Decidual stromal cells recruit Th17 cells into decidua to promote proliferation and invasion of human trophoblast cells by secreting IL-17

Abstract

T helper 17 (Th17) cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A (IL-17A) have been implicated in the pathogenesis of certain autoimmune diseases, several types of cancer and allograft rejection. However, the role of Th17 cells at the maternal/fetal interface remains unknown. Here, we demonstrate that Th17 cells are present in decidua and are increased in the peripheral blood of 10 clinically normal pregnancies based on intracellular cytokine analysis. Our results suggest a potential role of Th17 cells in sustaining pregnancy in humans. Furthermore, we demonstrate that decidual stromal cells (DSCs) but not trophoblast cells recruit peripheral Th17 cells into the decidua by secreting CCL2. The recruited Th17 cells promote proliferation and invasion and inhibit the apoptosis of human trophoblast cells by secreting IL-17 during the first trimester of pregnancy. These findings indicate a novel role for Th17 cells in controlling the maternal-fetal relationship and placenta development.

Figures

Figure 1

Th17 cells are increased in human pregnancy. The percentages of IL-17-expressing CD4+ T cells were analyzed by intracellular cytokine assays. The results were gated on CD4 and MACS-sorted peripheral blood and decidual CD4+ T cells were stimulated with PMA, ionomycin and BFA for 4 hours. The surface marker of Th17 cells were analyzed by FACS. (a) The proportion of Th17 cells in PBMC during human pregnancy is compared with that in non-pregnancy (n=10, left); the phenotype of first-trimester human peripheral blood Th17 cells (right). (b) The proportion of Th17 cells in human first-trimester decidua is compared with non-pregnant endometrium of secretory phase (n=10, left); the phenotype of first-trimester human decidual Th17 cells (right). (c) Comparison of CD161 on Th17 cells between PBMC and deciduae. (d) Comparison of Th17 cells between PBMC and decidua in first-trimester human pregnancy. Error bars indicate the s.e.m. The FACS picture represents an individual sample. BFA, brefeldin A; FACS, Fluorescence-activated cell sorting; MACS, magnetic affinity cell sorting; PBMC, peripheral blood mononuclear cell; PMA, phorbol myristate acetate; s.e.m., standard error of the mean; Th17, T helper 17.

Figure 2

DSCs recruit peripheral Th17 cells into decidua via secreting CCL2. (a) One case of chemotaxis for Th17 cells (left); fold increase in Th17 cells after treatment with different supernatants (right). (b) Specific brown-colored staining for CCL20 occurs in the membrane of villous cytotrophoblasts, syncytiotrophoblasts and invasive trophoblast cells in decidua. DSCs also express CCL20 moderately; glandular epithelium and ESCs of endometrium are weakly positive for CCL20. No background staining was observed in the isotype control. These results were highly reproducible in five independent experiments (including five placental samples), and the picture represents one sample (×200, left). Accumulated concentration of CCL2 and CCL20 in supernatants of primary DSCs was examined by ELISA (right upper). The level of CCL2 was higher than CCL20 from primary DSCs after 72 h of culture (right lower). (c) Fold change in Th17 cells after treatment with DSC supernatant with or without neutralizing antibody to CCL20 or CCL2. *P<0.05, **P<0.01; error bars: s.e.m. DSC, decidual stromal cell; ESC, endometrial stromal cell; DSC sup, DSCs supernatant; s.e.m., standard error of the mean; Th17, T helper 17; Tro-DSC sup, supernatant from the coculture of trophoblasts with DSCs; Tro. sup, trophoblasts supernatant.

Figure 3

Th17 cells promote invasion of first-trimester human trophoblasts through secreting IL-17. (a) Left: Expression of IL-17R on human first-trimester villi and decidua (left); expression of IL-17R on primary trophoblast, DSCs and human choriocarcinoma JEG-3. (×200, right). (b) Effect of Th17 cell-derived supernatants on the invasive capacity of trophoblasts with or without neutralizing antibody to IL-17. Upper: The neutralizing antibody to IL-17 completely inhibits trophoblast invasion increase induced by Th17 cell supernatants. Lower: Representative of three experiments (×200). (c) Effect of rhIL-17A on the invasive capacity of trophoblasts; error bars: s.e.m. Th17 sup, Th17 cell supernatants. DSC, decidual stromal cell; EVCT, extravillous cytotrophoblast; IL-17R, interleukin-17 receptor; s.e.m., standard error of the mean; Th17, T helper 17.

Figure 4

Th17 cells promote growth of first-trimester human trophoblasts by secreting IL-17. (a) Effect of Th17 cells-derived supernatants on the proliferation of trophoblasts with or without neutralizing antibody to IL-17. (b) The effect of different concentrations of rhIL-17A on the proliferation of trophoblasts. (c) Comparison of trophoblast proliferation induced by rhIL-17A or Th17 cell-derived supernatants. (d) Effect of rhIL-17 on the apoptosis of trophoblasts. (e) Effect of Th17 cell-derived supernatants on trophoblast apotopsis. Flow cytometry plot is a representative of three experiments. *P<0.05; **P<0.01; error bars: s.e.m. s.e.m., standard error of the mean; Th17, T helper 17; Th17 sup, Th17 cell-derived supernatant.

Figure 5

Roles of Th17 cells at the maternal/fetal interface. Th17 cells are recruited by DSC-secreted CCL2 into decidua and improve the growth and invasiveness of trophoblast cells through secreting IL-17 during the first trimester of human pregnancy. DSC, decidual stromal cell; Th17, T helper 17.