TGFb1 suppresses the activation of distinct dNK subpopulations in preeclampsia

Abstract

Background: Decidual natural killer (dNK) cells are the predominant lymphocytes accumulated at the maternal-fetal interface. Regulatory mechanism of dNK cells in preeclampsia, a gestational complication characterized by high blood pressure and increased proteinuria occurring after 20 weeks pregnancy, is not completely understood.

Methods: Multi-parameter flow cytometry is applied to investigate the phenotype and function of dNK cells freshly isolated from decidual samples or conditionally cultured by TGFb stimulation.

Findings: In preeclampsia, we documented elevated numbers of CD56⁺ CD3^- dNK cells in close proximity to Foxp3⁺ regulatory T (Treg) cells within the decidua. In vitro experiments using dNK cells from early gestation showed that dNK activation (IFNG, IL-8 and CD107a) can be downregulated by Treg cells. The expression of these markers by dNK cells was significantly lower in preeclampsia. We also observed a positive correlation between the expression of dNK activation receptors (NKp30 and NKG2D) and the expression of IFNG in specific dNK subsets. TGFb levels are increased in the decidua of preeclamptic pregnancies. We analyzed co-expression of activation (IFNG/IL-8/CD107a) and angiogenic (VEGF) markers in dNK cells. TGFb treatment reduced while blockade of TGFb increased co-expression of these markers.

Interpretation: Our findings suggest that elevated decidual TGFb1 supresses the activation of specific subsets of dNK which in turn contributes to the uteroplacental pathology associated with the onset of preeclampsia.

Keywords: IFN-gamma; Preeclampsia; TGF-beta; Treg cells; dNK cells.

Fig. 1

Phenotypic characteristics of decidual NK and Treg cells in preeclampsia. a) Percentage of CD56⁺CD3⁻ dNK cells and CD56 MFI in preeclampsia, preterm and normal term pregnancies. b) The expression level (%) of surface receptor NKp46, NKp44, NKp30, NKp80, 2B4 and NKG2D on dNK cells. c) Phenotypic character of decidual Treg cells by their CD4, CD25 and Foxp3 expression. d) Visual illustration of distinct sub-populations of CD45⁺ decidual lymphocytes in preeclampsia, preterm and term pregnancies using t-SNE mapping. e) Immunohistochemical staining for CD56 and Foxp3 expression in human deciduae. Spatial proximity of CD56⁺ dNK and Foxp3⁺ Treg (arrows) cells was found in deciduae of preeclampsia, preterm and normal term pregnancies. Bar = 50 μm. n = 61 (preeclampsia), 26 (preterm) and 23 (term). *, p < 0.05 when using Kruskal–Wallis test followed by Dunn's test.

Fig. 2

Functional characteristics of decidual NK in preeclampsia. a) Representative contour plots shown the function of CD56⁺CD3^- dNK cells by overlaying the expressions of IFNG, IL-8, VEGF and CD107a from preeclamptic, preterm and normal term pregnancies. Data are presented by boxplots and overlaid with mean values as blue diamond symbols. *p < 0.05 when using Kruskal–Wallis test followed by Dunn's test. b) Functional correlation between angiogenic and cytotoxic factors of dNK cells in preeclampsia, preterm and term pregnancies. n = 61 (preeclampsia), 26 (preterm) and 23 (term). The legend color shows the correlation coefficients and the corresponding colors. *p < 0.05 determined by Pearson correlation.

Fig. 3

Functional heterogeneity of dNK cells can be affected by autologous Treg cells and TGFb1. a) Decidual NK and Treg cells were isolated from early pregnancy and enriched by MACS. The purity of isolated population was assessed by flow cytometry. Representative plots of 15 independent experiments were shown. b) Function of dNK cells after co-culture with autologous Treg at ratio of 10:1 in the presence of rhIL12/15/18. *p < 0.05 when using Mann-Whitney U test. c) Correlation between angiogenic and cytotoxic factors of dNK cells with or with Treg cells. The legend color shows the correlation coefficients and the corresponding colors. *p < 0.05 determined by Pearson correlation. d) Freshly isolated dNK cells from early pregnancy were stimulated with rhTGFb1 (rhTGF) or TGF inhibitor (SB) in the presence of rhIL12/15/18. Ctrl, control group. e) The co-expression of VEGF with IFNG, IL-8 or CD107a was assessed by 2-D flow cytometry showing that TGFb blockade improved dNK function. n = 17. *, p < 0.05 when using Kruskal–Wallis test followed by Dunn's test. f) tSNE plots visualized that functional divergence of dNK cells were decreased by rhTGFb1, while improved by TGFb inhibition (SB).

Fig. 4

TGFb conditioned IFNG expression by dNK cells is associated with NK receptors. a) The expression of surface markers NKp46, NKp30 and NKG2D on dNK cells was analyzed after rhTGFb1 and SB431542 (SB) treatment. *p < 0.05 when using Kruskal–Wallis test followed by Dunn's test. b) The linear regression of IFNG expression to dNK receptor NKp46, NKp30 and NKG2D was evaluated. P values were determined by Pearson correlation. c) IFNG expression by dNK subsets was assessed according to their NKp46, NKp30 and NKG2D combinations using logic gating (i.e. positive or negative subsets). n = 9–18 per group. d) The distribution patterns of IFNG⁺ dNK clusters in response to TGFb stimulation. Three major IFNG clusters (red) can be identified by manual gating according to tSNE maps and their fold changes were calculated (e). n = 6. *p < 0.05 when using Kruskal–Wallis test followed by Dunn's test.

Fig. 5

The responsiveness of preeclamptic dNK to rhTGFb1 stimulation. a) The TGFb1protein secretion in decidual homogenates collected from preeclampsia and normal term pregnancy were measured by Bio-plex. n = 9 for each group. b) Functional changes of freshly isolated preeclamptic dNK cells stimulated by rhTGFb1 or SB431542 (SB). n = 6. c) Angiogenic function of dNK cells was changed by TGFb. Early gestation dNK cells were treated with rhTGFb1 or SB for 24 h, and then conditioned medium was collected for tube formation assay using HUtMEC. The representative images of 12 independent experiments were shown. *p < 0.05 when using Mann-Whitney U test.

Fig. 6

Immune regulation in decidual area during preeclampsia development. In the decidual zone of normal pregnancy, dNK cell mediated immune response and angiogenesis was delicately regulated by Treg cells via soluble TGFb1. However in preeclamptic decidua, higher amounts of TGFb produced by Treg cells could greatly impair the phenotypic and functional diversity of dNK subpopulations. This skewed immune response may further damage decidual angiogenesis and cause pathological pregnancy.

Supplementary Fig. 1

Gating strategy of flow cytometry analysis. a) Representative dot plots showing the gating strategy of flow cytometry analysis for dNK cells from a preeclamptic patient. Decidual samples were processed and stained as described in EXPERIMENTAL PROCEDURES. Followed with singlet events gating (SSC_PEAK/SSC_TOF and FSC_PEAK/FSC_TOF), viable CD45+ lymphocytes were identified. The CD56+CD3- dNK population and phenotype were examined according to the expression of surface marker NKp46, NKp44, NKp30, NKp80, 2B4 and NKG2D. b) Gating strategy for regulatory T (Treg) cells analysis. Viable CD45+CD3+ T cells were gated and analyzed to capture the different Treg subpopulations. The frequency of Treg cells was obtained according to their expression of CD4, CD25 and Foxp3. CD3+ T cells are back gated in black. CD4+ T cells are in green and Foxp3+ T cells in red.

Supplementary Fig. 2

Functional evaluation of dNK cells. a) Intrinsic dNK feature upon PMA stimulation. The expression of IFNG and CD107a by CD56+CD3- dNK cells isolated from preeclampsia and normal term pregnancy. Decidual leukocytes were incubated 4hr with Cell Stimulation Cocktail of phorbol 12-myristate 13-acetate (PMA) and ionomycin (eBioscience) prior to flow cytometry analysis. Data are presented by boxplots. n=8 (preeclampsia) or 9 (term). b) Different dNK subsets had distinct response to TGFb1 treatment. In response to different TGFb stimulation, the expression of IFNG by dNK cells was assessed according to their surface marker NKp46, NKp30 and NKG2D. n=9-18. *, p<0.05 using Mann-Whitney U test.

Supplementary Fig. 3

Classification of dNK changes induced by TGFb treatment. a) Principal components analysis (PCA) of NKp46, NKp30, NKG2D and IFNG expression by dNK cells under different TGFb situations. The coordination of variables was shown in the biplot. b) Variable correlation of top 3 components (PC1/2/3) obtained from PCA computation was graphed. Positive correlation was coloured in blue and negative is red.

Supplementary Fig. 4

TGFb1 treatment can retain CD56+ dNK cells. Decidual lymphocytes from early pregnancy were cultured with rhTGFb1 and SB431542 in the presence of rhIL2 (20ng/ml) and rhIL-15 (10ng/ml). After 7d cultures, cells were harvested and stained for flow cytometry analysis (a). The frequency (b) and MFI level (c) of CD56+ dNK cells were examined. *, p<0.05 when using Kruskal–Wallis test followed by Dunn's test.