Inhaled air pollution including emissions from diesel engines have been associated with a host of cardiovascular and respiratory diseases, imparting a significant strain on public health [3],[9]. Data presented here on DNA methylation changes in response to short-term exposure to DE demonstrated a potential epigenetic mechanism for biological responses to DE exposure. PCA enabled detection of DE signals from subject-level noise generated by demographic variables such as age, ethnicity, and BMI, and focused the investigation onto a smaller list of pertinent CpG sites. Furthermore, the DE-associated methylation changes in LINE1 and Alu CpG sites corroborated with previous research demonstrating sensitivity of repetitive elements to environmental exposure [16],[29]. Lastly, methylation change was also observed for miR-21, a microRNA associated with oxidative stress and allergic inflammation. Collectively, these results were consistent with our hypothesis that epigenetics servers as a potential avenue through which exposure to air pollution impacts biological systems.

Air pollution has long been linked to diseases including asthma and cardiovascular issues [1],[3],[32],[33]. What is still lacking in our understanding is defining the biological pathways that may be either a mediator or a consequence of the association. DE is a major source of fine particles that have been shown to impact gene expression, and change DNA methylation at inflammation associated sites [16],[28],[29],[31]. Our results support previous studies that have found an association between PM exposure and decrease in DNA methylation of target promoter, candidate genes and repetitive elements [16],[29],[30] We suggest that this could be explained through the effects of DE on the dynamics between DNA and DNA methyltransferases. First, ROS-induced oxidative damage to methyl-CpG binding protein recognition sequences could have inhibited the capacity for DNA methyltransferase to bind and methylate DNA [36]. Secondly, decrease in saturation of the DNA methyltransferase enzymes could potentially come into play, since studies have shown a dependent relationship between exposure to particles and decrease in mRNA transcripts of the DNA methyltransferases Dnmt1, Dnmt3a, Dnmt3b in mouse macrophages [37]. Decreased mRNA transcripts could lower the cellular concentration of methyltransferases and as a result decease the methylation level of CpG sites. Both of these scenarios could be part of the mechanism leading to loss of methylation that partially explains why the majority of the gene-specific sites found here showed decrease in methylation.

DAVID functional enrichment analysis of the 2827 DE-associated probes identified by PCA revealed enrichment of NFkB-related functions. NFkB is a redox-related transcription factor that activates a pro-inflammatory response to ROS induced oxidative stress [38]–[41]. NFkB has been shown to activate in response to DE exposure, increasing the downstream expression of inflammatory cytokines [42]. Besides NFkB, we also discovered enrichment of the MAPK protein kinase pathway. Previous studies have shown that MAP kinase is a stress-activated kinase pathway that can be induced by DE exposure [12],[43]. Given the proposed role of DNA methylation as a mediator between environment and gene expression, the observations made here further demonstrated that a tight circuitry exists between transcriptional pathways known to be involved in response to DE and DNA methylation changes elicited by DE exposure.

Results from PCA followed by regression modeling in this study revealed that in agreement with our hypothesis, DE was associated with changes in DNA methylation at genes that are known to be associated with inflammation and oxidative stress, most notably GSTP1[44],[45]. Studies investigating the effects of GSTP1 gene polymorphism found that subjects homozygous for the GSTP1 G allele have lower functional levels of the enzyme, and thus are at increased risk for oxidative stress, lung cancer, and asthma [44],[46],[47]. Since the GSTP1 associated probe (cg09038676) was located in the body of the GSTP1 gene, then its increase in DNA methylation would likely to be associated with increase in GSTP1 transcription, resulting in higher levels of the enzyme to combat the effects of oxidative stress. Interestingly, this effect was most profound for subjects with at least one A allele, suggesting that these individuals are less susceptible to the negative effects of DE exposure than individuals homozygous for the G allele, consistent with the literature [48]. Non-asthmatic controls were not included in this study, thus we cannot conclude that the changes observed are specific to asthmatics, but our finding bolsters mechanistic plausibility nonetheless. Furthermore, the significant baseline (FA) methylation difference between subject with and without the GSTP1 G allele substitution that cg09038676 could have been a methylation associated SNP; however, due to the fact that only 5 of 16 subjects had an A allele, we were not able to quantitatively assess this possibility.

Besides GSTP1, we also found decreased methylation in a probe residing within the body of HDAC9 (cg24458314), a class IIa histone deacetylase [49]. A study involving regulatory T (Treg) cells in mice showed that absence of HDAC9 enhances the suppressive ability of Treg cells, resulting in decreased immune responsiveness and inflammation [49]. Changes in DNA methylation of HDAC9 such as we observed could impact downstream gene expression that then modifies the allergic airway inflammation in response to DE exposure. Considering that Tregs constitute only a small fraction of PBMCs, we speculate this small change might indicate a profound effect on the Treg population; however, a more concrete conclusion could not be reached without independent examination of Treg cell-specific methylation.

Lastly, we discovered that cg05094429, which resides in the promoter region of CCR6 gene, decreased in methylation after exposure to DE. CCR6 is expressed by both Treg and Th17 cells, and it is a key regulator of the migration of these cells to sites of inflammation [50]. Lack of this protein in Th17 cells hampers the recruitment of both Th17 and Tregs [50]. Thus it is possible that decreased methylation of CCR6 in the promoter region could have resulted in increased expression of CCR6, eventually leading to increased presence of Th17 and Treg cells responding to DE induced inflammation. It should be noted an effect of ambient particulate matter on DNA methylation patterns has been previously documented in numerous studies [16],[28]–[31]. Therefore, despite the fact that most changes found here were small in magnitude, the overall findings reported here demonstrated an effect of DE that is concordant with existing observations and is impactful on the genomic scale.

Repetitive elements comprise of half of the genome and, under normal conditions, harbor higher DNA methylation in comparison to the rest of the genome [51]. Repetitive elements are activated during cellular stress, which in this case could be elicited by exposure to DE [52]. In accordance with past research on effects of particulate matter, we found both increase and decrease in Alu and LINE1 methylation after DE exposures [37]. In some cases, demethylation of Alu and LINE1 increases genomic instability, which could mean that DE exposure predisposes some cells to genomic rearrangements [53]–[55]. Furthermore, repeat elements also impact adjacent sequences; they can propagate the spread of DNA methylation to nearby sequences or serve as insulators [56],[57]. A recent study has shown that sequences close to a 3′ repeat element demonstrated better methylation stability [58] Therefore, it is conceivable that disruption of repetitive element DNA methylation due to DE exposure could be associated with genomic rearrangement as well as affecting methylation stability of nearby sequences causing changes in gene expression.

In this study we also examined the effects of DE on methylation of microRNAs, which are a class of small noncoding RNAs with functions in post-translational regulation of expressed genes that are important mediators of cellular processes [59]. Pollutants can cause microRNA dysregulation, which could lead to lung diseases and inflammation [19]. In particular, miR-21 is involved in oxidative stress and allergic inflammation, and has been shown to be up-regulated in asthma [19],[60]–[62]. Our results showing that DNA methylation was also altered after DE build upon a previous study on the same individuals that reported changes in miR-21 expression upon DE exposure [19], though this observation was associative and we cannot conclude that the changes in methylation are directly responsible for the changes in expression.

Lastly we were able to demonstrate that DE-associated changes lasted at least 30 hr post-exposure. This observation was consistent with reports showing that methylation changes elicited by environmental exposure could persist for days in the absence of persistent triggers, though the precise dynamics of this in the context of air pollution requires further study. These results should be interpreted in light of the limited sample size, lack of another dataset sufficiently similar to attempt validation, and the systemic nature of the tissue investigated. Nevertheless, this investigation presents a novel approach to analyzing the association between PM and genome-wide DNA methylation, and although we did not observe residual effects in the subjects after 2 weeks, long-term or repeated exposures to DE may lead to accumulative effects. Future studies could build upon the approach presented here to investigate similar events in airway tissues.