Progress 08/01/16 to 07/31/21
Outputs
Target Audience:The targets of this period includes threegraduate students (Morgan Collins, Cole McCutcheon, and Kathryn Brittain) and one postdoctoral researcher (Soo Hyun Ahn). These traineeshave been gaining technical experience, researching the published literature, designing experiments, and presentingtheir work. Work has been published in several fournals (Infection & Immunity), American Journal of Pathology, Frontiers in Immunology, PLoS One, and Scientific Reports.The work was also recently presented in part at the 2021 Society for the Study for Reproductive Investigation, the2021 American Society for Reproductive Immunology, and is scheduled to be presented at the 2021 Society for the Study of Reproduction conferences.The target audiences at these journals andconferences are biomedical scientists and trainees of thereproductive and veterinary sciences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project served as the dissertaion research for Sean Nguyen, PhD, who has now graduated and moved on as a data scientist at SG2 Ventures. The early years of the project employedundergraduates Jacob Greenberg and Ben Collaer, whoassisted Sean with the technical work and are now medical students. Each of these studentsgained professional experience by presentation of theirwork in poster and oral formats at several scientific conferences. Soo Hyun Ahn also has worked onthe new project involving progesterone action on theimmune system. Dr. Ahn broughtexpertise in immunology of pregnancy as well as in haslearned a number of new techniques and has submitted her work for funding at several institutions. Dr. Ahn has presented her work orally at the Society for Reproductive Investigation and the American Society for Reproductive Immunology. In both venues, Dr. Ahn received the Presidential award and theGusdon Award, respectively.We are preparing a K99 grant application for her transition into a faculty position. Additional graduate students, Morgan Collins, Cole McCutcheon, and Kathryn Brittain, are also members of thelab, and a DVM student, Emily Winn, did a summer research project in 2021. How have the results been disseminated to communities of interest?1. Sean Nguyen presented his work at the Michigan Alliance for Reproductive Technologies, the American Association of Reproductive Immunolgy, and the Society for for the Study of Reproduction. Dr. Nguyen published his work in Scientific Reports and in PLoS One. 2. Soo Hyun Ahn presented her work at the Michigan Alliance for Reproductive Technologies, the American Association of Reproductive Immunolgy, the Society for for the Study of Reproduction, the American Association for Reproductive Immunology, and the Society for Reproductive Investigation. She has published some of her work in Frontiers in Immunology, and is preparing an additional publication to be submitted in 2021. 3. Margaret Petroff presented the work at the International Federation of Placenta Associations and the Society for Reproductive Investigation. She also published work in the American Journal of Pathology and other peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Most of the major goals of this project are complete. Aim 1a: Quantification of fetus/placenta-derived EVs. This was completed in the first years of the project and published in PLoS One (2019) and Scientific Reports (2021). Aim 1b: Determine teh cellular identify of maternal EV targets of fetal/placental EVs. This was completed in the later years of the project and is published in Scientific Reports (2021). Aim 1c: Perform genome-wide RNA expression analysis of recombined cells. We reported (Scientific Reports, 2021) the presence of recombined cells in the maternal lung of mice treated with placental vesicles containing fetal genetic information, and further, that placental vesicles are capable of causing recombination of target cells in vitro. We did not opt to perform RNA sequencing on these cells at this stage, as the in vivo recombined cellsin the lung were extremely rareand would require a significant technical effort for their purification. Additionally, we are as yet unsure if the recombined cells are fetal or maternal in origin. New studies will be required to complete these studies. Aim 2: Elucidate the functional impact of fetal-placental EVs on thematernal immune system. We have begun functional studies to determine whether placental EVs can impact the response to influenza virus in vivo. Preliminary results suggest that vesicles may increase inflammatory cells in the lung, and that weight loss caused by the virus is exacerbated by EVs. The preliminary reports were presented at the 2021 Society for Reproductive Investigation conference; additional funding will be required to follow up on these findings. Aim 3. Identify the maternal cellular targets of fetus/placenta-derived EVs. This work was completed and published in Scientific Reports (2021). Under the reporting period, we also began a project examining the role of projesterone in immune function during pregnancy. This work is currently underway and we are nearing submission of our first manuscript on this study. A review article was published (Frontiers in Immunology, 2020).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Warren BD, Ahn SH, Brittain KS, Nanjappa MK, Wang H, Wang J, Blanco G, Sanchez G, Fan Y, Petroff BK, Cooke PS, Petroff MG. Multiple lesions contribute to infertility in males lacking autoimmune regulator. American Journal of Pathology, 2021. 191:1592-1609.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nguyen SL, Ahn SH, Greenberg JW, Collaer BW, Agnew DW, Arora R, Petroff MG.Integrins mediate placental extracellular vesicle trafficking to lung and liver in vivo. Scientific Reports, 2021. 11:4217.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ahn SH, Nguyen SL, Petroff MG. Exploring the origin and antigenic specificity of maternal regulatory T cells in pregnancy. Frontiers in Immunology, 2020. 11:1302.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Flaherty RA, Aronoff DM, Gaddy JA, Petroff MG, Manning SD. Distinct group B Streptococcus sequence and capsule types differentially impact macrophage stress and inflammatory signaling responses. Infection & Immunity, 2021. 89:e00647-20.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ticiani E, Gingrich J, Pu Y, Vettathu M, Davis J, Martin D, Petroff MG, Veiga-Lopez A. Bisphenol S and epidermal growth factor receptor signaling in human placental cytotrophoblasts. Environmental Health Perspectives, 2021. 129:27005.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
McCutcheon CR, Aronoff DM, Gaddy JA, Petroff MG, Manning SD. Composition of group B Streptococcal membrane vesicles varies in a sequence type dependent manner. bioRxiv, 2021. 2021.08.24.457602; doi: https://doi.org/10.1101/2021.08.24.457602
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The targets of this period includes one graduate student (Sean Nguyen) and one postdoctoral researcher. Thesetrainees have been gaining both technical experience,researching the published literature, designing experiments, andpresenting their work. In addition, the work was recently presented in part at the 2019Society for the Study of Reproduction, the 2019American Society for Reproductive Immunology, and the 2019InternationalFederation of Placenta Associationsconferences. Due to the global pandemic, the work was not presented at conferences outside Michigan State University in 2020, althoughit was presented in several onlineformats to students, faculty, staffat MSU. The work will be presented, either online or in person, at the2021International Federation ofPlacenta Associations, American Society for Reproductive Immunology, Society for Reproductive Investigation, and/orand the American Association of Immunogists, in addition to local conferences. The target audiences at theseconferences are biomedical scientists and trainees of the reproductive and veterinary sciences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project serves as the dissertation research for Sean Nguyen, a PhD student. Under my mentorship, Sean designed,oversaw, andperforms the technical aspects of the project. In addition, undergraduates Jacob Greenberg and Ben Collaerassisted Sean with the technical work in previous years. Sean gained additional professional development by participation of his work in posterand oral formats at two international and several local scientific conferences. Sean has now defended his dissertation and will be graduating in December 2020. Soo Hyun Ahn joined the lab in 2018to work onproject involving progesterone action on theimmune system. Dr. Ahn, who brings expertise in immunology of pregnancy as well as in analysis of gametes and zygotes, islearning a number of new techniques including isolation and analysis of thymic cells, RNASeq, flow cytometry, and Rcomputational analysis. To support her professional development, Dr. Ahn attended a grant writing course and has submitteda fellowship application to the Banting organization (Canada) and Lalor foundations. Dr. Ahn and I are also preparing a K99NIH application for her transition into a faculty position. Finally, I am currently interviewing potential graduate students and advertising for a postdoctoral scientist to train and assist with completion of experiments detailed in thenew NIH grants. How have the results been disseminated to communities of interest?Due to the pandemic, virtually all conferences we planned to attend in 2020 were cancelled, resultinginreduced presentation of the workin 2020. However, Margaret Petroff presented the work at an MSU seminar in the Reproductive and Developmental Sciences Program, and Sean Nguyen presented the work in a dissertation defense seminar. We have submitted Dr. Nguyen's work for publicationand expect to submit Dr. Ahn's work for publication within the next few months. What do you plan to do during the next reporting period to accomplish the goals?We expect that Dr. Nguyen's and Dr. Ahn's work will be published within the next year, and expect an additional publication related to Aire. We will be expanding this work to include generation of Aire-deficient mouse models in collaboration with scientists at MSU and with the transgenic animal core facility at MSU. We will continue also our collaboration with University of Michigan investigating effects of vesicles on influenza infection, and hopefully publish this work as well.
Impacts
What was accomplished under these goals?
Quantification of fetus/placenta-derived EVs is complete (Aim 1a), as is Aim 1b.We labeled purified placental EVs with a fluorescent dye, injected them into the blood of mice, and examined the tissuesby confocal microscopy and flow cytometry. We have identified the cellular targets of placental EVs in the lungs and liver to beinterstitial macrophages, whichplay a role in lung development, repair, homeostasis, and antigenpresentation. Many EV were also found in the maternal liver, specifically in macrophages. Quantitative analysis indicated thatthe targeting of these organs is specific to pregnancy: EVs derived from serum of pregnant, but not non-pregnant, femalestargeted the liver and lung. Further, this targeting was found to involve integrins. We also observed rare cells that may represent either fetal microchimeric cells orrecombined maternal cells, the latter of which would strongly suggest transfer of functional fetal RNA or protein to maternaltarget cells. As a proof of principle test of the latter, we cultured bone marrow derived dendritic cells derived from a Cre-transgenic reporter mouse with placental vesicles from a Cre mouse. We observed recombination by flow cytometry and by PCR of the DNA from the dendritic cells. In the future we will test this in vivo using an embryo transfer system. Finally, we are currently testing the function of placental vesicles in the lung throughcollaboration with scientists at University of Michigan. In women, pregnancy exacerbates influenza infection, and we tested the hypothesis that this could be mediated by placental vesicles. Nonpregnant female mice were treated withInfluenza A virus alone or influenza plus placental vesicles. Control groups included mice treated with vehicle alone or vesicles alone. Preliminary results from 5-6 mice per group indicate that vesicles alone increased inflammatory cells recovered from bronchioalveolar lavage (BAL), and further, that vesicles exacerbated weight loss caused by influenza. We are currently awaiting results from histopathology, cell analysis of BAL samples, and cytokine analysis, as well as one additional replicate. We also continued our project examiningthe role of progesterone in immune function during pregnancy. Previousstudies have shown that output of nascent T cells from the thymus, the organ in which T lymphocytes develop, isdramatically reduced in pregnancy and that progesterone action on the thymus is critical for optimal fertility. Themechanisms by which the thymus supports pregnancy, however, are unknown. We found that the nuclear progesteronereceptor, PGR, is highly upregulated during gestation and that pregnancy may be compromised in these mice. Single cell RNA sequencing has suggested upregulation of genes important in immune tolerance to the fetus. Encouragingly, we received two NIH grants to support this project and a related project, with examines the role of Autoimmune Regulator (Aire) in the thymus and reproductive organs in fertility.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Integrins mediate placental extracellular vesicle trafficking to lung and liver in vivo. https://www.biorxiv.org/content/10.1101/2020.09.22.309047v1
doi: https://doi.org/10.1101/2020.09.22.309047
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The targets of this period includes one one graduate student (Sean Nguyen) and one postdoctoral researcher.These trainees have been gaining both technical experience, researching the published literature, designing experiments, and presenting their work to local and national audiences. In addition, the work was recently presented in part atthe 2020 Society for the Study of Reproduction, the 2020 American Society for Reproductive Immunology, and the 2020 International Federation of Placenta Associations conferences. In the next year, the work will be presented at the 2020 Gordon Conference on Mammalian Reproduction, the 2020 International Federation of Placenta Associations, and if financially possible, the American Association of Immunolgists,in addition to local conferences. The target audiences at these conferences are biomedical scientists and trainees of the reproductive and veterinary sciences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project serves as the dissertaion research for Sean Nguyen, a PhD student. Under my mentorship, Sean designs andoversees or performs the technical aspects of the project. In addition, undergraduatesJacob Greenberg and Ben Collaer assisted Sean withthe technical work. Sean gained additional professional development by participation of his work in poster and oralformats at two international and several local scientific conferences. In January 2018, Soo Hyun Ahn joined the lab to work on the new project involving progesterone action on the immunesystem. Dr. Ahn, who brings expertise in immunology of pregnancy as well as in analysis ofgametes and zygotes, is learning a number of new techniques including isolation and analysis of thymic cells, RNASeq, flowcytometry, and R computational analysis. To support her professional development, Dr. Ahn attendeda grant writingcourse and has submitted a fellowship application to the Banting organization (Canada) and Lalor foundations. Dr. Ahn and I are also preparing a K99 NIH application for her transition into a faculty position. How have the results been disseminated to communities of interest?1. Sean Nguyen presented his work at theMichigan Alliance forReproductive Technologies, the American Association of Reproductive Immunolgy, and the Society for for the Study of Reproduction. 2. Soo Hyun Ahn presented her work at theMichigan Alliance forReproductive Technologies, the American Association of Reproductive Immunolgy, and the Society for for the Study of Reproduction. 3. Margaret Petroff presented the work at the International Federation of Placenta Associations. What do you plan to do during the next reporting period to accomplish the goals?Sean Nguyen's project is nearing completion. We will finalize this work, and perform an additional experiment to determine whether the rare cells observed are derived from thefetus or resulted from maternal recombination events. We expect at least two primary publications and one review article based on this work within the next year. In addition, we expect to be able to write a grant to expand upon the observations made during this project. Dr. Ahn's work is ongoing. We will continue analysis of fertility in the PGR knockout mice as well as cellular output from the thymus during pregnancy. We expect to begin RNA sequencing studies based on purification of thymic epithelial cells. We expect at least one primary publication and one review article within the next year.
Impacts
What was accomplished under these goals?
Our quantification of fetus/placenta-derived EVs is complete (Aim 1a), and we have made significant progress on Aim 1b. Welabeled purified placental EVs with a fluorescent dye, injected them into the blood of mice, and examined the tissues byconfocal microscopy and flow cytometry. We have identified the cellular targets of placental EVsin the lungs to be interstitial macrophages, which are beleived to play a role in lung development, repair, homeostasis, and antigen presentation. Many EV were also found in the maternal liver, specifically in macrophages. Quantitative analysis indicated that the targeting of these organs is specific to pregnancy: EVs derived from serum of pregnant, but not non-pregnant, females targeted the liver and lung. Further, this targeting is mediated by proteins associated with the surface of the EV, since pre-treatment of the EV with proteinase abolished organ targeting. We found that certain integrins are present within EV, and in vivo targeting to thelungand liver could be reduced by integrin-blocking peptides. Different integrins appear to mediate EV targeting to the lung and liver. Interestingly, we observedrare cells that may represent either fetal microchimeric cells or recombined maternal cells, the latter of which would strongly suggest transfer of functional fetal RNA or protein to maternal target cells. We also began a new project that examines the role of progesterone in immune function during pregnancy. Previous studieshave shown that output of nascent T cells from the thymus, the organ in which T lymphocytes develop, is dramaticallyreduced in pregnancy and that progesterone action on the thymusis be critical for optimal fertility. The mechanisms by whichthe thymus supports pregnancy, however, are unknown. We found that the nuclear progesterone receptor, PGR, is highlyupregulated during gestation as early as GD6.5, and isexpressed almost exclusivelywithin thymic epithelial cells. To investigate the functional significance of this observation, we generated a thymic epithelial cell-specific PGR knockout mouse. Initial observations revealed that pregnancy is compromised in these mice, and ongoing studies are examining the role of PGR in thymic epithelial cells during syngeneic and allogeneic pregnancies, the patterns of production of regulatory T cells by the thymus, and the dynamics and mechanisms by which the thymus regenerates following pregnancy. In addition, Dr. Ahn has developed techniques to purify thymic epithelial cells, which will be used for RNA sequencing analysis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Flaherty RA, Magel M, Aronoff DM, Gaddy JA, Petroff MG, Manning SD. Modulation of death and inflammatory signaling in decidual stromal cells following exposure to Group B Streptococcus. Infect Immun. 2019 Sep 23. doi: 10.1128/IAI.00729-19.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Flaherty RA, Borges EC, Sutton JA, Aronoff DM, Gaddy JA, Petroff MG, Manning SD. Genetically distinct Group B Streptococcus strains induce varying macrophage cytokine responses. PLoS One. 2019 Sep 19;14(9):e0222910. doi: 10.1371/journal.pone.0222910.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Abrahamson DR, Steenhard BM, Stroganova L, Zelenchuk A, St John PL, Petroff MG, Patarroyo M, Borza DB. Maternal alloimmune IgG causes anti-glomerular basement membrane disease in perinatal transgenic mice that express human laminin ?5. Kidney Int. 2019 Jul 10. pii: S0085-2538(19)30701-X. doi: 10.1016/j.kint.2019.06.014.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Nguyen SL, Greenberg JW, Wang H, Collaer BW, Wang J, Petroff MG. Quantifying murine placental extracellular vesicles across gestation and in preterm birth data with tidyNano: A computational framework for analyzing and visualizing nanoparticle data in R. PLoS One. 2019 Jun 18;14(6):e0218270. doi: 10.1371/journal.pone.0218270.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Warren BD, Ahn SH, McGinnis LK, Grzesiak G, Su RW, Fazleabas AT, Christenson LK, Petroff BK, Petroff MG. Autoimmune Regulator is required in female mice for optimal embryonic development and implantation. Biol Reprod. 2019 Jun 1;100(6):1492-1504. doi: 10.1093/biolre/ioz023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Soo Hyun Ahn, Sean L. Nguyen, Geoffrey R. Grzesiak, Tae Hoon Kim, Jae-Wook Jeong, and Margaret G. Petroff. Gestation-dependent upregulation of thymic progesterone receptor expression by maternal thymic epithelial cells in murine pregnancy. Society for the Study of Reproduction, San Jose, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Sean L Nguyen, Soo Hyun Ahn, Benjamin W Collaer, and Margaret G Petroff. Placental Extracellular Vesicles in Murine Pregnancy. Society for the Study of Reproduction, San Jose, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Sean L Nguyen, Soo Hyun Ahn, Benjamin W Collaer, and Margaret G Petroff. Placental Extracellular Vesicles in Murine Pregnancy. American Society for Reproductive Immunology. Grand Rapids, MI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Soo Hyun Ahn, Sean L. Nguyen, Geoffrey R. Grzesiak, Tae Hoon Kim, Jae-Wook Jeong, and Margaret G. Petroff. Increased expression of thymic progesterone receptor by maternal thymic epithelial cells in murine pregnancy. American Society for Reproductive Immunology. Grand Rapids, MI.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The targets of this period includes one postdoctoral researcher (Soo Hyun Ahn), one graduate student (Sean Nguyen), and twoundergraduate students (Benjamin Collaer and Elizabeth Horn). These students have been gaining both technical experience, researching the published literature, designing experiments, and presenting their work to local and national audiences. In addition, the work was recently presented in part at the Gordon Conference on Mammalian Reproduction, Michigan Alliance for Reproductive Technologies, and Phi Zeta Research day. The target audiences at these conferences are biomedical scientists andtraineesof the reproductive and veterinarysciences. Changes/Problems:We have added the new project on thymic function in pregnancy. This project has a compelling rationale, as thymic function appears to play an important role in fertility. Our future strategy will include examination of transgenic mice, as well as examination of multiple species, including large domestic animals, companion animals, and women with problematic obstetrical outcome. What opportunities for training and professional development has the project provided?This project serves as the dissertaion research for Sean Nguyen, a PhD student. Under my mentorship, Sean designs and oversees or performs the technical aspects of the project. In addition, Jacob Greenberg and Ben Collaer assisted Sean with the technical work. Sean gained additional professional development in September 2017 by participation of his work in poster format at an international and several local scientific conferences.Ben also gained professional experience by presenting his work orally at theMSU Undergraduate Research Forum. Both Sean and Ben continue to work on their projects. In January 2018, Soo Hyun Ahn joined the lab to work on the new project involving progesterone action on the immune system; she is working together with Elizabeth Horn, who is receiving training in polymerase chain reaction and immunofluorescence microscopy. Dr. Ahn,whobrings expertise in immunology of pregnancy as well as in analysis of gametes and zygotes,is learning a number of new techniques including isolation and analysis of thymic cells, RNASeq, flow cytometry, and R computational analysis. To support her professional development, Dr. Ahnis attending a grant writing course and has submitted a fellowship application to the Banting organization (Canada). How have the results been disseminated to communities of interest?1. Sean Nguyen presented his work atthe Gordon Conference on Mammalian Reproduction, the Michigan Alliance for Reproductive Technologies, and Phi Zeta Research Day. 2. Ben Collaer presented his work at the MSU Undergraduate Research Forum. 3. Soo Hyun Ahn presented her work at the Michigan Alliance for Reproductive Technologies and Phi Zeta Research Day. What do you plan to do during the next reporting period to accomplish the goals?Over the next year, we will analyze more tissues at additional time points, some of which are already collected. Additionally, we will commence experiments to determine the junctional conseqeunces of exosomes on maternal immune cells, and determine the RNA expression profiles of placental exosomes. In addition, we will examine the fertility patterns and immune cell profiles of mice lacking progesterone receptor in the thymus, and examine the thymic function across gestation in sheep.
Impacts
What was accomplished under these goals?
Our quantification of fetus/placenta-derived EVs is complete (Aim 1a), and we have made significant progress on Aim 1b. We labeled purified placental EVs with a fluorescent dye,injected them into the blood of mice, and examined the tissues by confocal microscopy. We have also counter-stained the tissues with two cellular markers, CD31, which marks endothelial cells, and completing the analysis of quantification of fetus/placenta-derived extracellular. We found that exosomes alight in organs including the liver, lung, and spleen. In the liver, the exosomes colocalize with macrophages; in the lung andspleenthe exosomes appear to colocalize with endothelial cells. Interestingly, the exosomes were targeted to the germinal centers in the spleen, but not to the red pulp, suggesting that they homed to immunological areas of this organ. Confocal microscopy and 3D reconstruction analysis revealed that macrophages internalize exosomes. We also began a new project that examines the role of progesterone in immune function during pregnancy. Previous studies have shown that output of nascent T cells from the thymus, the organ in which T lymphocytes develop,is dramatically reduced in pregnancy and that progesterone action on the thymusis be critical for optimal fertility. The mechanisms by which the thymus supports pregnancy, however,are unknown. We found that the nuclear progesterone receptor, PGR, is highly upregulated during gestation as early as GD6.5, and is expressed within thymic epithelial cells. Additionally the thymic epithelium is dramatically restructured during pregnancy. To further investigate, we have generated a murine model in which PGR is deleted from thymic epithelial cells. Further study will reveal the consequences of lack of progesterone function on these cells. Finally, we evaluated thymic function in human pregnancy, and our preliminary data suggest that as in mice,thymic output in women is dramatically reduced. To determine whether this phenomenon is conserved, we are furthering this line of investigation by analysis of sheep during pregnancy. In a collaborative and ongoing project, samples from ewes prior to and longitudonally across gestation have been collected. Sample collection is ongoing and we expect to collect late gestationas well as postpartum samples in the coming months. The assay for analysis of sheep samples is currently being developed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
DaSilva-Arnold SC, Zamudio S, Al-Khan A, Alvarez-Perez J, Mannion C, Koenig C, Luke D, Perez AM, Petroff M, Alvarez M, Illsley NP. Human trophoblast epithelial-mesenchymal transition in abnormally invasive placenta. Biol Reprod. 99:409-421.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Alam SMK, Jasti S, Kshirsagar SK, Tannetta DS, Dragovic RA, Redman CW, Sargent IL, Hodes HC, Nauser TL, Fortes T, Filler AM, Behan K, Martin DR, Fields TA, Petroff BK, Petroff MG. Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes. Reprod Sci. 2018 Feb;25(2):185-197
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Warren BD, McGinnis LK, Ahn SH, Grzesiak G, Su RW, Fazleabas AT, Christenson LK, Petroff BK, Petroff MG. Autoimmune Regulator (Aire) is required in female mice for optimal embryonic development and implantation. Biol Reprod (Submitted)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Warren BD, Petroff BK, Cooke PS, Ketumaranahalli M, Wang H, Wang J, Ahn SH, Blanco G, Sanchez G, Petroff MG. Multiple lesions contribute to infertility in males lacking Autoimmune Regulator (AIRE) protein. Sci Reports (Submitted)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Kshirsagar SK, Grzesiak G, Ahn SH, Nguyen SL, Petroff MG. Tumor rejection antigen P1A (TRAP1A) expression in murine pregnancy. Presented at the Society for the Study of Reproduction, New Orleans, LA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Nguyen SL, Greenberg J, Wang J, Wang H, Petroff MG. tidyNano: A computational framework for analyzing and visualizing nanoparticle data in R. Presented at the Gordon Conference on Mammalian Reproduction, Lucca, Italy; the Michigan Alliance for Reproductive Technologies, Detroit, MI; the MSU Phi Zeta Research Conference, East Lansing, MI
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Nguyen SL, Greenberg JW, Collaer BW, Petroff MG. Placental exosomes in murine pregnancy. Presented at the Integrative Pharmacological Sciences Training Program Annual Retreat, East Lansing.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Parasar P, Bernard MP, Kshirsagar SK, Grzesiak GR, Vettathu M, Martin D, Petroff MG. Phenotypic evaluation of leukocytes in maternal peripheral and uterine blood from normal term Cesarean section deliveries. Presented at the Michigan Alliance for Reproductive Technologies, Detroit, MI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Ahn SH, Nguyen SL, Grzesiak GR, Kim TH, Jeong JW, Petroff MG. Increased expression of nuclear progesterone receptor in murine thymus during pregnancy. Presented at the Michigan Alliance for Reproductive Technologies, Detroit, MI and Phi Zeta Research Day, East Lansing, MI
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience of this period includes three students: 1 graduate student (Sean Nguyen) and 2 undergraduate students (Jacob Greenberg and Benjamin Collaer. These students have been gaining both technical experience, as well as experience in researching the published literature, designing experiments, and presenting their work to local and national audiences. In addition, the work was recently presented at the American Society for Reproductive Immunology in September 2017. The target audience at this meeting is biomedical scientists, trainees, and clinicians studying the basic and translational biology of the placenta and maternal-fetal interactions. Changes/Problems:No major changes or problems have taken place. What opportunities for training and professional development has the project provided?This project serves as the dissertaion research for Sean Nguyen, a PhD student. Under my mentorship, Seandesigns and overseesor performs the technical aspects of the project. In addition, Jacob Greenberg assisted Sean with the technical work. Sean gained additional professional development in September 2017 by participation of his work in popter format at the American Society fro Reproductive Immunology, an international scientific conference with more than 100 attendees. Sean's poster was well-attended. Jacob also gained professional experience by his successful application forthe William Sayer Sholarship from the Department of Microbiology and Molecular Genetics, and also by presenting his work orally at the Undergraduate Research Forum. Jacob received a first place prize for work in Microbiology and Immunology for his presentation. Jacob graduate from this department in June 2017, has received a job as a Clinical Coordinator in New Orleans, Louisianna, and is currently applying for medical school (DO/PhD and MD/PhD programs). Finally, a new student has joined the lab, Ben Collaer. Ben is a student in the Lyman-Briggs Honors College, and has worked at least 10 hours per week in the Spring semester and Fall semesters of 2017. He is currently taking a Research for credit course in my lab, and is expected to present his work at the Undergraduate Research Forum in Spring 2018. His work has invovled validation of an in vitro placental exosome isolation method. How have the results been disseminated to communities of interest?1. Jacob Greenberg presented this work at the Undergraduate Research Forum in Spring, 2017. 2. Sean Nguyen presented this work at the American Society for Reproductive Immunology in September, 2017. What do you plan to do during the next reporting period to accomplish the goals?Over the next year, we will analyze more tissues at additional time points, some of which are already collected. Additionally, we will commence experiments to determine the junctional conseqeunces of exosomes on maternal immune cells, and determine the RNA expression profiles of placental exosomes. Some materials for these portions of the project are collected; all of the experimental techniques are validated.
Impacts
What was accomplished under these goals?
We have made significant progress on Aim 1, completing the analysis of quantification of fetus/placenta-derived extracellular vesicles. We have found that maternal murineplasma exosomes increase in pregnancy as early as gestation day 5. At this stage, maternal exosomes were nearly double that of non-pregnant mice; this was surprising since fetal-placental directcommunication with the maternal blood has not yet commenced at this stage; indeed, the embryo is still very small and is just starting the implantation into the uterine wall. Maternal exosomes continued to rise through mid-gestation, and also surprisingly, fell slightly just before parturition. As early as one day postpartum, maternal exosome levels returned to pre-pregnancy concentrations. We were able to show also that at least some of these vesicle were derived from the fetal-placental unit because maternal plasma, as well as purified maternal plasma exosomes, tested positive for a fetal-placental transgenic marker.These results were published in part (Biol. Reprod., 2017) and presented at the annual American Society for Reproductive Immunology meeting (Chicago, IL). We have also mad progress on Aim 1b/Aim 3. To accomplish this, we have isolated placental exosomes from cultured placental explants, labeled them with a red fluorescent dye, and injected the vesicles intravenously. Mice were sacrificed after 5 minutes or 30 minutes, and tissue sections were analyzed by confocal fluorescence microscopy. Although the labeled exosomes were undetectable at 5 minutes, we detected several exosomes after 30 minutes. This delayed timing likely is due to the time required for circulation of the exosomes. The tissues we found the exosomes in included the lung, liver, and less abundantly, the spleen and heart. Over the next year, we will analyze more tissues at additional time points, some of which are already collected. Additionally, we will commence expseiments to determine the junctional conseqeunces of exosomes on maternal immune cells, and determine the RNA expression profiles of placental exosomes. Some materials for these portions of the project are collected; all of the experimental techniques are validated.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Jasti S, Farahbakhsh M, Nguyen SL, Petroff BK, Petroff MG. Immune response to a model shared placenta/tumor-associated antigen reduces cancer risk in parous mice. Biol. Reprod. 96:134-144.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2017
Citation:
Petroff MG, Nguyen SL. Immunology of Pregnancy. Submitted to: Encyclopedia of Reproduction (Elsevier).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Nguyen SL, Greenberg JW, Collaer BW, Petroff MG. Placental Exosomes in Murine Pregnancy. American Journal of Reproductive Immunology (In press).
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Progress 08/01/16 to 09/30/16
Outputs
Target Audience:During this initial period, the target audience includes the two students working on the project in my lab, plus the other technical staff of my lab. Additionally, the work done to date was presented at the International Federation of Placenta Associations meeting in September 2016. The target audience at this meeting is biomedical scientists and clinicians studying the basic and translational biology of the placenta and maternal-fetal interactions. Changes/Problems:As mentioned, we have encountered two possible interpretations for observed preliminary results: maternal recombined (EV-targeted) cells, or alternatively, fetal microchimeric cells. To discriminate between these possibilities, we are considering using embryo transfer.In this approach, we will transfer CRE-expressing embryos into mTmG (non-recombined) dams.Instead of fluorescing green, fetal cells will be non-fluorescent, whilethe only green cells present should be maternal recombined, EV-targeted cells. This change in approach should not delay the timeline of the project; however, new animal care protocols will be submitted prior to initiation of this experiment. What opportunities for training and professional development has the project provided?This project serves as the prinicpal project for one PhD student, Sean Nguyen, as well as an undergraduate researcher, Jacob Greenberg. Mr. Nguyen performs all of the experiments in consultation with Dr. Petroff, who helps him with appropriate experimental design and technical approaches. Mr. Greenberg assists Sean with the technical aspects; because of technical proficiency gained by Jacob, he will soon be initiating his own, related project. Sean gained professional development experience in September 2016 by presenting his work in poster format at the International Federation of Placenta Associations, an international scientific conference with more than 300 attendees. Sean's poster was well-attended; additionally his poster was competitively judged. How have the results been disseminated to communities of interest?This work was presented in poster format at the International Federation of Placenta Associations, an international scientific conference with more than 300 attendees. The conference was held on Sept 13-16, 2016. In addition, Dr. Petroff presented portions of the work orally during the "Trophoblast-Decidual Interactions Workshop" during this same meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next year we expect to make additional progress on the aims described. Specifically, we will have quantified total EVs before, during and following gestation in the mouse. Additionally, we hope to add the quantification of fetally-derived EVs across gestation based on the fetus-specific expression of GFP. We will perform additional experiments in vitro to confirm the immune cell targets of EVs, and begin approaches to confirm the presence of maternal recombined (EV-targeted) cells v. fetal microchimeric cells.
Impacts
What was accomplished under these goals?
Fertility is a major determinant of productivity in animal agriculture in Michigan and worldwide, and our work aims to improve the reproductive performance of animals, and ultimately humans, by identifying the ways that cells of the mother and the fetus communicate with each other, and the factors that are critical in the establishment and maintenance of pregnancy, and reducing prenatal mortality. The placenta is critical in ensuring both the in utero health of animals, as well as their long-term health in postnatal and adult life. In this project, we are testing the hypothesis is that the fetus communicates with the mother by releasing material called extracellular vesicles into her blood, and that the mother communicates with the fetus by releasing extracellular vesicles into the uterus, where the fetus grows during pregnancy. We are testing this hypothesis by using mice that are genetically manipulated to express fluourescent colors that can be viewed under the microscope that indicate when cells are communicating. We expect that the results will provide new information on the fundamental ways in which the mother and baby talk to each other during pregnancy. We have made progress on Aim 1 of this project. In order to quantify the fetus/placenta-derived EVs in maternal plasma across gestation (Aim 1a), we collected 36 plasma samples from female mice. These samples are currently in storage until analysis by nanoparticle tracking, which will be performed with the help of a collaborator at University of Kansas Medical Center. Importantly, we have verified technical aspects of EV isolation using electron microscopy, nanoparticle tracking analysis, and immunoblot analysis. We now are confident that we can isolate sufficient quantities of EVs that contain the correct morphology, size, and biochemical markers from small (<1 milliliter) amounts of plasma of mice. Preliminary results from these samples indicate that there is a ~30% increase in total EVs (maternal and fetal) during pregnancy. We will soon be isolating the remaining plasma samples for finalization of these data. We have made progress in generating a system in which fetally-derived EVs can be distinguished from maternal EVs in the maternal plasma. Using the transgenic system described, we have found that fetally-derived EVs contain Green Fluorescent Protein (GFP), and can be specifically identified by Western blot analysis. This is important because there is no biochemical marker that specifically marks EVs coming from the fetus or placenta. Using GFP, we can assay maternal plasma EVs and quantify which of those come specifically from the fetus. Finally, we have made progress on Aim 1b. Using an in vitro approach, we have found that mouse immune cells, including macrophages, T cells and dendritic cells can take up EVs isolated from cultured placentas. This finding will give us clues as to what in vivo targets are expected to occur in vivo.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2016
Citation:
Lash et al., Trophoblast-Decidua Interactions Workshop Report. Trophoblast Research (Submitted).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Nguyen et al., Placental Exosomes as modulators of the maternal immune system during pregnancy. Placenta 45(2016):87.
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