Seminal fluid and cytokine control of regulatory T-cells in murine pregnancy.

Abstract

For successful pregnancy, the maternal immune system must tolerate the presence of a fetus that expresses alloantigens. The appropriate and timely acquisition of this state of tolerance is critical and emerging evidence suggests that it needs to be present from the time the embryo implants into the uterus. Recently it has been demonstrated that a subpopulation of lymphocytes termed CD4⁺CD25⁺ regulatory T cells (Treg cells) are required for immune tolerance of the fetus during pregnancy. Despite their importance the factors that control regulatory T cells during pregnancy, and in particular in the peri-implantation period, are poorly understood. Using mouse models we have assessed the role of the ejaculate and its components (sperm and seminal plasma) in coordinating Treg cells in the period prior to embryo implantation. We have also used mice with a null mutation in the interleukin 10 (IL-10) gene to assess the role of this cytokine in coordination of Treg cell populations in later pregnancy. Experiments in the peri-implantation period just prior to implantation (day 3.5 postcoitum) showed that there was a significant increase (approximately 2-fold; p<0.05) in the total number of (CD4⁺Foxp3⁺) Treg cells in the iliac lymph nodes (LNs) that drain the uterus, but not in the distal inguinal LNs. This appeared not to be the result of a selective expansion in Treg cells but due to expansion of the entire CD4⁺ cell pool, since the percent of CD4+ cells expressing Foxp3 in any of the lymphoid tissues studied did not increase in response to mating. In addition, there was a similar increase in the density of these cells in the uterus just prior to implantation at day 3.5pc (p<0.05). By using males deficient in the sperm or seminal plasma components of the ejaculate we could show that the increase in both the lymph node and uterine Treg cell populations occurred in response to seminal plasma. The role of seminal plasma in regulating expression of mRNAs encoding migratory molecules in the peri-implantation uterus, and the involvement of these genes in recruiting Treg cells following mating, was then assessed. We analysed the mRNAs for the chemokines Ccl4, Ccl5, Ccl19, Ccl22, the chemokine receptors Ccr4, Ccr5, Ccr7 and the integrin Cd103 using qRT-PCR. We showed a significant elevation in Ccl19 and Ccr5 mRNA at day 3.5pc following mating to intact males. However the increase in mRNA was independent of factors associated with seminal fluid and might instead be regulated by ovarian steroid hormones. Using IL-10 null mutant (IL-10-/-) mice it was then shown that the cytokine IL-10 is involved in controlling Treg cell numbers in mid gestation. At gestational day (gd) 9.5, in IL-10-/- mice, there was an approximate 40% elevation in the proportion of CD4⁺ cells expressing Foxp3 compared with wild‐type control mice (p<0.01). This was seen in both the iliac LNs and inguinal LNs. In addition, there was a greater than 10-fold increase (p<0.0001) in the total number of Treg cells in the uterine-draining iliac LNs of IL-10-/- mice compared to wild-type mice. This was not seen in the inguinal LNs. Experiments comparing allogeneic and syngeneic mated mice showed that the proportional changes seen in the CD4⁺ cell population was dependent on fetal alloantigens, although the elevation in total numbers still occurred in the absence of fetal alloantigens. This study begins to unravel the process by which Treg cell populations are expanded and recruited into the uterus prior to embryo implantation and later in gestation. A greater understanding of this process may aid in the diagnosis and prevention of a range of pregnancy pathologies associated with immune dysregulation, such as preeclampsia and recurrent spontaneous abortion.