The role of macrophages in vascular adaptation to pregnancy in mice

Abstract

Macrophages are abundant in female reproductive tissues and play crucial roles in the establishment of murine pregnancy through actions in the ovary, and potentially in the uterus. Within the ovary, macrophages support the vascular integrity of the corpus luteum allowing progesterone (P4) production. These ovarian macrophages have been shown to be associated with endothelial cells in the corpora lutea and express the angiogenic marker Tie2. This supports an angiogenic role for ovarian macrophages at the outset of pregnancy. On the other hand, the role of uterine macrophages during murine pregnancy is not fully understood. It has been postulated that uterine macrophages contribute to immune tolerance and tissue remodelling for embryo implantation, trophoblast invasion, and decidualisation. In addition, some studies have suggested that macrophages are involved in uterine vascular remodelling through interactions with endothelial cells, trophoblast cells, and other immune cell subsets including uterine natural killer (uNK) cells and regulatory T (Treg) cells. Therefore, studies within this thesis sought to investigate the role of uterine macrophages in promoting vascular adaptations to pregnancy. Previous studies with macrophage-deficient mice or mice transiently depleted of macrophages during pregnancy have shown pregnancy failure (Pollard et al., 1991, Care et al., 2013). Whilst these studies demonstrate macrophages as being indispensable for pregnancy success, the specific physiological role of uterine macrophages has not been confirmed. Studies within this thesis utilised the CD11b-DTR transgenic mouse model to deplete CD11b-expressing cells via administration of diphtheria toxin (DT). Administration of 25 ng/g DT to CD11b-DTR mice elicited >90% depletion of macrophages from the uterus and other tissues 24 h post-DT administration. Importantly, administration of DT to wild type mice had no effect on pregnancy or macrophage numbers. Data from chapter three demonstrated that macrophage depletion resulted in pregnancy failure from day 7.5 pc, 48 h post-DT administration. Macrophage depletion impaired decidualisation and reduced trophoblast invasion on day 7.5 pc. In addition, vascular remodelling within the uterus was reduced post-macrophage depletion. Pregnancy failure on day 7.5 pc was in part attributable to defects in corpus luteum structure and reduced serum P4. However, hormone supplementation in macrophage-depleted mice failed to fully restore viable pregnancy. This implies a role for macrophages other than in ovarian P4 production and presumably within the uterus during the peri-implantation phase of murine pregnancy. Whilst near-complete macrophage depletion resulted in pregnancy failure, an attenuated dose of DT was investigated to explore the effect of less extensive macrophage loss. Moderate macrophage depletion during the peri-implantation phase allowed a greater proportion of pregnancies to survive. However, there was a reduced number of viable fetuses and reduced fetal growth measured at day 17.5 pc. Associated with fetal growth restriction was an impairment in the placental labyrinth zone which had reduced densities of fetal capillaries. In addition, uterine vascular remodelling was impaired during mid-gestation after moderate macrophage depletion, similar to what was observed at the higher DT dose. To further investigate the involvement of macrophages in uterine vascular remodelling, macrophage or P4 replacement procedures were conducted in DT-treated CD11b-DTR mice. Macrophage replacement improved viable pregnancy rates and restored serum P4, decidualisation, trophoblast invasion, and to an extent, uterine vascular remodelling. Whilst P4 supplementation restored decidualisation, P4 administration failed to improve uterine vascular remodelling and trophoblast invasion after macrophage depletion. In addition, macrophage depletion caused reduced abundance of uNK cells, while P4 or macrophage administration restored uNK cell abundance. In order to interrogate macrophage-derived products as mediators of uterine vascular remodelling during early to mid-gestation, RNA profiling was performed in the decidua and myometrium on day 7.5 pc. Macrophage depletion resulted in substantial gene expression changes. A majority of these dysregulated genes could be restored by either P4 or macrophage administration. Importantly, macrophage administration restored a larger proportion of the genes dysregulated by macrophage depletion than did P4 supplementation. Notably, C1q genes were downregulated after macrophage depletion, independent of P4 administration, but macrophage replacement restored C1q expression. C1q has been shown to be an important factor for placental development and fetal growth with some evidence to suggest that decidual vascular remodelling is impaired in C1q-deficient dams. To investigate C1Q function in murine pregnancy, C1Q-deficient dams were utilised in allogeneic pregnancy. At late gestation, fetuses were growth restricted and placentas were hypertrophic in C1Q-deficient dams. Importantly, C1Q deficiency caused impaired decidual vascular remodelling and reduced uNK cell abundance. Wild type macrophage administration to C1Q-deficient dams restored decidual vascular remodelling and uNK cells, highlighting that macrophage-derived C1Q can facilitate uterine vascular remodelling during early to mid-gestation. The data presented in this thesis shows that macrophages play an indispensable role during the peri-implantation phase of murine pregnancy through effects in the uterus, in addition to their known roles within the ovary. Furthermore, macrophage-derived C1Q appears to be a regulator of uterine vascular adaptation during early to mid-gestation and facilitates pregnancy success. This indicates that macrophages and C1Q are essential factors in promoting adequate placentation and fetal growth, and should be further investigated in the context of human pregnancy and gestational disorders.