Role of Epigenetics in the Pathogenesis, Treatment, Prediction, and Cellular Transformation of Asthma

Asthma is a mysterious disease with heterogeneity in etiology, pathogenesis, and clinical phenotypes. Although ongoing studies have provided a better understanding of asthma, its natural history, progression, pathogenesis, diversified phenotypes, and even the exact epigenetic linkage between childhood asthma and adult-onset/old age asthma remain elusive in many aspects. Asthma heritability has been established through genetic studies, but genetics is not the only influencing factor in asthma. The increasing incidence and some unsolved queries suggest that there may be other elements related to asthma heredity. Epigenetic mechanisms link genetic and environmental factors with developmental trajectories in asthma. This review provides an overview of asthma epigenetics and its components, including several epigenetic studies on asthma, and discusses the epigenetic linkage between childhood asthma and adult-onset/old age asthma. Studies involving asthma epigenetics present valuable novel approaches to solve issues related to asthma. Asthma epigenetic research guides us towards gene therapy and personalized T cell therapy, directs the discovery of new therapeutic agents, predicts long-term outcomes in severe cases, and is also involved in the cellular transformation of childhood asthma to adult-onset/old age asthma.


Introduction
Asthma is well known as a noncommunicable, chronic, and heterogeneous inflammatory condition of the lower airway tract characterized by various clinical conditions that vary in severity and frequency [1,2]. Asthma can be observed in any age group. Some epidemiological studies have shown that asthma begins early in preschool age, although symptoms appear later in adult life [3] and, in some cases, may not appear.
The common asthma phenotypes are type 2 asthma (T2 high) and non-type 2 asthma (T2 low). T2 asthma includes early-onset allergic and late-onset nonallergic eosinophilic asthma. The common Th2 biomarkers used in clinical practice are mainly blood eosinophils, fractional exhaled nitric oxide (FeNO), and IgE levels. Interestingly, the majority of people with T2 asthma respond well to standard therapy with inhaled corticosteroids [4]. However, non-T2 asthma is a neutrophilic and paucigranulocytic heterogenous type, predominant in those with adult-onset and corticosteroidresistant (less responsive), and inflammation-driven through IL-17, IL-6, and IL-23. Further, it has airway smooth muscle or neural dysfunction and may be associated with comorbidities, such as obesity and gastroesophageal reflux disease [5,6]. The exact linkage between childhood and adult-onset/old age asthma remains unclear. Does adult-onset/old age asthma represent the persistence or relapse of childhood asthma?
Although we have ongoing research and an improved understanding, prevalence of asthma has been increasing in recent years [7] and affecting more than 339 million people worldwide [8], with 417,918 deaths globally in 2016 [9]. Asthma is a heritable disease [10], and approximately 60% of heritability has been found in several studies [11]. Interestingly, monozygotic twins are four times more likely to develop asthma than dizygotic twins (19% vs. 4.5%) [12]. Despite many studies, the complete natural history, pathogenesis, and heterogeneous phenotypes of asthma remain unclear. These unsolved questions and increasing asthma incidence suggest that there may be other elements related to asthma pathogenesis with heredity, leading to researchers investigating the connection between epigenetic changes and asthma.
Waddington described the term epigenetics more than half a century ago [13]. Later, Nanney described it as an unexplainable inherited phenomenon by conventional genetics [14]. In 2007, it was defined preciously using three criteria: (I) genetic changes without mutation, (II) initiation by a signal (extracellular signal), and (III) inheritance by mitosis or meiosis [15]. Currently, epigenetics is regarded as a significant influencing factor in asthma pathogenesis [16], and many epigenetic studies involving asthma are currently underway. Commonly studied epigenetic phenomena include DNA methylation, histone modification, and small noncoding RNA (miRNAs). We hope to obtain a clear understanding of asthma through epigenetic studies in the near future.
Despite extensive knowledge, there is still much unknown about asthma. There are many new etiologic factors, variations in presentation, and new genetic linkage with asthma. Therefore, we must ask if there are any new factors driving asthma development and progression besides the known factors and etiology. This review is aimed at providing an overview on asthma epigenetics and its components, including asthma epigenetic studies, and discuss potential epigenetic links between childhood asthma and adult-onset/old age asthma.

Can Asthma Epigenetics Be Considered a Hot
Research Topic?
The answer is yes. Asthma epigenetics have received immense interest because genetic and environmental factors cannot wholly and independently explain asthma etiology, heterogeneity, and phenotypes ( Figure 1). Researchers' search to find an alternative explanation for still mysterious phenotypes and heterogeneity of asthma made asthma epigenetics a hot topic. Moreover, the results are providing answers to the above queries.

DNA Methylation
Historically, DNA methylation in mammals was documented just after the discovery of DNA as a genetic component [17]. Methylation is the addition of a methyl group, by DNA methyltransferase, on cytosine at position 5 with the formation of 5-methylcytosine [18] where guanine nucleotides follow the cytosine nucleotide known as CpG [19]. Approximately, 60%-80% of CpG islands (cluster of CpG) are usually methylated in humans, but not in single CpG islands [20]. Interestingly, this equals to only 1% of bases and 5% of cytosine methylation across the gene [21]. CpG islands usually contain more than 200 bases with approximately 50% guanine plus cytosine (G+C) and a ratio of 0.6 or more [22]. Methylation of CpG island results in gene activation or inhibition but usually promotes repression, since the islands are located in the transcription start site (TSS) of genes [23]. Recently, our study found that methyl CpG binding domain protein 2-mediated Th17 differentiation in severe asthma is associated with IRF4 and SOCS3 expression, providing novel insight into epigenetic regulation and target for treatment in severe asthma [24,25]. A specific term "epimutation" is used in literature to denote the heritable genetic changes caused by DNA modifications [26]. Bisulfite conversion (sequencing) of DNA is the most commonly used technique for DNA methylation. Other assessment techniques include high-performance liquid chromatography, microarrays or bead chips, mass spectrometry, methyl sensitive cut counting (MSCC), and antibodies or protein that bind to methylated DNA [27]. To date, the DNA methylation techniques have only covered 450000 (450K Infinium Human Methylation Beadchip, Illumina Inc., CA, USA) and 850000 (Infinium Methylation EPIC Beadchip, Illumina, USA) CpG through the epigenomewide association study [28,29]. We hope that the methylation technique will be developed further to allow for the capture of more CpG positions at once.
3.1. DNA Methylation and Asthma. DNA methylation is the most studied mechanism in asthma. Alteration in DNA methylation status results in differential gene expression related to cytokines and transcription factors, resulting in various and distinct phenotypic presentations in asthma (Table 1 and Figure 2).
Hypermethylation of IL-2 Site1 (4q27) has been found in the cord blood of children with severe asthma who were followed up for 8 years [37]. Hypomethylation of IL5RA (3p26.2) in the blood is associated with asthma in teens [38], and IL-5 plays a crucial role in eosinophilopoiesis [39]. Hypermethylated GATA3 (location 10p14), a significant regulator of Th2 differentiation, is associated with reduced asthma risk in cord blood [40], while ZPBP2 (Zona Pellucida Binding Protein 2) hypomethylation results in asthma in a lymphoblastoid cell line at genetic location 17q12-q21 [41]. CYP26A1 (10q23.33), a regulator of the allergic immune response, was found to be hypermethylated in blood samples of patients with asthma [42]. Hypomethylation of TET1 (10q21.3) from the nasal epithelium results in asthma in children exposed to traffic-related air pollution [43]. TET1 converts 5-methylcytosine to oxymethylcytosine 2 Mediators of Inflammation along with 5-formylcytosine, 5-carboxylcytosine, and 5hydroxymethylcytosine, resulting in DNA demethylation. IL-36, IL-38 [44], and IL-3 are believed to be new members of the IL-1 family and are involved in the pathogenesis of asthma, and hypermethylation of IL1R2 (2q11.2) in blood shows asthma-related phenotypes [45]. These studies show the various phenotypic presentations of asthma through the methylation of specific asthma-related genes. Increased risk of wheezing, especially in girls, is associated with hypermethylation of the AXL gene (19q13.2) in CpG islands in newborn blood spots [46]. Hypermethylation of arginase 2 (14q24.1) results in decreased FeNO in the buccal cells [47], and airway epithelium hypomethylation of nitric oxide synthase 2 (17q11.2) and IL-6 (7p15.3) is associated with an increased concentration of FeNO [48].
The ADRB2 gene (5q31-33) sampled from blood is hypermethylated and associated with severe asthma, but nitrogen dioxide (NO 2 ), a known air pollutant, has been shown to alter this association [49]. Interestingly, one study has shown decreased dyspnea episodes in asthmatic children with ADRB2 hypomethylation sampled from blood and saliva [50]. Hypermethylation of protocadherin (PCDH20) has been found in the sputum of asthmatic adult smokers at the genetic locus 13q21.2 [51]. Epigenetic changes in the beta-2-adrenergic receptor (ADRB2) gene play a vital role in the phenotypic presentation of asthma.
Differently methylated regions are considered the beginning of development stages and with reprogramming progress [58]. Cadherin-related genes are differently methylated in children with atopy and atopic asthma [52]. Cadherinrelated genes CDH26 and CDHR3 (genetic loci 20q13.33 and 7q22.3, respectively) from the epithelial airway are hypomethylated in children with a history of atopy and can easily differentiate atopy with atopic asthma [52]. The noticeable hypomethylation has been found in genes C7orf50 and ZAR1 (genetic loci 7p22.3 and 4p11, respectively), which are sampled from cord blood and are associated with IgE levels [53]. STAT5A genes are downregulated, differently methylated, and associated with different asthma levels [54]. STAT5A genes in the region 17q21.2 are hypermethylated and associated with enhanced Th1 response and decreased eosinophil recruitment in the airway epithelium [54]. These methylation studies show the usefulness in identifying IgE association, eosinophil recruitment, Th1 response, and even differentiating between children with atopy and atopic asthma through the epigenetic changes observed in respective genes at respective loci.
Research has shown that asthmatics showed distinctive methylation differences associated with the WNT signaling gene in neutrophilic asthma and purine and calcium metabolism gene in eosinophilic asthma. Hypermethylation of WNT2 genes in the region 7q31.2 from blood samples is associated with neutrophilic asthma [55]. Similarly, a distinctive and specific methylation profile was observed with FeNO, eosinophil count, and inhaled corticosteroid in asthmatic adults [56]. Hypermethylation of ORMDL3 from endobronchial airway epithelial cells at the genetic locus 17q12-21 results in asthma in adults [56]. Hypermethylation   3 Mediators of Inflammation of IL-13 from airway epithelial cells at location 5q31 is seen in asthma [57]. From these studies, we can conclude that a distinctive and specific methylation pattern is associated with asthma phenotypes and endotypes.
3.2. Environmental Exposures, DNA Methylation, and Asthma Risk. The environment is considered a significant influencer of DNA methylation and asthma. Maternal tobacco smoking, air pollution, stress, heavy metal exposures, pesticides, microbes, and possibly some foods are the epigenetic influencer. Therefore, the epigenetic mechanism, through the influence of environmental factors, affects the gene transcription and results in a more complex phenotypic pattern and presentation of asthma and allergy [59].
Prenatal and early childhood maternal tobacco smoking is considered a risk factor for asthma, and epigenome-wide studies have shown variations in DNA methylation in many genes involved in asthma [60] along with fetal lung tissue and placental DNA methylation [61]. Nicotine exposure results in the generational transmission of asthma, showing epigenetic changes and DNA methylation, and H3 and H4 acetylation [62]. A 2017 study has highlighted the three CpG sites of methylation by NO 2 from cord blood samples and a dose-dependent NO 2 connection with pregnancy [63]. Mixtures of gases and particulate matter are the main components of air pollution. Air pollution induces DNA methylation from an early age to old age [64]. Research has reported hypomethylation of iNOS by particulate matter 2.5 (PM2.5) and particulate matter 10 (PM10) [65], methylation of ACSL3 by traffic-related air pollution (TRAP) [66], and hypermethylation of IL-4 and IFN-γ CpG sites by exhaust particulate matter (DEP) [67]. DEP is associated with increased IgE levels in asthma [67]. Together, prenatal nicotine exposure, air pollution, PM2.5, and DEP induce epigenetic changes in genes through DNA methylation, resulting in distinct asthma phenotypes.
A study in 2019 showed that lead and cadmium are associated with asthma and other allergic conditions, and an interestingly significant degree of airflow obstruction is associated with the serum level of these metals [68]. Vanadium induces DNA methylation in air pollution-related asthma [69]. Occupational pesticide exposure and differential DNA methylation are correlated with symptomatic airflow obstruction [70].
Environmental microbial agents play an immunemodulating role under many conditions. The clinical asthma characteristics and airway microbiota have shown a significant correlation between asthma control and various pathogens [71]. Rhinovirus induces IL-33-dependent type 2  Mediators of Inflammation asthma exacerbations [72], and rhinovirus-induced DNA methylation changes on many genes are linked with asthma, especially the substantial changes seen on gene SMAD3 [73]. Ovalbumin (OVA) and house dust mites (HDM) are the most common allergen used in the mouse model of allergic asthma for the induction of the Th2 pathway. Other asthma models use paramyxovirus, Chlamydia muridarum, Rotavirus 1 (RV1), and Haemophilus influenzae for the induction of non-Th2 inflammation. HDM exposure has been found to induce DNA methylation of various potentially important genes for allergic asthma development [74]. In allergic rhinitis, a close entity in asthma, DNA hypomethylation of the IL-13 gene was observed after HDM sensitization [75]. DNA methylation changes in several genes (FOXO1, RUNX1, SP1, and APP) are potentially crucial for the development of asthma in the OVA model [76].
The role of diet, folate, vitamin B12, vitamin C, calcium, vitamin D, fish oil, and antibiotics in pregnancy and asthma has been established [77][78][79][80]. The association between dietary intake and childhood asthma has identified that dietary intake contributes to DNA methylation [77]. The nutrients involved in the one-carbon metabolism pathway (selenium and several others) are associated with improved asthma status, but some dietary constituents are associated with both extensive and gene-specific methylation in pediatric asthma [77]. Novel epigenetic loci associated with folate and vitamin B12 have been noted in a large-scale epigenome-wide study in 2019, and a negative association between folate and DNA methylation was found [78]. These data show that dietary intake and some nutrients and vitamins are responsible for methylation changes in asthma-related genes and result in different phenotypes as well as remission of asthma.

Histone Modifications
Histones are a highly alkaline principal fundamental protein component of chromatin that acts as a spool for DNA winding. The unwound DNA length to width ratio would be beyond 10 million to 1. Therefore, it is considered a crucial element for genetic stability, packaging, and gene expression.
Histone modification, an epigenetic mechanism, is an influencing factor in the pathogenesis and development of  Figure 2: The role of epigenetics in the pathogenesis of asthma. An epigenetic mechanism links genetic and environmental factors with developmental trajectories in asthma. The most commonly studied asthma epigenetic phenomena are DNA methylation, histone modifications, and miRNAs. DNA methylation is the process of adding methyl groups, by DNA methyltransferase, on cytosine at position 5 with the formation of 5-methylcytosine [18] where guanine nucleotide follows the cytosine nucleotide known as CpG [19]. The methylation of CpG islands (clusters of CpG) results in gene activation or inhibition, but usually repression, because the islands are found almost near the genes' transcription start site (TSS) [23]. Histone modification usually occurs at N-terminal with possible modifications on each "basic" residue, but common residue targets for modifications are lysine, serine, arginine, and tyrosine threonine. Acetylation, methylation, phosphorylation, ubiquitination, and sumoylation are well-known histone modification mechanisms. Histone acetylation and histone methylation are the most studied and better known to us [82]. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) work in opposition to each other as acetylation by HATs favors gene expression and deacetylation by HDACs is responsible for gene silencing. The addition of phosphate, i.e., phosphorylation, is mediated by kinases, while phosphatases remove the phosphate. Similarly, histone ubiquitination is mediated by ubiquitin ligases and opposed by ubiquitin-specific peptidases, also known as deubiquitinating enzymes. Histone sumoylation is mediated by the histone sumoylation proteins, also known as the small ubiquitin-like modifier (SUMO) protein. A microRNA (miRNA) is a 22-25 nucleotide single-stranded small noncoding RNA molecule transcribed from the DNA. However, miRNAs are not translated into proteins and play a role in gene expression either by blocking or by altering mRNA translation stability [86]. Alteration in epigenetic status results in differential gene expression related to cytokines and transcription factors, resulting in various and distinct phenotypic presentations in asthma. Some T cell subsets and cytokine production and functions are also shown in the figure. miRNAs: micro-RNAs; Th cells: T helper cells.  Table 2). Histone modification usually occurs at the N-terminal with possible modifications on each "basic" residue, but common residue targets for modifications are lysine, serine, arginine, and tyrosine threonine. Acetylation, methylation, phosphorylation, ubiquitination, and sumoylation are well-known histone modification mechanisms. Histone acetylation and histone methylation are the most studied and are better documented [82].
Allfrey et al. have reported the first documented case of histone acetylation in 1964 from calf thymus [83], and the link between chromatin and histone acetylation was published in 1988 [84]. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) function in opposition to each other as acetylation by HATs favors gene expression and deacetylation by HDACs is responsible for gene silencing. The imbalance of HDACs and HATs is the basics for impaired gene expression and a factor that contributes to asthma [16]. The subgroups of HATs and HDACs are beyond the scope of this review and are not explained here. The addition of mono-, di-, or trimethyl groups to lysine or arginine residues of histone tails favors histone methylation through the mediator histone methyltransferases (HMTs), while histone demethylases (HDMs) oppose HMTs. Enzymes of HMTs ("writers") and HDMs ("erasers") collectively balance a dynamic and static histone methyl landscape.
The addition of phosphate, phosphorylation, is mediated by kinases, while phosphatases remove phosphate. Similarly, histone ubiquitination is mediated by ubiquitin ligases and opposed by ubiquitin-specific peptidases, also known as deubiquitinating enzymes. Histone sumoylation is mediated by the histone sumoylation proteins, also known as the small ubiquitin-like modifier (SUMO) protein. Interestingly, although the biological process of DNA methylation and histone methylation differs, they mutually interact with each other [85].
4.1. Histone Modifications and Asthma. HDAC2 reduction signifies the possibility that the poor effectiveness of corticosteroids and inflammatory genes may be regulated by deace-tylation of the glucocorticoid receptor through HDAC2 [87]. Activation of the Notch gene is crucial for Th2 response in asthma, and Notch 1 dysregulated signaling of T cells is observed through the hyperacetylation of H3K9, H3K14, H3K18, H3K27, and H3K16 and trimethylation of H3K4 and H3K79 [88]. Suppression of Notch1 expression by HAT inhibitors reduced Th2 cytokines, especially IL-4, IL-5, and IL-13 [88], and it may provide a therapeutic alternative for asthma. LAT is a protein-coding gene of T cells, and its deficiency enhances Th2 proliferation. LAT hypoacetylation results in the inhibition of LAT in an allergic asthma model [89]. Self-renewal and reprogramming of somatic cells are mediated by SOX2, and HDAC1 mediates the remodeling of the asthmatic epithelium, while its inhibition prevents airway remodeling through SOX2 [90].
Increased expression levels of IFN-γ, IL-4, IL-17A, IL-17F, and transcriptional factors of Th17 and Treg cells (RORγt and Foxp3) have been observed with H3K4 trimethylation (H3K4me3) [91]. Foxp3 and IL-13 gene acetylation is associated with childhood asthma [92]. Histone hyperacetylation of ORMDL3 is associated with asthma [93], and increased ORMDL3 expression is associated with airway remodeling and Ca 2+ oscillations. EGFR plays an important role in the growth and maintenance of airway epithelium. Delta Np63 induces epithelial shedding, and STAT6 is involved in chronic inflammation of the airway mucosa. TSSs of EGFR, STAT6, and Delta Np63 show increased acetylation of H3K18 in the airway epithelium of asthmatics [94].
Chemokine ligand-8, a neutrophil activator produced by macrophages, and H3K18 acetylation leads to increased secretion of this activator from airway smooth muscles [95]. CCR4 is a mediator for Th2 cell recruitment, and CCL5 is a leukocyte chemoattractant found to be high in asthma. CCR4 and CCL5 dimethylation (H3K4me2) at single nucleotide polymorphisms is associated with Th2 differentiation [96].   Mediators of Inflammation transmission of asthma in subsequent generations has been studied in an animal model [62,97]. HDAC2 downregulation results in the upregulation of IL-8, TNF, and GM-CSF and glucocorticoid inhibition in smokers [98]. A study on maternal E-cigarette exposure has also showed epigenetic and cognitive changes in offspring [99]. Interestingly, a multigeneration analysis of the Respiratory Health study showed that the father's environmental exposures before conception can influence offspring's respiratory health in later life [100]. There is stronger asthma morbidity in children than adolescents [101], and histone H3 modification at birth is associated with prenatal air pollution, which is a risk factor for later-life air pollution-associated diseases [102]. The association of traffic-derived particulate matter, mainly black carbon, and global histone H3 modification has been established [103]. Environmental microbes may play roles in the immune response's modulation of immune responses. Histone H4 acetylation of essential Th2 genes is modified by nematode infections, such as ascariasis and HDM [104]. Interestingly, Acinetobacter lwoffii F78 administration to experimental mice during the prenatal period has shown beneficial effects and prevented the asthma phenotype. In this case, H4 acetylation of IFN-γ and deacetylation of IL-4 promoter genes result in the downregulation of Th2 cytokines, showing transmaternal asthma protection [105]. Gut microbiota inhibits HDACs, resulting in Th1/Th17 effector cell polarization along with global hyperacetylation of intestinal macrophages, which produce butyrate and short-chain fatty acids that inhibit the proliferation and differentiation of Th2 cytokines in mice [106,107]. Interestingly, in our OVA asthma model, alanylglutamine reduced cytokine production and relieved asthma symptoms by regulating gut microbiota composition [108].
Nickel, a silver-colored metal found in a large amount in some grains, vegetables, and fruits, can cause allergies in susceptible people and are associated with asthma and wheezing [109]. Nickel-induced posttranscriptional modification continues even after exposure through an epigenetic mechanism [110], while nickel exposure with hypoxic insult enhances H3K9me2 and H3K9me3 by inhibiting HDMs [111]. Interestingly, it can induce histone ubiquitination. Folate is an essential component for the repair and synthesis of DNA and other genetic materials. GATA3 (H3/H4) and IL-9 (H4) acetylation was noted with very high maternal folate levels in CD4+ T cells from cord blood [112]. GATA3 is a transcription factor for Th2 cells, and IL-9 is produced by Th9 and Th2 cells. High folate levels during pregnancy indicate the possibility of enhancement of Th2 promoter genes [112].

miRNAs (Noncoding RNA)
In 1993, Victor Ambros' laboratory discovered miRNAs and simultaneously identified the miRNA target gene. miRNAs are approximately 22-25 nucleotide single-stranded small noncoding RNA molecules transcribed from the DNA; however, they are still not translated into proteins and have a role in gene expression either by blocking or by altering mRNA translation stability [86]. miRNAs bind to the 3 ′ untranslated region of the gene with complementary sequence interaction and repress its expression.

miRNAs and Asthma.
Although the discovery of miRNA is approaching thirty years, its role in asthma became a topic of investigation in the last decade. Many mouse and human studies have been conducted to identify the role of micro-RNAs via epigenetic changes in genes, and altered miRNA expression has been found in various lung diseases, including asthma [113,114] (Figure 2 and Table 3). In the following paragraphs, we review the epigenetic changes in asthma genes through miRNAs, resulting in various phenotypic presentations and remission of asthma along with the possibility of diagnostic biomarkers and therapeutic potential from different studies.
A recent study published in 2020 has shown that circulatory miRNA levels correlate with the clinical status in patients with allergic and nonallergic asthma [115], and circulatory miRNA expression with T cell cytokines characterizes asthma exacerbation [116]. Some studies have even postulated that miRNAs can be considered a noninvasive biomarker in the diagnosis and identification of allergic asthma [117]. miR-21 prevents the expression of IL-3, IL-5, and IL-12, while the removal of miR-21 induces the production of IFN-γ and IL-12 from dendritic cells with reduced IFN-γ production from CD4+ T cells [118]. miR-21 controls type 1/type 2 balance in steroid-insensitive, OVA, and HDM model type 2 high asthma and is highly expressed in asthmatic children, with an inverse association between IL-p35 and miR-21 [118][119][120]. A study published in 2020 has shown that LncRNA-CASC7 enhances corticosteroid sensitivity through miR-21 by inhibiting the PI3K/AKT pathway and highlighted a potential therapeutic option in severe asthma, and it may also serve as a predictor of inhaled corticosteroid treatment response over time in asthma [141]. Th2 cytokines, especially IL-5 and IL-13, are decreased by miR-146a on ILC2 by inhibiting IRAK1 [121], and miR-146a also alters neutrophil migration from bronchial epithelial cells in asthma [122]. Similarly, the let-7 family of miRNAs found exclusively in the asthmatic lungs modulates the IL-13 expression, and its inhibition inhibited the production of allergic cytokines [123]. Together, these studies suggest the role of miRNAs in type 1/type 2, steroid-insensitive asthma and guide the prediction of asthma and the possibility of future therapy.
The increased expression level of miR-1248 is believed to be involved in the pathogenesis of asthma. miR-1248 increases IL-5 expression by interacting with the 3 ′ UTR of the gene [124]. miR-126 promotes Th2 eosinophilic asthma, and antagomir-induced inhibition of miR-126 suppresses eosinophil recruitment [125]. Upregulated miR-126 in pediatric asthma correlates with immune imbalance and is postulated as a possible serum marker in the diagnosis and management of asthma [142]. miR-155 is the first identified and most studied miRNA to be linked to various diseases, including allergy and asthma. A Th2 association is seen with miR-221 and miR-155 along with other cells involved in allergic responses, such as mast cells [143], eosinophils 7 Mediators of Inflammation [126], and macrophages. However, it has recently been found that airway mir-155 also signifies Th1 cytokine polarization with viral respiratory infections [127]. Similarly, a study in 2020 explained that miR-1 is a direct inhibitor of eosinophilic response in asthma and chronic rhinosinusitis [128]. The miR-23-27-24 cluster controls T cell functions and differentiation, and miR-24 and miR-27 inhibit Th2 differentiation, resulting in inhibited IL-4 [130]. These observations signify the role of miRNAs in allergic, eosinophilic, and virus exacerbated asthma, as well as serum biomarkers in the diagnosis and prediction of pediatric asthma.
In asthma, miR-16 and lung function parameters are negatively correlated, and inhaled β2-agonist responses are altered by miR-16 through ADRB2 [131]. Circular RNA (cir-cHIPK3) is involved in the proliferation of smooth muscles and airway remodeling in asthma through the miR-326/STIM1 pathway [132]. Pulmonary macrophage polarization is mediated by miR-130a-3p and miR-142-5p and is associated with airway remodeling [144]. The Th2 cytokine level increases with increased airway expression of miR-19a through PTEN and deubiquitinase A20 [133], and exaggerated remodeling of the airway is noted with reduced miR-19a [134]. VEGF-A is overexpressed in sputum and serum of asthmatic patients, and lower expression of has-miR-15a is related to the increased expression of VEGF-A in CD4+ T cells [135]. Airway inflammation and remodeling are reduced by miR-19b through TSLP mediated STAT3 inhibition [136] and MMP-16 and ATG7 by miR-192-5p [137]. This mechanism may provide a therapeutic target for airway remodeling of asthma. Downregulation of miR-1 in the endothelium results in Th2 inflammation via VEGF, with increased expression of myeloproliferative leukemia virus oncogene [129]. Thus, research has shown the role of multiple miRNAs in airway inflammation and remodeling. miR-27-3p is an important player in experimental pediatric asthma that mediates immune reactions. SYK and EGFR genes are targets of miR-27-3p that influence the PI3K-AKT pathway and cytokine production [138]. An inverse correlation between IL-22 and expression of miR-323-3p has shown enhanced expression of miR-323-3p in IL-22 and IL-17 expressing cells with decreased IL-22 production and TGF-β suppression [139]. hsa-miR-20a-5p induces allergic inflammation by preventing HDAC4 expression and epigenetically upregulating IL-10 in activated human mast cell 1 [140].
Taken together, these studies have shown the role of multiple miRNAs in Th1/Th2 polarization, disease presentation, correlation of clinical status, severity, remission, noninvasive biomarker, therapeutic potential, and response to therapy in asthma.

Environmental Exposures, miRNAs, and Asthma Risk.
Some in vivo and in vitro studies have identified an association between environmental factors, miRNAs, and asthma. Different types of allergens, such as cigarette smoking, air pollution, agriculture pesticides, metals, and various microbes, induce epigenetic changes in genes through the miRNA and show various phenotypic presentations and even remission of asthma. This section reviews the association between environmental factors, miRNAs, and asthma. However, some of the associations have already been described in miRNAs and Asthma.
OVA induces experimental asthma by downregulating let-7 miRNAs and IL-13 expression [123]. Exogenous administration of let-7 miRNA results in reduced eosinophil recruitment [123]. HDM is an important triggering factor for asthma and is responsible for lung-specific miRNA expression. HDM triggers IL-33 release, which activates miR-20a-5p Allergic inflammation/asthma [140] 8 Mediators of Inflammation and proliferates ILC2, which ultimately releases Th2 cytokines, fulfilling the clinical parameter of allergic asthma. miR-155 knockout HDM sensitized mice displaced low levels of IL-33 and ILC2, suggesting that HDM induces asthma through the critical role of miR-155 [145]. An allergen mix, containing dust mite, ragweed, and aspergillus (DRA allergen), reduces the miR-451 levels in pulmonary macrophages, and depletion of miR-451 results in asthma through the expression of sirtuin 2 [146]. Interestingly, IgE is involved in the pathogenesis of asthma-related hypotension through NCX1 downregulation and miR-212-5p activation, as shown by the significant restoration of blood pressure after the knockdown of miR-212-5p in asthmatic mice [147]. OVA, HDM, and DRA allergen are involved in the pathogenesis of asthma through the epigenetic changes in miRNAs. miR-335-5p inhibits inflammatory cytokines, and cigarette smoking lowers the expression of miR-335-5p in parenchymal lung fibroblast [148]. The upregulation of miR-500 and miR-181 and downregulation of miR-128b and miR-218 are seen in the airway epithelium of smokers [149], suggesting a possible association with cigarette smoke. Environmental tobacco smoke (ETS) during pregnancy promotes epigenetic changes and transgenerational transmission of allergic asthma with increased miR-221 and miR-16 levels and reduced levels of miR-130a [131,150]. Maternal smoking increases AXL methylation and reduces miR-199a expression, resulting in the alteration of childhood respiratory symptoms [151]. Together, these studies have shown an association between smoking and miRNAs in childhood asthma.
The association between serum levels of heavy metals, such as cadmium, mercury, and lead, and asthma and allergic rhinitis has been established. miR-148a, miR-211, miR-520c-3p, and miR-572 are associated with lead poisoning and are biomarkers of lead susceptibility in the Chinese worker population [159]. miR-211 inhibits the TGF-β path-way via inhibin-β A [160]. Cadmium downregulates miR-30 through the upregulation of the transcriptional factor SNAIL and can be considered an important step in some lung diseases [161]. miRNA-30a-3p reduces eosinophil activity in asthma by targeting CCR3 [150,161]. Occupational exposure to mercury upregulated miR-92a-3p and miR-486-5p in females working in a mercury thermometer factory [162]. These studies have shown an association between environmental occupational exposure in asthma and the expression of multiple miRNAs.

Is There an Epigenetic Link between
Childhood Asthma and Adult-Onset/Old Age Asthma?
Asthma predominantly occurring during childhood is childhood asthma, while asthma occurring during adulthood and old age is termed adult-onset/old age asthma. The T2 phenotypes of asthma (T2 high) include early-onset allergic and late-onset nonallergic eosinophilic asthma. The common Th2 biomarkers used in clinical practice are mainly blood eosinophils, FeNO, and IgE levels. Interestingly, the majority of people with T2 asthma respond well to standard therapy with inhaled corticosteroids [4], whereas non-T2 (T2 low) asthma is still obscure, is a neutrophilic and paucigranulocytic heterogenous type predominant with adult-onset, is associated with corticosteroid resistance (less responsive), and is inflammation-driven through IL-17, IL-6, and IL-23 with airway smooth muscle or neural dysfunction and may be associated with comorbidities like obesity and gastroesophageal reflux disease [5,6]. However, we should not forget that these profiles are not strictly age-limited (children and adults may have eosinophilic, neutrophilic, or even paucigranulocytic asthma with overlapping steroid response), and specific and useful signature biomarkers are lacking for non-T2 asthma apart from the absence of T2 high inflammation. Together, these observations suggest the two phases of asthma in human life as childhood asthma and adult-onset/old age asthma.
Environmental factors are asthma epigenetic influencers that induce epigenetic changes in genes through DNA 9 Mediators of Inflammation methylation, histone modifications, and miRNAs, as reviewed in Sections 3.2, 4.2, and 5.2, respectively. Some environmental factors affect childhood asthma, while others are involved in the pathogenesis or remission of adult-onset/old age asthma through epigenetic changes; however, some overlap still exists in both. Table 4 shows the environmental exposures and the respiratory outcomes relating to childhood and adult-onset/old age asthma, including a study from an animal asthma model.
Maternal tobacco smoking, in utero nicotine exposure, maternal E-cigarette exposure, and even secondhand smoke (ETS) during pregnancy are associated with increased risk and rate of childhood asthma, elevated IgE levels, wheezing, alteration of childhood respiratory symptoms, and increased bronchial activity [57,97,120,148,149]. Prenatal air pollution is a risk factor for childhood asthma and a risk factor for later-life air pollution-related diseases [100]. Similarly, indoor air pollution aggravates childhood asthma [150]. Viral respiratory infections in children exacerbate asthma and increase Th1 polarization, while paramyxovirus, Chlamydia muridarum, Rotavirus 1, and Haemophilus influenzae induce non-Th2 inflammation [69,71,72,129]. Nutrients, such as folate (methyl group donors), vitamin B12, vitamin D, and selenium (one-carbon metabolism pathway), play important roles in childhood asthma pathogenesis. There are many conflicting studies on childhood asthma risk with maternal folate levels. One study has shown that high folate levels during pregnancy are associated with the possibility of enhanced Th2 promoter genes [110]. Interestingly, Acinetobacter lwoffii F78 prenatal administration prevents childhood asthma in an animal model [103]. Together, these environmental exposures induce epigenetic changes and play a role in the pathogenesis or remission of childhood asthma.
Multiple environmental factors are also associated with adult-onset/old age asthma. Occupational exposure to lead, cadmium, mercury, nickel, and pesticides results in asthma and symptomatic airflow obstruction in adults [66,68]. Rhinovirus and ascariasis are responsible for type 2 asthma and exacerbations in adults [70,102]. Genetic predisposition (asthmatic parents), mode of delivery, obesity, hormonal level, and hygiene are also related to childhood asthma as well as adult-onset/old age asthma.
Overall, human asthma studies and experimental asthma models have shown the involvement of various environmental factors in the pathogenesis of childhood and adult-onset/old age asthma [65, 103-106, 140, 152, 154] by inducing epigenetic changes through the genes (Sections 3.2, 4.2, and 5.2) and resulting in various phenotypic presentations of childhood and adult-onset/old age asthma.
Is there a link between childhood asthma and adultonset/old age asthma? Does adult-onset/old age asthma represent the persistence or relapse of childhood asthma? How does increased IgE, eosinophil-predominant, and steroidsensitive T2 asthma change into neutrophilic/paucigranulocytic, steroid-insensitive non-T2 severe asthma with age?

Conclusion and Perspectives
Asthma is a mysterious disease with heterogeneity in etiology, pathogenesis, and clinical phenotypes under genetic influences. Asthma heritability is established through genetic studies, and environmental factors are also influential in  Figure 3: The two peaks of human asthma and the role of epigenetics in the cellular transformation of childhood asthma to adult-onset/old age asthma. (a) The two peaks of human asthma are childhood asthma (↑ IgE, ↑ eosinophils, FeNO, corticosteroid sensitive, and T2 high) and adult-onset/old age asthma (neutrophilic/paucigranulocytic, corticosteroid less sensitive, and non-T2 asthma) with airway smooth muscle or neural dysfunction, possible association with comorbidities [5,6], and periods of remission in some cases. However, we should not forget that these profiles are not strictly age-limited (children and adults may have eosinophilic, neutrophilic, or even paucigranulocytic asthma with overlapping steroid response), and several other components of the immune, neuronal, and hormonal responses are also involved with asthma. (b) The two peaks of asthma (childhood asthma and adulthood as adult-onset/old age asthma). In general, there is male dominance in childhood asthma, with a shift to female dominance in later peak. Components of epigenetic mechanisms, as well as cells involved in asthma, are also shown, as their interaction is needed in asthma epigenetics. The long arrows in (a) show the possible point of commencement and involvement of epigenetic mechanisms, as we believe, in the cellular transformation of childhood asthma to adult-onset/old age asthma, although further epigenetic research is required in the future. IgE: immunoglobulin E; miRNAs: micro-RNAs; FeNO: fractional exhaled nitric oxide. 11 Mediators of Inflammation asthma. However, genetic and environmental factors cannot entirely explain the asthma endotypes and phenotypes.
Emerging evidence suggests that epigenetic mechanisms link genetic and environmental factors with asthma trajectories. Epigenetics is defined as the study of heritable phenotypic changes due to activation, inhibition, or repression of genes without changes in the underlying DNA environment. The most studied epigenetic asthma phenomena are DNA methylation, histone modifications, and small noncoding RNA (miRNAs).
Asthma epigenetics play an important role in immune response and upregulation of various cellular functions, including T cell differentiation, Th cell balance, changes in the expression of inflammatory genes resulting in asthma, and remission or protection in some cases. Asthma epigenetic studies can be a valuable approach to identify the increasing incidence of asthma and equally crucial for the study of environmental factors involved in asthma pathogenesis. Although more clinical, epidemiological, environmental, cellular, and interventional in vivo, in vitro, and human studies are needed, we believe that the asthma epigenetic mechanism can be a novel process for the identification of unsolved pathogenesis, new asthmatic loci, heterogeneity, and treatment options through the discovery of new drugs. Identification of epigenetic markers is vital for identifying asthma endotypes, phenotypes, personalized treatments, and prevention. In the future, we expect that the study of asthma epigenetics will guide us towards gene therapy, T cell therapy, and even provide directions for treating severe uncontrolled drug-insensitive asthma and predict longterm outcomes.
Given the queries raised, we believe that the epigenetic mechanisms play vital roles in the cellular transformation of childhood asthma (eosinophil-predominant, steroidsensitive T2 asthma) to adult-onset/old age asthma (neutrophilic/paucigranulocytic, steroid less-sensitive non-T2 severe asthma). However, we expect further epigenetic studies on this aspect in the future.
Although epigenetic studies of asthma are still at early stages, we hope that the mystery surrounding asthma will be solved to some extent via the integration of omics data via novel findings for improved phenotyping, diagnoses, and asthma treatments, as well as the mechanism that will eventually prevent its development. Together, in view of all asthma epigenetic studies, we can conclude that asthma epigenetics are a valuable, novel approach to solve the mysteries surrounding asthma and will guide us to the discovery of new therapeutic agents and prediction of long-term outcomes in severe cases and aid in the understanding of the cellular transformation of childhood asthma to adult-onset/old age asthma.

Additional Points
Points to Remember. (i) Genetic and environmental factors are unable to completely explain asthma pathogenesis and phenotypes. (ii) Asthma epigenetics has become a "hot topic" due to the search for alternative explanations for mysterious phenotypes and heterogeneity of asthma. (iii) DNA methylation, histone modifications, and miRNAs are the primary components of asthma epigenetics. (iv) Epigenetic mechanism links genetic and environmental factors with developmental trajectories and plays an important role in T cell differentiation, while influencing the Th cell balance, and epigenetic marks can be inherited to subsequent generations.
(v) Asthma epigenetics may provide a novel mechanism for identifying the increased incidence, pathogenesis, and heterogeneity of asthma and a novel mechanism to identifying new asthmatic loci, associated environmental factors, and treatment options by the discovery of new drugs. (vi) In the future, asthma epigenetics will direct us to T cell therapy, gene therapy, and personalized asthma therapy and will give long-term predictability in severe cases.
(vii) We believe that asthma epigenetics plays a vital role in the cellular transformation of childhood asthma to adultonset/old age asthma. However, further research must be conducted in this field. (viii) Although the asthma epigenetic field is very promising and is still at early stages, we hope that the mystery surrounding asthma will be explained shortly and ultimately lead us to improved phenotyping, diagnosing, and treating of asthma.