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. 2015 Aug 6;7(1):81.
doi: 10.1186/s13148-015-0113-1. eCollection 2015.

A varying T cell subtype explains apparent tobacco smoking induced single CpG hypomethylation in whole blood

Mario Bauer  1 , Gunter Linsel  2 , Beate Fink  1 , Kirsten Offenberg  1 , Anne Maria Hahn  3 , Ulrich Sack  4 , Heike Knaack  4 , Markus Eszlinger  5 , Gunda Herberth  1
Affiliations

A varying T cell subtype explains apparent tobacco smoking induced single CpG hypomethylation in whole blood

Mario Bauer et al. Clin Epigenetics. .

Abstract

Background: Many recent epigenetic studies report that cigarette smoking reduces DNA methylation in whole blood at the single CpG site cg19859270 within the GPR15 gene.

Results: Within two independent cohorts, we confirmed the differentially expression of the GPR15 gene when smokers and non-smokers subjects are compared. By validating the GPR15 protein expression at the cellular level, we found that the observed decreased methylation at this site in white blood cells (WBC) of smokers is mainly caused by the high proportion of CD3+GPR15+ expressing T cells in peripheral blood. In current smokers, the percentage of GPR15+ cells among CD3+ T cells in peripheral blood is significantly higher (15.5 ± 7.2 %, mean ± standard deviation) compared to non-smokers (3.7 ± 1.6 %). Treatment of peripheral blood mononuclear cell (PBMC) cultures with aqueous cigarette smoke extract did not induce a higher proportion of this T cell subtype.

Conclusions: Our results underline that DNA hypomethylation at cg19859270 site, observed in WBCs of smokers, did not arise by direct effect of tobacco smoking compounds on methylation of DNA but rather by the enrichment of a tobacco-smoking-induced lymphocyte population in the peripheral blood.

Keywords: Blood; CpG; DNA methylation; EWAS cell composition; GPR15; Tobacco smoking.

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Figures

Fig. 1
Fig. 1
Tobacco-smoking-dependent GPR15 gene expression in human peripheral white blood cells. Implemented were blood specimens from two different cohorts (Working Place Cohort (gray box plot) and Replication Cohort (black boxplot)). The GPR15 gene expression was significantly increased in smokers (a, b) as well as formerly smokers (a), cig/d, cigarettes per day. Boxes indicate the 25 to 75 % percentile and whiskers the non-outlier range. P values from unpaired Student’s t test
Fig. 2
Fig. 2
Percentage of GPR15 protein expressing lymphocytes in blood specimens of the Replication Cohort (91 non-smokers vs. 32 smokers in total). a Representative dot plots gated on lymphocytes show GPR15 expression in CD3+ T cells and CD19+ B cells in smoker and non-smoker. Percentages represent the frequency of these cells in the lymphocyte gate. b Cumulative data of the GPR15 expressing cells as percentage of CD3+ or CD19+ lymphocytes. P values from Mann-Whitney U Test
Fig. 3
Fig. 3
GPR15 gene expression in whole blood versus frequency of GPR15 expressing T lymphocytes. Percentage of GPR15+ of CD3+ T cells (a) and of CD19+ B cells (b). Correlation coefficient from linear regression analysis
Fig. 4
Fig. 4
Methylation of CpG site cg19859270 in PBMCs and sorted CD3+GPR15- and CD3+GPR15+ cells. CpG was analyzed by pyrosequencing of PBMCs and flow cytometric-sorted cells of non-smoker (white plots, n = 6) and smokers (gray plots, n = 6). P values from Mann-Whitney U Test
Fig. 5
Fig. 5
Influence of cigarette smoke extract (CSE) on GPR15 expression in PBMCs of randomly selected non-smokers (NS, n = 3) and smokers (S, n = 3). PBMCs were exposed in vitro for 5 days
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