week, medium was removed, and cells were fixed with 10% glutar- aldehyde before stained with 0.1% crystal violet solution (Beyotime, Shanghai, China). After staining, cells were washed with water and detected for OD595 for assessment of cell amounts to reflect proliferation.

Cytometric Bead Array
Amounts of cytokines in the serum were determined using the mouse Th1/Th2/Th17 cytometric bead array (CBA) kit (Pharmingen, San Diego, California, USA).

Statistics
Statistical significance differences were determined by Student
t tests. P values of <.05 were considered significant.

Results
Integrin avb3 Is Essential for Normal Pregnancy
In order to investigate the physiological role of integrin avb3 in implantation in mice, we employed small molecule inhibitor SB-273005, which was shown previously to reduce integrin avb3 expression in the endometrium. Here, we confirmed that SB-273005 injection reduced integrin avb3 expression both in the endometrium and in the spleen (Figure 1A). We found that compared with the normal gravid nodules observed in the con- trol pregnant mice, SB-273005 injection resulted in absent, smaller, or inhomogeneous gravid nodules, which indicated abnormal pregnancy (Figure 1B). This result showed that integ- rin avb3 is required for the formation of normal gravid nodules and for normal pregnancy.

Integrin avb3 Is Essential for Maintenance of Decidua Homeostasis During Pregnancy
We next set out to study the underlying mechanisms of integrin avb3-mediated protection of pregnancy. Histology analysis showed that SB-273005 injection led to disorganized decidua tissue in pregnant mice (Figure 2A). We showed that SB- 273005 injection reduced protein expression of integrin avb3 in the endometrium tissue of pregnant mice (Figure 2B). Such reduction in integrin avb3 expression was accompanied with decreased expression of cyclin D1, increased expression of

Inhibition of integrin avb3 in vivo by SB-273005 resulted in aggravated abortion in pregnant mice. A, Immunohistochemistry analysis of integrin avb3 expression in the endometrium or spleen of pregnant mice treated with DMSO or SB-273005. B, Mice pregnancy after treatment with DMSO or SB-273005. DMSO indicates dimethylsulfoxide. p21,24 and decreased phosphorylation levels of ERK, AKT, and FAK (Figure 2B). These results demonstrate that the cellular proliferation was suppressed in the decidua after integrin avb3 inhibition, suggesting that integrin avb3 is essential for the maintenance of decidua homeostasis. Integrin avb3 Is Essential for SPP1-Induced Proliferation of Villous Trophoblast Cells We showed that cellular proliferation in the decidua tissue was suppressed in the absence of integrin avb3 (Figure 2B). Since the proliferation of villous trophoblasts and their invasion into the decidua in critical for normal pregnancy,2 we wondered whether the proliferation of villous trophoblasts was compro- mised in the absence of integrin avb3, thus leading to abnormal pregnancy (Figure 1B). We first screened human villous tro- phoblast cell lines stably expressing shRNA targeting integrin av for a cell line with the most reductions in integrin av expres- sion both at mRNA levels (Figure 3A) and at protein levels (Figure 3B and C). Next, we set out to investigate the role of integrin avb3 in proliferation of villous trophoblasts by stimu- lating these cells with SPP1 (osteopontin), an ECM protein that serves as a ligand for integrins.25 Here, in villous trophoblast cell lines, we observed colocolization of SPP1 and integrin avb3 (Figure 3D). More importantly, we confirmed interaction between SPP1 and integrin avb3 in villous trophoblast cells by co-IP (Figure 3E). On stimulation with SPP1, robust prolifera- tion of villous trophoblasts was observed; however, silencing integrin avb3 in these cells antagonized this effect (Figure 3F and G). These data indicate that integrin avb3 is essential for SPP1-induced proliferation of villous trophoblast cells and sug- gest that compromised proliferation of villous trophoblasts after SB-273005 injection might contribute to the abnormal pregnancy we observed above (Figure 1B). Integrin avb3 Is Essential for Suppressing Pathogenic Natural Killer Cell Responses During Pregnancy The above results showed that homeostasis of the decidua tis- sue was compromised in the absence of integrin avb3. Given Inhibition of integrin avb3 in vivo by SB-273005 broke the homeostasis of the decidua in pregnant mice.

A, Histology analysis of deciduae in pregnant mice treated with DMSO or SB-273005. B, Western blot analysis of the indicated protein expression or phosphorylation of signaling molecules in the endometrium tissue of pregnant mice treated with DMSO or SB-273005. C, Statistical analysis using multiple mice from the indicated groups was shown for (B). *P < .05. (A-C) Data are representative of 3 independent experiments. DMSO indicates dimethylsulfoxide.that immune tolerance is linked to tissue homeostasis, we won- dered whether broken homeostasis of the decidua in the absence of integrin avb3 would also result in compromised immune tolerance in the maternal–fetal interface during preg- nancy. We examined peripheral blood in pregnant mice treated with inhibitor against integrin avb3 or DMSO and found that the percentages of NK cells, but not T cells or B cells, signif- icantly increased (Figure 4A). Furthermore, IFN-g levels in the serum of the inhibitor group were significantly elevated (Figure 4B). Considering that T cells were not increased (Figure 4A), the elevated serum levels of IFN-g might be the result of ele- vated activation of NK cells. These data indicate that systemic NK cell responses were enhanced in the absence of integrin avb3, which might at least in part contribute to the abnormal pregnancy observed after integrin avb3 inhibition in vivo.

Discussion
Invasion of villous trophoblasts into the decidua is essential for proper implantation and normal pregnancy.2 Such pro- cess involves intrinsic functions of trophoblasts, as well as interactions with various cells within the microenvironment, including immune cells. Our study shed light on both sides of the process by revealing the physiological functions of integrin avb3 in the regulation of decidua tissue homeosta- sis and pregnancy.
Integrins play important roles in cellular proliferation, migration, adhesion, and survival.26 Among many integrins, integrin avb3 is a target for antitumor treatment under clin- ical trials.26 The role of integrin avb3 in cellular proliferation has been studied.27 In our study, we showed that integrin avb3 is essential for osteopontin-mediated proliferation of Integrin avb3 is essential for SPP1-induced proliferation of HVT cells. A, HVT cells stably expressing shRNAs targeting integrin av, or control shRNA, were examined for expression of integrin av mRNA by PCR. B, HVT cells in (A) were examined for expression of integrin av protein by Western blot. C, HVT cells in (A) were examined for expression of integrin av protein by immunofluorescence. D, Immunofluor- escence showed that integrin avb3 colocalized with SPP1 in HVT cells. E, Immunoprecipitation using HVT cells showed that integrin avb3 interacted with SPP1. F, Proliferation of HVT cells stably expressing shRNA targeting integrin avb3 or control shRNA, as shown by crystal violet staining. G, Statistical analysis (F) shows relative proliferation of Human Villous Trophoblasts cells.

(A-G) Data are representative of 3 independent experiments. (A and G) Data are presented as the mean + SEM. *P < .05. PCR, polymerase chain reaction.
trophoblasts, since silencing integrin av in human villous trophoblasts resulted in arrested cell cycle in vitro. Also, our in vivo data showed that inhibition of integrin avb3 sup- pressed activation of cellular proliferation-associated signal- ing pathways in the decidua, possibly leading to abnormal pregnancy. These results further strengthen the role of integ- rins in regulating cellular proliferation.Besides intrinsic regulations of cellular functions, integrins are also involved in orchestrating immune responses through their roles in immune cell migration and cell–cell interac- tions.28 Integrin avb3 has been shown to facilitate TLR signal- ing.29 On the other hand, the role of integrins in maintenance of immune tolerance has not been investigated before. Our data showed that physiological inhibition of integrin avb3 led to abnormal pregnancy, accompanied with enhanced NK cell responses. NK cell–mediated immune injury has been exten- sively studied.30-32 It’s possible that the enhanced NK cell responses we observed might be pathogenic, contributing at least in part to the abnormal pregnancy after integrin avb3 inhibition. IFN-g is the typical effector cytokine produced by NK cells upon activation. IFN-g was reported to reduce expression of protein inhibitor of activated STAT3 (PIAS3),leading to increased phosphorylation of STAT3 and aggra- vated pathogenic inflammations.33 On the other hand, the elevated serum levels of IL-17 we observed was inconsistent with the elevated IL-17 levels in endometriosis as well, which activates STAT3 through IL-6. Activated STAT3 binds to and stabilizes HIF-1a without hypoxia.34 Altogether, these mechanisms contribute to the pathogenesis of endometriosis and poor pregnancy. However, the mechanisms underlying the enhanced NK cell responses are still elusive. Given that compromised proliferation and invasion of trophoblasts dur- ing implantation could result in local hypoxia, the resulting inflammation might account for the stimulus that activated NK cells for secondary injury. Further studies are required to reveal the details of NK cell–mediated immune pathology in the absence of integrin avb3.

Collectively, our study demonstrates the physiological func- tions of integrin avb3 in the maintenance of decidua home- ostasis and of immune tolerance, which is essential for normal pregnancy. Based on our study, further relevance studies in human genetics are required to confirm whether genetic defi- ciency in integrin avb3 could be considered as a risk factor for normal implantation and pregnancy. Enhanced NK cell responses after inhibiting integrin avb3 by SB-273005 in vivo. A, Percentages of T cell, B cells, NK cells, and CD16þ cells among NK cells in the peripheral blood of pregnant mice treated with DMSO or SB-273005. B, Serum concentrations of indicated cytokines from pregnant mice treated with DMSO or SB-273005. (A and B) Data are pooled from 3 independent experiments (n > 15) and are represented as the mean + SEM. *P < .05. DMSO indicates dimethylsulfoxide.

Declaration of Conflicting Interests
The author(s) declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This workwas supported by Expert Promotion plan of Longgang District from the government of Longgang District to Shaojuan Wang.

Supplemental Material
Supplementary material for this article is available online.

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