Effect of Granzyme K, FasL and Interferon-γ Expression in Placentas with Preeclampsia

Abstract

This study aimed to investigate the cytotoxic activity of decidual lymphocytes and the mRNA/protein expression of cytotoxic proteins in various cell types in the context of preeclampsia (PE) compared to those of healthy pregnancies. We analyzed fresh decidua basalis tissue and tissue embedded in paraffin (FFPE) from PE pregnancies (n = 15) and compared them with those of healthy pregnancies (n = 15) of the corresponding gestational age. Using double immunofluorescence staining, we observed differences in the intensity and distribution of staining for granzyme K (GZMK) and FasL in extravillous trophoblasts. RT-qPCR analysis of FFPE placental tissue showed that GZMK mRNA expression was statistically higher (p < 0.0001) in PE compared to that of healthy controls. On the contrary, there was a low expression (p < 0.001) of FasL mRNA in PE compared to controls, while there was no statistically significant difference for IFN-γ mRNA between PE and controls. Although the level of cytotoxic activity changed depending on the ratio of effector and target cells, there was no significant difference observed between PE and controls in this in vitro study. In conclusion, in PE, extravillous trophoblasts exhibited increased expression of GZMK and decreased expression of FasL. These changes may contribute to impaired trophoblast invasion. However, these alterations did not appear to affect the cytotoxic properties of decidual lymphocytes. Additionally, the possibility of cell sorter separation of decidual lymphocytes would greatly contribute to a better understanding of single cells' genetic profiles.

Keywords: FasL; granzyme K; interferon-γ; preeclampsia; trophoblast markers.

Figure 1

The histological appearance in PE placenta showed (A) increased perivillous and intravillous fibrin deposits, hyalinization change, thickened fetal blood capillaries, increased syncytial knots and (B) calcification. Extravillous trophoblasts (arrows) and villous trophoblasts (arrowheads). H&E stain. Scale bar—25 μm.

Figure 2

Double immunofluorescence of GZMK and CD8 (a,b), GZMK and CD56 (c) in PE and healthy control placenta (d). GZMK-positive cells in extravillous trophoblasts (arrows) and in villous trophoblasts (arrowhead). Scatter CD8 + T cells and CD56-positive cells (NK cells) can be seen in the decidua basalis in PE (*), while abundant CD8+ T cells can be seen in the decidua basalis in healthy control placenta (**). DAPI—nuclear stain; merged—all images in the row. Scale bar—100 μm (a,c,d) and 25 μm (b).

Figure 3

Double immunofluorescence of FasL and CD8 (a) as well as FasL and CD56 (b) in PE, FasL and CD8 in healthy control placenta (c) and IFN-γ and CD56 (d) in PE. FasL- and IFN-γ-positive cells in extravillous trophoblasts (arrows) and FasL in villous trophoblasts (arrowhead). Scatter CD8 + T cells and CD56-positive cells (NK cells) can be seen in the decidua basalis in PE (*), while abundant CD8 + T cells can be seen in the decidua basalis of healthy control placenta (**). DAPI—nuclear stain; merged—all images in the row. Scale bar—100 μm (a,d) and 25 μm (b,c).

Figure 4

RT-qPCR mRNA fold change of GZMK (a), FasL (b) and IFN-γ (c) from FFPE placental tissue of PE compared to healthy controls. The data were presented as mean ± SD. Mann–Whitney test was used for GZMK and IFN-γ and T-test for FasL; * < 0.01 and *** < 0.0001.

Figure 5

Flow cytometry measurement of cytotoxicity in control samples including only target cells K562 (RED R1) (a) or effector cells (GREEN R2) (b) using staining with PE-conjugated anti-CD45 and side scatter profiles to identify different cell populations and uptake of 7-AAD to detect cell death (M2). Effector (R2) and target cells (R1) were incubated overnight in mixed cultures according to a ratio of 12.5:1 (c) and a mixed culture ratio of 50:1 (d), which also display conjugates between targets and the effector, as identified by (R3). The percentage of dead cells in region R1 are calculated from a histogram showing 7-AAD uptake as measured by the intensity of red fluorescence in M2.

Figure 6

Cytotoxicity of decidual lymphocytes determined from R1 at E/T (effector/target) ratios of 12.5:1 and 50:1.