The genetic and epigenetic landscapes of the epithelium in asthma

Modification of DNA occurs through addition or removal of small covalent molecules such as methyl or acetyl groups. DNA methylation occurs when methyl groups are added by a DNA methyltransferase (DNMT), onto cytosine nucleotides that are followed by guanine residues (CpG sites). Gene promoters are relatively rich in CpG sites which are known as CpG islands. DNA methylation can lead to gene expression silencing through formation of 5-methyl-cytosine (5mC) [98].

Recent studies have characterised a number of DNA methylation signatures in epithelial cells of asthmatics, including cytokeratin 5 (KRT5) [99], signal transducer and activator of transcription 5A (STAT5A) [99], cysteine-rich protein 1 (CRIP1) [99], arginase 2 (ARG2) [100], IL-6 [98], inducible nitric oxide synthase (iNOS) [98] and ADAM33 [101] (Table 2).

KRT5 is a marker of basal cells and its expression is increased in the epithelium of asthmatics [5, 36]. In asthmatic children, KRT5 exhibits reduced methylation resulting in increased expression of this gene [97, 99]. Increased KRT5 may hence be associated with dysregulated epithelium differentiation. The STAT5A transcription factor is activated by different pro-Th2 cytokines (e.g. IL2, IL7, or TSLP) suggesting its significant role in promoting Th2 cell differentiation and responses [102] and epithelial cell proliferation [103]. CRIP1 has been reported to play a role in cell motility, adhesion, and structure through interaction with the cytoskeletal protein actin [104] and also translocates to the nucleus to facilitate protein interactions important for transcriptional regulation [105]. The promoters of STAT5A and CRIP1 are hyper-methylated in epithelium of asthmatic children [99], resulting in decreased expression of STAT5A, contrary to increased CRIP1 expression [99]. Further studies are therefore needed to understand the roles of STAT5A and CRIP1 in epithelial function.

ARG2, IL-6 and iNOS are three methylated genes that have been related to fractional exhalation of nitric oxide (FeNO) in asthmatic children [98, 100, 106]. Methylation of the ARG2 promoter in asthmatic children is associated with reduced FeNO [100]. However, asthmatic children with lower DNA methylation of the IL-6 and iNOS promoters in nasal epithelial cells had higher airway inflammation, as measured by increased FeNO [98]. Therefore, further investigation is required to determine the underlying biological mechanisms driving the association of these DNA methylations with FeNO and whether children with different degrees of asthma severity and symptom management have different levels of DNA methylation.

Furthermore, hyper-methylation on ADAM33 in bronchial epithelial cells is strongly associated with BHR, irrespective of asthma status [101]. This is in contrast to ADAM33 hypo-methylation in fibroblasts, which is speculated to be involved in airway remodelling [107]. This highlights the importance of cell specific epigenetic changes, as well as the potential challenges in developing novel therapeutics.

Recently, DNA methylation has been shown to occur in airway epithelial cells isolated from asthmatics after a single 24 h exposure to IL-13 [108]. Intriguingly, areas of methylation were mainly adjacent to asthma susceptibility genes and in particular genes related to fibrotic and inflammatory pathways (e.g. neutrophil cytosolic factor 2; NCF2, and MMP14) [108]. Global and gene specific methylation status in the airway epithelium however still requires further investigation before potential targets can be identified and trialled as therapies for asthma.

The DNA of each cell is packaged into nucleosomes where its 147 base pairs wrap around an octamer of four core histone (H2A, H2B, H3, and H4). The covalent alterations of the amino acid residues of core histone N-terminal tails are essential for modification of the chromatin structure and regulate gene expression. For example, the acetylation of lysine residues on histone tails via histone acetyltransferases (HATs), generally results in increased gene transcription whereas removal of the acetyl group via deacetylases (HDACs) leads to gene suppression. In contrast, methylation of histone tails can be both activating and suppressing depending on the particular residue. Methyl groups are added to lysine or arginine residues by histone methyltransferases (HMTs) and removed by histone demethylases (HDMs) [109, 110].

Increased HAT activity and reduced HDAC activity in biopsies from mild asthmatics have been reported to lead to the increased expression of multiple inflammatory genes [111]. Interestingly, these activities may be partially reversed by treatment with inhaled corticosteroids [111]. Furthermore, within the adult airway epithelium, elevated histone H3 lysine 18 (H3K18) acetylation and histone H3 lysine 9 trimethylation (H3K9me3) have been shown in asthmatics [109]. H3K18 acetylation increases the expression of ΔNp63, EGFR, and STAT6, which, are known to be altered in the epithelium of asthmatics [109]. Very few studies have investigated histone modifications in children with asthma. In one study, passive cigarette smoke reduced HDAC2 activity and protein expression via PI3K signalling in children with severe asthma. This is believed to suppress the anti-inflammatory effects of corticosteroid treatment [112] (Table 2).

Whether histone modifications in epithelial cells are major contributors in conferring asthma susceptibility and/or severity remains to be determined.

A number of classes of noncoding RNAs have been discovered in mammalian cells including long non-coding RNAs (lncRNAs), Piwi-interacting RNAs (piRNAs), and miRNAs.

lncRNAs are non-protein coding RNA transcripts longer than 200 nucleotides. There are approximately 15,000 lncRNAs discovered so far although only a small number of which have been shown to be biologically relevant. piRNAs are small non-coding RNAs of 26–31 nucleotides long, predominantly found in spermatogenic and ovarian cells [113]. Their functions have been linked to both epigenetic and post-transcriptional gene silencing [113]. piRNAs interact with piwi protein, a RNA-binding protein, which degrades target mRNAs to prevent protein translation [113]. There have been no studies that extensively investigate profiles of lncRNAs and piRNAs in airway epithelium of asthmatics. Only one study has shown that piR30840 directly targets and degrades IL-4 mRNAs leading to inhibition of the development of Th2 T-lymphocytes. Furthermore, the level of piR30840 is significantly reduced in serum from patients with asthma [113].

miRNAs are proposed to control expression of 30–60 % of human genes [114] and hence are crucial in most biological and pathological processes including cell proliferation, differentiation, apoptosis, carcinogenesis and immune responses [115‐117]. miRNAs are 20–24 nucleotides long and bind to the 3′ untranslated region (UTR) of target mRNAs resulting in their degradation or translational inhibition [118‐120]. Currently, there are over 1000 miRNAs identified in miRbase (www.​mirbase.​org), many of which have multiple binding partners and thus affect multiple pathways [118]. miRNAs may modulate protein synthesis at both initiation and post-initiation of translation [118]. miRNAs have also been shown to up-regulate some mRNA targets [120]. The balance between up- and down-regulation of miRNA plays a pivotal role in cell cycle and process of cell proliferation and regeneration, a process that is dysregulated in asthmatic epithelium. miRNAs are therefore interesting regulatory factors which may contribute substantially to airway epithelium abnormalities in asthmatics.

There are a limited number of studies examining miRNA expression in epithelium of asthmatics or non-asthmatics (Table 2). miRNA microarray performed on primary bronchial epithelial cells cultured at air-liquid interface (ALI) showed higher expression of let-7f, miR-181c* and miR-487b but lower expression of miR-203 in mild asthmatics compared with healthy controls [121]. miR-203 has been shown to play a potent role in the (keratinocyte) self-renewal program during epidermal differentiation by targeting p63, facilitating cell cycle exit and promoting differentiation [122]. Given that the epithelium of asthmatics expresses higher levels of p63 [36], it is intriguing to speculate that there is a direct link between miR-203 and p63 (Fig. 2). Recently, miR-203 has also been shown to inhibit airway smooth cell proliferation through targeting the non-receptor tyrosine kinase c-Abl (Abelson tyrosine kinase, Abl, ABL1) [123]. In particular, Abl kinases regulate cell-cell adhesion in epithelial cells and fibroblasts through cadherin-mediated adhesion signals via regulating the activities of the Rac and Rho GTPases [124]. Since, c-Ab1 plays important roles in regulation of the actin cytoskeleton and hence different cellular functions such as proliferation, cell adhesion and migration as well as growth and development [123], the reduced level of miR-203 in epithelial cells of asthmatics may promote cell proliferation, increase goblet cell hyperplasia and/or decrease ciliated cells. Genome wide profiling of bronchial epithelial brushings also revealed four members of the miR-34/449 family (miR-34b-5p, miR-34c-5p, miR-449a, and miR-449b-5p) were significantly suppressed in asthma [125]. Interestingly, no clear relationship was observed between these differentially expressed miRNA and serum IgE level in asthmatics [125], which indicates the role of these miRNAs at cellular and molecular levels rather than inflammatory and allergic responses. Furthermore, inhaled corticosteroids showed only minor effects on miRNA expression, and failed to restore miRNA levels to healthy control levels [125]. The miR-34/449 family are closely associated with regulation of epithelial cell proliferation and differentiation. Specifically, miR-449 is essential in regulation of airway ciliated cells by targeting NOTCH1 [126]. Notch signaling triggers airway mucous metaplasia and inhibits alveolar development (ciliated cells) [127]. The low level of miR-449 in epithelial cells of asthmatics may therefore shift the fate of these cells toward more mucous production (Fig. 2). Furthermore, miR-34/449-deficient mice suffer from primary ciliary dyskinesia (PCD). These abnormalities have been shown to be mediated by Cp110, a centriolar protein suppressing cilia assembly, that is a target of miR-34/449 [128]. A panel of miRNAs including miR-18a, miR-27a, miR-128 and miR-155 were also down-regulated in the epithelium of asthmatics. These miRNAs are involved in activation/signaling of IL-6 and IL-8 [129].

Recently, miR-19a was reported to be the only miRNA that differentiates severe from mild asthma. miR-19a is up-regulated in severe asthmatic epithelial cells and is not restored by corticosteroids [130]. Elevated levels of miR-19a further stimulate cell proliferation of epithelial cells by targeting TGF-β receptor 2 mRNA. Overexpression of miR-19a results in reduction in phosphorylated SMAD3 whereas suppression of miR-19a facilitates SMAD3 phosphorylation through TGF-β receptor 2 signaling and restore epithelial cells proliferation [130]. These may suggest the potential of miR-19a as a therapeutic target in airway epithelium of asthmatics to re-establish epithelial regeneration.

There are limited numbers of investigations on miRNAs expression in asthmatic epithelium of children, possibly due to difficulty in obtaining epithelial samples. Higher level of miR-148b; a member of miR-152 family, was reported in airway epithelial cells of adults with an asthmatic mother and correlates with sHLA-G levels in the BAL fluid of these subjects [131]. The prenatal effects of maternal asthma on the regulation of foetal genes in airway cells may persist well into adulthood through miRNA regulatory mechanisms.

Airway epithelial cells display dysregulted differentiation in asthma. It is therefore important to investigate potential miRNAs fundamental for lung development and epithelial cell homeostasis such as miR-17 family [132, 133]. miR-17 family consist of three paralog clusters of miR-17–92 (miR-17-5p, miR-18, miR-19b, miR-20a, miR-92, miR-19a and miR-17-3p), miR-106a–363 (miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92–2, and miR-363), and miR-106b–25 (miR-106b, miR-93, and miR-25) [134]. Among the miR-17 family, the miR-17-92 cluster and miR-106b have been shown to be essential in maintaining of the structural homeostasis of developing lung epithelium [132, 134]. miR-17-5p, miR-19b and miR-20 are increased in the epithelium and mesenchyme of the embryonic lung compared with fully developed lung [132]. Furthermore, miR-17, miR-20a and miR-106b modulate fibroblast growth factor (FGF)10–FGFR2b signaling by specifically targeting STAT3 and MAPK14, and altering E-cadherin distribution. This is vital for epithelial bud morphogenesis in response to FGF10 signaling [134]. STAT3 and MAPK14 signalling play important roles in airway homeostasis. STAT3 stimulates regeneration and multiciliogenesis by inhibition of the Notch pathway and direct regulation of genes such as Mcidas and Foxj1 [135]. MAPK14 (known also as p38α) has also been shown to regulate lung stem or progenitor cell proliferation and differentiation [136]. MAPK14 regulates C/EBP and HNF3b which are necessary for the differentiation of the stem cells into AT2 and Clara cells, while coordinately suppressing the regulators of stem and progenitor cells proliferation; cyclin D1 and EGFR [136]. These findings emphasise the role of these signalling pathways as well as their regulatory miRNAs in airway epithelial cells homeostasis, which is dysregulated in asthma. These pathways may initiate development and progression of asthma.