Cyclosporine A enhances Th2 bias at the maternal-fetal interface in early human pregnancy with aid of the interaction between maternal and fetal cells

Abstract

Our previous study has demonstrated that cyclosporine A (CsA) administration in vivo induces Th2 bias at the maternal-fetal interface, leading to improved murine pregnancy outcomes. Here, we investigated how CsA treatment in vitro induced Th2 bias at the human maternal-fetal interface in early pregnancy. The cell co-culture in vitro in different combination of component cells at the maternal-fetal interface was established to investigate the regulation of CsA on cytokine production from the interaction of these cells. It was found that interferon (IFN)-γ was produced only by decidual immune cells (DICs), and not by trophoblasts or decidual stromal cells (DSCs); all these cells secreted interleukin (IL)-4, IL-10, and tumor necrosis factor (TNF)-α. Treatment with CsA completely blocked IFN-γ production in DICs and inhibited TNF-α production in all examined cells. CsA increased IL-10 and IL-4 production in trophoblasts co-cultured with DSCs and DICs although CsA treatment did not affect IL-10 or IL-4 production in any of the cells when cultured alone. These results suggest that CsA promotes Th2 bias at the maternal-fetal interface by increasing Th2-type cytokine production in trophoblasts with the aid of DSCs and DICs, while inhibiting Th1-type cytokine production in DICs and TNF-α production in all investigated cells. Our study might be useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Secretion of cytokines in component cells at the maternal-fetal interface in early human pregnancy.

Primary human first trimester trophoblasts, decidual stromal cells, or decidual immune cells were cultured in 24-well plates for 24, 48, or 72 h. The supernatants were harvested and subjected to a cytokine ELISA assay. Data represent the mean ± SE of three independent experiments with a total of 17 villi and three deciduas, performed in triplicate wells with three different samples.

Figure 2. Characterization of cells in co-culture consisting of human primary trophoblasts, DSCs and DICs of early human pregnancy.

In the co-culture, the attached trophoblasts (A) and DSCs (B) were stained and recognized by CK7 and vimentin antibodies, resulting in specific brown staining in the membrane and cytoplasm, respectively. No specific staining was seen with the isotype-control (C). Immune cells in suspension were harvested and stained with CD45-FITC (D) or the isotype control (E), and then subjected to flow cytometry. The percentages of positive cells in the indicated cells were shown. Data represent the mean ± SE of three independent experiments with 14 villi and three deciduas, performed in triplicate wells with three different samples.

Figure 3. CsA promotes Th2 bias at the maternal-fetal interface in early human pregnancy.

A: Primary human trophoblasts, DSCs, DICs, or various co-culture combinations were treated with CsA (1.0 µM) or DMSO (1/1000, v/v) for 48 h. Before harvest, the cells were treated with phorbol myristate acetate (PMA) and ionomycin for 4 h. The supernatants were harvested and subjected to ELISA. Cytokine secretion into the culture media is shown. Data represent the mean ± SE of three independent experiments with 23 villi and three deciduas, performed in triplicate wells with three different samples. B. The ratios of IL-4/TNF-α and IL-10/IFN-γ in the co-culture were obtained from the mean values in 3A, and presented in 3B.

Figure 4. Effect of CsA on intracellular cytokine production in individual component cells at the maternal-fetal interface.

Primary human trophoblasts, decidual stromal cells, decidual immune cells, or various co-culture combinations were treated with CsA (1.0 µM) or DMSO (1/1000, v/v) for 48 h. Before harvest, the cells were treated with PMA, and ionomycin, or with brefeldin A for 4 h. The cells were labeled for the surface expression of CD45 (FITC) and for intracellular cytokeratin 7, vimentin, TNF-α, IFN-γ, IL-4, and IL-10 using PE-conjugated mAbs. The intracellular production of TNF-α, IFN-γ, IL-4, and IL-10 was analyzed by flow cytometry. The percentages of positive cells among the indicated cells are shown. Data represent the mean ± SE of three independent experiments with 29 villi and three deciduas, performed in triplicate wells with three different samples. *P