Reproductive Immuno-dysfunctional Diseases

Chronic endometritis in infertility: pathogenesis, epidemiology, diagnosisand treatment

K Kitaya

IVF Center, Oak Clinic, Osaka, Japan

Chronic endometritis (CE) is characterized by unusual plasma cell (PC) infiltration in the endometrial stroma. CE has long been ignored in gynecologic practice for its subtle symptoms, time-consuming examinationsand benign pathology. Recent studies, however, have suggested the potential involvement of CE in reproductive failure and obstetric/neonatal complications. It becomes important to gain and update the knowledge on CE both for researchers and physicians.

Effect of intravenous immunoglobulin on Th17 and Foxp3+ regulatory T cells in women with recurrent pregnancy loss

SK Lee1, JY Kim1, J Kwak-Kim2 , 3

1Department of Obstetrics and Gynecology, College of Medicine, Konyang University, Gasoowon-dong, Seo-gu, Daejeon, Korea; 2Reproductive Medicine, Department of Obstetrics and Gynecology, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, Vernon Hills, IL, USA; 3Department of Microbiology and Immunology, The Chicago Medical School at Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA

IL-17 secreting CD4+ T (Th17) cells, Foxp3+ regulatory T (Treg) cells, and a ratio of Th17/Treg cells have been proposed as new risk factors for recurrent pregnancy loss (RPL). Intravenous immunoglobulin G (IVIG) has immune modulatory functions in many autoimmune disorders. The role of IVIG in RPL women is still under investigation, even though meta-analysis in women with RPL showed limited effect on pregnancy outcome. We recently investigated whether IVIG has regulatory effect on the level of Th17 or Treg cells in women with idiopathic RPL. Peripheral blood was drawn from the human subjects during the early pregnancy on the day of IVIG treatment (400 mg/Kg) and 1 week after. The proportions of Th17 cells and Foxp3+ Treg cells were analyzed using flow cytometry. In the current study, the percentages of IL-17 producing T cells failed to decrease after IVIG treatment, but the number of Fopx3+ Treg cells increased significantly. In the same vein, the ratio of CD3+IL-17+ T/CD3+Foxp3+Treg cells was also down-regulated after IVIG treatment. The results suggest that dysregulated peripheral blood IL-17+ T and Foxp3+ Treg cell ratio can be modulated by IVIG treatment in pregnant RPL women with idiopathic etiology.Peripheral blood was drawn from the study subjects during early pregnancy on the day of IVIG treatment (400 mg/Kg) and another time 1 week later.

Treg cells and B cells in pre-eclampsia

A-H Liao1, B Zeng1, J Kwak-kim2, Y Liu1, M-X Tang1, Y-H Zhang1

1Family Planning Research Institute, Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Reproductive Medicine, Department of Obstetrics and Gynecology, Chicago Medical School at Rosalind Franklin University of Medicine and Science, Vernon Hills, IL, USA

Pre-eclampsia (PE), a common syndrome that adversely affects approximately 8% of the first pregnancies, is responsible for more than 75,000 maternal deaths annually worldwide, remaining a leading cause of both maternal and fetal morbidity and mortality. Even with intense research efforts, the underlying etiology of PE has not been fully elucidated. Increasing evidence has shown that abnormal activation of immune system plays a key role in the aetiology of PE. Regulatory T cells (Tregs), which have emerged as key players in allowing fetal survival within the maternal uterus. Deficiencies in the number or function of Tregs are associated with some reproductive pathologies, including infertility, recurrent spontaneous abortion, repeated implantation failure after IVF treatment, and particularly after PE. Recently, the concept of PE as an autoimmune disease has been introduced, which is characterized by the presence of some circulating autoantibodies. B lymphocytes are the major contributors of the pathogenesis of autoimmunity through autoantibody production. Our previous study had found that patients with PE increased circulating CD27+CD38 memory B cells and enhanced function of B cells (Liao et al. 2009). Recently, Jensen et al. (2012) reported that CD19+CD5+ B-1a B cells were responsible for autoantibody production during PE. However, the exact roles of B cells in the pathogenesis of PE are not fully understood, and even there is a dearth of literature on the related studies. To date, the information concerning the relationship between Tregs and B cells in the maternal circulation during normal pregnancy and PE has been extremely scarce. Recently published data indicated that Tregs can directly suppress the activity of B cells in vitro and in vivo (Zhao et al. 2006; Iikuni et al. 2009). At the upcoming conference, we will discuss our recent work on the dynamic changes and relationship of Tregs and B cell-subsets in peripheral blood from pre-eclamptic patients to gain a deeper insight into the pathogenesis of PE in a systemic immune environment.

Acknowledgments: This work was supported by research grants from the National Natural Science Foundation of China (No. 81070507).

Natural cytotoxicity receptors expression and cytokines production of natural killer cells in peripheral blood and peritoneal fluid in women with endometriosis

A Funamizu, A Fukui, K Fuchinoue, M Yokota, R Fukuhara, H Mizunuma

Department of Obstetrics and Gynecology of Medicine, Hirosaki, Japan

Problem: Endometriosis, a common disease in women of reproductive generation, can cause dysmenorrhea and infertility. However, the pathogenesis of endometriosis remains unknown. It has been suggested that immune and genetic factors are involved in the development of endometriosis. It was reported that the cytotoxicity of peritoneal fluid natural killer (NK) cells decreased and implantation of endometrial cells occurred in the abdominal cavity of women with endometriosis. As in the case of peritoneal fluid, the cytotoxicity of peripheral blood NK cells was reported to decrease. NK cells have inhibiting and activating receptors on their surface. It has been reported that the expression of inhibitory Killer Immunoglobulin-like Receptor (KIR) on peripheral blood and peritoneal fluid NK cells increased in women with endometriosis. However, there are only a few reports that show the relation between activating receptors of NK cells and endometriosis. The purpose of this study was to investigate the role of NK cells in the development of endometriosis by analyzing the expression of natural cytotoxicity receptors (NCRs) and cytokine production via NK cells in peripheral blood and peritoneal fluid.

Materials and Methods: NK cells in peripheral blood and peritoneal fluid from women with severe endometriosis (n = 18) and controls (n = 28) were collected at the time of operative laparoscopy. The expression of NCRs (NKp46, NKp44 and NKp30) and CD16 on NK cells (CD56dim and CD56bright) were analyzed using multi-color flow cytometry. The production of cytokines (IFN-, TNF-, IL-4, IL-10, GM-CSF, TGF-1) by NK cells was also analyzed.

Results: In peripheral blood NK cells, the expressions of NCRs and cytokines production were not different between women with and without endometriosis. In peritoneal fluid NK cells, the percentages of CD56+/NKp46+ cells (< 0.05) and CD56dim/NKp46+ cells (< 0.01) in severe endometriosis group were significantly higher than those in the controls. TNF- production by peritoneal fluid NK cells in severe endometriosis was significantly higher than that in the controls (< 0.05). There was a significant negative correlation between the percentages of CD56+/NKp46+ cells and CD56+/TNF-+ cells in severe endometriosis group (= −0.721, < 0.05).

Conclusions: Lower expression of NKp46 on peritoneal fluid NK cells would be involved in the decrease of NK cytotoxicity and higher TNF- production by peritoneal fluid NK cells. Peripheral blood NK cells would not reflect the condition of peritoneal fluid NK cells.

Potential role of circulating microRNAs as a biomarker for unexplained recurrent spontaneous abortion

W Qin, Y Tang, L Tang, N Yang, R Cui, L Wang

Family planning research institution of Guangdong province, Guangzhou, China

Unexplained recurrent spontaneous abortion (URSA), defined as three or more consecutive pregnancy losses before the 20th week of gestation, occurs in 1–5% of women of reproductive age. URSA is a complex disease resulting from an array of diverse etiologies. Its diagnosis remains difficult because of lacking specific indexes. MicroRNAs are small single-stranded RNA molecules which play an important role in the regulation of gene expression at the transcriptional level. Circulating microRNAs have been investigated in a wide variety of patient samples. Altered circulating microRNA expression in URSA patients was found using the miRCURY™ LNA Array (v.18.0), with 4 differentially up-regulated and 3 down-regulated microRNAs. These findings have been confirmed by real-time reverse transcription-polymerase chain reaction assays on select microRNAs, including miR-320b, miR-146b-5p, miR-204-3p and miR-22-5p.This is the first report on the expression of microRNAs altered in peripheral circulating of patients with URSA, suggesting that circulating microRNAs can provide a new promising diagnostic tool for URSA.

The work was supported by the National Natural Science Foundation of China (30901610) and Family Planning Science Foundation of Guangdong Province (2012221).

Pathogenesis of autoimmune-type recurrent spontaneous abortion in a novel mouse Model

S Xiao1,*, X Lu1,*, X Li3, L Zhang3, S Bao4, A Zhao1

1Department of Obstetrics and Gynecology, Renji Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China; 2Department of Obstetrics and Gynecology, Huaian Maternity and Child Health Hospital, Jiangsu, China; 3Department of Clinical Laboratory, Shanghai Changning Maternity and Infant Health Hospital, Shanghai, China; 4Experimental Animal Center of Shanghai, the Chinese Academy of Science, Shanghai, China

Objective: To establish a new mouse model for autoimmune-type recurrent spontaneous abortion (AI-RSA) and explore the potential role of anti-β2GP-1 antibodiesin AI-RSA.

Materials and Methods: We performed an intrauterine injection of human β2GP-1 into BALB/c mice and of unrelated protein, adjuvants, and normal saline (NS) into controls. The mean number of embryos implanted (MNEI), embryo loss rate (ELR), mean embryo bulk (MEB), and mean placental weight (MPW) were analyzed.

Results: Compared with the controls, BALB/c mice injected with human β2GP-1 showed increased anti-β2GP-1 antibody in the peripheral blood, accompanied by significantly higher ELR and lower MEB and MPW. Moreover, BALB/c mice immunized with human β2GP-1 exhibited hypercoagulability and vascular thrombus formation in the placenta. Electron microscopy confirmed the existence of platelet aggregation, mitochondrial swelling, and endothelial cell necrosis in the placentas of BALB/c mice immunized with human β2GP-1.

Conclusion: Intrauterine injection of human β2GP-1 successfully induced AI-RSA in mice, suggesting that increased anti-β2GP-1 antibody can independently induce hypercoagulability, vascular endothelial injury, and vascular thrombus formation in the placenta, leading to AI-RSA.

Key words: Abortion, β2GP-1, Rodent model, Anti-phospholipid antibody syndrome.

*These authors are similar in author order.

Effect of HLA-G on cell immune escaping and metastasis in ovarian carcinoma

A Lin, HH Xu, DP Xu, X Zhang, Q Wang, WH Yan

Medical Research Center, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai, Zhejiang, China

The non-classical HLA class I antigen HLA-G has been suggested to be part of strategy for tumor cells to avoid host immunosurveillance. Aberrant HLA-G expression was frequently detected in metastic or invasive tumor sites; however, the mechanism of HLA-G expression in malignancy metastasis has yet to be explored.

In the current study, we found that HLA-G expression decreased significantly NK cytotoxicity against the ovarian carcinoma cell line (HO-8910) engineered to express HLA-G (HO-8910-G), which probably resulted from trogocytosis, and matrix metalloproteinase-15 (MMP-15) expression was up-regulated in HO-8910-G cells. Furthermore, a strong correlation between HLA-G and MMP-15 expression was observed in a cohort of ovarian cancer samples. Knockdowning HLA-G-induced MMP-15 expression by slightly interfereing with RNA (siRNA) significantly decreased HO-8910-G cell migration potential and tumor metastasis. Collectively, our study indicated that HLA-G involved in tumor invasiveness or metastasis may rely on the NK cytotoxicity inhibition and induction of MMP-15 expression in ovarian cancer.

Uterine bleeding reduced by Shenghua Decoction by regulating T cell Paradigm in human decidua of RU486 medical abortion

X Li1, B Wang2, J Zhang3, F Qin3, L Wang1, Y Guo1, X Zhou1, C Yao1, G Jiang1

1Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Key Laboratory for Rare & Uncommon Disease, Key Laboratory of Biotechnology Drugs of the Ministry of Public Health, Laboratory for Immunopharmacology of State Administration of Traditional Chinese Medicine, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, China; 2Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China; 3Shengli Oilfield Central Hospital, Dongying, China

Ethnopharmacological Relevance: Excessive uterine bleeding is the most common and problematic side effect of RU486 medical abortion. Shenghua Decoction (SHD) is a well-known traditional Chinese herbal prescription for reducing uterine bleeding induced by RU486 medical abortion. However, its therapeutic mechanism still remains unclear. The Th1/Th2/Th17/Treg paradigm plays an important role in achieving maternal-fetal immunotolerance and its bias participates in RU486-induced abortion. Our previous research on mice had demonstrated that the uterine bleeding volume was negatively related to the proportions of Th1 and Th17 cells, but positively related to the proportions of Th2 and Treg cells. Additionally, Th1-type cytokine inducing effect was identified. Therefore, it was hypothesized that SHD could reduce the uterine bleeding in RU486 medical abortion by inducing Th1/Th2/Th17/Treg paradigm bias.

Objective: To explore the regulatory effect and mechanism of SHD on human decidual Th1/Th2/Th17/Treg paradigm for alleviating uterine bleeding in RU486 medical abortion.

Materials and Methods: Ninety women within 7 weeks of a normal intrauterine pregnancy, who elected for termination of pregnancy, were divided into three groups labeled as vacuum aspiration, RU486, and SHD-RU486. Duration of uterine bleeding was recorded and its volume was measured by alkaline hematin photometric. To determine the regulatory effect of SHD on Th1/Th2/Th17/Treg paradigm, the proportions of Th1/Th2/Th17/Treg cells in the decidua of different groups were analyzed using a FACScalibur. Correlation was analyzed to demonstrate the relationship between the Th1/Th2/Th17/Treg paradigm and uterine bleeding in RU486 medical abortion. Moreover, to explore the mechanism underlying the T-cell paradigm regulating of SHD, the mRNA and protein expressions of subset-specific transcription factors (T-bet, GATA-3, RORγt, and Foxp3) for the differentiation of Th1/Th2/Th17/Treg paradigm in human decidual CD4+ T cells were detected by reverse transcription-polymerase chain reaction (RT–PCR) assay and western blot analysis, respectively. Additionally, the mRNA expression of the characteristic cytokines of Th1/Th2/Th17/Treg paradigm (IFNγ, IL-4, IL-17A, TGF-β) was analyzed via RT – PCR assay.

Results: Compared with those of RU486 group, the uterine bleeding volume and duration reduced significantly in SHD-RU486 group, which demonstrated a negative correlation with the proportions of Th1 and Th17 cells, but a positive correlation with Th2 and Treg cells. SHD increased the proportions of Th1 and Th17 cells and decreased those of Th2 and Treg cells. Thus, the ratios of Th1/Th2 and Th17/Treg cells elevated markedly after SHD treatment. SHD promoted the mRNA as well as the protein expressions of subset-specific transcription factors for the differentiation of Th1 and Th17 subsets (T-bet and RORγt) while inhibiting those of Th2 and Treg cells (GATA-3 and Foxp3). Additionally, the mRNA expression of Th1- and Th17- type cytokines (IFNγ and IL-17A) was up-regulated, while that of Th2-type and Treg-produced cytokines (IL-4 and TGF-β) was down-regulated significantly after SHD administration.

Conclusion: Th1/Th2/Th17/Treg paradigm bias was involved in RU486 medical abortion, and SHD reduced the uterine bleeding efficiently by inducing Th1 and Th17 skews in the maternal-fetal of RU486 medical abortion patients. The regulatory effect of SHD on Th1/Th2/Th17/Treg paradigm in RU486 medical abortion can be attributed to the modulation of transcription and protein expression of subset-specific transcription factors for T-cell subsets differentiation and their characteristic cytokines.

This work was supported by the Natural Science Foundation of China (30901918 and 81373670), the Science and Technology Development Project of Shandong Province (2012GSF11908), the Doctor's Foundation of Shandong Province (BS2009YY041), the Natural Science Foundation of Shandong Province (ZR2010HQ066, ZR2010HQ043), the Science and Technology Development Grant of Family Planning Committee of Shandong Province ([2009]8), the Science and Technology Development Grant of the State Administration of Traditional Chinese Medicine of Shandong Province (2009-173, 2013-216), and the Science and Technology Star Grant of Jinan.

Keywords: Sheng-hua Decoction, RU486, uterine bleeding, T-cell paradigm, transcription factor, cytokine.

Roles of circulating monocyte subsets in normal pregnancy and pregnancy complications

M Tang, Y Zhang, A Liao

Family Planning Research Institute, Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

To accommodate the semi-allogeneic fetus during pregnancy, modifications of the maternal innate and adaptive immune response are required. A major alteration involves significant activation of the innate immune system as reflected by an increase in the number of peripheral blood monocytes. Previous studies have shown that these cells are progressively activated in the circulation during pregnancy. Human blood monocytes are a heterogeneous, constantly differentiating cell population. Based on the recent understanding by flow cytometry, human circulating monocytes are divided into 3 subsets, CD14hiCD16negclassical,” CD14hiCD16posintermediate” and CD14dimCD16posnon-classical” monocytes, each with distinct phenotypes and functions. The latter two subpopulations were also termed as “pro-inflammatory” monocytes, because they are characterized by high expression of MHC class II and by high production of TNF-α and IL-1β. Recently, Melgert et al. (2012) reported that the percentage of CD16pos monocytes was higher during pregnancy as compared to non-pregnant controls. Their results reinforced the concept that pregnancy is a pro-inflammatory condition. During pre-eclampsia (PE) and preterm labor (PTL) characterized by intense inflammatory response in circulation, the CD16pos monocyte subset was further increased. With the onset of pregnancy, monocytes are chemotactically recruited to the decidua by trophoblasts and developed into decidual macrophages (DM). Nevertheless, current reports noted that DM could be the key sources of monocyte chemokines. They primarily produced CCL2/MCP-1 (ligands of CCR2), which are the dominant chemokines recruiting monocytes to the utero environment throughout pregnancy. As indicated in recent reports, the three monocyte subsets exhibited distinct chemokine-receptor expression; the classical monocytes, a high level of CCR2, while the non-classical monocytes, a highest level of CX3CR1, and the intermediate monocytes, the highest level of CCR5 and CCR2. Other data demonstrated that pregnancy influenced the expression of peripheral monocyte chemokine receptors including CCR2, CCR5 and CXCR3. It seems clear that the influx of monocyte subsets must be tightly controlled, since elevated monocyte infiltration to the decidua can interfere with trophoblast differentiation and lead to pregnancy complications such as PE and recurrence spontaneous abortion (RSA). Additionally, DMs are categorized as pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. The classically activated M1 phenotype is related to inflammation as macrophages secrete IL-1, TNF-α and IL-12. However, alternatively activated M2 phenotype is characterized by increased IL-1 receptor antagonist and decreased IL-12 production. Thus, M2 phenotype can be anticipated during normal pregnancy. Alternatively, M1 phenotype could be a part of mechanism leading to pregnancy failure. Hence, the balance of M1/M2 macrophages may regulate and participate in the progression of gestation process. Better understanding the roles of these newly defined monocyte subsets in pregnancy maintenance will shed more light on the pathogenesis of pregnancy related diseases, including PE, RSA, and PTL.

Acknowledgments: This work was supported by the Graduates' Innovation Fund of Huazhong University of Science & Technology, China (NO. HF-11-27-2013).

Predictive value of fetal fibronectin on the embryonic loss of patients with recurrent spontaneous abortion in early pregnancy

S Fu, Y Liu, L Chen, L Zhu, J Tan, J Zhang, H Chen

Department of Obstetrics and Gynaecology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, China

Objective: The aim of the current study was to investigate the predictive value of fetal fibronectin (fFN) in embryonic loss of patients with recurrent spontaneous abortion (RSA) in early pregnancy.

Methods: Eighty-four patients with RSA in early pregnancy were selected as the test group, and 31 healthy women in early pregnancy, as the control, with the age and number of previous abortionsand other information recorded. These patients underwent an fFN test, and their pregnancy outcome was followed up until the 14th weeks.

Results: The incidence of spontaneous abortion was 20.24% in the test group and 9.68% in the control. The positive fFN [fFN (+)] rate was 57.14% in the test group and 12.90% in the control, indicating a statistically significant difference (< 0.01, χ2 = 17.89). The incidence of spontaneous abortions was 29.17% (14/48) in the fFN (+) group and 8.33% (3/36) in the fFN (−), indicating a statistically significant difference (< 0.05, χ2 = 5.53). The sensitivity, specificity, and positive and negative predictive values in the prediction of abortion in fFN (+) patients of the test group were 82.35%, 49.25%, 29.17%, and 91.67%, respectively.

Conclusion: If detected at an early stage of pregnancy, fFN in patients with RSA can be largely related to the prediction of abortion and facilitate the evaluation of pregnancy outcomes.

Key words: Fetal fibronectin, recurrent spontaneous abortion, early pregnancy.

Expression of peripheral CD4+CD25+CD127 regulatory T cells combined with level of MLC-BE to better evaluate the efficacy of paternal lymphocyte induced immunization in recurrent spontaneous abortion patients with unexpected cause

M Yuan, M Wang, Y Meng, Z Duan, D Li

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

Objective: The aim of this retrospective study was to investigate the positive conversion rate of the one-way mixed lymphocyte culture blocking efficiency (MLC-BE) in unexplained spontaneous abortion patients (URSA) in pre-treatment and post-treatment period. Meanwhile, in order to explore the more reliable and convenient monitoring indicators for accurate diagnosis and clinical assessment of paternal lymphocyte induced immunization, an investigation was made of the peripheral proportional changes of CD4+CD25+CD127 regulatory T cells of the same patients.

Methods: In the present study were enrolled 76 patients with URSA due to blocking antibody deficiency (with negative MLC-BE results), who had received active paternal lymphocyte immunotherapy after routine examinations to rule out other known causes for recurrent spontaneous abortion (RSA) and accepted the follow-up survey for pregnant results. The level of MLC-BE and the proportion of peripheral CD4+CD25+ CD127 regulatory T cells before and after paternal lymphocytes immunization were detected according to the operating instructions, respectively, the results of which were compared between the successfully pregnant and the failed group.

Results: Follow-up survey revealed that 59 of 76 couples conceived again, of whom 38 remained pregnant for 20 gestational weeks, 13 failed again in the first trimester, 8 were still under follow-up, and 17 still failed to conceive. After active immunization therapy, the successful pregnant rate of MLC-BE positive group was significantly higher than that of still MLC-BE negative group (86.2% versus 59.1%, P < 0.05). Analysis of the proportional expression of CD4+CD25+CD127 regulatory T cells in peripheral blood both before and after immunotherapy suggested a positive relationship between a higher level of Tregs and a successful rate of pregnant results, as indicated by the data that in the successfully pregnant women, the Tregs level was significantly elevated from 1.98 ± 1.09 to 3.40 ± 1.07 (P < 0.01); on the contrary, in those who failed again, there existed no statistical significance (1.84 ± 0.57 versus 2.07 ± 0.88, P > 0.05).

Conclusions: Comparison of the parameters demonstrated that a relatively higher MLC-BE level as well as the expression of CD4+CD25+CD127 regulatory T cells may contribute to the successful pregnant results. However, nearly 60% patients maintained pregnancy successfully despite the negative MLC-BE conversion rates after immunization; obviously MLC-BE was reluctant to be an ideal sensitive indicator for immunotherapy. On the other hand, the significant enhancement of the percentage of peripheral CD4+CD25+ CD127 Tregs after allogeneic lymphocyte therapy suggested that it may serve as a potential biomarker for monitoring the efficacy of therapy in URSA patients. A combination of the two parameters, which represented different aspects of clinical significance, may help better evaluate and explain the efficacy of paternal lymphocyte induced immunization in URSA patients.

Macrophages tolerance induced by indoleamine 2,3-dioxygenase-1 (IDO1) in endometrial stromal cells through interleukin-33 in progress of endometriosis

J Mei1, M Li1, L Jin1, D Li1, X Zhu1,2

1Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Shanghai Medical School, Fudan University, Shanghai, China; 2Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China

Objective: To examine the effect of indoleamine 2,3-dioxygenase-1 (IDO1) in endometrial stromal cells (ESCs) on macrophage phenotype and cytokines secretion, and to explore the mechanism involved.

Methods: Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were employed to detect the macrophage phagocytic ability, monocyte immunophenotype and cytokine secretion. Normal ESCs were transfected with plasmid pEGFP-N1-IDO1, vector-only plasmid or not, and co-cultured directly and indirectly with peripheral blood (PB)-derived monocytes (PBMC)/PBMC-driven macrophages. The supernatant of co-culture unit was determined by ELISA for cytokine production, macrophages for phagocytic ability and monocytes for phenotypes by flow cytometry.

Results: Compared with normal ESCs, the PBMC-driven macrophages co-cultured with overexpression (IDO1+) ESC displayed lower phagocytic ability. And PBMC co-cultured with IDO1+ ESC were identified with higher CD163, CD209 and lower HLA-DR, CD11c. Moreover, both intracellular expression and extracellular secretion of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), increased significantly, while IL-12p70 decreased in monocytes after co-cultured with IDO1+ ESC. However, no significant difference of macrophage phagocytic ability, monocyte immunophenotype and cytokine secretion was found between direct and indirect co-culture units. The lower phagocytic and alternatively activated macrophages induced by IDO1+ ESCs were reversed by IL-33 inhibitor, soluble ST2 (sST2).

Conclusion: Via IL-33, the higher expression of IDO1 in ESCs can contribute to truncated phagocytic ability of macrophage, and inducement of macrophage tolerance. And alternatively activated macrophages can further promote endometrial tissues growth, favoring the progression of endometriosis.

Effect of CpG-TLR9 pathway on pregnancy outcome in non-obese diabetic mice

Y Lin

International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

To investigate the relation between CpG-induced activation of innate immunity and pregnancy outcome, we mimiced TLR9 activation using CpG administration in pregnant wild-type (WT) and NK cell deficient NOD mice. We evaluated fetal resorption and preterm birth and performed flow cytometry and ELISA. CpG-induced fetal resorption or preterm birth was steadily observed onlyin NOD mice, but not in WT mice. Concurrently, CpG treatment triggered amplification of uterine macrophages and neutrophils. Moreover, CpG induced a substantial increase of serum mouse keratinocyte-derived cytokine (mKC) and TNF-α that were produced by uterine CD11b+F4/80+ cells, but not by NK or CD11b+Gr-1+ cells. In addition, the depletion of F4/80+ cells abrogated CpG-induced increase in TNF-α production, improving pregnancy outcomes in NOD mice treated with CpG. These results provide evidence that CpG-driven innate immune activation may lead to activation and amplification of macrophages followed by their migration to feto-maternal microenvironment, up-regulated TNF-α production and consequent adverse pregnancy outcomes.