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Background: Despite compelling evidence that cigarette smoking impacts the risk of developing multiple sclerosis (MS), little is known about smoking-associated changes in the primary exposed lung cells of patients. Objectives: We aimed to examine molecular changes occurring in bronchoalveolar lavage (BAL) cells from MS patients in relation to smoking and in comparison to healthy controls (HCs). Methods: We profiled DNA methylation in BAL cells from female MS ( n = 17) and HC ( n = 22) individuals, using Illumina Infinium EPIC and performed RNA-sequencing in non-smokers. Results: The most prominent changes were found in relation to smoking, with 1376 CpG sites (adjusted P Conclusions: Our study provides insights into the impact of smoking on lung inflammation and immunopathogenesis of MS.

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 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.

Background Twin studies are powerful models to elucidate epigenetic modifications resulting from gene–environment interactions. Yet, commonly a limited number of clinical twin samples are available, leading to an underpowered situation afflicted with false positives and hampered by low sensitivity. We investigated genome-wide DNA methylation data from two small sets of monozygotic twins representing different phases during the progression of rheumatoid arthritis (RA) to find novel genes for further research. Methods We implemented a robust statistical methodology aimed at investigating a small number of samples to identify differential methylation utilizing the comprehensive CHARM platform with whole blood cell DNA from two sets of twin pairs discordant either for ACPA (antibodies to citrullinated protein antigens)-positive RA versus ACPA-negative healthy or for ACPA-positive healthy (a pre-RA stage) versus ACPA-negative healthy. To deconvolute cell type-dependent differential methylation, we assayed the methylation patterns of sorted cells and used computational algorithms to resolve the relative contributions of different cell types and used them as covariates. Results To identify methylation biomarkers, five healthy twin pairs discordant for ACPAs were profiled, revealing a single differentially methylated region (DMR). Seven twin pairs discordant for ACPA-positive RA revealed six significant DMRs. After deconvolution of cell type proportions, profiling of the healthy ACPA discordant twin-set revealed 17 genome-wide significant DMRs. When methylation profiles of ACPA-positive RA twin pairs were adjusted for cell type, the analysis disclosed one significant DMR, associated with the EXOSC1 gene. Additionally, the results from our methodology suggest a temporal connection of the protocadherine beta-14 gene to ACPA-positivity with clinical RA. Conclusions Our biostatistical methodology, optimized for a low-sample twin design, revealed non-genetically linked genes associated with two distinct phases of RA. Functional evidence is still lacking but the results reinforce further study of epigenetic modifications influencing the progression of RA. Our study design and methodology may prove generally useful in twin studies. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0374-0) contains supplementary material, which is available to authorized users.