5. The genetic and epigenetic landscapes of the epithelium in asthma:
Genetic and environmental risk factors play important roles in the development of asthma. The interaction between those triggers and susceptible genetic factors can affect the epigenetic status of AECs [11]. This may result in functional and morphological remodeling of the airway epithelium causing distinct phenotypes of asthma [5]. Epigenetic factors that regulate the structure and function of airway epithelium is an attractive area for assessing asthma susceptibility with a focus on DNA methylation changes. Genome-wide association studies (GWAS) and whole genome sequencing (WGS) have provided evidence for the contribution of AECs in the development of asthma in addition to detecting genes related to asthma susceptibility. Elucidating the genetic and epigenetic landscape of epithelial cells in asthmatics may provide a scientific basis for further potential markers for the diagnosis and treatment of asthma.
5.1. Asthma susceptibility genes in airway epithelium :
There is little overlap in asthma susceptibility genes identified using different approaches. Early genetic studies on asthma-related genes identified several genes expressed in AECs including A disintegrin and metalloprotease 33 (ADAM33), the G protein-coupled receptor GPRA, protocadherin-1 (PCDH1), serine protease inhibitor Kazal type-5 (SPINK5), IL-1 receptor associated kinase-M (IRAKM), dipeptidyl-peptidase 10 (DPP10) and HLA-G [5]. Methodological advancements resulted in the detection of a completely different set of genes as asthma susceptibility genes expressed in airway epithelium such as IL-1 receptor-like 1 (IL1RL1) and IL18 receptor 1 (IL18R1), IL33, HLA-DQ, SMAD3, TSLP, ORM1-like 3 (ORMDL3) and gasdermin B (GSDMB) [5]. Collectively these genes are important in AEC damage, innate and adaptive immunity and airway inflammation. Furthermore, some of these gene products such as IL-33 and IL-18 can determine the phenotype of asthma [80, 81]. In previous gene expression and RT-qPCR studies, three genes were identified as being highly induced by IL-13 in AECs from subjects with asthma: POSTN, CLCA1, and SERPINB2. These genes are considered markers of T2 inflammation and are overexpressed in a specific subset of patients with asthma [82]. Related studies have shown that IRAKM, PCDH1, ORMDL3/GSDMB, IL-33, CDHR3 and CST1 are expressed by AECs. IRAKM may represent a potential biomarker for early onset of asthma [83] whereas PCDH1 may be a potential biomarker of asthma in both children and adults [84]. ORMDL3/GSDMB is more suitable for predicting asthma risk in children [85] and CDHR3 is associated with asthma in children with severe exacerbations. Finally, CST1 can differentiate asthmatics with exercise-induced bronchoconstriction (EIB) from those without EIB [86, 87].
5.2. Epigenetic regulatory factors in airway epithelium :
Environmental challenges can affect gene expression through three main mechanisms epigenetic mechanisms: DNA modifications, histone modifications and non-coding RNAs [5].
5.2.1. DNA modifications
Compared to healthy control subjects, a variety of asthma-associated genes linked to immunity were differentially methylated in nasal epithelial cells of atopic asthmatic children includingIFNGR2, HLKA-DPA1, LAG3, NFIL3, PRF1, TNFSF13. Other differentially methylated promoters highlighted genes involved in epigenetic regulation (ATXN7L1, H1F0, HIST1H1D, METTL1), airway obstruction (GABRG3) and obesity (C1QTNF1, GPC4) [88]. A number of DNA methylation signatures have also been identified in asthmatic AECs including cytokeratin 5 (KRT5) [89], signal transducer and activator of transcription 5A (STAT5A) [89], cysteine-rich protein 1 (CRIP1) [89], arginase2 (ARG2) [90], and so on.
5.2.2. Histone modifications
AECs in adult asthmatics express increased levels of histone H3 lysine 18 (H3K18) acetylation and histone H3 lysine 9 trimethylation (H3K9me3) [91] H3K18 acetylation increases the expression of ΔNp63, EGFR and STAT6, which are known to be altered in the epithelium of asthmatics [91]. In addition, the degree of acetylation of lysine 27 on histone 3 (H3K27ac; an active promoter and enhancer mark) is closely related to genes linked with T2high asthma [82].
5.2.3. Non-coding RNAs
A number of classes of non-coding RNAs exist in mammalian cells including long non-coding RNAs (lncRNAs), piwi-interacting RNAs (piRNAs), and miRNAs [5]. Among them, miRNAs are proposed to control the expression of 30–60% [92] of human genes and are closely related to the occurrence and development of asthma. Martinez-Nunez and colleagues [93] found that microRNAs-18a, -27a, -128 and -155 were down-regulated in asthmatic bronchial epithelial cells compared to cells from healthy donors. These miRNAs have an inhibitory effect on IL-8 and IL-6 gene expression. miR-19a is currently the only miRNA that differentiates severe from mild asthma [94]. It is up-regulated in severe asthmatic epithelial cells and further stimulates cell proliferation of epithelial cells by targeting TGF-β receptor 2 mRNA. Other miRNAs expressed in AEC with a potential role in asthma development include the miR-34/449 family and the miR-17 family [5]. Differentially expressed miRNAs in asthmatic AECs may be used as a potential biomarker for the ”endotype” classification of asthma and as such the miR-34/449 family is considered to be related to T2 asthma [95]. Interestingly, no clear relationship was observed between these differentially expressed miRNA and serum IgE level in asthmatics [96]. Furthermore, inhaled corticosteroids only had minor effects on miRNA expression and failed to restore miRNA levels to those seen in healthy control subjects [96].
Tissue compartments for biomarker assessment:
Various compartments can be used for the assessment of biomarkers and each compartment has its own strengths and weaknesses. Using the blood to identify biomarkers is minimally invasive (the procedure can be painful and difficult in some patients) and easy to realize in the clinical setting and requires minimal patient effort. In addition, blood can be collected across the age spectrum and is a cost-effective sample type [97]. However, peripheral blood does not necessarily reflect airway biology or provide disease-relevant mechanistic insight.
EBC has the advantage of being noninvasive and can offer real-time monitoring, simplicity and repeatability. However, there are also limitations, such as the lack of unified standards for the selection of biological indicators, collection methods and collection time window, the sensitivity of the detection reagent, and the inability to conduct anatomical localization of the gas passage. Nowadays, EBC is mainly used for research purposes and is not widely used in clinical practice. Studies have shown that leukotriene (LT) B4 and 8-isoprostane reflect airway inflammation and oxidative stress [98]. Horvath and co-workers [99] published a European Respiratory Society technical standard that provides technical norms and recommendations for the collection and analysis of EBC samples. Overall, EBCs have the potential to provide useful airway-associated information to enable the detection of disease progression and therapeutic efficay. The analysis of volatile organic compounds (VOCs) using electronic noses and/or mass spectrometry may become an increasingly important noninvasive means of assessing AEC function over time in all patients with asthma [100]. In recent years, induced sputum analysis has become a more common noninvasive method to evaluate airway inflammation in respiratory diseases. The safety and tolerability of sputum induction accounts for its popularity, whilst the technique has been standardized to improve the quality and reproducibility of specimens. However, due to the time and cost of sputum induction and the failure to achieve a suitable sample in every subject, its wide application in clinical practice is limited. More importantly, although the short-term reproducibility of induced sputum cell analysis appears to be good [101], inflammatory phenotypes are unstable and can change spontaneously or with changes in treatment, and a single induced sputum test cannot reliably predict persistent airway inflammatory phenotypes [102].
Bronchoalveolar lavage is an invasive technique with poor patient compliance, especially for critically ill patients, and it is difficult to accept repeated invasive examinations. Therefore, the application of this method in clinical practice is limited.
The nasal mucosa is a readily accessible site for the study of inflammatory processes. The fluid from nasal washings can reflect the intensity of the inflammatory process and provide a parallel between symptoms of the upper and lower respiratory tracts [103]. However, nasal epithelial cells and washes are not exactly the same as those from the airway of asthmatics, and there are differences in epithelial cell states and immune cell composition [104]. Further comparisons may reveal which aspects of nasal epithelial cell function provide insight into processes deeper within the airway.
Among the candidate biomarkers mentioned above there are various sampling compartments that may be used in future clinical applications. Reduced ezrin expression is detected in both EBC and serum of asthma patients [70, 72] and elevated levels of claudin 4 [105] and YKL40 are detected in asthmatic blood [77-79]. Epithelial brushings from patients with asthma have significantly lower claudin18 levels than healthy controls [106]. CCSP16 [67, 68], periostin [55-59], osteopontin [107-109], IL-33 [34, 110, 111], IL-25 [112, 113] and fibrinogen [114, 115] can all be measured in the sputum and blood of asthma patients. Except for CCSP16, the expression of all of these biomarkers is increased in asthma. In contrast, the levels of CCSP16 in asthmatic sputum and BAL are elevated compared to controls but levels are reduced in asthmatic blood. In addition, serum periostin is far less predictive of T2 asthma than sputum periostin [62]. TSLP [110, 116, 117] and osteopontin [118] expression levels are elevated in nasal secretions and in serum whilst that of MMP-9 [119, 120] is elevated in EBC and sputum. Reduced Sec14l3 [121] expression has only been studied in the BAL and lung tissue of asthmatic mice.