Characterisation of leukocytes in reproductive tissues before and after pregnancy

Reproduction is a crucial process, required for bringing the next generation into the world. In preparation for pregnancy, and throughout pregnancy itself, reproductive tissues recruit specific populations of immune cells that are thought to contribute in a variety of ways to successful reproduction. Pregnancy culminates in parturition, an inflammatory process characterised by an influx of inflammatory cells into reproductive tissues. Effective healing of reproductive tissues in the post-partum period is vital for continued reproductive success, and it too is thought to involve specific populations of immune cells. For leukocytes to effectively perform their functions in the reproductive system and elsewhere, migration to the right place at the right time is crucial. Key regulators of leukocyte homing are the chemokine family of chemoattractants and their G-protein coupled receptors. The chemokine network is complex and controls migration of leukocytes from the Bone Marrow (BM) into the blood and from the blood into tissues. Chemokines influence leukocyte position within tissues, and orchestrate their departure. Very little is known about the types of leukocytes present within reproductive tissues in the post-partum period, or the chemokines and receptors that could be involved in their migration. Exploring these processes is critical for an understanding of how tissues are repaired in readiness for subsequent pregnancies. In this thesis I have examined the leukocyte populations in reproductive and peripheral tissues of mice during the post-partum period and compared them to those found in Non-Pregnant (NP) mice. This analysis has encompassed a range of myeloid cell types, and also the complex populations of CD3+ cells that exist in reproductive tissues. I was also interested in how these cells are instructed to enter reproductive tissues, and in particular on the role of CC chemokine Receptor 2 (CCR2), a receptor associated with the recruitment of monocytes and T cells into tissues. My work has clearly identified cells expressing CCR2 both in reproductive tissues and elsewhere, and defined the impact of the genetic deletion of this receptor on leukocyte populations during the post-partum period. These experiments exploited a variety of standard techniques including histology, quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA) and Luminex, but they also required the development of challenging multiparameter flow cytometry protocols that allowed the simultaneous analysis, and definitive identification, of several leukocyte populations in various tissues and at specific reproductive time-points. Chapter 3 describes detailed experiments that focussed on characterising the myeloid cell populations across a variety of tissues in NP, 1 Day Post Partum (DPP) and 7DPP mice. Most strikingly, this revealed a profound accumulation of several myeloid cell populations in reproductive tissues at 1DPP, including inflammatory Ly6Chigh (hi) monocytes and neutrophils. Moreover, many of these myeloid cells expressed active CCR2 and remarkably CCR2 deletion was associated with a dramatic reduction in myeloid cell abundance in the uterine horn one day after birth. Thus, CCR2 appeared to be required for myeloid cell recruitment to the post-partum uterine horn. Chapters 4 and 5 describe changes in CD3+ cell populations over the post-partum period. Interestingly, the main finding from reproductive tissues was that the large majority of CD3+ cells lacked expression of CD4 and CD8, and were thus termed CD3+ Double-Negative (DN) cells. Three main CD3+ DN cell populations were described. CD3+CD25+NK1.1+TCRβ+ DN cells, likely to be Natural Killer T (NKT) cells, which were mainly found in reproductive tissues and blood. All tissues studied were found to contain CD3+NK1.1-TCRβ+ DN cells, likely to be ‘true’ DN T cells and CD3+NK1.1-TCRβ-TCRγδ+ DN cells, which were consistent with a γδ T cell phenotype. CD3+ DN cells were also found to increase in number at 1DPP, compared to NP tissues, driven by an increase in DN T cells. In contrast to myeloid cells CCR2 was not required for this change. However, at 1DPP there was a CCR2-dependent increase in the proportion of CD3+ DN cells in the blood. Finally, in Chapter 6, hormonal influences on leukocyte populations in reproductive and peripheral tissues were considered. This work had two major components: analysing sex differences in myeloid and T cell populations and exploring the effect that lactation has on these cell subsets over the post-partum period. Females were found to have an increased proportion of eosinophils in their blood, whereas males had a higher proportion of monocytes. I also found that female and male reproductive tissues, as well as peripheral tissues, have very similar CD3+ DN cell populations, suggesting that these cells serve roles in reproductive tissues that are not unique to one sex. Finally, CD3+ cell populations in the post-partum period were found to be controlled to some extent by lactation. Collectively, this work has significantly extended our understanding of leukocytes in various tissues in the post-partum period, and revealed the importance of chemokines in the regulation of these cells. It has laid the groundwork for future investigations aimed at dissecting the functions of these cells in reproductive tissues in the post-partum period.