吸烟可诱导肺泡灌洗液细胞中真 DNA 甲基化、羟甲基化和基因表达的改变。

BACKGROUND: While smoking is known to associate with development of multiple diseases, the underlying mechanisms are still poorly understood. Tobacco smoking can modify the chemical integrity of DNA leading to changes in transcriptional activity, partly through an altered epigenetic state. We aimed to investigate the impact of smoking on lung cells collected from bronchoalveolar lavage (BAL). METHODS: We profiled changes in DNA methylation (5mC) and its oxidised form hydroxymethylation (5hmC) using conventional bisulphite (BS) treatment and oxidative bisulphite treatment with Illumina Infinium MethylationEPIC BeadChip, and examined gene expression by RNA-seq in healthy smokers. FINDINGS: We identified 1667 total 5mC + 5hmC, 1756 5mC and 67 5hmC differentially methylated positions (DMPs) between smokers and non-smokers (FDR-adjusted P <.05, absolute Δβ >0.15). Both 5mC DMPs and to a lesser extent 5mC + 5hmC were predominantly hypomethylated. In contrast, almost all 5hmC DMPs were hypermethylated, supporting the hypothesis that smoking-associated oxidative stress can lead to DNA demethylation, via the established sequential oxidation of which 5hmC is the first step. While we confirmed differential methylation of previously reported smoking-associated 5mC + 5hmC CpGs using former generations of BeadChips in alveolar macrophages, the large majority of identified DMPs, 5mC + 5hmC (1639/1667), 5mC (1738/1756), and 5hmC (67/67), have not been previously reported. Most of these novel smoking-associating sites are specific to the EPIC BeadChip and, interestingly, many of them are associated to FANTOM5 enhancers. Transcriptional changes affecting 633 transcripts were consistent with DNA methylation profiles and converged to alteration of genes involved in migration, signalling and inflammatory response of immune cells. INTERPRETATION: Collectively, these findings suggest that tobacco smoke exposure epigenetically modifies BAL cells, possibly involving a continuous active demethylation and subsequent increased activity of inflammatory processes in the lungs. FUND: The study was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Stockholm County Council (ALF), the King Gustav's and Queen Victoria's Freemasons' Foundation, Knut and Alice Wallenberg Foundation, Neuro Sweden, and the Swedish MS foundation.

背景：虽然吸烟与多种疾病的发生有关，但其中的机制仍不清楚。吸烟会改变 DNA 的化学完整性，导致转录活性的改变，部分原因是表观遗传状态的改变。我们旨在研究吸烟对支气管肺泡灌洗液（BAL）中肺细胞的影响。

方法：我们使用常规亚硫酸氢盐（BS）处理和氧化亚硫酸氢盐处理，结合 Illumina Infinium MethylationEPIC BeadChip，对 DNA 甲基化（5mC）及其氧化形式羟甲基化（5hmC）进行了分析，并通过 RNA-seq 检测了健康吸烟者的基因表达。

结果：我们在吸烟者和非吸烟者之间鉴定了 1667 个总 5mC+5hmC、1756 个 5mC 和 67 个 5hmC 差异甲基化位置（DMPs）（FDR 调整后 P<.05，绝对 Δβ>0.15）。5mC DMPs 主要是低甲基化的，而 5mC+5hmC 则较少。相比之下，几乎所有的 5hmC DMPs 都是高甲基化的，这支持了这样的假设，即吸烟相关的氧化应激可以通过已建立的连续氧化导致 DNA 去甲基化，其中 5hmC 是第一步。虽然我们在肺泡巨噬细胞中使用前几代 BeadChips 证实了先前报道的与吸烟相关的 5mC+5hmC CpGs 的差异甲基化，但绝大多数鉴定的 DMPs（1639/1667，5mC+5hmC；1738/1756，5mC；67/67，5hmC）以前没有报道过。这些新的与吸烟相关的位点中的大多数都是 EPIC BeadChip 特有的，有趣的是，其中许多与 FANTOM5 增强子有关。影响 633 个转录本的转录变化与 DNA 甲基化图谱一致，并集中于参与免疫细胞迁移、信号转导和炎症反应的基因的改变。

解释：总的来说，这些发现表明，烟草烟雾暴露会使 BAL 细胞发生表观遗传修饰，可能涉及持续的主动去甲基化和随后增加肺部炎症过程的活性。

资助：该研究得到了瑞典研究委员会、瑞典心肺基金会、斯德哥尔摩郡议会（ALF）、古斯塔夫和维多利亚国王共济会基金会、Knut 和 Alice Wallenberg 基金会、神经瑞典和瑞典多发性硬化症基金会的支持。