Abstract
Uterine receptivity and embryo implantation are two main processes that need a finely regulated balance between pro-inflammatory and tolerogenic mediators to allow a successful pregnancy. The neuroimmune peptide vasoactive intestinal peptide (VIP) is a key regulator, and it is involved in the induction of regulatory T cells (Tregs), which are crucial in both processes. Here, we analyzed the ability of endogenous and exogenous VIP to sustain a tolerogenic microenvironment during the peri-implantation period, particularly focusing on Treg recruitment. Wild-type (WT) and VIP-deficient mice [heterozygous (HT, +/−), knockout (KO, −/−)], and FOXP3-knock-in-GFP mice either pregnant or in estrus were used. During the day of estrus, we found significant histological differences between the uterus of WT mice vs. VIP-deficient mice, with the latter exhibiting undetectable levels of FOXP3 expression, decreased expression of interleukin (IL)-10, and vascular endothelial growth factor (VEGF)c, and increased gene expression of the Th17 proinflammatory transcription factor RORγt. To study the implantation window, we mated WT and VIP (+/−) females with WT males and observed altered FOXP3, VEGFc, IL-10, and transforming growth factor (TGF)β gene expression at the implantation sites at day 5.5 (d5.5), demonstrating a more inflammatory environment in VIP (+/−) vs. VIP (+/+) females. A similar molecular profile was observed at implantation sites of WT × WT mice treated with VIP antagonist at d3.5. We then examined the ability GFP-sorted CD4+ cells from FOXP3-GFP females to migrate toward conditioned media (CM) obtained from d5.5 implantation sites cultured in the absence/presence of VIP or VIP antagonist. VIP treatment increased CD4+FOXP3+ and decreased CD4+ total cell migration towards implantation sites, and VIP antagonist prevented these effects. Finally, we performed adoptive cell transfer of Tregs (sorted from FOXP3-GFP females) in VIP-deficient-mice, and we observed that FOXP3-GFP cells were mainly recruited into the uterus/implantation sites compared to all other tested tissues. In addition, after Treg transfer, we found an increase in IL-10 expression and VEGFc in HT females and allowed embryo implantation in KO females. In conclusion, VIP contributes to a local tolerogenic response necessary for successful pregnancy, preventing the development of a hostile uterine microenvironment for implantation by the selective recruitment of Tregs during the peri-implantation period.
Highlights
The peri-implantation period requires a variety of cellular processes that are encompassed to ensure proper trophoblast growth and invasion with intense vascular remodeling in an immunotolerant microenvironment [1,2,3].Critical to implantation is an adequate decidual response associated with a threshold level of physiological inflammation that facilitates the generation of a receptive endometrium [4]
Considering the relevance of Tregs to sustain a tolerogenic microenvironment during the implantation period, we evaluated FOXP3 gene expression at estrus in the uterus and other relevant tissues, such as mesenteric lymph nodes (MLN) and inguinal lymph nodes (ILN)
FOXP3 expression was reduced in vasoactive intestinal peptide (VIP)−/− females in MLN, ILN compared with WT, while no differences in expression were found in the thymus (Figures 1B–D)
Summary
Critical to implantation is an adequate decidual response associated with a threshold level of physiological inflammation that facilitates the generation of a receptive endometrium [4]. Within days of conception, inflammation needs to be controlled to sustain decidualization and implantation progress [5, 6]. Regulatory T cells (Tregs, CD4+FOXP3+ cells) are critical for controlling inflammation in early pregnancy, establishing the receptive decidual environment necessary for placentation. Considerable evidence from in vivo and in vitro models demonstrates that Tregs are generated throughout pregnancy and are critical for implantation even before mating [7,8,9,10]. The adoptive cell transfer of Tregs (one injection prior to mating and the other at d2 of gestation) prevented the defective embryo implantation in this model [10]. Regarding the origin of these Tregs, the evidence pointed out that natural Tregs (thymus origin) were present in the thymus and in the lymph nodes draining the uterus at early pregnancy, and that an expanded population of FOXP3+ cells was generated in the periphery (induced Tregs) at later pregnancy stages [12]
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