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.