Antenatal Steroids and Cord Blood T-cell Glucocorticoid Receptor DNA Methylation and Exon 1 Splicing

Abstract

Antenatal administration of glucocorticoids such as betamethasone (BMZ) during the late preterm period improves neonatal respiratory outcomes. However, glucocorticoids may elicit programming effects on immune function and gene regulation. Here, we test the hypothesis that exposure to antenatal BMZ alters cord blood immune cell composition in association with altered DNA methylation and alternatively expressed Exon 1 transcripts of the glucocorticoid receptor (GR) gene in cord blood CD4⁺ T-cells. Cord blood was collected from 51 subjects in the Antenatal Late Preterm Steroids Trial: 27 BMZ, 24 placebo. Proportions of leukocytes were compared between BMZ and placebo. In CD4+ T-cells, methylation at CpG sites in the GR promoter regions and expression of GR mRNA exon 1 variants were compared between BMZ and placebo. BMZ was associated with an increase in granulocytes (51.6% vs. 44.7% p = 0.03) and a decrease in lymphocytes (36.8% vs. 43.0% p = 0.04) as a percent of the leukocyte population vs. placebo. Neither GR methylation nor exon 1 transcript levels differed between groups. BMZ is associated with altered cord blood leukocyte proportions, although no associated alterations in GR methylation were observed.

Keywords: Betamethasone; Epigenetics; Programming.

Fig. 1

Glucocorticoid receptor schematic. Black circles are methylatable CpGs in distal and proximal promoter regions assessed in this study. Solid lines indicate GR splice variants assessed in this study (1D, 1B and 1C)

Fig. 2

Sample gating strategy. A) Cell Type Enumeration. Side vs forward scatter plot showing gate around cells and excluding cell debris. Cell phenotyping by linage markers - Left to right, CD45 X side scatter for total immune cells; CD14 X side scatter - % monocytes, granulocytes, and lymphocytes; CD19 X CD3 Lymphocytes (B-cells and T-cells); CD8 X CD4 (Helper T-cells (Th) and Cytotoxic T-cells (Tc)). B) CD4+ T-cell isolation. Left to right, side vs forward scatter showing gate around cells and excluding cell debris, CD45 X side scatter for total immune cells, CD3 X CD4 for CD4⁺ T-cells.

Fig. 3

Box-plot showing CpG Methylation as average beta values (the relative quantity of methylation at an individual CpG locus ranging from 0 to 1 (unmethylated to completely methylated)) in CD4+ T-cells isolated from cord blood. Region covered includes the distal and proximal promoters through Exon 2 of the GR gene. Error bars are minimum and maximum percentage methylation, box is the upper and lower quartiles, and the line within the box is median. Grey box is BMZ (n=24), white box is placebo (n=20). No differences were observed between BMZ and placebo for % methylation in any region examined

Fig. 4

Box-plot showing levels of GR 1D, 1B, and 1C mRNA transcripts relative to total GR in cord blood CD4+ T-cells. Error bars are minimum and maximum transcript level, box is the upper and lower quartiles, and the line within the box is median. Grey box is BMZ (n=24), white box is placebo (n=23). No differences were observed between BMZ and placebo for any GR transcript levels examined