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An in vitro model of placental inflammation
Oral Presentations / Journal of Reproductive Immunology 111 (2015) 10–18
Fig. 1. Percentage of STBMs double positive for HLA-DR and PLAP obtained from uncomplicated pregnancies (controls), women with EOPET or LOPET. A significant aberrant expression of HLA-DR was found in EOPET women compared to controls (*P = 0.03).
the escape mechanisms of the semiallogeneic fetus from maternal immune system recognition and rejection. Since an exaggerate systemic inflammatory response is known to be a feature of pre-eclampsia (PET), aim of this study was to investigate the possible abnormal expression of MHC class II molecules, namely HLA-DR, in circulating syncytiotrophoblast microparticles (STBMs) from pre-eclamptic patients. Methods: STBMs obtained by dual placental perfusion after caesarean section from 9 women with early onset (≤34 weeks of gestation) PET (EOPET), 7 women with late onset (>34 weeks of gestation) PET (LOPET) and 12 women with uncomplicated pregnancies were analysed for HLA-DR and the syncytiotrophoblastspecific placental alkaline phosphatase (PLAP) expression by flow cytometry. Results: STBMs from women with EOPET showed a significant expression of HLA-DR coupled with PLAP compared to controls (P = 0.03). No significant difference was found between either controls and LOPET (P = 0.24) or EOPET and LOPET (P = 0.35) (Fig. 1). Conclusion: To our knowledge, this is the first observation of an aberrant expression of HLA-DR in STBMs from women with EOPET. This finding might lead the way to the identification of a novel immunological mechanism representing a co-cause or a consequence of the exaggerate pro-inflammatory status that is a feature of PET. More studies are needed to confirm this observation in a larger cohort of patients and to define the possible functional role of aberrant HLA-DR expressed on STBMs spread in maternal circulation in PET. http://dx.doi.org/10.1016/j.jri.2015.06.083 O14 Acetaminophen application during murine pregnancy impairs pathways of maternal adaptation to pregnancy K. Thiele 1,∗ , M. Emilia Solano 1 , K. Karimi 2 , G. Tiegs 2 , P.C. Arck 1 1
Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg, Germany 2 Institute of Experimental Immunology and Hepatology, Center for Experimental Medicine, University Medical Center Hamburg, Germany Objective: Epidemiological studies highlight the association between prenatal use of acetaminophen (APAP, i.e. Paracetamol®
15
or Tylenol® ) and an increased risk for asthma in the children later in life. We previously reported that APAP causes elevated frequencies of mature dendritic cells (DCs) in the uterus-draining lymph node of pregnant mice. Insights on possible effects of APAP on the adaptive immune response during pregnancy, for example regulatory T cells (Tregs) and B cells, are still unknown. Materials and methods: BALB/c-mated C57BL/6J and double knock-in FIR/Tiger reporter females were injected with vehicle (control group) or 250 mg/kg of APAP on gestation day (gd) 12.5. FIR/Tiger mice allow the immediate and simultaneous detection of FoxP3 and Interleukin 10 (IL-10) by flow cytometry. On gd 13.5, reproductive outcome was assessed and maternal immune cell subsets were analysed by flow cytometry in uterus-draining lymph nodes, spleen and uterus. Results: On gd 13.5, APAP-induced hepatotoxicity was confirmed by significantly elevated alanine aminotransferase levels. Litter size and fetal loss remained unaltered, but fetal weight was significantly reduced upon APAP-treatment. Increased frequencies of CD4+ FoxP3+ Tregs in lymph nodes were independently observed in C57Bl/6J and FIR/Tiger mice. Coincidentally, the frequency of IL10 was also increased. Further, CD19+ B220+ B cells were decreased in lymph nodes and spleen, whereas the frequency of IL-10 producing B cells increased upon APAP. In the uterus, an increased frequency of mature DCs was identified along with increased frequencies of Treg cells and IL-10 as observed in lymph nodes. Conclusions: Our data support that APAP interferes with maternal adaptation to pregnancy by reversing the tolerogenic phenotype of DCs at the feto-maternal interface. The increased frequencies of Tregs and the increased IL-10 production by Tregs and B cells in APAP-treated dams could be a protective mechanism against the immune response triggered by APAP administration, mounted to rescue pregnancy maintenance. http://dx.doi.org/10.1016/j.jri.2015.06.084 O15 An in vitro model of placental inflammation H. Derricott ∗ , S.L. Greenwood, R.L. Jones, A.E.P. Heazell Maternal & Fetal Health Research Centre, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK Introduction: Villitis of unknown etiology (VUE) and chronic intervillositis of unknown etiology (CIUE) are inflammatory conditions of the placenta associated with fetal growth restriction and stillbirth. They are characterised by infiltrates of monocytes/macrophages, CD4 and CD8 T cells. Diagnosis of VUE or CIUE occurs after post-natal histological analysis of the placenta; it is not yet known how these conditions impact upon placental function. We hypothesised that an excess of immune cells and their pro- or anti-inflammatory cytokines in the placental environment would detrimentally affect placental function. We aimed to develop a model of placental inflammation to investigate the potential consequences of VUE/CIUE. Methods: Biopsies of villous tissue were obtained from placentas of normal term pregnancies delivered by elective caesarean section. Matched maternal blood was obtained at the time of delivery. CD4 and CD8 T cells were isolated using a positive and negative antibody selection technique. Fragments of villous tissue were cultured with immune cells for 4 days (n = 5). In separate experiments (n = 5) villous fragments were cultured with pro-inflammatory cytokines interleukin (IL)-2 and IL-12 and in the absence of antiinflammatory cytokine IL-4. Placental function was assessed by system A uptake measurement and hCG hormone production.
16
Oral Presentations / Journal of Reproductive Immunology 111 (2015) 10–18
Results: Co-culture of explants with T cells results in a trend towards reduced system A activity. Syncytial function, assessed by hCG production, was unaffected by culture with immune cells. There was no systematic effect of culturing explants with cytokines. Discussion: Immune cells in the placental environment may have a detrimental effect on the nutrient transport capabilities of the organ as demonstrated by a reduction in system A activity. Culture with cytokines alone does not demonstrate the same effect, implying that an inflammatory milieu is required. Further work is required to fully assess the effect of the culture environment on cell turnover and hormone production. http://dx.doi.org/10.1016/j.jri.2015.06.085 O16 Statin treatment in a novel infection-induced mouse model of preterm birth A.K. Boyle 1,∗ , S.F. Rinaldi 1 , A.J. Thomson 2 , A.G. Rossi 3 , P.T. Saunders 3 , J.E. Norman 1 1 Tommy’s Centre for Maternal and Fetal Health at the MRC Centre for Reproductive Health, University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom 2 University/British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom 3 MRC Centre for Inflammation Research, University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom
Background: Preterm birth (PTB) is a leading cause of child death, affecting an estimated 11.1% of births annually, for which there are few therapies. Statins (3-hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors) are potent inhibitors of cholesterol biosynthesis but have pleiotropic effects such as immunomodulation, anti-inflammatory properties and oxidative stress reduction. Previous studies have shown that statins prevent PTB in a mouse model. We used an alternative, minimally invasive ultrasound-guided intrauterine LPS-induced mouse model of PTB to further investigate the effects of statins. Methods: On gestational day (D)16, C57Bl/6 mice received an intraperitoneal (IP) injection of pravastatin (10 g), simvastatin (20 g) or PBS (200 l). Mice were anaesthetised on D17 of pregnancy and the number of viable fetuses confirmed by ultrasound. LPS (20 g) or PBS, in a volume of 25 l, was injected into the uterus, between 2 gestational sacs using ultrasound guidance. The mice received a further IP PBS or statin treatment 2 h post-LPS injection (n = 5–6/group). Time to delivery (time from intrauterine injection to delivery of the first pup) and the number of live/dead pups was recorded. Percentage pup survival was calculated. Results: Mice receiving intrauterine injection of LPS delivered significantly earlier than those receiving intrauterine PBS (28.1 ± 10 h vs. 59.5 ± 2 h; mean ± SEM, p = 0.0022) and the percentage pup survival was significantly reduced (12.5% vs. 91%, p < 0.0001). Statin treatment before and after LPS injection did not significantly alter time to delivery compared to the vehicle treated LPS group (pravastatin: 27.1 ± 9 h, simvastatin: 17.9 ± 1 h) or alter percentage pup survival (pravastatin: 20%, simvastatin: 0%). Conclusion: Treatment with pravastatin or simvastatin does not delay PTB or improve pup survival in a mouse model of PTB at the doses described. Refinement of the model will be conducted in an attempt to further examine the potential effects of statin treatment. http://dx.doi.org/10.1016/j.jri.2015.06.086
O17 The inflammosome: A new player from innate immunity in the pathogenesis of recurrent pregnancy loss N. Di Simone ∗ , C. Tersigni, S. D’Ippolito, F. Di Nicuolo, R. Castellani, M. Veglia, G. Scambia Department of Obstetrics and Gynaecology, Università Cattolica del Sacro Cuore, Rome, Italy Objectives: Recurrent pregnancy loss (RPL) is defined as three or more consecutive miscarriages. Although RPL has been attributed to various anatomic, hematologic, hormonal, immune and genetic defects, in several cases (30–40%), the etiology remains unknown. An abnormal expression of cytokines might be one of the mechanism that adversely affects endometrial receptivity in women with RPL. NALP-3 inflammosome is an intracellular multi-protein complex, belonging to innate immunity system, that, in response to pathogens or cellular danger signals, activates caspase-1 and increases IL-1ß and IL-18 levels. These events are necessary for the induction of further systemic responses and to spreading of inflammation. Aims: To evaluate the expression and activation of NALP-3 inflammosome in endometrial samples from women with unexplained RPL compared to fertile women (controls). Methods: After informed consent, endometrial samples were obtained from 30 women with unexplained RPL and 10 fertile women undergoing diagnostic mini-invasive hysteroscopy during the implantation window (days 19 to 24). Endometrial lysates were evaluated for NALP-3 inflammosome expression by Western blot analysis and for NALP-3 activation by caspase-1, IL-1ß and IL-18 levels (ELISA). Results: NALP-3 inflammosome expression was increased in endometrial samples obtained from women with RPL compared to controls (p < 0.01). The intracellular cascade following NALP-3 assembly was significantly activated in endometrial samples from women with RPL, showing increased levels of caspase-1 (p < 0.01), IL-1ß and IL-18 (p < 0.001), compared to controls. Conclusion: These results demonstrate that endometrial inflammosome activation is present in women with idiopathic RPL. The high expression of endometrial NALP3 and the increased levels of interleukins in RPL patients support a key role of these proteins during implantation. Even if further studies are needed, the NALP3 inflammosome might represent a novel family of clinical biomarkers or therapeutic targets in idiopathic RPL. http://dx.doi.org/10.1016/j.jri.2015.06.087
Fig. 1. Percentage of STBMs double positive for HLA-DR and PLAP obtained from uncomplicated pregnancies (controls), women with EOPET or LOPET. A significant aberrant expression of HLA-DR was found in EOPET women compared to controls (*P = 0.03).
the escape mechanisms of the semiallogeneic fetus from maternal immune system recognition and rejection. Since an exaggerate systemic inflammatory response is known to be a feature of pre-eclampsia (PET), aim of this study was to investigate the possible abnormal expression of MHC class II molecules, namely HLA-DR, in circulating syncytiotrophoblast microparticles (STBMs) from pre-eclamptic patients. Methods: STBMs obtained by dual placental perfusion after caesarean section from 9 women with early onset (≤34 weeks of gestation) PET (EOPET), 7 women with late onset (>34 weeks of gestation) PET (LOPET) and 12 women with uncomplicated pregnancies were analysed for HLA-DR and the syncytiotrophoblastspecific placental alkaline phosphatase (PLAP) expression by flow cytometry. Results: STBMs from women with EOPET showed a significant expression of HLA-DR coupled with PLAP compared to controls (P = 0.03). No significant difference was found between either controls and LOPET (P = 0.24) or EOPET and LOPET (P = 0.35) (Fig. 1). Conclusion: To our knowledge, this is the first observation of an aberrant expression of HLA-DR in STBMs from women with EOPET. This finding might lead the way to the identification of a novel immunological mechanism representing a co-cause or a consequence of the exaggerate pro-inflammatory status that is a feature of PET. More studies are needed to confirm this observation in a larger cohort of patients and to define the possible functional role of aberrant HLA-DR expressed on STBMs spread in maternal circulation in PET. http://dx.doi.org/10.1016/j.jri.2015.06.083 O14 Acetaminophen application during murine pregnancy impairs pathways of maternal adaptation to pregnancy K. Thiele 1,∗ , M. Emilia Solano 1 , K. Karimi 2 , G. Tiegs 2 , P.C. Arck 1 1
Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg, Germany 2 Institute of Experimental Immunology and Hepatology, Center for Experimental Medicine, University Medical Center Hamburg, Germany Objective: Epidemiological studies highlight the association between prenatal use of acetaminophen (APAP, i.e. Paracetamol®
15
or Tylenol® ) and an increased risk for asthma in the children later in life. We previously reported that APAP causes elevated frequencies of mature dendritic cells (DCs) in the uterus-draining lymph node of pregnant mice. Insights on possible effects of APAP on the adaptive immune response during pregnancy, for example regulatory T cells (Tregs) and B cells, are still unknown. Materials and methods: BALB/c-mated C57BL/6J and double knock-in FIR/Tiger reporter females were injected with vehicle (control group) or 250 mg/kg of APAP on gestation day (gd) 12.5. FIR/Tiger mice allow the immediate and simultaneous detection of FoxP3 and Interleukin 10 (IL-10) by flow cytometry. On gd 13.5, reproductive outcome was assessed and maternal immune cell subsets were analysed by flow cytometry in uterus-draining lymph nodes, spleen and uterus. Results: On gd 13.5, APAP-induced hepatotoxicity was confirmed by significantly elevated alanine aminotransferase levels. Litter size and fetal loss remained unaltered, but fetal weight was significantly reduced upon APAP-treatment. Increased frequencies of CD4+ FoxP3+ Tregs in lymph nodes were independently observed in C57Bl/6J and FIR/Tiger mice. Coincidentally, the frequency of IL10 was also increased. Further, CD19+ B220+ B cells were decreased in lymph nodes and spleen, whereas the frequency of IL-10 producing B cells increased upon APAP. In the uterus, an increased frequency of mature DCs was identified along with increased frequencies of Treg cells and IL-10 as observed in lymph nodes. Conclusions: Our data support that APAP interferes with maternal adaptation to pregnancy by reversing the tolerogenic phenotype of DCs at the feto-maternal interface. The increased frequencies of Tregs and the increased IL-10 production by Tregs and B cells in APAP-treated dams could be a protective mechanism against the immune response triggered by APAP administration, mounted to rescue pregnancy maintenance. http://dx.doi.org/10.1016/j.jri.2015.06.084 O15 An in vitro model of placental inflammation H. Derricott ∗ , S.L. Greenwood, R.L. Jones, A.E.P. Heazell Maternal & Fetal Health Research Centre, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK Introduction: Villitis of unknown etiology (VUE) and chronic intervillositis of unknown etiology (CIUE) are inflammatory conditions of the placenta associated with fetal growth restriction and stillbirth. They are characterised by infiltrates of monocytes/macrophages, CD4 and CD8 T cells. Diagnosis of VUE or CIUE occurs after post-natal histological analysis of the placenta; it is not yet known how these conditions impact upon placental function. We hypothesised that an excess of immune cells and their pro- or anti-inflammatory cytokines in the placental environment would detrimentally affect placental function. We aimed to develop a model of placental inflammation to investigate the potential consequences of VUE/CIUE. Methods: Biopsies of villous tissue were obtained from placentas of normal term pregnancies delivered by elective caesarean section. Matched maternal blood was obtained at the time of delivery. CD4 and CD8 T cells were isolated using a positive and negative antibody selection technique. Fragments of villous tissue were cultured with immune cells for 4 days (n = 5). In separate experiments (n = 5) villous fragments were cultured with pro-inflammatory cytokines interleukin (IL)-2 and IL-12 and in the absence of antiinflammatory cytokine IL-4. Placental function was assessed by system A uptake measurement and hCG hormone production.
16
Oral Presentations / Journal of Reproductive Immunology 111 (2015) 10–18
Results: Co-culture of explants with T cells results in a trend towards reduced system A activity. Syncytial function, assessed by hCG production, was unaffected by culture with immune cells. There was no systematic effect of culturing explants with cytokines. Discussion: Immune cells in the placental environment may have a detrimental effect on the nutrient transport capabilities of the organ as demonstrated by a reduction in system A activity. Culture with cytokines alone does not demonstrate the same effect, implying that an inflammatory milieu is required. Further work is required to fully assess the effect of the culture environment on cell turnover and hormone production. http://dx.doi.org/10.1016/j.jri.2015.06.085 O16 Statin treatment in a novel infection-induced mouse model of preterm birth A.K. Boyle 1,∗ , S.F. Rinaldi 1 , A.J. Thomson 2 , A.G. Rossi 3 , P.T. Saunders 3 , J.E. Norman 1 1 Tommy’s Centre for Maternal and Fetal Health at the MRC Centre for Reproductive Health, University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom 2 University/British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom 3 MRC Centre for Inflammation Research, University of Edinburgh, Queen’s Medical Research Institute, Edinburgh, United Kingdom
Background: Preterm birth (PTB) is a leading cause of child death, affecting an estimated 11.1% of births annually, for which there are few therapies. Statins (3-hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors) are potent inhibitors of cholesterol biosynthesis but have pleiotropic effects such as immunomodulation, anti-inflammatory properties and oxidative stress reduction. Previous studies have shown that statins prevent PTB in a mouse model. We used an alternative, minimally invasive ultrasound-guided intrauterine LPS-induced mouse model of PTB to further investigate the effects of statins. Methods: On gestational day (D)16, C57Bl/6 mice received an intraperitoneal (IP) injection of pravastatin (10 g), simvastatin (20 g) or PBS (200 l). Mice were anaesthetised on D17 of pregnancy and the number of viable fetuses confirmed by ultrasound. LPS (20 g) or PBS, in a volume of 25 l, was injected into the uterus, between 2 gestational sacs using ultrasound guidance. The mice received a further IP PBS or statin treatment 2 h post-LPS injection (n = 5–6/group). Time to delivery (time from intrauterine injection to delivery of the first pup) and the number of live/dead pups was recorded. Percentage pup survival was calculated. Results: Mice receiving intrauterine injection of LPS delivered significantly earlier than those receiving intrauterine PBS (28.1 ± 10 h vs. 59.5 ± 2 h; mean ± SEM, p = 0.0022) and the percentage pup survival was significantly reduced (12.5% vs. 91%, p < 0.0001). Statin treatment before and after LPS injection did not significantly alter time to delivery compared to the vehicle treated LPS group (pravastatin: 27.1 ± 9 h, simvastatin: 17.9 ± 1 h) or alter percentage pup survival (pravastatin: 20%, simvastatin: 0%). Conclusion: Treatment with pravastatin or simvastatin does not delay PTB or improve pup survival in a mouse model of PTB at the doses described. Refinement of the model will be conducted in an attempt to further examine the potential effects of statin treatment. http://dx.doi.org/10.1016/j.jri.2015.06.086
O17 The inflammosome: A new player from innate immunity in the pathogenesis of recurrent pregnancy loss N. Di Simone ∗ , C. Tersigni, S. D’Ippolito, F. Di Nicuolo, R. Castellani, M. Veglia, G. Scambia Department of Obstetrics and Gynaecology, Università Cattolica del Sacro Cuore, Rome, Italy Objectives: Recurrent pregnancy loss (RPL) is defined as three or more consecutive miscarriages. Although RPL has been attributed to various anatomic, hematologic, hormonal, immune and genetic defects, in several cases (30–40%), the etiology remains unknown. An abnormal expression of cytokines might be one of the mechanism that adversely affects endometrial receptivity in women with RPL. NALP-3 inflammosome is an intracellular multi-protein complex, belonging to innate immunity system, that, in response to pathogens or cellular danger signals, activates caspase-1 and increases IL-1ß and IL-18 levels. These events are necessary for the induction of further systemic responses and to spreading of inflammation. Aims: To evaluate the expression and activation of NALP-3 inflammosome in endometrial samples from women with unexplained RPL compared to fertile women (controls). Methods: After informed consent, endometrial samples were obtained from 30 women with unexplained RPL and 10 fertile women undergoing diagnostic mini-invasive hysteroscopy during the implantation window (days 19 to 24). Endometrial lysates were evaluated for NALP-3 inflammosome expression by Western blot analysis and for NALP-3 activation by caspase-1, IL-1ß and IL-18 levels (ELISA). Results: NALP-3 inflammosome expression was increased in endometrial samples obtained from women with RPL compared to controls (p < 0.01). The intracellular cascade following NALP-3 assembly was significantly activated in endometrial samples from women with RPL, showing increased levels of caspase-1 (p < 0.01), IL-1ß and IL-18 (p < 0.001), compared to controls. Conclusion: These results demonstrate that endometrial inflammosome activation is present in women with idiopathic RPL. The high expression of endometrial NALP3 and the increased levels of interleukins in RPL patients support a key role of these proteins during implantation. Even if further studies are needed, the NALP3 inflammosome might represent a novel family of clinical biomarkers or therapeutic targets in idiopathic RPL. http://dx.doi.org/10.1016/j.jri.2015.06.087