Smoking effect on DNA methylation in human blood cell types

Tobacco smoking alters DNA methylation profiles of immune cells which may underpin some of the pathogenesis of smoking-associated diseases. However, approaches linking smoking-driven epigenetic effects in specific immune cell types with disease risk are limited. We isolated six leukocyte subtypes, CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells, from whole blood of healthy adult smokers and nonsmokers for epigenome-wide association study (EWAS). As part of the Epigenetic Biomarkers of Tobacco Smoke Exposure project, a group of healthy volunteers (68 smokers and 74 nonsmokers) was recruited at the NIEHS Clinical Research Unit (protocol 10-E-0063) between March 2013 and January 2018 from the Raleigh, Durham and Chapel Hill, NC area. All nonsmokers were self-reported as not having smoked >100 cigarettes in their lifetime. Smokers reported their average daily cigarette consumption for the past 3 months [Su D et al. 2016]. For each individual, granulocytes were isolated directly from whole blood using CD15 Dynabeads (Invitrogen, Waltham, MA), magnetic beads covalently coupled with an anti-human CD15 antibody, according to the manufacturer’s protocol. Additionally, whole blood was layered on Histopaque-1077 Ficoll medium in Accuspin™ Tubes (Sigma-Aldrich, St. Louis, MO) and density gradient centrifugation was performed to isolate the mononuclear layer. Peripheral blood mononuclear cells (PBMCs) were then counted for viability and incubated with CD34 MicroBeads magnetic beads (Miltenyi Biotec, San Jose, CA). CD34 negative cells were collected in the flowthrough, recounted on a Cellometer Auto T4 Bright Field Cell Counter (Nexcelom, St. Lawrence, MA), and incubated with CD14 Dynabeads to isolate CD14+ monocytes according to the manufacturer’s protocol. Flowthrough (CD14-) cells were again counted and split by volume. One half of the cells were incubated with CD19 PanB Dynabeads for isolation of CD19+ B cells. The other flowthrough half was split in two, with half incubating with CD4 Dynabeads (for CD4+ T cells) and half incubating in CD8 Dynabeads (for CD8+ T cells). All flowthrough (CD19-, CD4-, or CD8- cells) was then combined and incubated with CD56 MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol to isolate CD56+ NK cells.