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\begin{document}

\title{Inhibition of CpG methylation improves the barrier integrity of
bronchial epithelial cells in asthma.}



\author[1]{Paulina Wawrzyniak}%
\author[1]{Krzysztof Krawczyk}%
\author[2]{Acharya Swati}%
\author[1]{Ge Tan}%
\author[1]{Marcin Wawrzyniak}%
\author[3]{Emmanuel Karouzakis}%
\author[4]{Anita Dreher}%
\author[5]{Bogdan Jakiela}%
\author[6]{Can Altunbulakli}%
\author[5]{Marek Sanak}%
\author[7]{Liam O'Mahony}%
\author[2]{Kari Nadeau}%
\author[3]{Cezmi Akdis}%
\affil[1]{Swiss Institute of Allergy and Asthma Research}%
\affil[2]{Stanford University}%
\affil[3]{University of Zurich}%
\affil[4]{Swiss Institute of Allergy and Asthma Research (SIAF)}%
\affil[5]{Jagiellonian University Medical College}%
\affil[6]{Swiss Institute of Allergy and Asthma Researc}%
\affil[7]{University College Cork National University of Ireland}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\emph{To the Editor:~}

\emph{}Asthma is a complex and heterogeneous chronic airway inflammatory
disease with the involvement of environmental factors through epigenetic
mechanisms.\textsuperscript{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.\textsuperscript{2}Epigenetics is defined by heritable changes in
gene expression without changes in the DNA sequence.\textsuperscript{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.\textsuperscript{4} Their \emph{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).\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{9}

Paulina Wawrzyniak\textsuperscript{1}, PhD,

Krzysztof Krawczyk\textsuperscript{1,3}, MSc,

Swati Acharya\textsuperscript{5}, PhD,

Ge Tan\textsuperscript{1,7}, PhD,

Marcin Wawrzyniak\textsuperscript{1}, PhD,

Emmanuel Karouzakis\textsuperscript{4}, PhD,

Anita Dreher, Sci. Tech.,

Bogdan Jakiela\textsuperscript{2}, MD, PhD,

Can Altunbulakli\textsuperscript{1}, PhD,

Marek Sanak\textsuperscript{2}, MD, PhD,

Liam O`Mahony\textsuperscript{1,6}, PD, PhD,

Kari Nadeau\textsuperscript{5}, MD, PhD,

Cezmi A. Akdis\textsuperscript{1}, MD

\textsuperscript{1}Swiss Institute of Allergy and Asthma Research
(SIAF), University of Z\selectlanguage{ngerman}ürich, Davos, Switzerland, Christine Kühne-Center
for Allergy Research and Education (CK-CARE)

\textsuperscript{2}Department of Medicine, Jagiellonian University
Medical College, Krakow, Poland

\textsuperscript{3}Faculty of Biology and Environmental Protection,
Department of Cellular Immunology, Lodz, Poland

\textsuperscript{4}Department of Rheumatology,~University Hospital of
Zurich

\textsuperscript{5}Departament of Medicine, Stanford University, United
States

\textsuperscript{6} Department of Medicine and School of Microbiology,
APC Microbiome Ireland, University College Cork, Cork, Ireland.

\textsuperscript{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.

\textbf{Word count:} 765

\textbf{Keywords:} asthma\textbf{,} tight junction, CpG methylation, DNA
methyltransferases,

\textbf{Abbreviations:}

ALI: air-liquid interface,

BEGM: bronchial epithelial growth medium,

DNMT: DNA methyltranferase

FITC: fluorescein isothiocyanate,

HBEC: human bronchial epithelial cells,

LINE-1: long interspersed nuclear element-1

5-mc: 5-methylcytosine

TER: transepithelial electric resistance,

ZO: zonula ocludens

\textbf{FIGURE LEGENDS} :

\textbf{Figure 1} . \textbf{Increased global methylation in bronchial
epithelial cells in asthma}

\begin{enumerate}
\tightlist
\item
  Global LINE-1 methylation levels in bronchial epithelial cells from
  healthy control (n=4) and asthma patients (n=3) on the baseline. (B)
  Representative immunofluorescence staining and data showing the mean
  intensity of ALI cultures of differentiated bronchial epithelial cells
  from controls (n=5) and asthma patients (n=8) for 5-methylcytosine
  (5mc). Data are presented as mean±SD, (**P \textless{} 0.01), Mann
  Whitney U test.
\item
  \textbf{Figure 2. DNA methyltransferase inhibitor decreases global
  methylation and improve the barrier integrity of bronchial epithelial
  cells in asthma}
\end{enumerate}

Representative immunofluorescence staining of 5-methylcytosine (5mc) and
data showing the mean intensity in asthmatic ALI cultures of HBEC
treated with 8 \selectlanguage{greek}µ\selectlanguage{english}M DNA methyltransferase inhibiitor, SGI-1027 for 48
hours, (n=8). (B) Increased TER in ALI cultures of HBEC from asthmatic
individuals treated with 8 \selectlanguage{greek}µ\selectlanguage{english}M of DNA methyltransferase
inhibitor-SGI-1027, n=5 per group. (C) The decreased paracellular flux
of 4kDa FITC-dextran in response to 48 hours treatment of 8 \selectlanguage{greek}µ\selectlanguage{english}M DNA
methyltransferase inhibitor SGI-1027, n=5 per group. (D) Western blots
of DNMT1, ZO-1 and claudin-18 from ALI cultures of asthmatic HBEC
treated with DNA methyltransferase inhibitor-SGI-1027 in diferent doses
(2 \selectlanguage{greek}µ\selectlanguage{english}M, 4 \selectlanguage{greek}µ\selectlanguage{english}M, 8 \selectlanguage{greek}µ\selectlanguage{english}M, 16 \selectlanguage{greek}µ\selectlanguage{english}M), n=5. Data represent mean\selectlanguage{ngerman}±SD, (*\emph{P}
\textless{} 0.05), (**P \textless{} 0.01), Mann Whitney U test.

References:

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and endotypes. \emph{Allergy.} 2012;67(7):835-846.

2. Heijink IH, Kuchibhotla VNS, Roffel MP, et al. Epithelial cell
dysfunction, a major driver of asthma development. \emph{Allergy.} 2020.

3. Paivandy A, Grujic M, Rafati N, Pejler G. DNA demethylation regulates
gene expression in IgE-activated mouse mast cells.
\emph{Allergy.}2020;75(7):1776-1780.

4. Zhang H, Kaushal A, Merid SK, et al. DNA methylation and allergic
sensitizations: A genome-scale longitudinal study during
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5. Lyko F. The DNA methyltransferase family: a versatile toolkit for
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6. Yoo J, Choi S, Medina-Franco JL. Molecular modeling studies of the
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One.}2013;8(4):e62152.

7. Wawrzyniak P, Wawrzyniak M, Wanke K, et al. Regulation of bronchial
epithelial barrier integrity by type 2 cytokines and histone
deacetylases in asthmatic patients. \emph{J Allergy Clin
Immunol.}2017;139(1):93-103.

8. Sugita K, Soyka MB, Wawrzyniak P, et al. Outside-in hypothesis
revisited: The role of microbial, epithelial, and immune
interactions.\emph{Ann Allergy Asthma Immunol.} 2020.

9. Tough DF, Tak PP, Tarakhovsky A, Prinjha RK. Epigenetic drug
discovery: breaking through the immune barrier. \emph{Nature Reviews
Drug Discovery.} 2016;15:835.\selectlanguage{english}
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