Interleukin-17-producing decidual CD4+ T cells are not deleterious for human pregnancy when they also produce interleukin-4

Abstract

Background: Trophoblast expressing paternal HLA-C antigens resemble a semiallograft, and could be rejected by maternal CD4+ T lymphocytes. We examined the possible role in human pregnancy of Th17 cells, known to be involved in allograft rejection and reported for this reason to be responsible for miscarriages. We also studied Th17/Th1 and Th17/Th2 cells never investigated before. We defined for the first time the role of different Th17 subpopulations at the embryo implantation site and the role of HLA-G5, produced by the trophoblast/embryo, on Th17 cell differentiation.

Methods: Cytokine production by CD4+ purified T cell and T clones from decidua of normal pregnancy, unexplained recurrent abortion, and ectopic pregnancy at both embryo implantation site and distant from that site were analyzed for protein and mRNA production. Antigen-specific T cell lines were derived in the presence and in the absence of HLA-G5.

Results: We found an associated spontaneous production of IL-17A, IL-17F and IL-4 along with expression of CD161, CCR8 and CCR4 (Th2- and Th17-type markers) in fresh decidua CD4+ T cells during successful pregnancy. There was a prevalence of Th17/Th2 cells (producing IL-17A, IL-17F, IL-22 and IL-4) in the decidua of successful pregnancy, but the exclusive presence of Th17 (producing IL-17A, IL-17F, IL-22) and Th17/Th1 (producing IL-17A, IL-17F, IL-22 and IFN-γ) cells was found in the decidua of unexplained recurrent abortion. More importantly, we observed that Th17/Th2 cells were exclusively present at the embryo implantation site during tubal ectopic pregnancy, and that IL-4, GATA-3, IL-17A, ROR-C mRNA levels increased in tubal biopsies taken from embryo implantation sites, whereas Th17, Th17/Th1 and Th1 cells are exclusively present apart from implantation sites. Moreover, soluble HLA-G5 mediates the development of Th17/Th2 cells by increasing IL-4, IL-17A and IL-17F protein and mRNA production of CD4+ T helper cells.

Conclusion: No pathogenic role of decidual Th17 cells during pregnancy was observed. Indeed, a beneficial role for these cells was observed when they also produced IL-4. HLA-G5 could be the key feature of the uterine microenvironment responsible for the development of Th17/Th2 cells, which seem to be crucial for successful embryo implantation.

Keywords: Ectopic pregnancy; IL-17; IL-4; Pregnancy; Spontaneous abortion; Th17.

Fig. 1

Associated spontaneous production of both IL-17A and IL-4 and expression of CD161, CCR8 and CCR4 by fresh decidua CD3+CD4+ T cells in successful pregnancy. a Unstimulated decidual and PB CD4+ T cells purified from the same 9 pregnant women were cultured for 24 h and IL-4, IL-17A, IL-17F and IL-22 production (mean ± SEM) was measured. b RT-PCR of unstimulated decidual and PB CD4+ T cells purified from the same 3 pregnant women for IL-17A, IL-4 and RORC was performed. c, d Th1 cells express CXCR3, whereas Th2 cells express CCR4, CCR8 and CRTH2 and Th17 cells express CD161, IL-23 receptor (IL-23R) CCR6 and CCR4. The associated expression of molecules expressed by Th17 cells and by Th1 and Th2 cells in fresh unstimulated CD4+ T cells purified from decidua and PB of the same pregnant women were analyzed by 4-color (CD3, CD4, CD161 and CCR4 or CCR8 or CXCR3) flow cytometry. c is a representative experiment and d data are represented as mean ± SEM. For a, b and d the statistical analysis was performed with Wilcoxon test

Fig. 2

Significant increase of both IL-4 and IL-17A production by CD4+ T cell clones in decidua compared to PB in successful pregnancy. 172 and 55 CD4+ T cell clones were respectively generated from decidual biopsies and peripheral blood obtained from 4 pregnant women who underwent an elective termination of pregnancy. IL-4, IL-17A, IL-17F, IL-22, and IFN-γ (mean ± SEM) were measured in the supernatant by multiplex bead-based assay. The statistical analysis was performed with Wilcoxon test

Fig. 3

Prevalence of Th17/Th2 cells in the decidua of successful pregnancy but of Th17 and Th17/Th1 cells in the decidua of unexplained recurrent abortion. a Analysis of the percentages of Th1-, Th2-, Th0-, Th17-cells and of Th17/Th1, Th17/Th2 and Th17/Th0 in CD4+ T cell clones derived from the decidua of four women with normal pregnancy, and from the decidua obtained from four women suffering from unexplained recurrent abortion. Data are represented as mean ± SEM. The statistical analysis was performed with Chi-square test. b 54 and 103 CD4+ T cell clones were generated respectively from the decidua of 4 women with normal pregnancy, and from the decidua obtained from 4 women suffering from unexplained recurrent abortion. IL-17A, IL-17F and IL-22 (mean ± SEM) were measured in the supernatant by multiplex bead-based assay. The statistical analysis was performed with Wilcoxon test

Fig. 4

Th17/Th2 cells exclusively found at implantation site of ectopic pregnancy. a Percentage of Th1-, Th2-, Th0- and Th17-cells, and of Th17/Th1 and Th17/Th2 and Th17/Th0 among the CD4+ T cell clones derived from the implantation site of embryo (N = 133) and those distant from the implantation site in the same fallopian tube (N = 62) of 3 women suffering from ectopic pregnancy. Data are represented as mean ± SEM. b Levels of IL-4, IL-17A, IL-17F and IL-22 produced by the CD4+ T cell clones at the implantation site (N = 133) and distant from the implantation site (N = 62). Data are represented as mean ± SEM. c mRNA levels of IL-4, GATA-3, IL-17A, ROR-C and IFN-γ in the biopsies taken at embryo implantation site and distant from the implantation site in the fallopian tube of three women suffering from ectopic pregnancy. For a, c the statistical analysis was performed with Chi-square test. For b the statistical analysis was performed with Wilcoxon test

Fig. 5

Soluble HLA-G5 mediates the development of Th17/Th2 cells by increasing IL-4 and IL-17A production by the CD4+ T helper cells. a IL-4, IL-17A, IL-17F and IFN-γ (mean ± SEM) were measured by bead-based assays in the supernatants of SK-specific T cell lines derived in the presence and in the absence of HLA-G5. b mRNA levels (mean ± SEM) of IL-4, IL-17A, IL-17F, IL-23R and IFN-γ were measured by RT-PCR in SK-specific T cell lines derived in the presence and in the absence of HLA-G5. c CD4+ T cell clones were derived from the 4 SK-specific T cell lines derived in the presence of HLA-G5 (N = 87) and from the 4 SK-specific T cell lines derived in the absence of HLA-G5 (N = 68) of 4 donors and the levels of IFN-γ, IL-4, IL-22, IL-17A and IL-17F produced by the T cell clones were measured. The percentages of Th17/Th1 and Th17/Th2 and Th17/Th0 and pure Th17 CD4+ T cell clones derived from the SK-TCL modulated in presence or absence of HLA-G5 were analysed. Data are represented as mean ± SEM. d The levels of IL-4, IL-17A, IL-17F and IL-22 produced by the Th17/Th2 T cell clones in the SK-specific T cell lines derived in the absence and in the presence of HLA-G5 were measured by bead based assays. Data are represented as mean ± SEM. For a, b and d the statistical analysis was performed with Wilcoxon test. For c the statistical analysis was performed with Chi-square test