Female Reproductive Immunology


Placenta specific protein 1 (PLAC1) promoting invasion and migration of human trophoblast cells

W Chang1 , 2, Q Yang2, H Zhang3, H-Y Lin1, Z Zhou1 , 3, X Lv1,4, C Zhu1, Y Zhang5, L Xue2, H Wang1

1State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 2College of Veterinary Medicine, Hunan Agricultural University, Changsha, China; 3Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China; 4Graduate School of Chinese Academy of Sciences, Beijing, China; 5Department of Immunology, Peking University Health Science Center, Beijing, China

PLAC1, a placenta-specific gene, is implicated in the development of placenta in both humans and mice. However, the precise role of PLAC1 in placental trophoblast function remains unclear. In this study, the localization of PLAC1 in human placental tissues and its physiological significance in trophoblast invasion and migration were investigated through technical studies including real time RT-PCR, in situ hybridization (ISH), immunohistochemistry (IHC), and functional studies by utilizing cell invasion and migration assays in trophoblast cell line HTR8/SVneo as well as the primary inducing extravillous trophoblast cells (EVTs). The results showed that PLAC1 was mainly detected in the trophoblast columns (TC) and syncytiotrophoblast (ST) of the first trimester human placental villi, as well as in the EVTs which invaded into the maternal decidua. Knockdown of PLAC1 by RNA interference suppressed significantly the invasion and migration of HTR8/SVneo cells and shortened the distance of the outgrowth of the induced EVTs from the cytotrophoblast column of the explants. All the data suggest that PLAC1 can play an important role in human placental trophoblast invasion and migration.

Crosstalk of decidual T cells and trophoblasts

D-J Li

Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

There is a precise intercellular crosstalking at maternal-fetal interface, especially between decidual immunocytes and trophoblasts in pregnancy. The decidual T cells are recruited from peripheral into decidua by trophoblasts-secreted CXCL16 which increases the proliferation of γδ T cells, and downregulates the granzyme B activity in γδ T cells. The decidual γδ T cells promote proliferation and inhibit apoptosis, and enhance invasiveness of trophoblast mainly through IL-10 secretion in the early human pregnancy. The trophoblast-derived TSLP can instruct decidual DCs that result in differentiation and expansion of regulatory T cells (Treg) from naïve T cells. Tregs in the decidua promote and inhibit invasiveness of trophoblasts by secreting IL-10 and TGF-β1, respectively, and the interaction of cell surface molecules between both cells is also involved in modulation of the trophoblast invasion. The CD4+CD25+ Tregs may upregulate HLA-G expression in trophoblasts which may be involved in regulating the phenotype and functions of decidual NK cells. The DSCs-secreted CCL2 recruits Th17 cells into the decidua. Th17 cells induce the proliferation and invasion, and inhibit apoptosis of trophoblast mainly through IL-17 secretion.

Recent studies on the functions of endometrial NK cells in human reproduction

A Fukui

Department of Obstetrics and Gynecology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan

Natural Killer (NK) cells play an important role in human pregnancy and their systemic regulation contributes to reproductive success. The regulation of uterine and circulating peripheral blood NK cells has been associated with reproductive immunology such as recurrent pregnancy loss (RPL), implantation failure or preeclampsia. No doubt there are an abnormal number and ratio of NK cells, abnormal expression of their surface receptors and their production of abnormal cytokines in such reproductive failures. Thus the appropriate regulation of immune cells, especially NK cells, can lead to reproductive success.

The main population of uterine NK cell is CD16/CD56bright cytokine producing cells, and for peripheral blood NK cell it is CD16+/CD56dim cytotoxic NK cell. We have previously reported that the percentages of CD16+/CD56dim NK cells in peripheral blood and endometrium in IVF-ET cycle were significantly higher and that of CD16/CD56btight NK cells was markedly lower in reproductive failures than in the successful group. For the NK cell surface markers, natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We reported that CD56+/NKp46+ cells were notably decreased in women with RPL as compared with those of normal controls. During pregnancy, pregnant women who had a history of PRL had significantly decreased percentages of CD56bright/NKp46+ cells. Moreover, the proportion of CD56bright/IFN-+/TNF-+ cells was significantly higher in women with RPL and implantation failures than in the normal controls.

To know the role of NKp46 expression in cytokine-producing NK cells, we investigated the IFN-, TNF-, IL-4, IL-10, and TGF-1 expressions on NK cells using flow cytometry. NKp46 expression was found to be associated with a higher frequency of cytokine-producing NK cells, including not only CD56bright NK cells but also CD56dim NK ones. The percentage of TNF-+ and IL-10+ NK cells in CD56+/NKp46+ NK cells in the uterine endometrium showed a significant correlation with those of the peripheral blood in all subpopulations. The expression of NKp46 can be involved in cytokine production of CD56+ NK cells in peripheral blood and uterine endometrium.

Recently, a new type of NKp46+ NK cell, which produces IL-22, was identified as NCR22 or NK22. IL-22 production via NCR22 cells was reported to mediate immune defense in the skin, lung and intestine. IL-22 producing NK cells were present in uterine endometirum and NCR22 cells might play a key role in reproduction. Many interesting questions, however, remain unanswered regarding the functions of NCR22 cells and the role of IL-22 in reproductive failure and other diseases. Our studies have shown that the production of NK22 cells was significantly higher in women with RPL than in the normal controls. In women with RPL, moreover, there were negative correlations between peripheral blood CD56+/IL-22+ cells, CD56bright/IFN-+ cells and CD56bright/TNF-+ cells. Similarly, negative correlation was observed between endometrial CD56+/IL-22+ cells and CD56bright/IFN-+. IL-22 producing NK cells may be associated with the production of cytokines in both peripheral blood and endometrium.

In conclusion, there are abnormal expressions of NK cell surface antigens and dysregulation of NK cell cytotoxicity and cytokine production in women with reproductive failures.

The role of placental tryptophan catabolism

P Sedlmayr

Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria

The first and rate-limiting step of tryptophan oxidation, initiating the kynurenine pathway of tryptophan catabolism, is mediated by the enzymes tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2). Whereas data on expression of IDO2 and TDO in the human placenta are limited, there is ample, partly conflicting evidence on expression and localisation of IDO1 in the placenta. IDO1 has antibacterial, antiparasitic and immunoregulatory properties, it has been implicated in inducing activation of Treg cells and mediating feto-maternal tolerance. IDO1 is localized in surface and glandular epithelium of the uterus and may contribute to the antiinfections mechanisms preventing ascending infections of the female genital tract. During pregnancy vascular endothelium on both sides of the fetomaternal interface increasingly expresses IDO1. As kynurenine, a degradation product of tryptophan, has vasodilatory properties, this suggests a role of endothelial IDO1 in mediating placental perfusion and growth. Moreover, the role of endothelial cells as antigen-presenting cells will deserve attention in this context.

Anti-inflammatory effect of a Chinese herbal medicine on atherosclerosis via estrogen receptor β mediating nitric oxide production and NF-κB suppression in endothelial cells

L Wang1,*, X-M Qiu1,*, Q Hao1, D-J Li1,2

1Laboratory for Reproductive Immunology, Department of Obstetrics and Gynecology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, Shanghai, China 2Department of Obstetrics and Gynecology, The Affiliated Hospital, Hainan Medical College, Haikou, China

Bu-Shen-Ning-Xin Decoction (BSNXD) administration has alleviated the early pathologic damages of atherosclerosis by inhibiting the adhesion molecule expression, upregulating the estrogen receptor (ER) β expression in endothelial cells, and increasing the serum nitric oxide (NO) level without any effect on serum lipid status, endometrium and fat deposition in liver in ovariectomized rabbits. The BSNXD-derived serum increases ER β expression in the human umbilical vein endothelial cells (HUVECs), decreases malondialdehyde (MDA) production, and upregulates eNOS expression to increases NO synthesis through ERβ-dependent pathway. NO not only suppresses the LPS-induced NF-κB transcription in HUVECs, but decreases apoptosis of endothelial cells. The BSNXD-derived serum decreases monocyte chemoattractant protein-1 production, and suppresses cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) expression in HUVECs injured by oxidized low-density lipoproteins (ox-LDL), and these effects can be abolished by ERβ antagonist (R,RTHC) and NO synthase inhibitor (L-NAME). The BSNXD-derived serum-treated HUVECs supernatant reduces CCR2, LFA-1 and VLA-4 expression in monocytes cell line U937 cells, which in turn inhibits adherence of U937 to injured endothelial cells. NO synthesis increases, and MDA production decreases through ERβ-mediated pathway that suppresses apoptosis and NF-κB activity in endothelial cells that downregulates adhesion molecules expression on endothelial cells via ERβ/NO/NF-κB pathway, and in turn leukocyte adhesion, which suggests the potential value of BSNXD in prophylaxis atherosclerosis.

*These authors contributed equally to this work.

Osteoclastogenesis inhibited by traditional Chinese herbal formula via estrogen receptor alpha suppressing RANKL-induced NFATc1 and NF-κB signaling pathway in ovariectomized mice

L Wang, X-M Qiu, Y-D Wang, D-J Li

Laboratory for Reproductive Immunology, Hospital & Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, Shanghai, China

Summary

In the present study, we investigated the pharmacological effect of Bu-Shen-Ning-Xin Decoction (BSNXD) on ovariectomy-induced osteoporosis in mice. We found that BSNXD produced an inhibitory effect on osteoclast differentiation by abrogation of RANKL-induced NFATc1 expression.

Introduction

BSNXD, a traditional Chinese medicinal composition, has been used as a remedy for postmenopausal osteoporosis, but the molecular mechanism of the efficiency on bone metabolism is little known. We sought to identify whether BSNXD could regulate osteoclast differentiation.

Methods and Results

Mid-dose and high-dose BSNXD administration ameliorated the osteoporotic phenotype of ovariectomized (OVX) mice, as evidenced by an increase in bone mineral density (BMD), bone volume, and a decrease in osteoclast bone resorption as revealed by microradiographic and histological analysis, respectively. We then investigated the effect in vitro of mid-dose BSNXD-derived serum on osteoclastogenesis. BSNXD-derived serum dose-dependently suppressed RANKL-activated osteoclastogenesis. This suppressive effect could be reversed by estrogen receptor (ER)α antagonist Methyl-piperidino-pyrazole (MPP), but not by ERβ antagonist R, R-tetrahydrochrysene (R,RTHC) or androgen receptor (AR) antagonist, flutamide (FLUT). Meanwhile, the BSNXD-derived serum did not change the Csf1R+ RANK+ osteoclast precursor population, but suppressed the RANKL-induced NF-κB transcription in osteoclasts, reversed by ERα antagonist MPP. BSNXD-derived serum inhibited accumulation of the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) in osteoclast precursor cells, which is a key transcription factor for the differentiation of osteoclasts, whereas PLC-γ2 phosphorylation was unaffected. The inhibitory effect of the BSNXD-derived serum on NFATc1 expression was abolished by ER α antagonist (MPP).

Conclusions

These results suggest that BSNXD presents an inhibitory effect on osteoclast differentiation via abrogation of RANKL-induced NFATc1 expression as downstream of ERα, and may serve as a promising therapy for bone loss in postmenopausal osteoporosis.

Key words

traditional Chinese herbal formula, osteoclastogenesis, estrogen receptor alpha, NF-κB, NFATc1, BMD.

Regulatory T cells and inflammation contributing to the pathogenesis of endometriosis by promoting angiogenesis

X Wang, D Li

Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Shanghai Medical College, Fudan University, Shanghai, China

Background

Although a series of studies have shown that the frequency of Treg cells increases significantly in the peritoneal fluid of patients with endometriosis, it remains unclear how Treg plays a role in the disease. The aim of this study was to investigate the functional regulation of Treg cells and its effect on angiogenesis in the ectopic milieu.

Methods

The human endometrial stromal cells (ESCs) were isolated from women with endometriosis. Macrophage and Treg cells were purified from healthy human peripheral blood using MACS separation kits. Enzyme-linked immunosorbent assay was used to quantify the concentration of CCL22, CCL17, TGF-β1, IL-8 and VEGF in the culture supernatant; transwell chemotaxis assay, to analyze the chemoattractant for Treg cells; MLC, to detect the immune suppression of Treg cells; Immunochemistry, to demonstrate the translation of smad2 and p-smad2; in cell western, evaluate the expression of smad2/p-smad2, p38/p-p38, ERK/p-ERK and JNK/p-JNK; flow cytometry, to analyze the expression of Stat 3, Stat 6, FOXP3, CD357, CD39, CD73 and CTLA-4 on Treg cells.

Results

ESCs co-culture with macrophage promoted the secretion of CCL22 and CCL17, which were further enhanced by estrogen and progesterone. The co-culture unit attracted Treg cells by secreting CCL22 and CCL17, strengthening their immune suppression, such as stimulating TGF-β1 secretion, up-regulating Stat 3 and Stat 6 expression in Treg cells and increasing their inhibitory effect on CD4+T cells’ proliferation. The co-culture unit also enhanced the expression of FOXP3, CD39, CD73 and CTLA-4 in Treg cells. A higher level of smad2/p-smad2 immunoreactivity was observed in eutopic endometrium than in the normal endometrium. Treg activated p38/ERK-smad2 signal pathway in ESC cells by secreting TGF-β1, IL-1β, TNF-α playing a synergistic role. TGF-β1 and inflammatory cytokines IL-1β or TNF-a promoted synergistically the expression of pro-angiogenic factors IL-8 and VEGF in ESCs via the signal pathway.

Conclusion

ESCs and macrophage interaction can present chemotaxis to Treg cells by secreting high levels of CCL17 and CCL22, which enhances the immune suppression of the cells. Treg cells can promote angiogenesis in ectopic milieu by secreting TGF-β1, IL-1β or TNF-α can play a synergistic role in it, which contributes to the pathogenesis of endometriosis.

Key words

endometriosis, Treg cells, angiogenesis, TGF-β1, inflammation cytokines.

The modulation of DHEA on MSC differentiating into osteoblast

X-M Qiu, L Wang, D-J Li

Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, Shanghai, China

Introduction

Postmenopausal osteoporosis (PMO), a metabolic bone disease, is characterized by a decrease of bone mass after menopause. We have found that DHEA level decreases in postmenopausal women when compared to that of the fertile women, which suggests the potential clinical benefit of DHEA as therapeutics of PMO. However, both the molecule and biochemical mechanisms by which DHEA exerts its effect on bone metabolism remains unknown.

Objective

To investigate the mechanism of DHEA on the osteoblast differentiation from MSC.

Methods

Under the osteoblast(OB) differentiation inducing condition, DHEA was added to the cell culture system, including MSC, co-culture of BMM and MSC, Treg and MSC, MSC, BMM and Treg. Real time RT-PCR analysis was applied to Runx2, Collagen1, Osterix, Osteocalcin, RANKL mRNA expression; ALP activity, to OB quantity; and Alizarin Red O, to adipocyte numbers. IGF-1, PI3K and mTOR inhibitor were used to investigate the regulation on the differentiation. Under the adipocyte differentiation inducing condition, DHEA was added to the MSC culture, or the correspondent co-culture of MSC and BMM. C57BL/6 mice were ovariectomized for the foundation of osteoporosis, and then afforded with DHEA (OVX+DHEA group), 17β-estradiol (OVX+E2 group) and Saline (OVX group), respectively. The femur and spleen were collected in 12 weeks of the administration. Femur was measured for morphometry of bone tissue by micro-CT and bone tissue chemical method. The immune cells from spleen were analyzed by flow cytometry (FCM).

Results

DHEA increased OB production in the culture of MSC; in the co-culture of BMM and MSC, Treg and MSC, MSC and BMM and Treg, DHEA increased osteoblastogenisis. DHEA raised the expression of Runx2, osterix, collagen1, osteocalcin mRNA, and decreased the RANKL mRNA expression, and then increased the ALP activity in OB, and bone nodule number. Under the adipocyte differentiation inducing condition, both in the MSC culture or the co-culture, DHEA promoted adipocyte quantity; PPARγ expression. With the same number of MSCs, in either OB differentiation or adipocyte differentiation inducing condition, DHEA increased the ration of OB numbers to adipocyte numbers, which implies that the effect of DHEA on OB differentiation is dominant. DHEA promotes BMD level; DHEA decreased the percent of CD19+B cells, CD14+monocytes, CD8+T cells in spleen, and increased the percent of FoxP3+Treg, but had no effect on the percent of CTLA-4+ cells.

Conclusions

DHEA can promote MSC differentiating into OB, antagonist the downregulation of BMM on MSC differentiating into OB, and enhance the upregulation of Treg on the MSC differentiating into OB by increasing the expression of Collagen1, Runx2, osteocalcin, OSX, and decreasing the expression of RANKL mRNA expression. DHEA actions are enhanced by IGF-1, independent of PI3K-mTOR signal pathway. DHEA could improve the bone tissue morphometry of the PMO model and play a regulatory role in immunity through FoxP3+ Treg.

New Insights into mucosal immune responses against viral infection in the female genital tract

C Kaushic

McMaster University, Department of Pathology and Molecular Medicine, Ontario, Canada

The genital epithelial cells (GECs) comprise the first line of defense in the female reproductive tract, recognizing and initiating innate immune responses to harmful viruses and bacteria. These cells are the first to come in contact with pathogens and their interactions with different sexually transmitted viruses are distinct, based on their ability to recognize and respond to viral components and the reciprocal strategies developed by viruses to subvert the immune responses. We have extensively examined and described the GEC interactions with two sexually transmitted viruses, HIV-1 and HSV-2, which pose significant health problems, globally. Our studies show that HIV-1 does not directly infect GECs. Instead, TLR2 and TLR4 on surface of GECs recognize the HIV-1 surface glycoprotein, gp120, and induce an innate immune response mediated by NF-kB activation. The ensuing induction of pro-inflammatory cytokines, specifically TNF-alpha, acts on epithelial tight junctions to impair the mucosal barrier, allowing translocation of luminal virus and bacteria. This could potentially benefit HIV-1 by facilitating infection and subsequent initiation of immune activation due to microbial translocation, seen in HIV-infected individuals. On the other hand HSV-2, unlike HIV-1, directly infects and replicates in GECs and has therefore developed alternate strategies to subvert the host innate immunity. The GECs recognize and initiate innate immunity by activating both pro-inflammatory cytokine and Type I IFN response following HSV-2 infection. HSV-2 viral host shutoff (vhs) protein significantly down regulates GEC responses resulting in higher viral replication. Since our studies indicate that inflammation promotes mucosal barrier disruption and replication of both HIV-1 and HSV-2, we are currently testing strategies that enhance the epithelial barrier functions and suppress local inflammation to determine if treatment with these can prevent sexually transmitted viral infections or replication.

Interplay between sex hormones, mucosal immunology and susceptibility to HIV infection in the human female reproductive tract

CR Wira, JV Fahey, M Rodriguez-Garcia, Z Shen

Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA

Problem

Effectiveness of immune protection in the human female reproductive tract (FRT) is essential for protection against HIV-1 and other sexually transmitted infections (STI) which compromise reproductive health and threaten the lives of women worldwide. We hypothesized that sex hormones (estradiol and progesterone) produced during the menstrual cycle alter immune function to optimize the chances for successful implantation. By acting directly on immune cells and indirectly through the regulation of cytokines, chemokines and growth factors made by the epithelial cells and fibroblasts from the upper and lower FRT, immune effectiveness can be precisely regulated to optimize the chances for fertilization without compromising maternal protection. Despite extensive studies, relatively little is known about the complex interactions between the mucosal immune system and the role of sex hormones throughout the FRT.

Methods

Epithelial cells, fibroblasts and immune cells were isolated from uterine endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients.Studies were performed with the Dartmouth College Institutional Review Board approval. Women undergoing hysterectomies at Dartmouth-Hitchcock Medical Center (Lebanon, NH) gave written informed consent before surgery. Approval to use tissues was obtained from the Committee for the Protection of Human Subjects (CPHS). The cells were isolated and grown in wells or inserts and treated with estradiol and progesterone in culture. A variety of immunological assays were carried out using real time PCR, ELISA analysis and flow cytometry to examine the effects of sex hormones on immune function. For HIV studies, isolated cells were infected with HIV-1BaL (R5), after which cell cultures were maintained for 6–7 days, prior to intracellular p24 measurement.

Results

Analyses of mRNA, proteins secreted and immune function indicated that estradiol and/or progesterone acted directly to: (a) stimulate and inhibit the secretion of antimicrobials by uterine and vaginal epithelial cells, respectively; (b) enhance anti-HIV activity in secretions; (c) reduce HIV infection of CD4+ T cells and macrophages; (d) increase fibroblast secretion (SDF-1) and (e) enhance HGF and TGF production in fibroblasts and epithelial cells. These hormones also acted indirectly through TGF, cytokines, chemokines and growth factors made by epithelial cells and fibroblasts to: (a) reduce intracellular IFN protein in uterine NK cells; (b) down-regulate DC-SIGN expression on immature dendritic cells (iDC); (c) inhibit iDC HIV trans-infection to HIV target cells in the FRT; and (d) reduce HIV infection of CD4+T cells in a way that varies with the site analyzed in the FRT.

Conclusions

Estradiol and progesterone are capable of modifying the mucosal landscape of the upper and lower FRT. These studies indicate that sex hormones influence HIV-infection by acting directly on immune cells as well as indirectly by altering the microenvironment in the FRT. Further, the analysis of immune function in the upper and lower FRT indicates that hormone actions are distinct and vary with the site analyzed. These results identify a level of complexity in immune regulation in the FRT not widely recognized that should provide an insight into the mechanisms involved in susceptibility to and transmission of HIV and other STI.

Supported by NIH Grant AI071761 and AI102838 (CRW).

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