Since the pathogenesis of atopic dermatitis could not be explained only by a population genetic and phenotypic profiles, epigenetic regulator factors have been considered. Epigenetics is the study of inherited changes in gene expression that are not related to changes in its nucleotide sequence. One of the main classical regulatory mechanisms in human cells is DNA methylation. It is not clear how permanent modifications caused by this process are and whether it is possible to affect them by changing the activity of enzymes that trigger remodeling reactions. In this chapter we analyze all recent studies in this field. We focus more on methylation of innate and adaptive immune factors, with an emphasis on T-lymphocyte genes such as CD3, CD4, and CD8.
- atopic dermatitis
- DNA methylation
- genome-wide methylation analysis
- immune system
Atopic dermatitis (AD) is a chronic recurrent inflammation of the skin, characterized by impairment of the epidermal barrier that entailing its further dysfunction. The predisposition to IgE-mediated hypertension contributes to such a malfunction, realized in sensitization to surrounding allergens . This pathology is also characterized by infiltration and accumulation of type 2 T helper cells (Th2) and eosinophils . Atopic dermatitis is a multifactorial disease. The main triggers are various genetically predetermined defects of the epidermal barrier and the immune system influenced by environment .
Thus, the study of tissues and cells transcriptome involved in the pathogenesis of the disease is one of the best options for detecting molecular signs of complex diseases such as AD . In one of these studies, it was found that the expression of a large number of genes which were responsible for terminal differentiation of keratinocytes was reduced in case of AD compared with healthy controls. These genes include filaggrin (
Loss of function mutations in the gene encoding
However, genetic changes solely do not fully shed light on the molecular mechanisms involved in the pathogenesis of AD. Therefore, epigenetic mechanisms involved in the genomic adaptation according to environmental conditions may possibly explain how environmental exposure affects the risk of allergy development.
Epigenetic mechanisms, in particular methylation, play a key role in immune regulation and are influenced by a variety of environmental factors leading to persistent molecular changes in genes. The methylation process involves the addition of a methyl group to the cytosine (C5 position; 5-methylcytosine, 5mC). DNA methylation occurs primarily in the context of CpG dinucleotides and is the main epigenetic modification involved in the regulation of chromatin structure and gene expression .
2. Targeted methylation studies
2.1 DNA methyltransferase studies
The main enzymes responsible for the methylation process in humans are DNA methyltransferases 1, 3a, and 3b (DNMT1, DNMT3a, and DNMT3b). It is generally accepted that DNMT3a and DNMT3b are
In the context of the AD study, only DNA methyltransferase-1 (DNMT-1), an enzyme that catalyzes the methylation of cytosine bases in CpG islands, has so far been considered. Nakamura et al. for the first time carried out an indirect assessment of methylation status in patients with atopic dermatitis by measuring the expression of messenger RNA (mRNA) of DNA methyltransferase-1 in peripheral blood mononuclear cells (PBMC) by quantitative RT-PCR. Although the expression level of
It is common knowledge that many local factors, such as skin impairment, play an important role in the development of AD [12, 13]. However, Th2-infiltration in response to penetration of allergens and production of cytokines by infiltrated cells (for example, IL-4 and IL-5) plays the key role in the development of IgE-mediated response and chronic inflammation involving eosinophils [14, 15, 16]. It is assumed that in these processes DNA hypomethylation contributes to the hyperreactivity of Th2 cells in response to allergens and, as a consequence, cytokine-mediated IgE production. It has also been suggested that IL-4-mediated IgE production in patients with high serum IgE levels is associated with DNA hypomethylation in B cells [16, 17]. In this study the decrease in
On the other hand, the lack of significant differences while comparing
methylation studies FCER1G
Based on the opinion that overexpression of the high-affinity IgE receptor on monocytes and dendritic cells contributes to the pathogenesis of AD, a group of scientists studied the epigenetic mechanism of deregulation of high-affinity IgE receptors – FcεRI .
Liang et al. measured the methylation level of total DNA of monocytes from 10 patients with AD and 10 healthy people from the control group. Bisulfite sequencing was used as the main method to determine the methylation status of the
Thus, total hypomethylation in CD14+ monocytes in patients with AD was revealed, as well as locus-specific hypomethylation of the
Atopic monocytes with high FcεRI levels are thought to play an important role in the pathogenesis of AD. This is due to the fact that monocytes carrying FcεRI can differentiate into inflammatory dendritic epidermal cells (IDECs), which intensify allergic inflammatory reactions in the skin by stimulating T cells, and are also involved in the transition to the chronic course of AD with a predominance of Th2 [19, 20]. In this study, it was shown for the first time that changes in the epigenetic regulation of the
TSLP methylation studies
Thymic stromal lymphopoietin (TSLP) plays an important role in maintaining T-cell homeostasis and, apparently, is of great importance in the development of allergic symptoms, especially in atopic dermatitis and asthma [21, 22]. Human TSLP is overexpressed in keratinocytes of patients with acute and chronic AD. However, the mechanism of such TSLP expression remains unclear. The question is whether TSLP expression is regulated by modification of aberrant DNA methylation of
It is known that the TSLP protein cannot be found in healthy skin, in skin lesions in patients with nickel contact dermatitis or in patients with disseminated lupus erythematosus, as well as in intact skin in patients with AD; however, increased levels of TSLP expression are observed in both acute and chronic atopic skin lesions [21, 24]. TSLP overexpression in keratinocytes can activate myeloid dendritic cells by enhancing the surface expression of CD54, CD80, CD83, CD86 molecules and MHC class II molecules on myeloid dendritic cells , which lead to Th2-inflammatory reactions .
Luo et al. measured the levels of mRNA and TSLP protein in samples of affected skin from 10 atopic children and 10 healthy people from the control group, using quantitative RT-PCR and immunohistochemistry . Bisulfite sequencing was performed to determine the methylation status of the
As a result, the levels of expression of mRNA and TSLP protein relative to β-actin were significantly higher in affected skin regions of patients with AD compared with healthy controls. In addition, hypomethylation of the promoter region of the
It can be concluded that DNA demethylation of the specific regulatory region of the
In another study, the authors tried to reveal the effect of prenatal smoking on DNA methylation in case of atopic disorders . Methylation differences associated with exposure to tobacco smoke were initially identified with the use of Illumina Infinium 27 K methylation kits in 14 children in a Taiwanese study cohort. Information on the course of the disease and possible risk factors was collected. Cord blood levels of cotinine were measured in order to represent prenatal smoking. CpG loci, in which statistically significant differences in methylation were found, were validated by methylation-dependent fragment separation (MDFS). Differential methylation in three genes (
Thus, it can be assumed that changes in
The results of Wang et al. study suggest that prenatal exposure to tobacco smoke is associated with a risk of atopic dermatitis, possibly through DNA methylation.
methylation studies MICAL3
Cho et al. conducted a research to assess the role of 25-hydroxyvitamin D (25[OH]D) deficiency in cord blood in comparison with postnatal 25[OH]D levels in AD development during the first 3 years of life and found out how 25[OH]D deficiency affects the DNA methylation profile of cord blood leukocytes .
Severe 25[OH]D deficiency in cord blood was associated with a higher risk of atopic dermatitis diagnosing precisely at the age from 2 to 3. Comparison of differentially methylated CpG sites in accordance with moderate and insufficient 25[OH]D levels in cord blood revealed the common
To reproduce the mechanism of atopic dermatitis associated with ROS, using the example of
methylation studies HBD-1
Noh et al. described patterns of DNA methylation of human β-defensin-1 (HBD-1), a unique antimicrobial peptide expressed in various tissues, including the skin . HBD-1 may be associated with a variety of innate immune system defects in the AD pathogenesis. A possible mechanism for the decrease in
To identify specific CpG sites that play a significant role in HBD-1 expression in NHEK cells, bisulfite genomic sequencing of the region upstream of the proximal site of the
Thus, epigenetic modulation of the
3. Genome-wide DNA methylation
3.1 In naive CD4+
Both atopic dermatitis and psoriasis are characterized by a targeted immune response via polarized CD4+ T cells. During the polarization of naive CD4+ T cells, DNA methylation plays an important role in the regulation of gene transcription. Taken into consideration the similarity of immune response of atopic dermatitis and psoriasis, Han et al. conducted a study of the global DNA methylation profile in naive CD4+ T cells in patients with AD and psoriasis, as well as in healthy people using the ChIP-seq method. DNA hypomethylation (more than 4 times) was found in T-cell samples isolated from patients with psoriasis and healthy people in 26 genome sites ranging in size from 10 to 70 kb. These regions were mostly pericentromeric on 10 different chromosomes and randomly overlaid with various defining epigenome signals, such as histone modifications and binding sites for transcription factors (according to the ENCODE project), which implied the potential influence of epigenetic regulation in the development of psoriasis .
To determine whether naive CD4+ T cells from patients with AD or psoriasis have DNA methylation patterns different from those of healthy people, complex genome-wide CpG methylation profiling was performed. The uniquely mapped regions coincided with strong histone modification signals such as H3K4Me1, H3K27Ac, and H3K4Me3, as well as with transcription factor binding sites in various cell lines.
It appears that hypomethylation in some pericentromeric regions of naive CD4+ T cells may be a sign of psoriasis, but not atopic dermatitis. It is not yet clear what exact role epigenetic changes of these regions play in the development of T cells. However, these data show for the first time the importance of such changes in the development of immune-mediated skin diseases .
The X chromosome encodes many of immune genes, which show a higher hypermethylation pattern than other genes. It is known that abnormalities, such as inactivation of the X chromosome, can contribute to the impairment of self-structures recognition and, ultimately, lead to autoimmunity . In addition, DNA methylation is involved in the initiation of the X chromosome inactivation, as well as in the stable maintenance of the gene silencing state . These studies suggest that DNA methylation may affect gene expression on the X chromosome or the development of T cells in psoriasis. It was found that DNA methylation is dramatically increased in the promoter region of genes on the X chromosome in patients with psoriasis. The binding sites for CDPCR3, GATA3, BRN2, and other transcription factors were identified as slightly enriched. The data obtained on epigenome changes in T cells show that naive CD4+ T cells may be involved in the development of atopic dermatitis or psoriasis even before antigenic stimulation. This may be due to the effects of various environmental factors.
3.2 Tissue-specific patterns
To determine the tissue-specific differences in DNA methylation associated with AD, the research group of Rodriguez et al. examined the DNA of whole blood, T cells, B cells, as well as the affected and unaffected epidermis of atopic patients and healthy people from the control group . To identify functional associations, they studied the expression profiles of epidermal mRNA.
Whole-genome methylation analysis was performed using Human Methylation27 BeadCheap. The results for epidermal tissue were different from those for blood cells. To determine the intraindividual and interindividual differences in DNA methylation, the researchers identified a pairwise correlation of methylated regions in the same tissue in samples from patients of similar sex and age, as well as between different tissues in the same person. In whole blood, T cells, and B cells, there were no significant differences in genome DNA methylation in the pathology group as compared with and the control group, and in general, intraindividual differences in DNA methylation were greater than those between individuals. A clear link was shown in case of comparing similar tissue in different individuals for different CpG sites, which partially correlated with altered levels of gene transcripts, mainly related to the processes of epidermal differentiation (
Regarding differentially methylated CpG islands in the epidermal tissue, 9 regions were identified as reliably associated with atopic dermatitis: in the
Based on analysis of whole genome mRNA expression (using HumanHT-12v3 Expression BeadChip), significant differences were revealed in seven transcripts when comparing samples of the affected skin of patients with AD and the skin of healthy people.
From nine selected pairs of differentially methylated regions / differentially expressed transcripts using the EpiTYPER system and quantitative PCR, the following combinations associated with the development of AD were successfully validated:
Olisova et al. carried out a genome-wide study of DNA methylation using the Illumina Infinium Human Methylation450 BeadChip technology . When comparing the affected and unaffected skin areas in atopic patients, no difference in the methylation profile was found. This suggests that epigenetic changes affected the entire skin as a whole, although they have not yet appeared in clinically intact skin areas. However, when comparing the affected skin with the skin of healthy volunteers, differentially methylated genes of the TSS200 and TSS1500 regions were isolated, whose protein products were involved in the pathogenesis of atopic dermatitis and related processes: steroid hormone biosynthesis and cell metabolism (
3.3 Replicated methylation
Another genome-wide epigenetic study examined differences in DNA methylation in atopic dermatitis together with herpetic eczema (HE), and revealed how methylation changes in patients with atopic dermatitis, complicated or uncomplicated HE .
490 significantly differentially methylated CpG sites were identified. Many of these were associated with indicators of disease severity, especially with the level of eosinophils (431/490 sites). One CpG region was replicated and was significantly differentially methylated based severity and phenotype.
The authors found replication for one CpG region associated with total serum IgE in the
4. Epigenetic regulation of immune system factors
It is known that abnormal epigenetic regulation of immune factors and skin barriers contribute to the pathogenesis of AD. During the development of immune system cells, epigenetic mechanisms are involved in specific changes in the variants of immune response . Here are some examples.
Regulatory T cells (Tregs) play an important role in early immune programming and the formation of an adequate immune response in relation to pro-allergic or tolerant conditions. Tregs are best characterized by the expression of transcription factor 3 (Foxp3), which is important for the induction and stability of Tregs . Foxp3 is controlled by DNA methylation of its transcriptional regulatory regions. Naturally induced by TGF-β Foxp3+ Tregs indicate stable expression of Foxp3, which is associated with selective demethylation of an evolutionarily conserved element at the Foxp3 locus - a Treg-specific demethylated region. Inhibition of DNA methylation by azacytidine, even in the absence of exogenous TGF-β, not only promotes induction of Foxp3 expression
In the neonatal immune system, epigenetic regulation can be shifted away from Th1-mediated immunity in order to prevent dangerous cellular immune responses to the developing fetus. The IFN-γ gene (
MicroRNAs (miRNAs) are short, single-stranded RNA molecules that function with their associated proteins and cause the degradation of targeted mRNAs, inhibiting their translation. miRNAs play an important role in a wide range of biological processes, including proliferation, differentiation, determination of cell development, apoptosis, signal transduction, and organ development. Some miRNAs are expressed specifically for each type of cells and tissues and contribute to the maintenance of cell identity. Tissue-specific miRNAs function at various levels of gene regulation, ranging from control of targeted effector genes, incompatible with the differentiated state, to control over the levels of transcriptional regulators and alternative pre-mRNA splicing. This multilevel regulation of miRNAs influences the gene expression program of differentiated cells . miRNAs, including miR-21, miR-146, and miR-223, activated in patients with allergic disorders, are also activated in the skin of patients with AD . A study by Herberth et al. showed that maternal exposure to tobacco during pregnancy correlated with high levels of miRNA-223 and low Treg cell levels, which predisposed children to atopic dermatitis during the first 3 years of life . Sonkoly et al. found that miR-155 was one of the most activated miRNAs in lesional skin samples from atopic patients in comparison with skin samples from healthy people. It has been found that local exposure of relevant allergens to intact skin of patients with AD induces miR-155 expression. miR-155 suppresses cytotoxic T lymphocyte – associated protein 4 - CTLA-4, which negatively regulates the function of T cells. This suppression of CTLA-4, in turn, enhances the T cell proliferative response, which can then lead to a long-term chronic inflammatory state .
There is not much evidence on the role of epigenetic mechanisms of innate and adaptive immunity regulation in the pathogenesis of atopic diseases, as these mechanisms have been studied recently. The described candidate genes involved in pathological processes such as dysfunction of the epidermal barrier, enhanced transmission of Th2 immunity signals, weakened innate immune responses, etc. play an important role in the pathogenesis of AD. Epigenetic studies also indicate modifications in genes involved in these mechanisms. Dysfunction of the epithelial barrier and immune response reactions together trigger the development of atopic dermatitis.
New insights on epigenetic and immunological markers associated with the risk of development of atopic dermatitis will help to create new prognostic approaches in the management of patients with atopic pathology. In this regard, it is important to have a complete understanding of the pathogenic mechanisms of an allergic disease.
Conflict of interest
The authors declare no conflict of interest.