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Higher levels of peripheral natural killer cells in patients with primary recurrent miscarriage are associated with altered cytokine profile
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Abstracts / Journal of Reproductive Immunology 115 (2016) 71–90
P2-23 Glucocorticoid receptors are upregulated and histone proteins responsible for rapid gene activation are downregulated in IUGR placentas Stefan Hutter, Simone Hofmann, Christina Kuhn, Julia Messner, Ulrich Andergassen, Doris Mayr, Sven Mahner, Petra Arck, Udo Jeschke Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University Hospital, Munich, Germany Study hypothesis: The purpose of this study was to analyze the expression of both glucocorticoid receptor (GR) subtypes in placentas of pregnancies complicated by Intrauterine Growth Restriction (IUGR) and investigate the impact of GR changes on histone protein modification. Materials and methods: We screened for the fetal gender specific expression of GR␣ and GR in 14 IUGR and 15 normal control placentas, equally divided in both gender. The expression of the histone proteins H3K4me3 and H3K9ac was analyzed in the same panel and finally in trophoblast tumor cells BeWo after stimulation with prednisolone in vitro. Results: In female IUGR placentas, upregulation of GR␣ was found in the Extravillous Trophoblast (EVT) and GR upregulation was found in both the syncytiotrophoblast and the EVT in male IUGR placentas. Upregulation of GRs is accompanied with gender specific and trophoblast subtype specific inhibition of histone protein modification. H3K4me3 is down regulated in female syncytiotrophoblast and H3K9ac is downregulated in male EVT. Conclusion: Elevated cortisol level and or reduced 11-HSD2 activity leads to a gender specific upregulation of GRs in IUGR placentas. These changes are accompanied with histone modifications that are specific for fetal gender and for the trophoblast subtype. Histone modifications may be responsible for general epigenetic changes found in the IUGR offspring. http://dx.doi.org/10.1016/j.jri.2016.04.242 P2-24 GnRH analogues induce pro-inflammatory changes in T cell immunity Nayoung Sung, Maria D. Salazar, Li Wu, Annie Skariah, Svetlana Dambaeva, Kenneth Beaman, Alice Gilman-Sachs, Joanne Kwak-Kim Chicago Medical School at Rosalind Franklin University of Medicine and Science, Chicago, OH, USA Introduction: The potential function of Gonadotropin-releasing hormone (GnRH) and GnRH analogues in the immune system has been studied. In this study, we aim to investigate the effect of GnRH analogues in human immunity, such as the alteration of T cell immunity, in-vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were treated with different doses of GnRH agonist (Buserelin acetate) or antagonist (Cetrorelix acetate), 0.1, 1, 5 and 10 M for 4 h. TH1, TH2 and TH17 cell levels and the intracellular NF-kB signaling pathway in T lymphocytes were analyzed by flow cytometry. Results: TNF-␣+/IL-10+ T helper (TH) cell ratios were increased in PBMCs treated with 1, 5 and 10 M GnRH agonist for 4 h culture when compared to controls. IFN-␥+/IL-10+ TH cell ratios were significantly increased with 0.1, 1, 5 and 10 M GnRH agonist as compared with controls. When GnRH-agonist and -antagonist effects were compared, TNF-␣+, IFN-␥+ and IL-10+ producing TH
cell levels, and IFN-␥+/IL-10+ TH cell ratios were significantly different. The proportion of pNF-kB+ TH cells were decreased in PBMCs treated for 4 h with 0.1 and 1 M of GnRH analogues but increased in PBMCs with 0.1 M GnRH analogues for 48 h as compared to untreated controls. Conclusions: GnRH analogues led to pro-inflammatory changes in TH cells, the effect varied depending on GnRH analogues and doses. However, the changes were not explained with NF-B signaling pathway. http://dx.doi.org/10.1016/j.jri.2016.04.243 P2-25 Higher levels of peripheral natural killer cells in patients with primary recurrent miscarriage are associated with altered cytokine profile Franziska Müller, Ruben Kuon, Kilian Vomstein, Thomas Strowitzki, Volker Daniel, Bettina Toth Department of Gynecological Endocrinology and Fertility Disorders, Ruprecht-Karls University Heidelberg, Heidelberg, Germany Objectives: Natural killer cells (NK cells) are associated with recurrent miscarriage (RM). Elevated levels of NK cells as well as cytokine dysbalances have been described in recent studies. However, controversial debates are ongoing. The aim of this study was to identify factors regulating NK cell levels with focus on a high sensitive cytokine assay. Methods: In total n = 91 couples with ≥3RM were identified. RM subgroups consisted of n = 53 primary RM (pRM) and n = 38 secondary RM (sRM). Peripheral blood was drawn in non-pregnant patients and CD56+CD16+ NK cells were determined using fourcolor fluorescence flow cytometry. Cytokines were analyzed using LUMINEX technique and highly sensitive cytokine assays (R&D systems). Results: In our subset of RM patients, higher levels of CD56+CD16+ were observed in pRM vs sRM patients (/l and %, mean ± SD 290 ± 119, 15.2 ± 5.8 vs 219 ± 126, 11.6 ± 4.8, p = 0.002, p = 0.008). RANTES (CCL5) and TNF were higher in pRM patients (both p < 0.05). Ratios of cytokines reflecting TH1/TH2 profile showed no significant difference. Conclusion: NK cells as well as RANTES and TNF differed significantly between pRM and sRM indicating possible effects of previous live birth. However, no significant differences were present with regard to cytokine ratios reflecting TH1/TH2 profile. We hypothesis that NK cells as well as RANTES and TNF might reflect immmunological changes in pRM and sRM patients and help to identify patients which might profit from immunomodulatory treatment. http://dx.doi.org/10.1016/j.jri.2016.04.244 P2-26 Human serum – An alternative growth supplement for cell culture? Julia Heger, Jana Pastuschek, Karolin Fröhlich, Lisa M. Görke, Sven Peters, Udo R. Markert, André Schmidt University Hospital Jena, Department of Obstetrics, Placenta Lab, Jena, Germany Purpose: Most experimental setups include the use of fetal calf serum (FCS) as medium supplement for cell and tissue culture. Due to its unstable availability, biosafety, potential interaction of human
Abstracts / Journal of Reproductive Immunology 115 (2016) 71–90
P2-23 Glucocorticoid receptors are upregulated and histone proteins responsible for rapid gene activation are downregulated in IUGR placentas Stefan Hutter, Simone Hofmann, Christina Kuhn, Julia Messner, Ulrich Andergassen, Doris Mayr, Sven Mahner, Petra Arck, Udo Jeschke Department of Gynaecology and Obstetrics, Ludwig-Maximilians-University Hospital, Munich, Germany Study hypothesis: The purpose of this study was to analyze the expression of both glucocorticoid receptor (GR) subtypes in placentas of pregnancies complicated by Intrauterine Growth Restriction (IUGR) and investigate the impact of GR changes on histone protein modification. Materials and methods: We screened for the fetal gender specific expression of GR␣ and GR in 14 IUGR and 15 normal control placentas, equally divided in both gender. The expression of the histone proteins H3K4me3 and H3K9ac was analyzed in the same panel and finally in trophoblast tumor cells BeWo after stimulation with prednisolone in vitro. Results: In female IUGR placentas, upregulation of GR␣ was found in the Extravillous Trophoblast (EVT) and GR upregulation was found in both the syncytiotrophoblast and the EVT in male IUGR placentas. Upregulation of GRs is accompanied with gender specific and trophoblast subtype specific inhibition of histone protein modification. H3K4me3 is down regulated in female syncytiotrophoblast and H3K9ac is downregulated in male EVT. Conclusion: Elevated cortisol level and or reduced 11-HSD2 activity leads to a gender specific upregulation of GRs in IUGR placentas. These changes are accompanied with histone modifications that are specific for fetal gender and for the trophoblast subtype. Histone modifications may be responsible for general epigenetic changes found in the IUGR offspring. http://dx.doi.org/10.1016/j.jri.2016.04.242 P2-24 GnRH analogues induce pro-inflammatory changes in T cell immunity Nayoung Sung, Maria D. Salazar, Li Wu, Annie Skariah, Svetlana Dambaeva, Kenneth Beaman, Alice Gilman-Sachs, Joanne Kwak-Kim Chicago Medical School at Rosalind Franklin University of Medicine and Science, Chicago, OH, USA Introduction: The potential function of Gonadotropin-releasing hormone (GnRH) and GnRH analogues in the immune system has been studied. In this study, we aim to investigate the effect of GnRH analogues in human immunity, such as the alteration of T cell immunity, in-vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were treated with different doses of GnRH agonist (Buserelin acetate) or antagonist (Cetrorelix acetate), 0.1, 1, 5 and 10 M for 4 h. TH1, TH2 and TH17 cell levels and the intracellular NF-kB signaling pathway in T lymphocytes were analyzed by flow cytometry. Results: TNF-␣+/IL-10+ T helper (TH) cell ratios were increased in PBMCs treated with 1, 5 and 10 M GnRH agonist for 4 h culture when compared to controls. IFN-␥+/IL-10+ TH cell ratios were significantly increased with 0.1, 1, 5 and 10 M GnRH agonist as compared with controls. When GnRH-agonist and -antagonist effects were compared, TNF-␣+, IFN-␥+ and IL-10+ producing TH
cell levels, and IFN-␥+/IL-10+ TH cell ratios were significantly different. The proportion of pNF-kB+ TH cells were decreased in PBMCs treated for 4 h with 0.1 and 1 M of GnRH analogues but increased in PBMCs with 0.1 M GnRH analogues for 48 h as compared to untreated controls. Conclusions: GnRH analogues led to pro-inflammatory changes in TH cells, the effect varied depending on GnRH analogues and doses. However, the changes were not explained with NF-B signaling pathway. http://dx.doi.org/10.1016/j.jri.2016.04.243 P2-25 Higher levels of peripheral natural killer cells in patients with primary recurrent miscarriage are associated with altered cytokine profile Franziska Müller, Ruben Kuon, Kilian Vomstein, Thomas Strowitzki, Volker Daniel, Bettina Toth Department of Gynecological Endocrinology and Fertility Disorders, Ruprecht-Karls University Heidelberg, Heidelberg, Germany Objectives: Natural killer cells (NK cells) are associated with recurrent miscarriage (RM). Elevated levels of NK cells as well as cytokine dysbalances have been described in recent studies. However, controversial debates are ongoing. The aim of this study was to identify factors regulating NK cell levels with focus on a high sensitive cytokine assay. Methods: In total n = 91 couples with ≥3RM were identified. RM subgroups consisted of n = 53 primary RM (pRM) and n = 38 secondary RM (sRM). Peripheral blood was drawn in non-pregnant patients and CD56+CD16+ NK cells were determined using fourcolor fluorescence flow cytometry. Cytokines were analyzed using LUMINEX technique and highly sensitive cytokine assays (R&D systems). Results: In our subset of RM patients, higher levels of CD56+CD16+ were observed in pRM vs sRM patients (/l and %, mean ± SD 290 ± 119, 15.2 ± 5.8 vs 219 ± 126, 11.6 ± 4.8, p = 0.002, p = 0.008). RANTES (CCL5) and TNF were higher in pRM patients (both p < 0.05). Ratios of cytokines reflecting TH1/TH2 profile showed no significant difference. Conclusion: NK cells as well as RANTES and TNF differed significantly between pRM and sRM indicating possible effects of previous live birth. However, no significant differences were present with regard to cytokine ratios reflecting TH1/TH2 profile. We hypothesis that NK cells as well as RANTES and TNF might reflect immmunological changes in pRM and sRM patients and help to identify patients which might profit from immunomodulatory treatment. http://dx.doi.org/10.1016/j.jri.2016.04.244 P2-26 Human serum – An alternative growth supplement for cell culture? Julia Heger, Jana Pastuschek, Karolin Fröhlich, Lisa M. Görke, Sven Peters, Udo R. Markert, André Schmidt University Hospital Jena, Department of Obstetrics, Placenta Lab, Jena, Germany Purpose: Most experimental setups include the use of fetal calf serum (FCS) as medium supplement for cell and tissue culture. Due to its unstable availability, biosafety, potential interaction of human