Palmitic Acid Upregulates CD96 Expression to Mediate Maternal-Foetal Interface Immune Tolerance by Inhibiting Cytotoxic Activity and Promoting Adhesion Function in Human Decidual Natural Killer Cells

Abstract

Decidual natural killer cells (dNK cells) are an essential component of the immune cells present at the maternal-foetal interface during early pregnancy, and they play a vital role in various physiological processes. Abnormalities in the ratio or function of dNK cells have been linked to recurrent miscarriages. CD96 has been previously shown to regulate NK cell function in the tumour microenvironment; however, its role and mechanism at the maternal-foetal interface remains unclear. The present study aimed to investigate the immunomodulatory role of CD96 in dNK cells and its function at the maternal-foetal interface. Immunofluorescence staining and flow cytometry were used to detect the expression of cellular markers such as CD96. Furthermore, the secretory function, adhesion-function-related molecules, and cell proliferation markers of CD96+ and CD96- dNK cells were detected using flow cytometry. In addition, we performed cell culture experiments via the magnetic bead sorting of NK cells to detect changes in the expression of the aforementioned functional molecules in dNK cells after the CD96 blockade. Furthermore, we examined the functional characteristics of dNK cells after palmitic acid treatment at a concentration of 10 μM. We also examined the changes in dNK cell function when subjected to the combined effect of palmitic acid and CD96 antagonists. The results indicated that CD96, TIGIT, CD155, and CD112 were highly expressed at the maternal-foetal interface, with dNK cells predominantly expressing CD96, whereas TIGIT was mainly expressed on T cells, and CD155 and CD112 were mainly present in metaphase stromal and trophoblast cells. CD96+ dNK cells displayed low cytotoxic activity and a high adhesion phenotype, which mediated the immunosuppressive effect on dNK cells at the maternal-foetal interface. Palmitic acid upregulated CD96 expression on the surface of dNK cells in the coculture system, inhibiting dNK cell activity and increasing their adhesion molecule expression. CD96 antagonist treatment blocked the inhibitory effect of trophoblasts on dNK cells, resulting in enhanced cytokine secretion and reduced adhesion. The results of this study provide valuable insight into the immunomodulatory role of CD96 in dNK cells and its mechanism at the maternal-foetal interface, particularly in metaphase NK cells. This study sheds light on the mechanisms of immune regulation at the maternal-foetal interface and their implications for the study of recurrent miscarriages of unknown origin.

Keywords: CD96; dNK cells; immune adhesion; maternal–foetal immunology; recurrent spontaneous abortion.

Figure 1

Immunofluorescence staining of the maternal–foetal interface: (A) Immunofluorescence staining of villi in normal pregnancy and spontaneous abortion and vimentin-labelled chorionic stromal cells. (B) Immunofluorescence staining of normal endometrium, decidua of normal pregnancy, and decidua of spontaneous abortion and vimentin-labelled DSCs or ESCs. (C) Immunofluorescence staining of normal endometrium, decidua of normal pregnancy, and decidua of spontaneous abortion and NCAM1-labelled dNK cells. All fields of view were shot under 400× lenses. VIM: vimentin; SA: spontaneous abortion; NE: normal endometrium; NP: normal pregnancy. *: p < 0.05; **: p < 0.01.

Figure 2

Expression of CD96 and CD155 at the decidual interface in normal pregnancy: (A) Gating strategies for NK cells, T cells, and total immune cells. (B) Histogram of differences in the expression of CD96 and TIGIT in different cell subsets in decidual and endometrial tissues of normal pregnancy. (C) Differences in the average fluorescence intensity of CD96 expression in different subpopulations of cells. (D) Differences in the mean fluorescence intensity of TIGIT expression in different cell subpopulations. (E) Entrapment gating strategies for stromal cells in deciduous tissue and endometrial tissue. (F) Histogram of differences in the expression of CD155 and CD112 in DSCs and ESCs. (G) Statistics of the difference in the expression of CD155 and CD112 in DSCs and ESCs. DSCs: deciduous stromal cells; ESCs: endometrial stromal cells. TL: total lymphocyte; DSC: decidual stromal cell; ESC: endometrial stromal cell; MFI: mean fluorescence intensity. ns: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 3

Functional molecule expression on CD96+ dNK cells and CD96− dNK cells: (A) The population and proportion of NK cells in primary cells. (B) The expression of CD96 on NK cells circled in (A). (C) Histogram of the expression intensity of the three adhesion factors: CD54, CD62E, and CD106, as tested via flow cytometry. (D) The mean fluorescence intensity of the three adhesion factors was measured via flow cytometry, and the differences were calculated. (E) Histogram of the expression intensity of IFN-γ and granzyme B as measured via flow cytometry. (F) The mean fluorescence intensity of the two cytokines was measured via flow cytometry, and the difference was calculated. MFI: mean fluorescence intensity. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 4

After the addition of CD96 antagonists, the function of NK cells in the coculture system decreased significantly. (A) Cell fluorescence staining assay to detect the adhesion of NK cells to DSCs before and after the addition of antagonists (400× magnification). (B) Cell fluorescence staining to detect the adhesion of NK cells to HTR8 cells before and after the addition of antagonists (400× magnification). (C) Number of dNK cells adhered to the surface of DSCs. (D) Number of dNK cells adhered to the surface of HTR8. (E) Flow cytometry to detect the effect of adding antagonists on the expression of NK cell adhesion molecules. (F) Flow cytometry to detect the expression intensity histogram of three cytokines. (G) Flow cytometry was used to detect the average fluorescence intensity of the three cytokines and count their differences. MFI: mean fluorescence intensity. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 4

After the addition of CD96 antagonists, the function of NK cells in the coculture system decreased significantly. (A) Cell fluorescence staining assay to detect the adhesion of NK cells to DSCs before and after the addition of antagonists (400× magnification). (B) Cell fluorescence staining to detect the adhesion of NK cells to HTR8 cells before and after the addition of antagonists (400× magnification). (C) Number of dNK cells adhered to the surface of DSCs. (D) Number of dNK cells adhered to the surface of HTR8. (E) Flow cytometry to detect the effect of adding antagonists on the expression of NK cell adhesion molecules. (F) Flow cytometry to detect the expression intensity histogram of three cytokines. (G) Flow cytometry was used to detect the average fluorescence intensity of the three cytokines and count their differences. MFI: mean fluorescence intensity. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 5

Palmitic acid may inhibit NK cell activity through CD96 in dNK cells. (A) Effect of palmitic acid on CD155 expression in HTR8 T cells. (B) Effect of palmitic acid on CD155 expression in HTR8 cells cocultured with dNK cells. (C) Effect of palmitic acid on CD96 expression in dNK cells. (D) Effect of palmitic acid on CD96 expression in dNK cells cocultured with HTR8 cells. (E) ROS content of dNK cells under different conditions. (F) Ratio of JC-1 polymer/monomer of dNK cells under different conditions. (G) Histogram of the expression intensity of the three adhesion molecules detected via flow cytometry. (H) The mean fluorescence intensity of the three adhesion molecules was measured via flow cytometry, and the differences were calculated. (I) Histogram of the expression intensity of the three adhesion cytokines detected via flow cytometry. (J) The mean fluorescence intensity of the three cytokines were measured via flow cytometry, and the differences were calculated. PA: palmitic acid; MFI: mean fluorescence intensity. ns: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 6

Low-dose palmitic acid upregulates the surface expression of CD155 on trophoblast cells and CD96 on decidual natural killer (dNK) cells, resulting in decreased expression of cytotoxic cytokines in dNK cells. Moreover, it leads to an increase in inhibitory cytokine IL-10 and adhesion molecules ICAM1, VCAM1, and SELE. These changes signify the transition of dNK cell function towards an immune-tolerant phenotype. Additionally, there is a significant decrease in the expression of the proliferation marker Ki-67 in dNK cells. These findings suggest the potential involvement of these factors in the regulation of early-pregnancy maternal–foetal immune tolerance.