THE IMPORTANCE OF MHC-INDEPENDENT NATURAL KILLER CELL RECEPTORS IN THE IMMUNOREGULATION OF MURINE PREGNANCY

Numerous 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.

EXAMINATION OF THE EFFECTS OF VALPROIC ACID EXPOSURE OR P300 PROTEIN INHIBITION ON THE BALANCE OF APOPTOSIS AND PROLIFERATION IN P19 EMBRYONAL CARCINOMA CELLS

Valproic acid (VPA), a commonly-used anticonvulsant drug, is associated with increased risk of fetal malformations, including neural tube defects (NTDs). Previous in vivo studies determined that VPA-exposed embryos with a NTD had altered expression of several proteins regulated by p300, a histone acetyltransferase (HAT) protein. p300 is capable of acetylating histones and non-histone proteins through its HAT activity, allowing it to transcriptionally regulate genes as well as modulate the stability and activity of specific proteins. NFκB, Stat3 and Egr1, all of which function as transcription factors, are regulated by p300 through its HAT activity. Together, these proteins all play an important role in maintaining the balance of apoptosis, proliferation and differentiation, the regulation of which is extremely important for proper embryonic development. The studies in this thesis utilized P19 embryonal carcinoma (EC) cells in order to determine the effects of VPA exposure on the expression of p300 and the aforementioned transcription factors, as well as apoptosis and proliferation, in vitro. P19 EC cells were exposed to C646, a selective p300 inhibitor, in order to assess whether the effects observed as a result of VPA exposure were due to p300 protein degradation. It was found that VPA exposure for 24 hours in P19 EC cells in vitro resulted in a significant decrease in p300 protein expression. VPA exposure also significantly decreased NFκB protein expression, while resulting in increased Stat3 protein expression. However, Stat3 acetylation and phosphorylation, which both contribute to Stat3 activation, were significantly decreased as a result of VPA exposure. p300 inhibition resulted in a significant decrease in NFκB, similar to what was observed as a result of VPA exposure, which suggests that VPA-mediated degradation of p300 may play a role in reduced NFκB protein expression following VPA exposure. Conversely, Stat3 protein expression, acetylation and phosphorylation were not significantly changed as a result of p300 inhibition, suggesting that p300 degradation does not play a role in VPA’s effects on Stat3 protein expression and activation. VPA exposure also resulted in a significant increase in apoptosis, while p300 inhibition did not significantly increase apoptosis. These data suggest that p300 degradation plays a role in VPA-mediated teratogenicity, and that VPA may target other cellular mechanisms in order to exert its teratogenic effects.