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

Abstract

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 (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) 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. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (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.

Single-cell analyses identify tobacco smoke exposure-associated, dysfunctional CD16+ CD8 T cells with high cytolytic potential in peripheral blood

“…In addition to DNA damage, smoking alters the epigenome and transcriptome of human blood leukocytes Su et al, 2016;Wan et al, 2018). In Su et al (2016), we demonstrated that many changes identified in isolated cell fractions, which correspond to major immune populations, were distinct from each other and whole blood. For example, ITGAL, which is expressed in T cells and involved in inflammation , had significantly decreased methylation in smokers' T cells but not in whole blood or isolated cell fractions.…”

“…Several genes found to be altered by at least two methods include LGALS1, ADAM8, and CLDND1, which were significantly increased in bulk RNAseq and scRNAseq data; GPR15, which was increased in the bulk RNAseq and microarray data; and NDFIP1, which was significantly decreased in the bulk RNAseq and scRNAseq data ( Figures 5A, 5B, 5D, and S5B -S5E). ITGAL, a smoking methylation biomarker (Su et al, 2016), was only found to be significantly increased by scRNAseq (logFC = 0.25, q = 1.7 x 10 -26 ), and was also elevated in NK-like subset (smokers: logFC = 0.45, q = 6.53 x 10 -29 ; nonsmokers: logFC = 0.54, q = 1.50 x 10 -20 ).…”

“…All donors were recruited with written informed consent under approved human IRB protocols (NIEHS 10-E-0063) by the NIEHS Clinical Research Unit between March 2013 to August 2017 from the Raleigh, Durham and Chapel Hill, NC area (Su et al, 2016;Wan et al, 2018). Whole blood was obtained from healthy (without acute disease according to self-reported medical histories) from nonsmokers, not having smoked >100 cigarettes in their lifetime, and smokers who reported their average daily cigarette consumption for the past 3 months.…”

DNA methylation of membrane‐bound catechol‐O‐methyltransferase in Malaysian schizophrenia patients

“…Based on our findings, there was no relation between the COMT methylation levels and smoking in schizophrenia patients. This may be due to the lack of information obtained from the patients regarding whether or not they were chronic smokers; a previous study showed that long‐term, heavy smokers produce epigenetic effects not seen in light smokers …”

Is Cellular Heterogeneity Merely a Confounder to be Removed from Epigenome-Wide Association Studies?

“…Longitudinal and Mendelian randomization analysis suggests that increased HIF3A DNA methylation is a consequence of increasing adiposity [27], maybe mediated by cell fate changes, although the association survives adjustment for gross cell changes and methylation levels predict Type 2 diabetes risk [28]. Also, the example of DNA methylation associating with smoking discussed above has also been shown to be at least partially mediated by cell fate changes caused by smoking, leading to differential cellular heterogeneity within the blood samples of smokers and non-smokers [29].…”