CD39 abrogates platelet-derived factors induced IL-1β expression in the human placenta

Figures

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  FIGURE 1. Placental CD39 and CD73 expression over human gestation. Expression of CD39, encoded by ENTPD1, and CD73, encoded by NT5E, was analyzed in first trimester (FT) as well as third trimester (term) placental tissue on mRNA (A–C) and protein level (D–G). CD39 is increasing towards term on gene expression (FT n = 186, mean gestational week 7.72 ± 1.7 vs. term n = 9, mean gestational week 35.48 ± 2.64) (A, C) and on protein level (FT n = 21, mean gestational week 9.27 ± 2.15 vs. term n = 5, mean gestational week 35.89 ± 2.18) (D, E, G). Gene expression (B, C) and protein levels (D, F, G) of CD73 are decreasing at term of pregnancy. Immunoblotting for placental CD39 and CD73 (D). Band density of CD39 and CD73 was normalized to GAPDH. YWHAZ was used as reference gene. Data are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0002, ****p ≤ 0.0001.

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  FIGURE 2. CD39 and CD73 are expressed in key placental cell types. Serial sections of human first trimester (A, B, GA 7 + 5) and term (D, E, GA 39 + 0) placental villous tissue were subjected to immunochistochemistry. CD39 staining (A, D) revealed positive signals on cytotrophoblasts (black arrowhead) as well as on the syncytiotrophoblast, with higher intensity towards the apical side (white arrowhead). Positive staining for CD73 (B, E) was only found in the syncytiotrophoblast. Negative control for rabbit immunoglobulin fraction gave no staining on first trimester (C) and term placental tissue (F). Scale bar represents 50 μm. CD39 and CD73 was investigated in primary term trophoblast (PT, n = 6), arterial endothelial cells (ECA, n = 3), venous endothelial cells (ECV, n = 3) and Hofbauer cells (HBC, n = 3) from healthy term pregnancies (G). CD39 and CD73 were detected in all cell types, but predominantly in ECVs. Band densities of CD39 (H) and CD73 (I) were normalized to β-actin. Data are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01.

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  FIGURE 3. CD39 is upregulated in preeclampsia. Placental villi from healthy (C, n = 12, mean gestational week 38.64 ± 0.56) and preeclamptic (PE, n = 12, mean gestational week 35.34 ± 3.70) patients were subjected either to gene expression (A, B) or immunoblot analyses (E–G). ENTPD1 (A) and NT5E (B) were significantly upregulated in preeclamptic cases compared to controls. ENTPD1 and NT5E were also analyzed from a previously published microarray dataset (Herse et al., 2012) in controls (n = 23) and PE (n = 26) cases (C, D). Immunoblot analysis of CD39 (E, F) showed a slight increase in preeclamptic cases, whereas protein analysis of CD73 (E, G) revealed no difference between C and PE. Protein levels were normalized to β-actin. YWHAZ and GAPDH were used as reference genes for gene expression analysis. Data are presented as mean ± SEM. ****p ≤ 0.0001.

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  FIGURE 4. Effect of oxygen on placental CD39 expression. Human first trimester (A–C) and term (D–F) placental explants were incubated for 24 h under indicated oxygen concentrations. Gene expression analysis of ENTPD1 in first trimester tissue (n = 3, mean gestational week 7.48 ± 0.91) revealed a trend towards higher expression upon increasing oxygen concentrations (A). Protein levels of CD39 were analyzed via immunoblot in first trimester tissue (B, C) (n = 6, mean gestational week 7.90 ± 0.78). Healthy term placental explants were analyzed after incubation on mRNA (D) as well as on protein (E, F) level (n = 5, mean gestational week 38.49 ± 0.54). Values from control samples - first trimester (incubated at 2.5% O2, A–C) and term placentas (incubated at 8% O2, D–F)—were set to 1. Band density was normalized to β-actin (B, C, E, F). YWHAZ and TBP served as reference genes for gene expression analysis (A, D). Data are presented as mean ± SEM.

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  FIGURE 5. Platelet-derived factors induce placental IL-1β expression. Placental explants from first trimester (A–D) and term (E–H) were incubated for 24 h in presence or absence of a platelet releasate (PR; pool of 10 healthy pregnant women at term). First trimester explants were incubated at 2.5% O2 (A–D), whereas placental villi from third trimester were incubated at 8% O2 (E–H). After incubation with platelet releasate, placental villi from first trimester (A, B, n = 9; mean gestational week 7.66 ± 0.88) and term (E, F, n = 5; mean gestational week 38.49 ± 0.54) were subjected to gene expression analysis for ENTPD1 and IL-1β. Immunoblot analysis revealed a trend towards higher levels of CD39 upon platelet releasate treatment in early (C, D; n = 6; mean gestational week 7.90 ± 0.78) and late (G, H; n = 5; mean gestational week 38.49 ± 0.54) cases. Band density was normalized to β-actin (C, D, G, H). YWHAZ and TBP served as reference genes for gene expression analysis (A, B, E, F). Data are presented as mean ± SEM. ***p ≤ 0.0002.

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  FIGURE 6. Platelet releasate-induced IL-1β upregulation is abrogated by CD39 overexpression. BeWo cells were transfected with pCD39 or an empty vector for 24 h and were afterwards subjected to qPCR and protein analysis (A–D). Gene expression analysis of ENTPD1 revealed a strong upregulation in pCD39-transfected samples (A). NT5E was not significantly affected by CD39 overexpression (B). Immunoblot analysis confirmed CD39 overexpression in BeWo cells (C, D). Band density was normalized to GAPDH (C, D). Extracellular ATP levels in CD39 overexpressing and control BeWo cell cultures were measured in presence of platelet releasate (PR; pool of 10 healthy pregnant women at term) for 5 h via luminescence measurement (E). BeWo cells were incubated with platelet releasate for 24 h and subsequently subjected to qPCR of endogenous ENTPD1 (F). CD39 was overexpressed in BeWo cells for 24 h and cells afterwards incubated in presence or absence of platelet releasate for another 24 h. Cells were subjected to gene expression analysis for IL-1β (G). For gene expression analysis YWHAZ and GAPDH were used as reference genes. Data are presented as means ± SEM from three (B–D), four (E), and five (A, F, G)…