Mother-to-child transmission (MTCT) of HIV accounts for more than 90% of pediatric infections worldwide, yet the mechanism of vertical transfer remains unknown. The feline immunodeficiency virus (FIV)-infected cat is a cost-effective, small-animal model of HIV pathogenesis and MTCT, which produces a high rate of reproductive failure and fetal infection in litters delivered at early- and late-term gestation. Our previous data suggest that FIV infection may dysregulate placental cytokines and compromise pregnancy. We hypothesized that FIV-infection may cause dysregulation of placental cytokine expression, and aberrant expression of these cytokines may potentiate inflammation and transplacental infections. The purpose of this project was to evaluate feline placental immunopathology at the whole and cellular levels during early- and late-term gestation to understand how lentiviruses may perturb placental immune parameters. To determine whether placentas were vulnerable to FIV infection, we quantified the expression of the FIV receptors, CD134 and CXCR4, in RNA extracted from late-term placental tissue. We found higher expression of CD134 and CXCR4 in placentas from successful pregnancies. To evaluate relative cytokine expression in randomly-sampled, whole placental specimens, we quantified representative pro- and anti-inflammatory cytokines and a chemokine. IL-6 and IL-12p35 were increased in early-gestation, FIV-infected queens; IL-6 was increased in late-gestation, FIV-infected queens. To evaluate placental immunopathology at the cellular level, we developed a novel immunohistochemistry method to identify trophoblastic cells selectively. Trophoblasts were collected using laser capture microdissection, and RNA was extracted from captured cells. We detected expression of several anti- and pro-inflammatory cytokines and the chemokine receptor CXCR4 (the FIV co-receptor) in trophoblasts at both stages of gestation. However, we failed to detect expression of other cytokines and CD134, the FIV primary receptor. FIV infection slightly lowered expression of all cytokines at both early and late pregnancy, although only the decrease in IL-5, from early pregnancy, and IL-4 and IL-12p35, from late pregnancy, reached significant levels. Fetal non-viability was associated with decreased trophoblast expression of IL-4, IL-6, IL-12p35, and CXCR4 at early gestation and decreased expression of IL-4, IL-12p35, IL-12p40 at late gestation. Collectively, these data indicate that FIV infection negatively impacts pregnancy outcome and alters placental immunomodulation.

The etiology of many gestational disorders is still unknown. However, insufficient trans-placental passage of nutrients and wastes due to poor placentation is characteristic of several pathologies and may be due, in part, to altered function of placental mitochondria. Mitochondrial activity is essential in pregnancy because it sustains the metabolic activity of the placenta throughout gestation. Exposure to stressors that perturb processes governing placentation, including maternal drug use, can negatively impact fetal development. Cannabis use is prevalent during pregnancy. The psychoactive constituent, delta-9-tetrahydrocannbinol (THC), can cross the placenta to affect placental and fetal physiology. Importantly, cannabinoid receptors have been reported on trophoblast cells, and on mitochondria which are abundant in placentae. It has been reported that THC may target the mitochondria in various tissue types, including placental tissue, and alter its function. However, few studies have addressed the physiological control of mitochondria within the placenta, an organ that is critical for fetal growth and pregnancy maintenance. I investigated the role of mitochondria in trophoblast differentiation and syncytialization using rotenone, a complex I inhibitor. Subsequently, I investigated the role of THC on two important aspects of placentation – invasion and syncytialization – using placental trophoblast cells HTR8/SVneo and BeWo, respectively. In response to rotenone and THC, there was increased ROS production, oxidative stress, and altered transcriptional markers favouring mitochondrial fragmentation. Treatment with 20µM THC for 48 hours led to reduced mitochondrial respiration, ATP production and loss of mitochondrial membrane polarity. Critically, these THC-induced mitochondrial changes occurred concomitant with evidence of reduced trophoblast invasion and syncytialization. Furthermore, THC exposure reduced levels of human chorionic gonadotropin, human placental lactogen and insulin-like growth factor 2, which are growth factors necessary for fetal development. Placental mitochondrial dysfunction, particularly when THC-induced, may be critical in a range of gestational disorders which have important implications for maternal and fetal/offspring health. / Dissertation / Doctor of Philosophy (Medical Science) / Cannabis is commonly used by pregnant women. Fetal exposure to cannabis and its components can impair fetal growth and neurological development. These negative fetal outcomes may be the result of poor placental formation, due to placental cell exposure to cannabis and its psychoactive component, delta-9-tetrahydrocannabinol (THC). Importantly, THC can also target intracellular organelles, like the mitochondria which are known as the “powerhouses” of the cell. Few studies have investigated the direct effects of THC on placental development. The purpose of this study was to determine how THC exposure to placental cells may alter their function. We found that THC impaired processes that allow placental attachment to the uterus and form a protective barrier, and compromised mitochondrial function, which are important for placental formation. These findings serve to inform scientists and doctors, thus stimulating the creation of new ideas and methods to further explore the impact of THC on pregnancy outcomes.

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 1997. / Typescript. Bibliography: leaves [222]-256.

Les Brucella sont des bactéries intracellulaires responsables de la brucellose, une zoonose mondiale qui cause des avortements chez les animaux, entrainant d'énormes pertes économiques dans les élevages, et des problèmes de santé de longue durée chez l'Homme. Contrairement aux animaux, il existait jusqu’à présent peu de preuves que les infections à Brucella pouvaient causer des complications obstétriques chez la femme. Des récentes études épidémiologiques ont cependant démontré une augmentation significative des risques de complications (fausses couches, mort in utéro, accouchement prématuré) chez les femmes enceintes infectées par Brucella. De plus, il a été montré que plusieurs espèces zoonotiques de Brucella sont capables d’infecter des cytotrophoblastes (CTB) et des trophoblastes extravilleux (EVT) humains, deux types de cellules ayant des fonctions immunitaires et hormonales essentielles pendant le développement du placenta. Dans ce travail, nous avons étudié les conséquences de l’infection des trophoblastes humains par Brucella, du côté du pathogène mais aussi du côté de l’hôte. Nous avons évalué le comportement intracellulaire de différentes souches de Brucella, représentant différentes espèces, hôtes ou symptômes associés. Nous n'avons trouvé aucune corrélation entre la capacité de réplication dans les trophoblastes et l’association des souches avec des complications obstétricales chez leur hôte respectif. Nous nous sommes également intéressé à des souches récemment isolées chez des babouins, après une infection placentaire ayant causé la mort in utéro de leur fœtus. Nous avons montré que ces souches sont capables d’infecter les trophoblastes humains et affectent certaines de leurs propriétés qui sont essentielles lors du développement placentaire. Nous avons également commencé à caractériser des structures intracellulaires atypiques dans lesquelles Brucella semble pouvoir survivre dans certains trophoblastes. Du côté hôte, nous avons analysé le rôle de la protéine eucaryote CD98hc dans l'infection les trophoblastes. Nous avons montré que CD98hc est importante pour l'infection des trophoblastes humains par Brucella, comme cela avait été montré précédemment dans d'autres types cellulaires, et que l’infection modifie le niveau de cette protéine dans les trophoblastes. L'infection des trophoblastes humains par Brucella pourrait donc altérer leurs fonctions au cours du développement placentaire, entraînant ainsi des complications pendant la grossesse.Les résultats obtenus dans ce travail contribuent à une meilleure compréhension des mécanismes pouvant causer des complications obstétricales chez la femme enceinte infectée par brucella et fournissent des informations importantes pour la prise en charge clinique de la brucellose pendant la grossesse. / Brucella are intracellular bacteria responsible for brucellosis, a worldwide zoonotic disease associated with infectious abortions in animals, which causes huge economical losses in the livestock industry and long lasting health problems in humans. In contrast to animals, evidence of Brucella infections cause obstetric complications in humans is scarce. However, epidemiological studies have shown significant increases in the risk of adverse pregnancy outcomes (miscarriage, stillbirth, preterm delivery) in pregnant women infected with Brucella. Moreover, several zoonotic Brucella species were shown to infect efficiently human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT), two types of cells with essential immune and hormonal functions during placental development. In the present study, we studied the effect of Brucella infection in human trophoblasts, from both the bacterial and the host sides. We evaluated the intracellular behavior of different Brucella strains, representing different species, hosts or associated symptoms. We found no correlation between the bacterial replication rate in trophoblasts and whether the strains were associated with pregnancy complications in their respective host. Importantly, we show that strains isolated from female baboons after stillbirth can infect human trophoblasts and affect some of their properties that are essential during placental development. We also started the characterization of atypical intracellular structures in which Brucella seem to be able to survive in certain types of trophoblasts. From the host side, we analyzed the role of the eukaryotic protein CD98hc in trophoblast infection. We found that CD98hc is important for infection of trophoblasts by Brucella, as shown previously in other cell types, and that infection affects the level of the protein in trophoblasts. Infection of human trophoblasts by Brucella could thus affect their function during placental development, leading to complications in pregnancy.The results obtained in this work contribute to a better understanding of the mechanisms that could lead to obstetric complications in Brucella infected pregnant women and provide important information for the clinical management of brucellosis during pregnancy.

<p>The tolerance towards the semi-allogenic foetus is obtained through both systemic and local changes in the maternal immune response. Locally, in the decidua, the cell composition differs from that found in the blood; natural killer (NK) cells and macrophages being the major cell types. Decidual macrophages (dMØ), which are alternatively activated, and trophoblasts, placental cells of foetal origin, are believed to participate in the foetal tolerance at the foetal-maternal interface. To test the recruitment ability of macrophages and trophoblasts, and to test if these cells are responsible for the special cell composition in the decidua, a migration assay was established. In this migration assay peripheral blood mononuclear cells (PBMC) were allowed to migrate through Matrigel-coated transwell inserts into lower wells containing a recruiting stimulus. After testing several conditions, a protocol was established for further use. The results showed that <em>in vitro</em> alternatively activated macrophages, which display many of the surface markers as dMØ, hold a recruiting ability and recruit monocytes. Further there was an indication that trophoblasts also hold a recruiting ability. Neither cell types were shown to recruit NK cells. In conclusion, this study presents a suitable protocol for assessing chemotactic factors and different cell type’s ability to recruit cells from blood. Although the experiments need to be repeated and extended and the recruitment ability of dMØ needs to be evaluated in detail before a final conclusion can be drawn, the preliminary data indicated that macrophages and trophoblasts can recruit monocytes.</p>

The placenta is a highly vascularized, temporary organ developed in pregnancy that is composed of both maternal and fetal cells. It plays a pivotal role in gestational health by facilitating embryo implantation and fostering nutrient exchange between mother and fetus. Placental malformation and the diffusion of harmful exogenous substances through the placental barrier can cause pregnancy complications and, in more severe cases, death of the mother or the fetus. Further, the placenta undergoes profound morphological and functional changes throughout pregnancy. Establishing models to mimic these phenomena at different stages of pregnancy informs prescription drug safety and expedites the development of placental disease treatments. Mouse models are often used to simulate human fetal development despite major interspecies differences. These limitations drive researchers to developing in vitro models consisting of human-derived cells. This thesis presents three 3D vascularized placental models utilizing human placental stem cells (PSCs) and human umbilical vein endothelial cells (HUVECs) which can model multiple placental phenomena across early- and late-stage pregnancy. The first model features a 3D fibrin hydrogel network with self-assembled vasculature and a monolayer of syncytialized human trophoblastic stem cells (STs) serving as a platform for barrier studies at the maternal-fetal interface. By tuning trophoblast differentiation and vascularization of this model to mimic the early- and late-stage placenta, it was revealed that placental barrier permeability was dependent on placental maturity and that the vascular barrier is also a critical determinant of what molecules can be passed from the mother to the fetus. The design and manufacturing of this model were then streamlined to meet the demands of large-scale drug studies in the second placental barrier model. Placental invasion into the maternal decidua is carefully orchestrated by multiple cell types to prevent over- and under-invasion, both of which can be dangerous to the mother and fetus. Understanding the biochemical and environmental cues that permit this healthy invasion can allow for improved diagnostics and treatments of placental diseases, such as preeclampsia and placenta accreta. Thus, the third model presented herein is a placental invasion model with chorionic villus-like structures seeded with invasive extravillous cytotrophoblasts (EVTs) and a perfusable vascular channel. Collectively, these models facilitate the exploration of placental morphogenesis and function throughout various stages of pregnancy. They offer a valuable tool for probing placental dysfunctions and assessing drug safety, ultimately contributing to advancements in fetal-maternal health. / Thesis / Doctor of Philosophy (PhD) / The placenta is an essential organ in pregnancy and is responsible for a variety of phenomena that assure the survival of the fetus. However, many women suffer from negative pregnancy outcomes due to placental disorders, such as preeclampsia, or due to the crossing of unsafe compounds through the placenta to the fetus. Trophoblasts are the most notable placental cell type originating from the fetus and they have the capacity to mature into more specialized subtypes that are responsible for placental barrier function and placental development via invasion into the maternal tissue. In this work, we have designed three systems that either model placental barrier function or trophoblast invasion by culturing primary endothelial cells with differentiated trophoblast cells on a gel-based device. Using the barrier models, it is possible to assess the rate of transport of different compounds that may be present in the mother’s blood to the fetus, to assess their safety. Whereas the invasion model has the capacity to model the genesis of the placenta and therefore may be used to shed light on the causes for placental dysfunctions at the early stage of pregnancy.

Este estudo determinou a expressão gênica e a localização tecidual de receptores do fator de inibição da migração de macrófagos (MIF) na interface materno-placentária em camundongos aos 7,5, 10,5, 13,5 e 17,5 dias de gestação (ddg). Observou-se expressão gênica dos receptores na decídua durante todo o período estudado. CD74 e CD44 foi imunolocalizado em células deciduais, endoteliais e leucócitos, sugerindo que estas populações são presuntivos alvos do diálogo parácrino trofoblasto-decídua. No compartimento fetal, a expressão de CD74 ocorreu aos 7.5 e 17,5 ddg e de CD44, durante a gestação. RNAm dos receptores, mas não imunolocalização destes foi observada no trofoblasto aos 7,5 ddg, sugerindo mecanismos reguladores pós-transcricionais. Como a sinalização mediada por MIF requer CD74 para ativação de CD44, a baixa expressão de CD74 aos 10,5 e 13,5 ddg sugere sinalização limitada. Os resultados sugerem que a placenta fetal tem populações específicas expressando CD44-CD74 no final da gestação, o que pode ser determinante para a ativação celular mediada pelo MIF. / The study determined the gene expression and tissue localization of the macrophage migration inhibitory factor (MIF) receptors (CD74-CD44) at the maternal placental interface in mice, on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. We observed the gene expression of the receptors to the decidua in all gestation days. CD74-CD44 were immunolocalized in decidual and endothelial cells and, leukocytes, suggesting these cells are presumptive targets for trophoblast-decidual paracrine dialogue. In the fetal compartment, expression of CD74 occurred on gd7.5 and 17.5 and of CD44 during all pregnancy. mRNA for both receptors, but not immunolocalization was detected on the gd7.5-trophoblast, suggesting post-transcriptional regulatory mechanism. As MIF-mediated signaling requires CD74 for activation of CD44, low expression of CD74 on gd10.5 and 13.5 suggests restriction of MIF effect. The results suggest fetal placenta has specific populations expressing CD74-CD44 in the final pregnancy, which can be a determining factor in mechanisms of cellular activation mediated by MIF.

Este estudo determinou a expressão gênica e a localização tecidual de receptores do fator de inibição da migração de macrófagos (MIF) na interface materno-placentária em camundongos aos 7,5, 10,5, 13,5 e 17,5 dias de gestação (ddg). Observou-se expressão gênica dos receptores na decídua durante todo o período estudado. CD74 e CD44 foi imunolocalizado em células deciduais, endoteliais e leucócitos, sugerindo que estas populações são presuntivos alvos do diálogo parácrino trofoblasto-decídua. No compartimento fetal, a expressão de CD74 ocorreu aos 7.5 e 17,5 ddg e de CD44, durante a gestação. RNAm dos receptores, mas não imunolocalização destes foi observada no trofoblasto aos 7,5 ddg, sugerindo mecanismos reguladores pós-transcricionais. Como a sinalização mediada por MIF requer CD74 para ativação de CD44, a baixa expressão de CD74 aos 10,5 e 13,5 ddg sugere sinalização limitada. Os resultados sugerem que a placenta fetal tem populações específicas expressando CD44-CD74 no final da gestação, o que pode ser determinante para a ativação celular mediada pelo MIF. / The study determined the gene expression and tissue localization of the macrophage migration inhibitory factor (MIF) receptors (CD74-CD44) at the maternal placental interface in mice, on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. We observed the gene expression of the receptors to the decidua in all gestation days. CD74-CD44 were immunolocalized in decidual and endothelial cells and, leukocytes, suggesting these cells are presumptive targets for trophoblast-decidual paracrine dialogue. In the fetal compartment, expression of CD74 occurred on gd7.5 and 17.5 and of CD44 during all pregnancy. mRNA for both receptors, but not immunolocalization was detected on the gd7.5-trophoblast, suggesting post-transcriptional regulatory mechanism. As MIF-mediated signaling requires CD74 for activation of CD44, low expression of CD74 on gd10.5 and 13.5 suggests restriction of MIF effect. The results suggest fetal placenta has specific populations expressing CD74-CD44 in the final pregnancy, which can be a determining factor in mechanisms of cellular activation mediated by MIF.

The tolerance towards the semi-allogenic foetus is obtained through both systemic and local changes in the maternal immune response. Locally, in the decidua, the cell composition differs from that found in the blood; natural killer (NK) cells and macrophages being the major cell types. Decidual macrophages (dMØ), which are alternatively activated, and trophoblasts, placental cells of foetal origin, are believed to participate in the foetal tolerance at the foetal-maternal interface. To test the recruitment ability of macrophages and trophoblasts, and to test if these cells are responsible for the special cell composition in the decidua, a migration assay was established. In this migration assay peripheral blood mononuclear cells (PBMC) were allowed to migrate through Matrigel-coated transwell inserts into lower wells containing a recruiting stimulus. After testing several conditions, a protocol was established for further use. The results showed that in vitro alternatively activated macrophages, which display many of the surface markers as dMØ, hold a recruiting ability and recruit monocytes. Further there was an indication that trophoblasts also hold a recruiting ability. Neither cell types were shown to recruit NK cells. In conclusion, this study presents a suitable protocol for assessing chemotactic factors and different cell type’s ability to recruit cells from blood. Although the experiments need to be repeated and extended and the recruitment ability of dMØ needs to be evaluated in detail before a final conclusion can be drawn, the preliminary data indicated that macrophages and trophoblasts can recruit monocytes.

La fièvre Q se traduit par de graves conséquences obstétricales chez la femme enceinte. La voie principale de la contamination humaine par Coxiella burnetii, l’agent de la fièvre Q, est constituée des aérosols provenant des placentas d’animaux infectés. La nature des cellules placentaires cibles de C. burnetii reste totalement inconnue. J’ai montré que C. burnetii infecte les trophoblastes des lignées BeWo et JEG et que les bactéries se répliquent fortement dans les cellules BeWo et survivent dans les cellules JEG. Une analyse par microarray montre que C. burnetii induit une réponse inflammatoire dans les cellules BeWo qui lui est spécifique. Ces résultats suggèrent que les trophoblastes pourraient constituer une niche pour C. burnetii. Les macrophages placentaires pourraient également servir de réservoir pour C. burnetii. J’ai montré que les macrophages placentaires CD14+ sont des macrophages qui présentent des caractéristiques phénotypiques, transcriptionnelles et fonctionnelles différentes de celles des monocytes circulants et des macrophages dérivés de monocytes. Ils forment en outre des cellules géantes multinucléées qui pourraient réguler l’activité cytolytique des macrophages dans le contexte placentaire puisque les macrophages placentaires CD14+ ne sont ni de type M1 ni de type M2. / In pregnant women Q fever presents obstetrical complications. The principal way of human contamination by Coxiella burnetii, the agent of Q fever, is due to aerosols from placentas of infected animals. The nature of placenta cells that are targeted by C. burnetii remains unknown. I showed that C. burnetii infects BeWo and JEG trophoblastic cells and that organisms intensively replicated in BeWo cells and survived in JEG cells. A microarray analysis showed that C. burnetii induced a specific inflammatory response in BeWo cells. These results suggest that trophoblasts may serve as a reservoir for C. burnetii. Placenta macrophages placentaires may also targeted by C. burnetii. I showed that placenta CD14+ macrophages were characterized by phenotypic, transcriptional and functional properties different from those of circulating monocytes and monocyte-derived macrophages. In addition, placenta CD14+ macrophages differentiate into multinucleated giant cells that may regulate the cytolytic activity of macrophages in the placenta context since placenta CD14+ macrophages were not polarized in M1 or M2 macrophages. While M1/M2 polarization of macrophages is well established, that of monocytes remains an important question. We activated monocytes with canonical agonists of M1 and M2 profiles in macrophages using microarrays. The early response, 6 hours, of monocytes corresponded to a type M1/M2 response but the delayed response, 18 hours, did not correspond to the M1/M2 dichotomy, demonstrating a new level of heterogeneity of myeloid cells.