ItemEvaluation of Carbon Monoxide as a Therapeutic for PreeclampsiaMcRae, Karalyn; Anatomy and Cell Biology; Smith, Graeme NPreeclampsia (PE), a hypertensive complication of pregnancy, is one of the leading causes of maternal and neonatal morbidity and mortality. The etiology of PE is not well-understood, however it is widely accepted to occur in two stages: poor uteroplacental perfusion and placental ischemia, which subsequently leads to a systemic maternal inflammatory response and multi-organ effects. Although cigarette smoking during pregnancy is known to cause aberrant placental development and adverse pregnancy outcomes, smoking reduces the risk of PE by 33% in a dose-dependent manner. The same risk reduction does not hold true for users of smokeless tobacco products. This has lead to the notion that carbon monoxide (CO), one of the byproducts of cigarette combustion, may confer this decrease in PE risk. Endogenously, CO is produced through the degradation of heme by heme oxygenase (HO), and it has been suggested that modulation of the HO/CO system may attenuate the development or progression of PE. The studies presented herein demonstrate that low doses of CO, delivered through gaseous CO or CO donor molecules, may exhibit physiological benefits and have therapeutic potential during complicated pregnancies. Firstly, the challenges associated with reproducing the AdsFlt-1-induced mouse model of PE for studying the effects of CO in complicated pregnancies were investigated. Clinically, inhaled CO was shown to moderately increase biological CO above endogenous levels, and increase vascular responses in the cutaneous microvasculature. In mice, it is demonstrated that the CO-releasing molecule (CORM-A1) is able to transiently increase biological CO levels. CORM-A1 delivered in mid-gestation, resulted in increased CO, without adverse maternal or fetal effects, indicating the potential for CORM-A1 as a novel method to deliver CO during pregnancy. Collectively, the data presented in this thesis verify that low dose CO may have therapeutic properties to improve endothelial dysfunction, increase placental perfusion, and reduce progression of the clinical signs of PE. ItemPlacental Growth Factor Modulates Vascularization and Neuroanatomy in MiceKay, Vanessa; Anatomy and Cell Biology; Tayade, Chandrakant; Croy, B. AnnePreeclampsia (PE) is a common pregnancy complication and may result in fetal stress with lifelong consequences for cognition. Offspring cognitive alterations are associated with distorted neuroanatomy and cerebrovasculature. Although the pathophysiology is complex, reduced expression of pro-angiogenic factors may result in impaired fetal vascularization, altered brain structure and impaired cognition. In particular, low concentrations of placental growth factor (PGF) may alter neural development. Pgf -/- mice were used to interrogate the role of PGF in central nervous system vascularization, appropriate neuroanatomy and behaviour. Subsequently, Pgf -/- mice were used to investigate the postnatal period as a window for intervention. Pgf -/- mice were hypothesized to have impaired cerebrovascular development with permanent deviations of the cerebrovasculature, neuroanatomy and behaviour. Adult deficits were hypothesized to be correctable with an early postnatal PGF treatment. Whole-mount immunofluorescence of the brain and retina examined vascularization and circle of Willis connectivity. Micro-CT imaging visualized the entire cerebrovasculature in adult mice. Magnetic resonance imaging determined the volume of 62 unique structures in the adult mouse brain. Behavioural testing of adult mice examined spatial learning, object recognition, depressive-like behaviour and anxiety-like behaviour. As a postnatal treatment, recombinant PGF was administered to Pgf -/- mice from postnatal day 1-10 before examination of the cerebrovasculature, neuroanatomy and behaviour in adulthood. Pgf -/- mice exhibited delayed vascularization of the hindbrain, less connectivity of the circle of Willis and altered arteriovenous organization in the retina. In adulthood, more small-diameter cerebral vessels were present and 10/62 structures in the brain had smaller volume. On behavioural testing, Pgf -/- mice exhibited poorer performance on spatial learning tests but better performance on depressive-like behaviour and object recognition tests relative to Pgf +/+ controls. Postnatal PGF treatment altered adult neuroanatomy and behaviour but not the cerebrovasculature. In particular, treatment with 70 pg/g PGF partially corrected alterations seen in Pgf -/- mice. Overall, PGF is important for cerebrovascular development, adult neuroanatomy and cognitive behaviour in mice. PGF deficiency may be one factor in a PE-gestation that leads to altered offspring brain development. Finally, the postnatal period is a potential time for intervention in offspring after PE-impacted gestation. ItemThe Role of Interleukin-17A in the Pathogenesis of EndometriosisAhn, SooHyun; Anatomy and Cell Biology; Tayade, ChandrakantEndometriosis is an estrogen-dependent, chronic inflammatory disease characterized by the ectopic presence of hormonally-responsive endometrial epithelial glands and stroma in the pelvic cavity. Theorized to arise from the phenomenon of retrograde menstruation whereby menstrual tissue is refluxed into the peritoneal cavity through the fallopian tubes, the pathogenesis of endometriosis is not only characterized by peritoneal inflammation, but its development has also been linked to abnormal immune system response that is unable to clear away menstrual debris in the pelvic cavity. Numerous studies have shown elevated levels of inflammatory cytokines and abnormally activated immune cells in the peritoneal fluid and peripheral blood of women with endometriosis. However, a comprehensive overview of immune dysfunction and inflammation in women with endometriosis is lacking. Therefore, we conducted a targeted transcriptomic profiling on the molecular pathways of inflammation and immune cell signaling to uncover pathways associated to endometriosis pathogenesis. We hypothesized that genes encoding for pro-inflammatory cytokines and immune cell activation will be abnormally expressed in patient tissue samples compared to controls. We report that indeed, genes involved in inflammation and immune cell signaling are aberrantly expressed in women with endometriosis. Further, we explored the potential involvement of interleukin-17A (IL-17A) in the pathogenesis of endometriosis. In diseases with chronic inflammation, IL-17A is known to exacerbate disease progression by promoting inflammatory cytokine production and neutrophil recruitment via the production of IL-6, IL-8, GRO-α, and G-CSF. We hypothesized that IL-17A will also exacerbate endometriosis development by promoting local inflammation. We found increased concentration of IL-17A in plasma of women with disease. It can also promote inflammatory cytokines (G-CSF, IL-6, and IL-8) in vitro. In mouse model of endometriosis, intraperitoneal injection of IL-17A did not promote proliferation of vascularization of lesions compared to controls. We also observed increased G-CSF and CD11b+Ly6C+ inflammatory monocytes in mice treated with IL-17A. As a whole, we demonstrate how eutopic endometrium and ectopic endometriotic lesions are molecularly distinct entities compared to fertile endometrium. Further, we provide novel findings on the contribution of IL-17A in peritoneal inflammation, macrophage infiltration and polarization using mouse model of endometriosis. ItemThe Importance of MHC-Independent Natural Killer Cell Receptors in the Immunoregulation of Murine Pregnancy(2016-08-19) Felker, Allison; Anatomy and Cell Biology; Croy, B. AnneNumerous leukocyte populations are essential for pregnancy success. Uterine natural killer (uNK) cells are chief amongst these leukocytes and represent a unique lineage with limited cytotoxicity but abundant angiokine production. They possess a distinct phenotype of activating and inhibitory receptors that recognize major histocompatibility complex (MHC) molecules, such as the killer immunoglobulin like receptors (KIRs; mouse Ly49), and MHC-independent activating receptors, including the aryl hydrocarbon receptor (AHR) and natural cytotoxicity receptor 1 (NCR1). While the roles of MHC-dependent receptors are widely addressed in pregnancy, MHC-independent receptors are relatively unstudied. This thesis investigated the roles of MHC-independent receptors in promotion of mouse pregnancy and characterized early leukocyte interactions in the presence and absence of NCR1. It was hypothesized that loss of MHC-independent receptors impairs uNK cell development resulting in aberrations in leukocyte function and decidual vasculature. Implantation sites from Ahr-/- and Ncr1Gfp/Gfp mice were assessed using whole mount in situ immunohistochemistry (WM-IHC) and histochemical techniques. Leukocyte interactions identified during preliminary WM-IHC studies were confirmed as immune synapses. The novel identification of immune synapses in early mouse pregnancy compelled further examination of leukocyte conjugates in wildtype C57BL/6 and Ncr1Gfp/Gfp mice. In Ahr-/- and Ncr1Gfp/Gfp mice, receptor loss resulted in reduced uNK cell diameters, impaired decidual vasculature, and failures in spiral artery remodeling. Ahr-/- mice had severe fertility deficits whereas Ncr1Gfp/Gfp mice had increased fetal resorption indicating differing receptor requirements in pregnancy success. NCR1 loss primarily affected uNK cell maturation and function as identified by alterations in granule ultrastructure, lytic protein expression, and angiokine production. Leukocyte conjugates were frequent in early C57BL/6 decidua basalis and included uNK cells conjugating first with antigen presenting cells and then with T cells. Overall conjugate formation was reduced in the absence of NCR1, but specific uNK cell conjugations were unaffected by receptor loss. While KIR-MHC interactions are associated with numerous pregnancy complications in humans, the role of other uNK cell receptors are not well characterized. These results illustrate the importance of MHC-independent receptors in uNK cell activation during early pregnancy in mice and encourage further studies of pregnancy complications that may occur independently of maternal KIR-MHC contributions. ItemThe Transcription Factor NFIL3 Is Not Required For Uterine Natural Killer Cell Differentiation But Contributes To Placental And Conceptus Development(2015-09-26) Redhead, MacKenzie; Anatomy and Cell Biology; Croy, B. AnneUterine natural killer (uNK) cells are the most abundant lymphocyte in early human and mouse decidua. UNK cell functions have been deduced by histopathologic comparisons of implantation sites between alymphoid (NK-T-B-), NK cell reconstituted alymphoid (NK+T-B-) and normal NK+T+B+ mice. Circulating (c)NK cells are reported to be absent from C57BL/6 mice genetically ablated for the transcription factor Nfil3 (cNK-T+B+) and these mice experience midgestation Th17 cell-mediated fetal loss when mated by BALB/c males. Recently, tissue resident subsets of NK cells have been described in this mouse. A histological time-course examination of syngeneic Nfil3-/- male x Nfil3-/- female and allogeneic BALB/c-Tg(UBC-GFP)30Scha/J male x Nfil3-/- female pregnancy (gestation day; GD 6.5-15.5) was performed to determine the consequences of a maternal cNK-T+B+ phenotype on pregnancy with an Nfil3-/- or Nfil3+/- conceptus. Whole mount immunofluorescence (WM-IF) was used as an additional technique to provide greater insights at midgestation. Nfil3 is a pleiotrophic factor; to examine Nfil3-dependent uNK cell-specific effects, pregnancies in Nfil3-/- bone marrow engrafted alymphoid mice (BME) were studied. UNK cells were less frequent in Nfil3-/- pregnancies and viability of Nfil3-/- implantation sites (IS) did not differ from wild type (WT) matings. Early gestation pathologies included impaired antimesometrial decidualization (AMD) and delays in lumen closure and embryo development (ED). WM examination revealed an enrichment of CD45+CD11c+ cells at GD 8.5 in Nfil3-/- IS compared to WT. Mid-late gestation placental development, including labyrinth vascular space area and interhemal membrane width, deviated significantly less from WT in allogeneic compared to syngeneic matings. BME and alymphoid placental development was similar to WT. Spiral artery (SA) remodeling did not occur in any experimental mating. Results of the present investigation suggest Nfil3-independent uNK cells do not promote AMD or GD 8.5 ED and cannot remodel SAs. Nfil3 plays a role in placental tissue layer (specifically labyrinth) development possibly through trophoblast-dependent mechanisms. The absence of Nfil3 may promote recruitment or differentiation of CD45+CD11c+ cells in the early gestation implantation site. Although not an effective model for studying NK-T+B+ pregnancy, the Nfil3-/- mouse deepens the understanding of innate lymphoid cells and their respective subsets.