Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes

<div><p>Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking–associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (<i>p</i> <1.2x10^-7, Bonferroni correction). Several CpGs (<i>MTSS1</i>, <i>NKX6-2</i>, <i>BTG2</i>) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. <i>AHRR</i> (cg05575921), <i>ALPPL2</i> (cg21566642), <i>GFI1</i> (cg09935388), <i>IER3</i> (cg06126421) and <i>F2RL3</i> (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast <i>GPR15</i> (cg19859270) was highly significant in T and B cells and <i>ITGAL</i> (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.</p></div

Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes - Fig 5

<p>Comparison of smoking-associated CpG methylation (A-C) and gene expression (D-F) differences between cell lineages. Methylation levels were measured on 450K arrays and averages for nonsmokers and smokers are displayed (A) cg05575921 (<i>AHRR</i>), (B) cg19859270 (<i>GPR15</i>) and (C) cg03636183 (<i>F2RL3</i>). Gene expression level (D) <i>AHRR</i>, (E) <i>GPR15</i> and (F) <i>F2RL3</i> for each cell type was measured by RT-PCR and averages are shown based on smoking status. Expression is represented by fold change difference between smokers relative to the average of nonsmokers after normalization to β-actin. Bar = Mean ± Standard Error; *<i>p</i>≤0.05, **<i>p</i>≤0.001, Student’s t-test.</p

Characteristics of whole blood DNA methylation study participants.

<p>Characteristics of whole blood DNA methylation study participants.</p

RRBS demonstrates regional impact and lineage-specific smoking DMRs within <i>MYO1G</i> and <i>ITGAL</i>.

<p>(A) Increased levels of CpG methylation were observed in <i>MYO1G</i> across a ~500-bp region with greater effects in myeloid cell types. (B) At cg22132788 the pattern of change across cell types closely matches 450K array results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166486#pone.0166486.s003" target="_blank">S3C Fig</a>). (C) RRBS reveal hypomethylation in B cells that was not detected in 450K results. (D) T cell subsets show loss of methylation consistent with 450K array results.</p

GREAT Analysis of 738 Smoking Associated CpGs.

<p>GREAT Analysis of 738 Smoking Associated CpGs.</p

Clustered significant CpGs based on methylation effect size and significance in individual cell types.

<p>(A) Larger smoking effect sizes in myeloid cells (monocytes and granulocytes, green boxes) drive methylation effect size in whole blood (column one). (B) p-values for (A) show that significant CpG changes in whole blood are not always observed in PBMC (column two), although GPR15 is an exception to this (blue box). (C) Clustering CpGs by similarity of p-value patterns among cell types reveals cell-type specific smoking-associated CpGs (grey boxes). Most of these are not significant in PBMC or whole blood.</p

Distribution of major leukocyte cell types in whole blood.

<p>Distribution of major leukocyte cell types in whole blood.</p

Effects of current smoking on <i>AHRR</i> cg05575921 methylation among monocytes, granulocytes, B cells and T cells.

<p>(A) Log2-transformed cotinine levels (ng/ml) were regressed with methylation beta-value measured for cg05575921 in each blood cell type. (B) Strong concordance of DNA methylation at cg05575921 (<i>AHRR</i>) between CD14+ monocytes and CD15+ granulocytes within individual subjects.</p