Epigenetics provides a promising approach for understanding complex diseases like asthma. The epigenetic disruption of gene expression is now known to play an equally important role as genetic predispositions (such as polymorphism, mutation, deletion, and insertion) in the development of disease [Jiang et al., 2004; Godfrey et al., 2007]. Exposures to environmental stimuli, such as hypoxia, hypo-or hyper-nourishment, infection, hormones, drugs, or allergens, allow epigenetic reprogramming in cells, resulting in permanent alterations in the structure or function of specific organ systems [Tang et al., 2007]. This phenomenon is linked to gene activation, gene silencing, and chromosomal instability without altering DNA sequences. To date, there are three known epigenetic mechanisms: DNA methylation, histone modification, and noncoding RNAs involved in gene regulation.

Recent evidence has shown that epigenetic modifications via DNA methylation can provide a possible mechanistic explanation for the link between exposure to environmental pollutants and asthma [Perera et al., 2009; Kabesch et al., 2010]. DNA methylation refers to the covalent addition of a methyl group derived from S-adenosyl-l-methionine to the fifth carbon of the cytosine ring to form the fifth base, 5-methyl cytosine (5-mC) [Costello et al., 2001]. DNA methylation at the 5’ CpG island (CGI; CG-rich region with >60% GC content and an obs/exp ratio >0.6 in the 5’ flanking region of the gene promoter) alters gene transcription machinery and eventually leads to a decrease in gene expression. DNA methylation patterns have been shown to be affected by postnatal or adult exposure to diesel exhaust [Liu et al., 2008, Perera et al., 2009; Tang et al., 2012], black carbon [Belinsky et al., 2002], particulate matter [Tarantini et al., 2009], benzene [Bollati et al., 2007], and cigarette smoke [Pulling et al., 2004]. Our group employed an innovative epigenetic profiling approach to investigate allergen-induced airway hyper-responsiveness (AHR). We showed that acute exposure to house-dust-mite (HDM) extracts induced AHR that was associated with epigenetic modulation of genes related to airway smooth muscle (ASM) cell proliferation and contraction [Shang et al., 2013]. We speculated that these epigenetic changes in the lungs could persist over longer time spans with repeated exposure to HDM and eventually lead to airway remodeling and AHR. Advanced sequencing technology now enables the demonstration of the epigenetic changes that reprogram the lungs in response to environmental allergens in a high throughput manner. In the present study, we utilized methylated DNA immunoprecipitation-next generation sequencing (MeDIP-seq), which is a technique utilizing methylated DNA enrichment with the aid of methylation-binding domain (MBD) proteins followed by a single-run of massive parallel sequencing (also called next generation sequencing, NGS) on lung tissue DNA from saline- and HDM-exposed mice. Using bioinformatics analysis, we demonstrated a set of specific genes that are epigenetically reprogrammed by HDM and associated with an increase in AHR.

In urban areas, HDM is one of the most common allergens. It has been postulated that chronic exposure to HDM extracts induces airway remodeling and AHR via immune mechanisms. For example, HDM extracts can stimulate dendritic cells to secret Il6 and induce T-helper cell (Th)2 and Th17 differentiation, which contribute to AHR [Krishnamoorthy et al., 2008]. It has been proposed that HDM extracts can destroy airway epithelial and ASM cells by its proteolytic activity. Furthermore, HDM has been found to induce changes in ASM responsiveness via ERK1/2 signaling [Grunstein et al., 2005]. In our experimental asthma model, we found an increase in Th2 cytokine levels and the numbers of inflammatory cells in response to HDM. Furthermore, we observed thickening of the bronchial smooth muscle and increased levels of Pcna, Sma, and Camk2d gene expression in HDM-exposed mice. These findings confirm that repeated exposures to HDM can induce airway inflammation and ASM remodeling, resulting in increased AHR. There is an agreement that epigenetic regulation interacts with environmental exposure and its dysregulation leads to asthma pathogenesis [Perera et al., 2009; Kabesch et al., 2010; Niedzwiecki et al., 2012; Shang et al., 2013]. DNA methylation at 5’ CGIs alters gene transcription machinery and eventually leads to decreases in gene expression. Gene dysregulation further affects cellular functions. Global DNA methylation levels assayed by methylation sensitive pyrosequencing or PCR of the repetitive elements LINE and Alu have been found to be decreased in response to prenatal tobacco smoke [Breton et al., 2009] and polya-crylic aromatic hydrocarbons (PAHs) [Herbstman et al., 2012]. In these studies, reduced global DNA methylation was associated with an increased risk of childhood asthma. Our results support these studies by showing a decrease in global DNA methylation in mice with AHR. Furthermore, we demonstrated that there was an increase in 5-hmC levels in the lung DNA of HDM-exposed mice, indicating that hydroxymethylation at CpGs may occur concurrently with the removal of DNA methylation.

A number of studies show epigenetic modulation of B cell and Th2 phenotypes in both allergic and non-allergic asthma [Kwon et al., 2008; Pascual et al., 2011; Brand et al., 2012; Tang et al., 2012]. Few studies have demonstrated genome-wide DNA methylation changes in the lungs and their link to structural remodeling and hyper-responsiveness of the airway. In the present study, we demonstrated that repeated allergen exposure induced epigenetic changes in the lung genome, associated with increased AHR. By using MeDIP-seq and pathway analysis, we identified sets of genes differentially methylated in mice with repeated HDM challenges. Methylome research coupled with NGS could be an innovative and promising approach for understanding the pathogenesis of complex diseases with proper bioinformatics tools and data mining analysis [Robertson et al., 2007; Brunner et al., 2009; Grimm et al., 2013]. A genome-wide methylation profiling of the bronchial mucosa of asthmatics identified 48 genes that were hypomethylated in the bronchial mucosa of atopic asthmatics, and six genes that were hypermethylated compared to those of nonatopic asthmatics [Kim et al., 2013]. With GO analysis, these hypomethylated genes are assigned to hormone metabolic processes and growth factor receptor binding. The authors suggested the use of their selected genes for future asthma research. Our genome-wide methylation study has revealed that key pathways could be epigenetically regulated via DNA methylation in response to HDM exposure. We further demonstrated the epigenetic changes in well-defined airway cells, MASMCs. Phenotypic changes of ASM, including ASM hypertrophy, hyperplasia, and/or migration, were demonstrated in the thickening of the airway walls and increased ASM mass [Bentley et al., 2008; Hirota et al., 2009; Al Heialy et al., 2011]. These effects contribute to further narrowing of the airway and reduced airflow, leading to AHR [Siddiqui et al., 2008; Janson et al., 2010; Hamid et al., 2012]. Epigenetic mechanisms, including DNA methylation, histone modifications, and miRNA, have been shown to regulate the gene transcription of specific signaling pathways to maintain and alter ASM cell phenotypes that may contribute to asthma pathogenesis [Clifford et al., 2013]. Previously, we utilized one of the methylation profiling techniques, methylation sensitive restriction fingerprinting, and demonstrated that acute exposure to HDM induced methylation changes in Pde4d, Slc8a3, and Pom121l2. Dysregulation of these genes have been found to contribute in ASM proliferation and contraction, which may lead to increased ASM hyper-responsiveness [Shang et al., 2013]. In the present study, we demonstrated aberrant methylation of Tgfb signaling genes in lung tissues and specifically in MASMCs from HDM-exposed mice.

Tgfβ2, a member of the Tgfb family of cytokines, regulates cell proliferation, differentiation, adhesion, and migration by transducing its signal through combinations of transmembrane type I and type II receptors (Tgfbr1 and Tgfbr2) and their downstream effectors, the Smad proteins. In airway cells, Smad2/3 proteins act as transcriptional activators, which transduce Tgfb-regulated gene transcription intra-cellularly. Tgfβ2 has been shown to play a role in the regulation of airway inflammation and remodeling in both human and murine models of asthma [Chu et al., 2004; Balzar et al., 2005; Hatsushika et al., 2007; Bottoms et al., 2010; Lopez-Guisa et al., 2012]. In bronchial biopsies from asthmatic subjects, there was an increase in Smad2 expression [Sagara et al., 2002]. Inhibition of Smad3 has been reported to suppress ovalbumin-induced airway remodeling [Le et al., 2007]. Royce et al. also suggested that Tgfb2, Smad2, and Smad3 are key regulators of fibrosis in airway remodeling [Royce et al., 2012]. Ski, which is co-repressor of Smad2/3 complexes, represses Tgfb signaling [Groneberg et al., 2004]. Ski expression has been shown to inhibit vascular smooth muscle cell proliferation by suppressing Tgfb/Smad3 signaling but up-regulating p38 signaling [Li et al., 2013]. Activation of Tgfb signaling in OVA-sensitized and -challenged rats has been found to contribute to the pathogenesis of airway remodeling through the reduction of Ski proteins [Li et al., 2010]. We found CpG site-specific hypomethylation at Tgfβ2, Smad2 and Smad3, and Ski hypermethylation in lung and MASMCs, which may be associated with HDM-induced AHR. Other indirect interactions with Tgfb signaling are suggested to be epigenetically regulated and associated with increased AHR. Il6r functions as a regulator of inflammation and has been shown to be associated with asthma risk factors [Ferreira et al., 2011]. In our model, HDM induced Il6r demethylation in murine lungs and MASMCs, suggesting the epigenetic effect of HDM on Il6r-related AHR. On the other hand, calcium signaling is one of the key mediators in airway contraction, remodeling, and inflammation [Jude et al., 2008]. Hypomethylation of Trpm2, Slc22a15 and Cartpt (calcium signaling gene) was found in HDM-exposed mice. In addition, Trpm2, but not Slc22a15 or Cartpt, was hypomethylated in MASMCs. Although Trpm2 has been shown not to be involved in airway inflammation in OVA-induced allergic asthma in mice [Sumoza-Toledo et al., 2013], how Trpm2 contributes to ASM functions or airway structural remodeling has not been studied. In summary, we demonstrated that the most common allergen, HDM, induced the dysregulation of a specific set of genes via epigenetic modifications in the lungs. Specifically, the differentially methylated genes are highly enriched in the Tgfb signaling pathway, and these epigenetic changes may modulate the ASM cell functions and lead to AHR.

There are sets of functional components that modulate the epigenetic information embedded in the chromatin of gene promoters in response to environmental stimuli. Down-regulation of Mbd2, Mbd3, and Dnmt3a was observed in HDM-exposed mice. Mbd2 and Mbd3 can bind to methylated DNA and recruit enzymes, like Dnmt3a, to catalyze DNA methylation [Guibert et al., 2013]. We observed a significant decrease in global DNA methylation in HDM-exposed mice. Reductions in levels of Mbd2, Mbd3, and Dnmt3a may explain this phenomenon, and it is also possible that Mbd2, Mbd3, and Dnmt3a may act on specific gene promoters or affect DNA demethylation machinery. HDM-exposed mice showed increases in Tet1, Aid, and Smug1 expression and these proteins might contribute to global and gene-specific demethylation. Tet proteins convert 5-mC into 5-hmC; 5-hmC has been recently demonstrated to be a stable epigenetic mark and contributes to the differentiation of embryotic stem cells during development [Tahiliani et al., 2009; Wu et al., 2011]. Hydroxymethylation at 5’ CpGs contributes to gene transcription by influencing DNA demethylation and/or the recruitment of transcription complexes. Both Aid and Smug1 are also involved in demethylation. Aid activates the deamination of 5-mC, while Smug1 catalyzes base-excision repair to exchange cytosine [Krokan et al., 2002; Zhu, 2009; Gavin et al., 2013]. Taken together, our results suggested that HDM might trigger global and gene-specific DNA methylation machinery either thru the inhibition of DNA methylation, hydroxymethylation, or the passive/active demethylation via DNA repair, resulting in increased gene transcription. How these epigenetic machineries contribute to gene transcription and its functions in the airways remains unanswered and requires further study.

Advanced epigenetic technology, bioinformatics support, and proper validation assays will likely increase our ability to reveal markers for the prevention, diagnosis, and prognosis of asthma. Herein, we demonstrated the epigenetic changes associated with allergic AHR in an environmentally and clinically relevant experimental asthma model in a step-by-step manner. We first utilized a high-throughput and unbiased DNA methylation profiling approach to explore the genome-wide methylation changes in mouse lungs in response to the environmental allergen, HDM. Next, we chose two commercially available databases (CLC Genomic Workbench and IPA) to analyze the raw NGS data and map out biological networks that can be validated in subsequent studies. Then, we validated the gene expression and promoter methylation of 19 genes from the top “connected” network determined by IPA (Tgfb signaling pathway) by RTPCR and quantitative MSPCR respectively. The primers used in the MSPCR are designed to target the specific CpG sites showing the highest peak signal from NGS data for the potential candidate genes determined from the MeDIP-seq data. Furthermore, we correlated the gene expression levels of the candidate genes with their methylation status (ρ value) in order to associate DNA methylation changes with gene expression changes in the lungs from mice with allergic AHR (Table III). While bisulfite genomic sequencing may be the gold standard approach to examine the DNA methylation status of individual CpG sites, our current approach allows us to generate a candidate gene list by MeDIP-seq and validate the potential candidates in another set of HDM-exposed mice or well-defined airway cells like MASM cells (Table V) in a time- and cost-effective manner.

In the present study, we utilized lung tissues for the methylome analysis in order to get a sufficient amount of genomic DNA for MeDIP-seq. However, we do acknowledge the heterogeneity of cells (including MASMCs, airway epithelial cells, fibroblasts, goblet cells and immune cells) in the lung tissues may have influenced our results. Epigenetic changes induced by HDM exposure in the lung or airway may involve gene dysregulation in all or a subset of these cell types. We validated the gene expression and promoter methylation status of potential MeDIP-seq candidates in both lung tissues and MASMCs cells. We examined the epigenetic changes in MASMCs for test of concept because ASM is one of the key components in AHR. We clearly demonstrated that many of the candidate genes identified using the lung tissue also undergo DNA methylation and expression changes in MASMCs from exposed mice, suggesting that the methylation changes demonstrated are occurring in a relevant cell type to AHR. However, further validation of methylation status in different cell types from the mouse lung is recommended allowing for an even clearer understanding of the pathogenesis of asthma. Future experiments like gene knock-in or knock-out assays in mouse lung are needed to demonstrate the underlying mechanisms of how epigenetic changes of potential candidates alter ASM functions and AHR. Herein, we presented differentially methylated genes, which are highly enriched in the Tgfb signaling pathway, in HDM-exposed lung and MASMCs as compared to that of saline-control. A better understanding of the regulation of Tgfb signaling in transcription would provide more specific targets for therapeutic intervention of asthma [Howell et al., 2006; Halwani et al., 2011]. All together, our current findings have provided insight into experimental designs needed to demonstrate the epigenetic changes involved in asthma pathogenesis.