Associated production of IL-17 and IL-4 and expression of Th17 and Th2-type molecules by decidual CD3+CD4+ T cells in successful pregnancy
Unstimulated decidual and peripheral blood CD4+CD3+ 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 was measured in the corresponding cell culture supernatants (Fig. 1a). A significant increase of IL-4 and IL-17A release (p = 0.028 and p = 0.027, respectively) was observed in the culture supernatants of freshly isolated, unstimulated in vitro decidual CD4+ T cells compared to those of peripheral blood CD4+ cells from the same pregnant women (Fig. 1a). By contrast, no significant increase of IL-17F and IL-22 production was detected in the same culture supernatants of decidual CD4+ T cells compared to those of peripheral blood CD4+ cells from the same pregnant women. These data were confirmed by the increased levels of mRNA for IL-4, IL-17A and RORC (transcriptional factor of Th17 cells) expressed by freshly isolated, unstimulated decidual CD4+ T cells compared to those of peripheral blood CD4+ cells from the same 3 pregnant women (Fig. 1b). These results show a spontaneous, associated production of both IL-17A and IL-4 by fresh decidual CD3+CD4+ T cells during normal pregnancy.
Th1 cells express CXCR3, whereas Th2 cells express CCR4, CCR8 and CRTH2 [41–43]. Recent reports [10, 12] suggest that Th17 cells express CD161, IL-23 receptor (IL-23R) CCR6 and CCR4, as Th2 cells, but not CXCR3 in human adult peripheral blood or CCR6 in human decidua. We compared the associated expression of molecules expressed by Th17 cells and molecules expressed by Th1 and Th2 cells by fresh unstimulated CD3+CD4+ T cells purified from decidua and peripheral blood of the same pregnant women (Fig. 1c, d). Using multicolor flow cytometry analysis, we found that the percentage of CD3+CD4+ cells expressing CD161 (p = 0.027) and the mean fluorescence intensity (MFI) of CD161 were increased in the decidua compared to peripheral blood of the same 8 pregnant women (Fig. 1c). More importantly, this increased expression of CD161 by decidua CD3+CD4+ cells is associated with the increased expression of CCR4 and CCR8 by decidual CD3 + CD4+ cells compared to the peripheral blood CD3+CD4+ cells (Fig. 1d). These results indicate that decidual CD4+ T cells spontaneously expressed on their cell membrane molecules that characterize Th17 and Th2 cells.
Increased IL-4, IL-17A, IL-17F and IL-22 production by decidual CD4+ T cell clones in successful pregnancy
172 and 55 CD4+ T cell clones were respectively generated from decidual biopsies and peripheral blood obtained from 4 pregnant women (with normal pregnancy) who voluntarily underwent an elective termination of pregnancy. IL-4, IL-17A, IL-17F, IL-22 and IFN-γ were measured in the supernatant of the CD4+ T cell clones by multiplex bead-based assay.
In normal pregnancy, decidua CD4+ T cell clones produce higher levels of IL-4 (p = 0.0000004), a Th2-type cytokine, IL-17A (p = 0.015), IL-17F (p = 0.023) and IL-22 (p = 0.006), three Th17-type cytokines, compared to peripheral blood T cell clones (Fig. 2). By contrast, IFN-γ production by T cell clones was decreased (p = 0.0001) in the decidua compared to peripheral blood (Fig. 2).
These data confirm the associated production of IL-4, IL-17A, IL-17F and IL-22 by decidual CD4+ T cells in normal pregnancy, thus showing an association between Th2-and Th17-type cytokines by decidual CD4+ T cells in normal pregnancy.
Prevalence of Th17/Th2 CD4+ cells in the decidua of successful pregnancy whereas of Th17 and Th17/Th1 cells in the decidua of unexplained recurrent abortion
It has been reported that in “inevitable” spontaneous abortion with genital bleeding and in unexplained spontaneous abortion, the number of decidual Th17 cells increased compared to normal pregnancy [31, 33]. Previously, we showed that in normal pregnancy both IL-17A, IL-17F and IL-22 are produced by decidua CD4+ T helper cells in association with IL-4. However, these previous experiments did not show whether the Th17-type cytokines (IL-17A, IL-17F and IL-22) and the Th2-type cytokine, IL-4, are produced by two different decidual CD4+ T cell subsets or if the same CD4+ T cell simultaneously produces the Th17-type and the Th2-type cytokines. In fact, part of human IL-17A-producing cells were found to also produce IL-4 (these cells were named Th17/Th2 [35] and other cells together with IL-17 can produce interferon (IFN)-γ (these cells were named Th17/Th1 [6]. To investigate the possibility that in normal pregnancy the same CD4+ cell subset can produce IL-17 and IL-4, we analyzed not only the percentages of Th1-, Th2-, Th0- and Th17-cells, but also the percentages of Th17/Th1 (producing IL-17A,IL-17F, IL-22 and IFN-γ), Th17/Th2 (producing IL-17A, IL-17F, IL-22 and IL-4) and Th17/Th0 (producing IL-17A, IL-17F, IL-22, IL-4 and IFN-γ). CD4+ T cell clones were derived from the decidua of 4 women with normal pregnancies, who underwent an elective termination of pregnancy, and from the decidua obtained from 4 women suffering from unexplained recurrent abortion (Fig. 3). We found that 26 % of the whole CD4+ T cell clones generated from normal pregnancy produce IL-17 (54/208 T cell clones), whereas 59 % of the whole CD4+ T cell clones generated from unexplained recurrent abortion produce IL-17 (103/174 T cell clones). Thus, according to what was reported by Wang [31] and Nakashima [33], it seems that the percentage of the whole IL-17-producing T cells, without any Th17-type subpopulations analysis, is higher in unexplained recurrent abortion compared to normal pregnancy (p = 0.000001). We found that all the Th17, Th17/Th2 and Th17/Th1 T cell clones produced IL-22. There is no significant difference between the percentage of Th1, Th0 and Th17/Th0 CD4+ T cell clones generated from the decidua of normal pregnancy and those generated from spontaneous abortion (Fig. 3a). In contrast, the percentage of “proper” decidual Th17 (producing only IL-17A, IL-17F and IL-22) (p = 0.000001) and decidual Th17/Th1 (producing IFN-γ plus IL-17A, IL-17F and IL-22) T cell clones (p = 0.00001) were significantly higher in unexplained recurrent abortion compared to normal pregnancy (Fig. 3a). Noteworthy is the observation that no “proper” Th17 and Th17/Th1 T cell clones were ever detected in normal pregnancy decidua. However, the percentages of Th2 (p = 0.00001) and Th17/Th2 (producing IL-4 plus IL-17A, IL-17F and IL-22) (p = 0.001) T cell clones were significantly higher in the decidua of normal pregnancy compared to decidua of women suffering from unexplained recurrent abortion (Fig. 3a).
Measuring the levels of IL-17A and IL-17F produced by the whole IL-17-producing CD4+ T cell clones obtained from normal pregnancy (54 T cell clones) and by the whole IL-17-producing CD4+ T cell clones obtained from unexplained recurrent abortion (103 T cell clones), we did not find a significant increase in IL-17A and IL-17F production by CD4+ T cell clones generated from the decidua from unexplained recurrent abortion compared to the IL-17A and IL-17F production by CD4+ T cell clones generated from the decidua of successful pregnancy (Fig. 3b). Similarly we did not find a significant increase in IL-17A and IL-17F production by CD4+ T cell clones generated from peripheral blood of successful pregnancy and recurrent spontaneous abortions (Additional file 1). These results also indicate that spontaneous recurrent abortions are not necessarily associated with increased levels of IL-17A and IL-17F produced by CD4+ T cells. Moreover, we measured the levels of IL-17 produced by Th17/Th2 T cell clones in normal pregnancy (18 % of T cell clones) and unexplained recurrent abortion (4.5 % of T cell clones), and we found that the levels of IL-17A produced by Th17/Th2 cells in successful pregnancy is higher (9507 ± 7000 pg/ml) compared to the levels of IL-17A produced by Th17/Th2 cells in unexplained recurrent abortion (137 ± 80 pg/ml) (p = 0.0001). These results confirmed that high IL-17 production is not associated with spontaneous recurrent abortion.
We demonstrated that IL-17A, IL-17F, IL-22, together with IL-4, were produced by the same decidual CD4+ T cell subpopulation (the Th17/Th2 cells) in normal pregnancy and not by two different CD4+ T cell subsets. Thus, IL-17 production by decidual CD4+ T cells does not seem to be associated with spontaneous abortion or unexplained spontaneous abortion, as was reported [31, 33]. IL-17 produced by decidual CD4+ T cells, if associated with IL-4 production, is not deleterious for pregnancy outcome.
Th17/Th2 CD4+ T cells are exclusively present at the implantation site of ectopic pregnancy
Decidual Th17/Th2 cells seem to be important for normal pregnancy development. We wondered whether these cells were present at the implantation site of the embryo and thus could have an important role for embryo implantation. To answer this question, we performed the same kind of cytokine analysis in ectopic tubal pregnancies.
We evaluated not only the percentage of Th1-, Th2-, Th0- and Th17-cells, but also the percentages of Th17/Th1 (producing IL-17A, IL-17F, IL-22 and IFN-γ), Th17/Th2 (producing IL-17A,IL-17F, IL-22 and IL-4) and Th17/Th0 (producing IL-17A, IL-17F, IL-22, IL-4 and IFN-γ) among the CD4+ T cell clones derived from the implantation site of the embryo (N = 133) and those distant from the implantation site in the same fallopian tube (N = 62) of 3 women suffering from ectopic pregnancy.
There is no significant difference in the percentage of pure Th2 and pure Th0 CD4+ T cell clones generated from the implantation site and distant from the implantation site (Fig. 4a). At the implantation site the percentage of Th17/Th2 (p = 0.000001) and of Th17/Th0 (p = 0.000001) CD4+ T cell clones is higher than those clones distant from the implantation site. Conversely, the percentage of Th1(p = 0.00001), pure Th17 (p = 0.00001) and Th17/Th1 (p = 0.000001) is higher apart from the implantation site compared to the embryo implantation site where these 3 types of CD4+ T cells are not present (Fig. 4a). Thus, Th17/Th1 cells are present only outside the implantation site or, as seen above, in decidua of recurrent spontaneous abortions. In other words, it seems that Th17/Th1 and pure Th17 cells, together with Th1 cells, are observed at locations where no implantation of the embryo occurs or when the implantation failed or is not maintained. By contrast, Th17/Th2 are prevalent in normal pregnancy and exclusively present at the implantation site of the embryo.
The levels of IL-4 (p = 0.00000001), IL-17A (p = 0.003), IL-17F (p = 0.00001) and IL-22 (p = 0.00002), produced by the CD4+ T cell clones at the implantation site are higher than the levels of these cytokines distant from the implantation site (Fig. 4b), indicating that there is an increase of production of Th2-type and Th17-type cytokines at the implantation site.
We confirmed these results by determining mRNA expression in Fallopian tube tissue taken at the embryo implantation site and tissue sampled distant from the implantation site of 3 women suffering from ectopic pregnancy (Fig. 4c). At the implantation site, the levels of mRNA for Th2-type molecules (IL-4 and GATA3) and for Th17-type molecules (IL-17A and RORC) were increased compared to the mRNA levels for these molecules distant from the implantation site. In contrast, distant from the implantation site mRNA production of IFN-γ is increased compared to those expressed at the embryo implantation site (Fig. 4c).
Soluble HLA-G5 mediates the development of Th17/Th2 cells by increasing IL-4 and IL-17A production of the CD4+ T helper cells
We can speculate about the origin of the factor(s) present in the uterine microenvironment that is able to induce Th17/Th2 cells by increasing the production of both IL-4 and IL-17A of the CD4+ T helper cells. Years ago, we reported that progesterone could induce Th2 responses [44]. Recently it has been shown that progesterone inhibits IL-17 production [45]. Thus, progesterone cannot be the factor responsible for Th17/Th2 cell development. Very recently, we showed that soluble HLA-G5 induces an increased production of IL-4 by CD4+ T helper cells [40]. We wondered if HLA-G5 could also induce IL-17A production by the CD4+ T cells and could be the factor responsible for the development of Th17/Th2 cells at the implantation site in normal pregnancy.
To investigate the possible influence of HLA-G5 on IL-4 and IL-17 production of antigen-specific T cells, we generated streptokinase (SK)-specific T cell lines (TCL) from 5 donors cultured in the absence or presence of HLA-G5. As a control, peripheral blood mononuclear cells from the same donors were stimulated with SK in the presence of IL-4, a powerful inducer of Th2 differentiation [46] and IL-12, a potent inducer of Th1 differentiation [47], which indicate that SK-specific TCL are modulated in our culture conditions (data not shown). When we measured the cytokines present in the supernatants of SK-specific T cell lines, we found a significant increase of IL-4 secretion (p = 0.0001) by the SK-specific T cell lines in response to IL-4, and a significant increase of IFN-γ (p = 0.006) in response to IL-12 (data not shown). This suggests that the culture conditions were satisfactory for the modulation of the T cell line cytokine profile.
A statistically significant increase of IL-4 (p = 0.0002), IL-17A (p = 0.005) and IL-17F (p = 0.028) was observed with the SK-specific T cell lines generated in the presence of HLA-G5 1 µg/ml (Fig. 5a). In contrast, IFN-γ production in response to HLA-G5 was not statistically significant (Fig. 5a). We then analyzed the cytokine mRNA levels of SK-specific T cell lines by RT-PCR (Fig. 5b). A statistically significant increase of IL-4 (p = 0.042), IL-17A (p = 0.043), IL-17F (p = 0.042) and IL-23R (receptor expressed by Th17 cells) (p = 0.043) mRNA expression was observed with the SK-specific T cell lines generated in the presence of HLA-G5 compared to the SK-specific T cell lines generated in the absence of HLA-G5 (Fig. 5b). By contrast, no significant differences were observed for IFN-γ mRNA expression between the T cell lines generated in the presence or in the absence of HLA-G5 (Fig. 5b). These findings confirmed the results obtained at the protein level.
We derived CD4+ T cell clones from each of the SK-specific T cell lines of 4 donors (68 T cell clones in the absence of HLA-G5 and 87 T cell clones in the presence of HLA-G5). Subsequent analysis of their ability to produce IFN-γ, IL-4, IL-22, IL-17A and IL-17F (Fig. 5c) was performed. We analyzed the percentages of Th17/Th1 (producing IL-17A, IL-17F, IL-22 and IFN-γ) and Th17/Th2 (producing IL-17A, IL-17F, IL-22 and IL-4) and Th17/Th0 (producing IL-17A, IL-17F, IL-22, IL-4 and IFN-γ) and pure Th17 CD4+ T cell clones derived from the SK-TCL modulated in the presence or absence of HLA-G5 (Fig. 5c, d).
The percentage of Th17/Th2 CD4+ T cell clones increases in the presence of HLA-G5 (p = 0.000001) (Fig. 5c). However, the percentage of Th17/Th1 (p = 0.005) and Th17/Th0 (p = 0.05) T cell clones is decreased in the presence of HLA-G5, but there is no difference in the percentage of Th17 clones in the absence or in the presence of HLA-G5 (Fig. 5c).
The small percentage of Th17/Th2 T cell clones in the absence of HLA-G5 (Fig. 5c), prompted us to analyze the levels of Th2- and Th17-type cytokines produced by the Th17/Th2 T cell clones in the absence and in the presence of HLA-G5. We measured the cytokines present in the supernatants of CD4+ T cell clones derived from the SK-specific T cell lines (Fig. 5d). We found a significant increase in the secretion of IL-4 (p = 0.04) and IL-17A (p = 0.016) by the Th2/Th17 T cell clones derived from SK-TCL generated in the presence to HLA-G 5, compared to Th2/Th17 clones derived from SK-TCL without HLA-G5. By comparison, the levels of IL-17F and IL-22 were not significantly modified (Fig. 5d).
The above findings indicate that HLA-G5 increases the production of both IL-17 and IL-4 by antigen-specific T cells in response to HLA-G5, thus upregulating the development of Th17/Th2 cells found at the site of embryo implantation (Additional file 1: Figure S1).