Deletion of atypical chemokine receptor 3 (ACKR3) increases immune cells at the fetal-maternal interface

Abstract

Establishment of immune cell populations and adaptations in immune cells are critical aspects during pregnancy that lead to protection of the semi-allogenic fetus. Appropriate immune cell activation and trophoblast migration are regulated in part by chemokines, the availability of which can be fine-tuned by decoy receptors. Atypical chemokine receptor 3 (ACKR3), previously named C-X-C chemokine receptor 7 (CXCR7), is a chemokine decoy receptor expressed in placenta, but little is known about how this receptor affects placental development. In this study, we investigated the phenotypic characteristics of placentas from Ackr3^-/- embryos to determine how Ackr3 contributes to early placentation. In placentas from Ackr3^-/- embryos, we observed an increase in decidual compaction and in the size of the uterine natural killer cell population. Ackr3 knockdown in trophoblast cells led to a decrease in trophoblast migration. These findings suggest that this decoy receptor may therefore be an important factor in normal placentation.

Keywords: Chemokines; Decoy receptors; Immune cells; Placenta.

Figure 1.

(A) The placental morphology, labyrinth height, and labyrinth length in placentas of Ackr3^−/− embryos is similar to Ackr3^+/+ at e14.5 (per genotype, n = 6–7 total placentas, unpaired t-test with Welch’s correction). (B) A decrease in clusters of trophoblast cells (arrows) and an increase in fetal sinus area are observed in labyrinth of Ackr3^−/− embryos compared to Ackr3^+/+ at e14.5 (per genotype, n = 6–7 total placentas, unpaired t-test with Welch’s correction). (C) Reduction of Ackr3 in extravillous trophoblast cells (HTR8/svNeos) in vitro causes a decrease in trophoblast migration. Scale bars: 50 μm. The space/area left in each transwell was calculated to assess overall migration and cell coverage using Fiji. Overall trophoblast migration was determined using an unpaired two-tailed t-test with Welch’s correction. *P < 0.05.

Figure 2.

(A) The placental morphology, labyrinth height and labyrinth length in placentas of Ackr3^−/− embryos is similar to Ackr3^+/+ at e12.5 (per genotype, n = 5–6 total placentas, unpaired t-test with Welch’s correction). (B) Embryonic deletion of Ackr3 at e12.5 causes an increase in cellular density in the decidua (DEC) compared to wildtype controls. Scale bar, 40 μm. (C) Cxcl10 gene expression decreases in e12.5 placentas of Ackr3^−/− embryos compared to Ackr3^+/+ embryos. Gene expression of additional cytokines is similar in e12.5 placentas of Ackr3^+/+ and Ackr3^−/− embryos. Data graphed as 2^−ΔΔCT with mean ± SEM. *P < 0.05.

Figure 3.

(A) Flow cytometry of e12.5 placentas in Ackr3^+/+ and Ackr3^−/− embryos. Placentas from Ackr3^−/− embryos have an increase in uterine natural killer (uNK), and dendritic cells, and a trending increase of macrophages. Immune cell analysis was calculated as a percentage of gated cells and analyzed using an unpaired t-test with Welch’s correction. *P<0.05, **P<0.01. (B) Uterine natural killer (uNK) cells (denoted in green) increase in e12.5 placentas of Ackr3^−/− embryos compared to wildtype counterparts (left panel). Co-localization of dendritic cells (CD11c, denoted in magenta) and uNK cells decrease in embryos null of Ackr3 (right panel). Scale bars: 50 μm. Decidua, DEC, junctional zone, JZ.

Figure 4.

(A) H&E staining of spiral arteries in Ackr3^+/+ and Ackr3^−/− embryos at e12.5 (per genotype, n = 5–6 total placentas, unpaired t-test with Welch’s correction). (B) Quantification of the luminal area and wall thickness (ratio of vessel wall to lumen area) of spiral arteries in Ackr3^+/+ and Ackr3^−/− embryos at e12.5. (C) At e12.5 smooth muscle coverage (αSMA, green) is similar between Ackr3^+/+ and Ackr3^−/− embryos (per genotype, n = 5–6 total placentas).