To the Editor:
Asthma is a complex and heterogeneous chronic airway inflammatory
disease with the involvement of environmental factors through epigenetic
mechanisms.1 Accordingly, repeated injury, repair and
regeneration of the airway epithelium following exposure to
environmental factors and inflammation results in histological changes
and functional abnormalities in the airway mucosal epithelium, which are
associated with the pathophysiology of asthma.2Epigenetics is defined by heritable changes in gene expression without
changes in the DNA sequence.3 Regulation of gene
expression is mediated by different mechanisms such as DNA methylation,
histone modifications and RNA-associated silencing by small non-coding
RNAs. CpG sites are dinucleotides consisting of guanine and cytosine
concentrated in clusters referred to CpG islands found at important
regulatory sites, such as promoter and enhancer
regions.4 Their de novo methylation occurs in
response to various cellular stressors and signals by DNA
methyltransferases (DNMT3a and 3b), which add a methyl group to position
5 of cytosine residues at the CpG site. During DNA replication both of
the separated strands of DNA carry one methylated cytosine to be used as
a template for duplication. Daughter DNA duplex strands will thus be
hemi-methylated, which is recognized by a different DNA
methyltransferase isoform (DNMT1).5 Because DNA
methylation is a reversible process, the DNMTs are considered as a
therapeutic target. Several DNMT inhibitors have been identified
recently, among the non-nucleoside inhibitors, 4-aminoquoline-based
inhibitors, such as SGI-1027 showed potent inhibitory activity. SGI-1027
occupies the binding site of DNMTs resulting in the prevention of access
of target DNA to the substrate binding pocket.6
We have demonstrated in previous studies from our laboratory that human
primary bronchial epithelial cells (HBEC) isolated from patients with
asthma showed lower barrier integrity compared to
controls.7 To investigate the level of global
methylation in HBEC, we investigated control and asthma samples for the
long interspersed nuclear element-1 (LINE-1) methylation levels (Figure
1A). HBEC from asthma patients showed a tendency for higher global
methylation levels, together with higher expression of 5-methylcytosine
(5-mc) in immunofluorescence staining (Figure 1B). Next, we performed
methylation profiling (Illumina Infinium EPIC array) to investigate
genes methylated in ALI cultures of HBEC. Interestingly, in a highly
methylated group of top 100 genes, we found many genes associated with
cell growth, ion transport, and cytoskeletal remodeling (Figure S1). We
kept our attention on the methylated epigenetic and tight junction (TJ)
genes and further focused on TJs, especially zonula occludens and
claudins which showed higher methylation in contrast to occludin, which
was not methylated (Figure S2). As higher methylation levels were
observed in HBEC of asthmatic origin, we inhibited the DNA
methyltransferase enzyme with a specific inhibitor, SGI-1027, to
demonstrate the role of CpG methylation on epithelial barrier integrity.
ALI cultures were treated with the DNA methyltransferase inhibitor for
72 hours. Significantly decreased expression of 5-mc was observed after
48 hours of DNA-methyltransferase inhibition, demonstrating that the
methylation of 5-methylcytosine (5-mc) in bronchial epithelium was
reversed (Figure 2A). This prompted us to investigate the changes
triggered by the inhibitor in epithelial cells. Further experiments
showed increased transepithelial electric resistance (TER) in bronchial
epithelial cells, in ALI from asthmatic donors after 48 hours of DNMT
inhibition (Figure 2B). The link between barrier integrity and TER
results were confirmed by the significantly decreased paracellular
passage of FITC-labelled 4kD dextran after inhibition of DNMTs (Figure
2C). The reconstitution of TER in asthmatic ALI was associated with
decreased protein DNMT1 expression and increased ZO-1 and claudin-18
proteins (Figure 2D). We also observed increased claudin-4, but not
occludin expression upon DNMT inhibition (Figure S3). Increased
expression of ZO-1 with an intact and honeycomb-like structure in the
immunofluorescence staining of bronchial epithelial cells confirmed the
effect on protein expression of bronchial epithelial barrier in asthma
donors (Figure S4).
Defective epithelial barrier has been established in asthma in addition
to several chronic inflammatory diseases.8 Direct
targeting of the epithelial barrier leakiness for the treatments
represents an important target, however so far there is no treatment
possibility targeting epigenetic mechanisms. The present study
demonstrates an increased global methylation level in HBEC from
asthmatic individuals. CpG methylation of specific genes is essential
for the defect of epithelial barrier integrity, which is reversed upon
DNMT inhibition. The inversion of CpG methylation, restores leakiness in
the epithelium in asthma by increasing TER, decreasing paracellular flux
and improves the structure of bronchial epithelial cells by increasing
the expression of TJ proteins. The better understanding of the
importance of epigenetic memory in chronic tissue inflammatory diseases
together with the availability of treatment modalities targeting
epigenetic mechanisms and transition of these molecules into the
clinical studies may lead to curative treatment of allergic and
autoimmune inflammatory diseases.9
Paulina Wawrzyniak1, PhD,
Krzysztof Krawczyk1,3, MSc,
Swati Acharya5, PhD,
Ge Tan1,7, PhD,
Marcin Wawrzyniak1, PhD,
Emmanuel Karouzakis4, PhD,
Anita Dreher, Sci. Tech.,
Bogdan Jakiela2, MD, PhD,
Can Altunbulakli1, PhD,
Marek Sanak2, MD, PhD,
Liam O‘Mahony1,6, PD, PhD,
Kari Nadeau5, MD, PhD,
Cezmi A. Akdis1, MD
1Swiss Institute of Allergy and Asthma Research
(SIAF), University of Zürich, Davos, Switzerland, Christine Kühne-Center
for Allergy Research and Education (CK-CARE)
2Department of Medicine, Jagiellonian University
Medical College, Krakow, Poland
3Faculty of Biology and Environmental Protection,
Department of Cellular Immunology, Lodz, Poland
4Department of Rheumatology, University Hospital of
Zurich
5Departament of Medicine, Stanford University, United
States
6 Department of Medicine and School of Microbiology,
APC Microbiome Ireland, University College Cork, Cork, Ireland.
7 Functional Genomics Center Zurich, ETH
Zurich/University of Zurich
Corresponding author:
Paulina Wawrzyniak
Swiss Institute of Allergy and Asthma Research (SIAF), University of
Zürich, Davos, Switzerland
Obere Strasse 22,
7270 Davos, Switzerland
Tel: +41 81 410 08 48
Fax: +41 81 410 08 40
paulina.wawrzyniak@uzh.ch
Conflict of interest:
The authors declare that they have no conflicts of interest.
Founding sources:
Supported by Swiss National Science
Foundation grants 310030_156823,
and 320030_176190.
Word count: 765
Keywords: asthma, tight junction, CpG methylation, DNA
methyltransferases,