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Preeclampsia serum disrupts the autophagy pathway cooperated with endoplasmic reticulum stress
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Abstracts / Journal of Reproductive Immunology 112 (2015) 121–140
Postpartum acute myometritis as a pathological feature in postpartum hemorrhage of unknown etiology
interstitial cells. Moreover, in comparison with the control, steroid hormone- and/or drug metabolism-related gene expressions were changed in murine testis. At doses that do not induce morphological changes, acetamiprid exposure was found to undergo various changes in gene expression.
Naoaki Tamura ∗ , Mustari Farhana, Hiroaki Itoh, Kazuhiro Sugihara, Naohiro Kanayama
http://dx.doi.org/10.1016/j.jri.2015.09.009
03
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Japan Objective: Postpartum hemorrhage (PPH) is a major cause of maternal death over the world. Atonic bleeding is the most frequent case of PPH, however, the pathophysiology has not been clarified. Particularly in Japan, we currently assume these conditions as clinical amniotic fluid embolism. In the present study, we analyzed pathological changes and identified immune cells in myometrium in PPH of unknown etiology. Methods: Myometrium tissues were from 34 cases of PPH of unknown etiology, 15 cases of control pregnant women and 18 cases after delivery women without PPH. HE, Alcian Blue and immunohistochemical stainings with anti-C5a receptor, anti-mast cell tryptase, anti-neutrophil elastase, anti-CD68, anti-CD3, and anti-ZnCP1 antibodies were performed to count positive stained cells. The Ethics Committee of Hamamatsu University School of Medicine approved all the procedures of this study. Results: Myometrium from PPH presented stromal edema infiltrated with immune cells and C5aR positive cells. Mast cells, neutrophil and macrophage were significantly increased in myometrium in PPH but not in controls. T cells were not present in all subjects. ZnCP1 were positive in the 70% of PPH cases. Conclusion: Myometrium edema due to postpartum acute myometritis in the absence of an infective etiology may be a histological characteristic of PPH of unknown etiology. http://dx.doi.org/10.1016/j.jri.2015.09.008 04 Acetamiprid affect the steroid synthesis system in murine testis Hayato Terayama 1,∗ , Hitoshi Endo 2 , Teruhisa Kanazawa 1 , Masayuki Tatemichi 2 , Kou Sakabe 1 1 Department of Anatomy, Tokai University School of Medicine, Kanagawa, Japan 2 Department of Community Health, Tokai University School of Medicine, Kanagawa, Japan
Neonicotinoids such as acetamiprid belong to a new and widely used one class of pesticides, and are thought to be safe to humans. With mimic chemical structures to nicotine, neonicotinoids also share agonist activity at nicotine acetylcholine receptor (nAchR). However, it has recently reported that neonicotinoids are implicated in a number of human health disorders. Also, a various disorders associated with muscloskeletal and neuro-muscular tissues were induced by high dose of neonicotinoids in experimental animals. Actually, nAChR is expressed in not only the nervous system but also the reproductive system. To address this issue, animal experiments were designed to determine why reproductive systems in response to acetamiprid treatment. The results showed that exposure to acetamiprid-containing water for 3 or 7 days (centuple of NOAEL/day) caused a decrease of body weight, but not affect the testicular histological state in 10 weeks age of A/J mice. Testicular concentrations of AP was significantly higher than the control group. And, it found that nAChR␣7 and 2␣4 is localized testicular
05 Role of iNKT cells in the miscarriages of pregnant mice induced by adoptive transfer of ␣-GalCer-activated DEC-205+ DCs Y. Negishi 1,2,∗ , T. Ichikawa 1,2 , T. Takeshita 2 , H. Takahashi 1 1
Department of Microbiology and Immunology, Nippon Medical School, Japan 2 Department of Obstetrics and Gynecology, Nippon Medical School, Japan Introduction: Dendritic cells (DCs) are one of the key players for successful pregnancy. We have reported previously that DEC205+ DCs dominancy in the pregnant mice induced marked fetal loss (Negishi et al., Immunobiol. 2012). Furthermore, DEC-205+ DCs activated by ␣-galactosylceramide (␣-GalCer) did stimulate invariant natural killer T (iNKT) cells. Based on these findings, we have investigated the role of DEC-205+ DCs primed by ␣-GalCer for murine pregnancy. Methods: Bone marrow cells were obtained from female C57BL/6 mice and cultured for 6 days in the presence of 2.0 g/ml ␣-GalCer. The cultured cells were stained by the antiDEC-205 monoclonal antibody and isolated magnetically. Sorted cells were administered intravenously into the pregnant mice (C57BL/6 × C576BL/6) on gestation day 7.5. Results: A number of miscarriages was observed when DEC205+ DCs were transferred. The transferred cells facilitated iNKT cell and NK cell activation but not CD8+ T and CD4+ T cell accumulation into the implantation sites of miscarriages. In addition, the intracellular cytokine productions of iNKT cells were higher than those of NK cells. Conclusions: iNKT cells may have the central roles in the fetal loss induced by DEC-205+ DCs stimulated by ␣-GalCer. Understanding the correlations between glycolipid antigens and innate immune cells might offer a new notion about the mechanisms of miscarriages. http://dx.doi.org/10.1016/j.jri.2015.09.010 06 Preeclampsia serum disrupts the autophagy pathway cooperated with endoplasmic reticulum stress Akitoshi Nakashima 1,2,∗ , Tomoko Shima 1 , Akemi Ushijima 1 , Aiko Aoki 1 , Kumiko Inada 1 , Arihiro Shiozaki 1 , Osamu Yoshino 1 , Surendra Sharma 2 , Shigeru Saito 1 1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toyama, Japan 2 Department of Pediatrics, Women & Infant Hospital, Brown University, Japan
Purpose: We have reported that impaired autophagy contributes to the pathophysiology of preeclampsia. This paper shows the mechanism how autophagy is inhibited in preeclamptic placenta.
Abstracts / Journal of Reproductive Immunology 112 (2015) 121–140
Materials and methods: We used placental tissues and serums from preeclampsia patients with informed consent. In addition, an autophagy-deficient extravillous trophoblast cell line which is established in our department is used for some in vitro study. Proteostat dye® is also used for detecting aggregated proteins. Results: Lysosome functions are necessary for the autophagy pathway, an important mechanism for protein quality control. First of all, we found the decreased expression of lysosome-associated membrane proteins (LAMP1 & LAMP2), which is related with the number of lysosome, in the trophoblast layer in the placenta of preeclamptic cases. On the other hand, aggregated proteins were increased in the preeclamptic placenta. Secondarily, ER stress inducers, brefeldin A and tunicamycin, suppressed the LAMP1 and LAMP2 expressions in the autophagy-deficient trophoblasts, resulting in suppressing the autophagy in trophoblasts. Thus, ER stress was one of the causes of the autophagy suppression in preeclamptic placenta. We then took notice of transcriptional factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis. TFEB activates the lysosome biogenesis and functions. The expression of TFEB was significantly decreased in the preeclamptic placenta than that in normal placenta. It is reported that mammalian TOR (mTOR) inhibits TFEB activity via blocking the translocation to the nuclei of TFEB. Finally, we found that preeclamptic serum activated mTOR, resulting in suppressing the TFEB function. Taken together, not only ER stress but also preeclamptic serum cooperatively impaired homeostasis in placenta of preeclamptic cases via suppressing autophagy and lysosome functions. Conclusions: We found that failure of autophagy pathway by ER stress was involved in the placental disorder in preeclampsia. Overexpression of TFEB was known to improve the symptoms in a neurodegenerative disease of mice model. TFEB, which could activate autophagy pathway, would be a new possible therapeutic option for preeclampsia. http://dx.doi.org/10.1016/j.jri.2015.09.011 07 2-glycoprotein I complexed with MHC class II molecules are involved in the pathogenesis of antiphospholipid syndrome Kenji Tanimura 1,2,∗ , Yasuhiko Ebina 2 , Tatsuya Atsumi 4 , Hideto Yamada 2 , Hisashi Arase 1,3 1
Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Japan 2 Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Japan 3 Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Japan 4 Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Japan Antiphospholipid syndrome (APS) is characterized by thrombosis and pregnancy complications, which is associated with antiphospholipid (aPL) antibodies. It has been known that several MHC class II gene polymorphisms are associated with APS, although mechanisms how MHC class II gene polymorphisms regulate APS still have remained unclear. 2-glycoprotein I (2GPI) is the main phospholipid-binding molecule recognized by aPL antibodies. It is also unclear how 2GPI is recognized by aPL antibodies. On the other hand, Arase et al. has recently found that IgG heavy chain/MHC class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we found
123
that intracellular 2GPI is transported to the cell surface by MHC class II molecules. Furthermore, human anti-cardiolipin monoclonal antibody (EY2C9) as well as autoantibodies in APS patient’s sera specifically recognized 2GPI complexed with MHC class II molecules of APS-susceptible alleles (HLA-DR7). In addition, 100 of the 120 APS patients (83.3%) analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize 2GPI/HLA-DR7 complexes in the absence of phospholipids. Indeed, 2GPI/MHC class II complexes were detected in endothelial cells of vessels in uterine decidual tissues obtained from APS patients with spontaneous abortion. Furthermore, EY2C9 mediated complement-dependent cytotoxicity against cells expressing 2GPI/HLA-DR7 complex. These data suggest that 2GPI/MHC class II complexes are involved in pathologies of APS and detection of autoantibodies against 2GPI/HLA-DR7 complex could contribute for better recognition of patients with APS. http://dx.doi.org/10.1016/j.jri.2015.09.012 08 Role of IL-33 and cathepsin G in cervical mucus of patients with recurrent miscarriage Shinobu Goto 1,∗ , Yasuhiko Ozaki 1 , Yuuki Obayashi 1 , Satomi Kurakane 2 , Mayumi Sugiura-Ogasawara 1 1 Department of Obstetrics and Gynecology, Nagoya City University Hospital, Japan 2 Department of Obstetrics and Gynecology, Nagoya City East Medical Center, Japan
Objectives: Interleukin-33 (IL-33) is a member of IL-1 family of cytokines, which plays a major role in inflammatory, infectious and autoimmune diseases. IL-33 has been also reported to be released from human endometrial stromal cells upon decidualization and acts as one of the key regulator for reception of embryo. In our previous study, we have been reported that cathepsins-cystatins system cause endometrial dysfunction for early pregnancy. IL-33 is processed into active forms by cathepsin G. In this study, we investigated the existence and contribution of IL-33 and cathepsin G in early pregnancy of patients with recurrent miscarriage. Methods: Deciduas and villis were surgically collected from patients with recurrent miscarriage under the diagnosis of missed abortion. All samples were collected with informed consent. Immunohistochemistry was performed using antibodies against IL-33 and cathepsin G. The expression of IL-33 and cathepsin G in decidua was measured using SDS-PAGE and western blot analysis, and compared between patients with fetal chromosomal abnormality (n = 19) and without chromosomal abnormality (n = 15). The concentrations of IL-33 and cathepsin G in cervical mucus of early pregnancy in patients with recurrent miscarriage were measured by ELISA and the pregnancy outcome (miscarriage group: n = 19, Live birth group: n = 20) was compared prospectively. Results: Staining of IL-33 and cathepsin G was observed in the cytoplasm of stromal and epithelial cells in deciduas and villis using immunohistochemistry. The expression of IL-33 and cathepsin G in decidua, semi-quantified by SDS-PAGE and western blot analysis was not significantly different between patients with or without chromosomal abnormality. The concentration of IL-33 and cathepsin G in cervical mucus of patients who developed miscarriage (IL-33: 49.78 ± 29.07 pg/mL, cathepsin G: 0.11 ± 1.57 ng/mL) was significantly higher than in those who had a live birth (IL-
Abstracts / Journal of Reproductive Immunology 112 (2015) 121–140
Postpartum acute myometritis as a pathological feature in postpartum hemorrhage of unknown etiology
interstitial cells. Moreover, in comparison with the control, steroid hormone- and/or drug metabolism-related gene expressions were changed in murine testis. At doses that do not induce morphological changes, acetamiprid exposure was found to undergo various changes in gene expression.
Naoaki Tamura ∗ , Mustari Farhana, Hiroaki Itoh, Kazuhiro Sugihara, Naohiro Kanayama
http://dx.doi.org/10.1016/j.jri.2015.09.009
03
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Japan Objective: Postpartum hemorrhage (PPH) is a major cause of maternal death over the world. Atonic bleeding is the most frequent case of PPH, however, the pathophysiology has not been clarified. Particularly in Japan, we currently assume these conditions as clinical amniotic fluid embolism. In the present study, we analyzed pathological changes and identified immune cells in myometrium in PPH of unknown etiology. Methods: Myometrium tissues were from 34 cases of PPH of unknown etiology, 15 cases of control pregnant women and 18 cases after delivery women without PPH. HE, Alcian Blue and immunohistochemical stainings with anti-C5a receptor, anti-mast cell tryptase, anti-neutrophil elastase, anti-CD68, anti-CD3, and anti-ZnCP1 antibodies were performed to count positive stained cells. The Ethics Committee of Hamamatsu University School of Medicine approved all the procedures of this study. Results: Myometrium from PPH presented stromal edema infiltrated with immune cells and C5aR positive cells. Mast cells, neutrophil and macrophage were significantly increased in myometrium in PPH but not in controls. T cells were not present in all subjects. ZnCP1 were positive in the 70% of PPH cases. Conclusion: Myometrium edema due to postpartum acute myometritis in the absence of an infective etiology may be a histological characteristic of PPH of unknown etiology. http://dx.doi.org/10.1016/j.jri.2015.09.008 04 Acetamiprid affect the steroid synthesis system in murine testis Hayato Terayama 1,∗ , Hitoshi Endo 2 , Teruhisa Kanazawa 1 , Masayuki Tatemichi 2 , Kou Sakabe 1 1 Department of Anatomy, Tokai University School of Medicine, Kanagawa, Japan 2 Department of Community Health, Tokai University School of Medicine, Kanagawa, Japan
Neonicotinoids such as acetamiprid belong to a new and widely used one class of pesticides, and are thought to be safe to humans. With mimic chemical structures to nicotine, neonicotinoids also share agonist activity at nicotine acetylcholine receptor (nAchR). However, it has recently reported that neonicotinoids are implicated in a number of human health disorders. Also, a various disorders associated with muscloskeletal and neuro-muscular tissues were induced by high dose of neonicotinoids in experimental animals. Actually, nAChR is expressed in not only the nervous system but also the reproductive system. To address this issue, animal experiments were designed to determine why reproductive systems in response to acetamiprid treatment. The results showed that exposure to acetamiprid-containing water for 3 or 7 days (centuple of NOAEL/day) caused a decrease of body weight, but not affect the testicular histological state in 10 weeks age of A/J mice. Testicular concentrations of AP was significantly higher than the control group. And, it found that nAChR␣7 and 2␣4 is localized testicular
05 Role of iNKT cells in the miscarriages of pregnant mice induced by adoptive transfer of ␣-GalCer-activated DEC-205+ DCs Y. Negishi 1,2,∗ , T. Ichikawa 1,2 , T. Takeshita 2 , H. Takahashi 1 1
Department of Microbiology and Immunology, Nippon Medical School, Japan 2 Department of Obstetrics and Gynecology, Nippon Medical School, Japan Introduction: Dendritic cells (DCs) are one of the key players for successful pregnancy. We have reported previously that DEC205+ DCs dominancy in the pregnant mice induced marked fetal loss (Negishi et al., Immunobiol. 2012). Furthermore, DEC-205+ DCs activated by ␣-galactosylceramide (␣-GalCer) did stimulate invariant natural killer T (iNKT) cells. Based on these findings, we have investigated the role of DEC-205+ DCs primed by ␣-GalCer for murine pregnancy. Methods: Bone marrow cells were obtained from female C57BL/6 mice and cultured for 6 days in the presence of 2.0 g/ml ␣-GalCer. The cultured cells were stained by the antiDEC-205 monoclonal antibody and isolated magnetically. Sorted cells were administered intravenously into the pregnant mice (C57BL/6 × C576BL/6) on gestation day 7.5. Results: A number of miscarriages was observed when DEC205+ DCs were transferred. The transferred cells facilitated iNKT cell and NK cell activation but not CD8+ T and CD4+ T cell accumulation into the implantation sites of miscarriages. In addition, the intracellular cytokine productions of iNKT cells were higher than those of NK cells. Conclusions: iNKT cells may have the central roles in the fetal loss induced by DEC-205+ DCs stimulated by ␣-GalCer. Understanding the correlations between glycolipid antigens and innate immune cells might offer a new notion about the mechanisms of miscarriages. http://dx.doi.org/10.1016/j.jri.2015.09.010 06 Preeclampsia serum disrupts the autophagy pathway cooperated with endoplasmic reticulum stress Akitoshi Nakashima 1,2,∗ , Tomoko Shima 1 , Akemi Ushijima 1 , Aiko Aoki 1 , Kumiko Inada 1 , Arihiro Shiozaki 1 , Osamu Yoshino 1 , Surendra Sharma 2 , Shigeru Saito 1 1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toyama, Japan 2 Department of Pediatrics, Women & Infant Hospital, Brown University, Japan
Purpose: We have reported that impaired autophagy contributes to the pathophysiology of preeclampsia. This paper shows the mechanism how autophagy is inhibited in preeclamptic placenta.
Abstracts / Journal of Reproductive Immunology 112 (2015) 121–140
Materials and methods: We used placental tissues and serums from preeclampsia patients with informed consent. In addition, an autophagy-deficient extravillous trophoblast cell line which is established in our department is used for some in vitro study. Proteostat dye® is also used for detecting aggregated proteins. Results: Lysosome functions are necessary for the autophagy pathway, an important mechanism for protein quality control. First of all, we found the decreased expression of lysosome-associated membrane proteins (LAMP1 & LAMP2), which is related with the number of lysosome, in the trophoblast layer in the placenta of preeclamptic cases. On the other hand, aggregated proteins were increased in the preeclamptic placenta. Secondarily, ER stress inducers, brefeldin A and tunicamycin, suppressed the LAMP1 and LAMP2 expressions in the autophagy-deficient trophoblasts, resulting in suppressing the autophagy in trophoblasts. Thus, ER stress was one of the causes of the autophagy suppression in preeclamptic placenta. We then took notice of transcriptional factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis. TFEB activates the lysosome biogenesis and functions. The expression of TFEB was significantly decreased in the preeclamptic placenta than that in normal placenta. It is reported that mammalian TOR (mTOR) inhibits TFEB activity via blocking the translocation to the nuclei of TFEB. Finally, we found that preeclamptic serum activated mTOR, resulting in suppressing the TFEB function. Taken together, not only ER stress but also preeclamptic serum cooperatively impaired homeostasis in placenta of preeclamptic cases via suppressing autophagy and lysosome functions. Conclusions: We found that failure of autophagy pathway by ER stress was involved in the placental disorder in preeclampsia. Overexpression of TFEB was known to improve the symptoms in a neurodegenerative disease of mice model. TFEB, which could activate autophagy pathway, would be a new possible therapeutic option for preeclampsia. http://dx.doi.org/10.1016/j.jri.2015.09.011 07 2-glycoprotein I complexed with MHC class II molecules are involved in the pathogenesis of antiphospholipid syndrome Kenji Tanimura 1,2,∗ , Yasuhiko Ebina 2 , Tatsuya Atsumi 4 , Hideto Yamada 2 , Hisashi Arase 1,3 1
Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Japan 2 Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Japan 3 Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Japan 4 Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Japan Antiphospholipid syndrome (APS) is characterized by thrombosis and pregnancy complications, which is associated with antiphospholipid (aPL) antibodies. It has been known that several MHC class II gene polymorphisms are associated with APS, although mechanisms how MHC class II gene polymorphisms regulate APS still have remained unclear. 2-glycoprotein I (2GPI) is the main phospholipid-binding molecule recognized by aPL antibodies. It is also unclear how 2GPI is recognized by aPL antibodies. On the other hand, Arase et al. has recently found that IgG heavy chain/MHC class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we found
123
that intracellular 2GPI is transported to the cell surface by MHC class II molecules. Furthermore, human anti-cardiolipin monoclonal antibody (EY2C9) as well as autoantibodies in APS patient’s sera specifically recognized 2GPI complexed with MHC class II molecules of APS-susceptible alleles (HLA-DR7). In addition, 100 of the 120 APS patients (83.3%) analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize 2GPI/HLA-DR7 complexes in the absence of phospholipids. Indeed, 2GPI/MHC class II complexes were detected in endothelial cells of vessels in uterine decidual tissues obtained from APS patients with spontaneous abortion. Furthermore, EY2C9 mediated complement-dependent cytotoxicity against cells expressing 2GPI/HLA-DR7 complex. These data suggest that 2GPI/MHC class II complexes are involved in pathologies of APS and detection of autoantibodies against 2GPI/HLA-DR7 complex could contribute for better recognition of patients with APS. http://dx.doi.org/10.1016/j.jri.2015.09.012 08 Role of IL-33 and cathepsin G in cervical mucus of patients with recurrent miscarriage Shinobu Goto 1,∗ , Yasuhiko Ozaki 1 , Yuuki Obayashi 1 , Satomi Kurakane 2 , Mayumi Sugiura-Ogasawara 1 1 Department of Obstetrics and Gynecology, Nagoya City University Hospital, Japan 2 Department of Obstetrics and Gynecology, Nagoya City East Medical Center, Japan
Objectives: Interleukin-33 (IL-33) is a member of IL-1 family of cytokines, which plays a major role in inflammatory, infectious and autoimmune diseases. IL-33 has been also reported to be released from human endometrial stromal cells upon decidualization and acts as one of the key regulator for reception of embryo. In our previous study, we have been reported that cathepsins-cystatins system cause endometrial dysfunction for early pregnancy. IL-33 is processed into active forms by cathepsin G. In this study, we investigated the existence and contribution of IL-33 and cathepsin G in early pregnancy of patients with recurrent miscarriage. Methods: Deciduas and villis were surgically collected from patients with recurrent miscarriage under the diagnosis of missed abortion. All samples were collected with informed consent. Immunohistochemistry was performed using antibodies against IL-33 and cathepsin G. The expression of IL-33 and cathepsin G in decidua was measured using SDS-PAGE and western blot analysis, and compared between patients with fetal chromosomal abnormality (n = 19) and without chromosomal abnormality (n = 15). The concentrations of IL-33 and cathepsin G in cervical mucus of early pregnancy in patients with recurrent miscarriage were measured by ELISA and the pregnancy outcome (miscarriage group: n = 19, Live birth group: n = 20) was compared prospectively. Results: Staining of IL-33 and cathepsin G was observed in the cytoplasm of stromal and epithelial cells in deciduas and villis using immunohistochemistry. The expression of IL-33 and cathepsin G in decidua, semi-quantified by SDS-PAGE and western blot analysis was not significantly different between patients with or without chromosomal abnormality. The concentration of IL-33 and cathepsin G in cervical mucus of patients who developed miscarriage (IL-33: 49.78 ± 29.07 pg/mL, cathepsin G: 0.11 ± 1.57 ng/mL) was significantly higher than in those who had a live birth (IL-