Here we performed a functional characterization of uDCs with respect to their involvement in embryo implantation using an in vivo cell ablation strategy. Depletion of uDCs resulted in severe impairment of the implantation process and led to embryo resorptions. Rather than being involved in tolerance establishment, uDCs seem to regulate maternal receptivity through direct involvement in decidual tissue remodeling and specifically angiogenesis. Flow cytometric, histological, and in vivo characterization of uDCs during implantation and later pregnancy stages revealed distinct phenotypes and microanatomical localizations, suggesting discrete functions with pregnancy progression. Indeed, depletion of uDCs immediately after the conclusion of the implantation process did not lead to embryo loss. At the time of implantation (E5.5), uDCs exhibited an immature phenotype, characteristics of mucosal DCs, and could be subdivided into two subpopulations according to CD11b and CD8α expression. Moreover, uDCs at E5.5 were found in close proximity to blood vessels. To study the functional role of uDCs in implantation, we used a conditional ablation strategy to deplete the cells before the embryo makes its first contact with the uterus and throughout the entire implantation window (E4 until E5.5). uDC ablation resulted in embryo resorption. The remainder of our study was devoted to the investigation of the physiological and molecular mechanisms that underlie this unique requirement of uDCs for embryo implantation.
The presence of immune cells at the IS has long been associated with the response of the maternal immune system to the semiallogeneic fetus (22
). However, presentation of fetal antigen was reported to begin only at mid-gestation, in association with the endovascular invasion of placental trophoblasts and the hematogenous release of placental debris (31
). Accordingly, our results that focus on the implantation window suggest that uDCs are not required for tolerance establishment. Thus, uDC ablation also resulted in embryo resorption in syngeneic and T cell–deficient pregnancy models. Moreover, depletion of uDCs impaired artificial decidualization induced in pseudopregnant mice in the absence of an embryo.
The maintenance of pregnancy and specifically the decidualization process requires P4, a principal steroid hormone secreted by the corpus luteum (32
). However, in our model, the embryo loss induced by uDC depletion could not be rescued by P4 administration and is, therefore, unrelated to corpus luteum insufficiency. Interestingly, we rather found that the uDC ablation directly affected three critical parameters of the decidualization process: proliferation, differentiation, and angiogenesis. Immunohistochemical analysis of uDC-depleted ISs for phospho–histone H3 and Cx43 revealed an impairment of proliferation and decidual differentiation. Along with reduced expression of lectin in the uDC-depleted IS, noninvasive functional analysis of angiogenesis by dynamic macromolecular contrast-enhanced MRI revealed significant perturbances within the 2 days of the implantation window in uDC-depleted ISs. Angiogenesis during normal embryo implantation is characterized by increased vascular permeability on E4.5, followed by a rise in blood volume on E5.5. By the end of the implantation process (E5.5) and along with the attenuation of vascular permeability, blood vessels undergo maturation (28
). Upon uDC depletion, the formation of new blood vessels in the decidua was significantly reduced on E5.5, and vascular maturation was impaired, showing higher permeability per blood vessel, a characteristic of the initiation of the implantation process (E4.5). uDC ablation thus resulted in a delayed decidual angiogenic response. Given the limited time window in which the uterus is receptive toward the implanting embryo (33
), impaired angiogenesis results in a failure of the embryo to implant into the uterine wall (34
). In support of a suggested role for uDCs in decidual vascular development, uDCs are associated with blood vessels, as exhibited in the functional analysis using MRI and fluorescence microscopic data.
Physiological angiogenesis is a fine balance between pro- and antiangiogenic factors (34
). Vascular plasticity during angiogenesis is driven by VEGF, which is a potent vascular permeability factor. In pregnant mice, VEGF expression is observed in the luminal epithelium as early as day 1. VEGF is also expressed by stromal cells (35
), as well as uNKs and macrophages (7
). In light of the abundance of VEGF expression in the decidua, VEGF levels require local modulation to facilitate tissue remodeling and allow decidual transformation. One of the key molecules in the control of VEGF activity is a physiological VEGF trap in the form of a secreted VEGF receptor 1 (Flt1) (29
) that locally sequesters the proangiogenic factor to execute its effect on vascular permeability. Interestingly, our expression analysis of fractionated decidual tissue revealed that in the IS, sFlt1 is expressed by uDCs but not by uNKs. Moreover, the critical importance of this uDC-derived sFlt1 in the local control of angiogenesis is highlighted by the fact that immunoreactivity to anti-sFlt1 antiserum in the outer decidual rim, i.e., the exact location of the bulk of uDCs, was significantly reduced in uDC-depleted ISs. Our data thus strongly suggested that a key role of uDCs in decidua formation is to fine-tune angiogenesis by specifically attenuating vascular permeability via secretion of sFlt1. In the absence of uDCs, sFlt1 is missing, vascular permeability is not attenuated, and subsequent vascular maturation at the boundaries of the IS do not occur.
Our expression analysis of decidual cells further revealed that uDCs express the cytokine TGF-β1. Moreover, although TGF-β1 expression was shared with uNKs, the analysis of uDC-depleted ISs revealed a general reduction in the expression level of this cytokine. Notably, our data do not exclude the possibility that TGF-β1 expression by uNKs could also be driven by uDC-uNK crosstalk. The reduction of TGF-β1 in uDC-depleted IS might have multiple consequences. First, as TGF-β1 can promote endothelial cell survival (37
), TGF-β1 that is directly produced by uDCs could be critically required to locally quench a potential proapoptotic activity that has been reported for sFlt1 (as it sequesters VEGF, an endothelial cell survival factor) (38
) and thus provide crucial protection for the uDC-associated endothelial cells. This may ensure that the VEGF trap sFlt1 expressed by uDCs will only act to sequester VEGF activity as a permeability factor but will be less effective in causing endothelial cell death in the presence of TGF-β1.
Second, TGF-β is known to directly affect vascular maturation through its effect on endothelial–smooth muscle cell interactions (30
). Vascular maturation at the IS mostly occurs at the edges of the IS, and the observed global reduction of TGF-β1 in uDC-depleted ISs could thus have a major effect on the ability of these blood vessels to mature. Finally, the absence of TGF-β1 was previously proposed to have a direct effect on the proliferation and decidualization of stromal cells (30
). However, this might be secondary to the angiogenic disorder, since tissue growth and remodeling are critically dependent on proper vascular expansion.
Taken together, our results suggest that uDCs fine-tune angiogenesis in the decidua through provision of two critical factors, sFlt1 and TGF-β1, that act together to promote coordinated vascular expansion and maturation. In their absence, vascular permeability is increased, blood volume is reduced, and vessel maturation is attenuated, resulting in a fatal delay of the angiogenic reaction. This leads to faulty tissue remodeling, which causes the embryo loss. The specific localization of uDCs in the outer decidual rim and in proximity to blood vessels supports their role in attenuating permeability, promoting vascular survival and vessel maturation, which occur mainly at the edges of the IS. Of note, however, our data do not exclude more complex settings in which uDCs would provide multiple crucial factors to the decidualization and subsequent angiogenesis involving additional cell types.
In summary, we demonstrated a role for uDCs in successful embryo implantation, which is directly associated with adequate decidualization by coordinating the synchronization between uterine receptivity and embryo development. These results provide a new perspective on DC functions during pregnancy beyond their anticipated tolerogenic effect, suggesting a more active role in tissue renewal and specifically in angiogenesis.
In terms of medical implications, the temporal and spatial ablation of uDCs provides a unique experimental model to study this important phase of pregnancy, which will help to understand human infertility where implantation is impaired. Specifically, our results may assist in resolving the limited implantation success of embryos transferred following in vitro fertilization.