\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Type 2 cytokine-induced microRNA-29s suppressed both soluble ST2 release
and IFNAR1 expression in human bronchial epithelial cells}



\author[1]{Arisa Igarashi}%
\author[1]{Kenji Matsumoro}%
\author[1]{Akio Matsuda}%
\affil[1]{National Research Institute for Child Health and Development}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup








\sloppy


To the Editor:

Bronchial asthma is characterized by restricted airflow due to chronic
airway inflammation, and frequent lower respiratory viral infections in
early life are a significant risk factor for development of the disease.
Previous studies demonstrated that anti-viral interferon (IFN)
production, including of IFN-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}β \selectlanguage{english}and IFN-\selectlanguage{greek}λ, \selectlanguage{english}by leukocytes and
bronchial epithelial cells can be impaired in asthma
patients.\textsuperscript{1} An epidemiological study found that
allergic sensitization precedes wheeze during asthma development in
children, suggesting that Type 2 (T2) conditions play a key role in the
impaired anti-viral IFN production. Furthermore, a prospective cohort
study showed that, regardless of the type of virus, each successive
lower respiratory viral infection with wheeze increases the risk of
asthma by about 1.5 fold.\textsuperscript{2} However, we still don't
have a full understanding of the precise mechanism(s) of how respiratory
viral infections under T2 conditions lead to development of asthma.

A meta-analysis of large-scale genome-wide association studies revealed
that both \emph{IL-33} and its receptor, \emph{IL-33
receptor}(\emph{IL-33R} ; also known as \emph{ST2} ), are closely
associated with asthma development.\textsuperscript{3} Indeed, IL-33
expression was reportedly increased in rhinovirus-infected bronchial
epithelial cells and correlated significantly with the disease severity
of asthma.\textsuperscript{4} This suggests that virus-induced IL-33 in
the airway may be fundamentally involved in the mechanistic links
between viral infection and development and/or exacerbation of asthma.
In addition, impairment of anti-viral IFN production was reported to
cause necrosis---but not apoptosis---of the virus-infected
epithelium,\textsuperscript{5} which results in release of bioactive
IL-33.

MicroRNAs (miRNAs) are small, non-coding RNA molecules (containing about
22 nucleotides) that are found in diverse organisms. miRNAs regulate
expression of a broad spectrum of target genes through RNA silencing
and/or post-transcriptional regulation. Among them, microRNA-29a
(miR-29a) was induced by respiratory syncytial virus (RSV) infection in
a human lung adenocarcinoma cell line, A549, and suppressed expression
of IFN (\selectlanguage{greek}α, β \selectlanguage{english}and \selectlanguage{greek}ω\selectlanguage{english}) receptor 1 (IFNAR1).\textsuperscript{6} Furthermore,
miR-29a regulated the expression of soluble ST2 (sST2), a decoy receptor
for IL-33, in human tenocytes.\textsuperscript{7} Based on those earlier
findings, we focused on miR-29 in the present study. We hypothesized
that T2 cytokine induces miR-29 expression in bronchial epithelial
cells, leading to suppression of both sST2 release and IFNAR1 expression
by epithelial cells, and culminating in asthma development and/or
exacerbation.

Based on that hypothesis, we first examined whether T2 cytokine and
inflammatory cytokine induced sST2 production in a human bronchial
epithelial cell line, BEAS-2B. The detailed methods are described in
Supporting Information. Specific ELISA showed that IL-4 and TNF-\selectlanguage{greek}α
\selectlanguage{english}synergistically induced sST2 release from BEAS-2B, in a dose-dependent
manner (Figure 1A). Next, to examine the effects of miR-29
overexpression or inhibition on that cytokine-induced sST2 release,
BEAS-2B cells were first transfected with miR-29 mimics or inhibitors
for 24 hours and then stimulated with a combination of IL-4 and TNF-\selectlanguage{greek}α
\selectlanguage{english}for 48 hours.

The human miR-29 family consists of three mature members, i.e., miR-29a,
miR-29b, and miR-29c. These miR-29s are encoded by the miR-29a/b-1
cluster on chromosome 7q32.3 and the miR-29c/b-2 cluster on chromosome
1q32.2, respectively (Figure 1B).\textsuperscript{8} The three family
members share an identical seed sequence (Figure 1B), and their
functional properties are thought to be similar. We examined the effects
of miR-29a and miR-29b in this study. ELISA of the culture supernatants
showed that inhibition of miR-29a or miR-29b significantly enhanced
cytokine-induced sST2 release (Figure 1C). In contrast, overexpression
of miR-29a or miR-29b almost completely inhibited that release,
indicating that these miR-29s regulate sST2 release from bronchial
epithelial cells under T2 conditions. Of note, neither inhibition nor
overexpression of miR-29a or miR-29b had any effects on the protein
levels of the ST2 receptor in the BEAS-2B cells (Figure 1D, upper
panel). These results suggest that T2 cytokine-induced miR-29 plays a
critical role in IL-33-dependent allergic inflammation through
regulation of sST2 release from bronchial epithelial cells.

Furthermore, transfection of either miR-29a or miR-29b inhibitors
significantly enhanced IFNAR1 protein expression in the BEAS-2B cells
(Figure 1D, middle panel), which is consistent with earlier findings for
miR-29a in A549 cells.\textsuperscript{6} Conversely, transfection of
miR-29 mimics resulted in reduced IFNAR1 expression in BEAS-2B cells,
suggesting that overproduction of miR-29s in bronchial epithelial cells
may lead to suppression of antiviral responses by IFNs. Thus, we found
that miR-29s simultaneously regulate the expression of both sST2 and
IFNAR1 in bronchial epithelial cells. Our findings suggest the
possibility that T2 cytokine-induced miR-29s in airway epithelial cells
are key players in the development and/or exacerbation of asthma
triggered by respiratory viral infections through both decreasing
IFN-regulated antiviral activities and exacerbating IL-33-dependent
allergic inflammation.

miRNAs are released from cells into the extracellular environment via
exosomes, which can then fuse with target cells. This process can
deliver various proteins and nucleic acids, including miRNAs, into even
distant target/receiving cells.\textsuperscript{9} We, therefore,
examined whether exosomes similarly export miR-29s from bronchial
epithelial cells. BEAS-2B cells were stimulated with a combination of
IL-4 and TNF-\selectlanguage{greek}α \selectlanguage{english}for 48 hours, and exosomal fractions were collected from
the culture supernatants. Although qPCR detected both miR-29a and
miR-29b in the exosomes even without that T2 cytokine stimulation
(control), both of their copy numbers were significantly increased by
that stimulation (Figure 2A). Furthermore, Western blot analysis also
found that expression of CD81, an exosome marker, was enhanced by the
cytokine stimulation (Figure 2B). These results suggest that T2
cytokine-stimulated epithelial cells release more exosomes containing
more miR-29s than unstimulated cells.

This study has several limitations. First, no functional experiments
were performed in this study to confirm the effects of the changes in
sST2 release or IFNAR1 expression. In addition, we did not measure the
expression levels of miR-29s in clinical samples.

Figure S1 summarizes our findings as a schematic illustration of
bronchial epithelial cells. Based on those findings, we hypothesize that
elevated nasal, bronchial and/or exosomal levels of miR-29s in infancy
may be useful biomarker(s) for predicting later development of asthma,
and further studies are needed. Our data suggest a new perspective that
miRNAs are crucially involved in the association between viral infection
and asthma development. We believe that our research has great
significance in pointing to a novel direction for further studies and
the existence of a new key player, i.e., miRNAs, in the relationship
between viral infections and asthma development.

\textbf{Acknowledgments}

This work was supported in part by Grants-in-Aid for Young Scientists
(B) from JSPS KAKENHI (15K19439 to A.I.) and grants from National Center
for Child Health and Development (\#27-15 and \#2020B-4 to K.M.). We
thank Mr. Lawrence W. Stiver (Quality Translation Co., Ltd.; Tokyo,
Japan; qualityt@gol.com) for proofreading our English.



\textbf{Supporting Information}

Additional supporting information may be found online in the Supporting
Information section at the end of the article.

\textbf{ORCID}

\emph{Akio Matsuda} https://orcid.org/0000-0002-7781-0613

\emph{Kenji Matsumoto} \url{https://orcid.org/0000-0002-2630-6927}

\par\null

\textbf{Correspondence}

Akio Matsuda, Department of Allergy and Clinical Immunology, National
Research Institute for Child Health and Development, 2-10-1 Okura,
Setagaya-ku, 157-8535, Tokyo, Japan. Phone: +81-3-5494-7120 (ext. 4955);
Fax: +81-3-5494-7173; E-mail:
\href{mailto:matsuda-a@ncchd.go.jp}{\nolinkurl{matsuda-a@ncchd.go.jp}}

\textbf{Authorship}

AI and AM designed the study, contributed to data collection and wrote
the manuscript. KM interpreted the results and critically reviewed the
manuscript. All authors read and approved the final manuscript.\textbf{}

\textbf{CONFLICTS OF INTEREST}

The authors have no conflicts of interest directly relevant to the
contents of this article.

\textbf{FUNDING INFORMATION}

This work was supported in part by Grants-in-Aid for Young Scientists
(B) from JSPS KAKENHI (15K19439 to A.I.) and grants from National Center
for Child Health and Development (\#27-15 and \#2020B-4 to K.M.).



\textbf{REFERENCES}

1. Edwards MR, Strong K, Cameron A, et al. Viral infections in allergy
and immunology: How allergic inflammation influences viral infections
and illness. \emph{J Allergy Clin Immunol.} 2017;140(4):909-920.

2. B\selectlanguage{ngerman}ønnelykke K, Vissing NH, Sevelsted A, Johnston SL, Bisgaard H.
Association between respiratory infections in early life and later
asthma is independent of virus type. \emph{J Allergy Clin
Immunol.}2015;136(1):81-86.

3. Torgerson DG, Ampleford EJ, Chiu GY, et al. Meta-analysis of
genome-wide association studies of asthma in ethnically diverse North
American. \emph{Nat Genet.} 2011;43(9):887-892.

4. Jackson DJ, Makrinioti H, Rana BM, et al. IL-33-dependent type 2
inflammation during rhinovirus-induced asthma exacerbations in
vivo.\emph{Am J Respir Crit Care Med.} 2014;190(12):1373-1382.

5. Wark PA, Johnston SL, Bucchieri F, et al, Asthmatic bronchial
epithelial cells have a deficient innate immune response to infection
with rhinovirus. \emph{J Exp Med.} 2005;201(6):937-947.

6. Zhang Y, Yang L, Wang H, Zhang G, Sun X. Respiratory syncytial virus
non-structural protein 1 facilitates virus replication through
miR-29a-mediated inhibition of interferon-alpha receptor. \emph{Biochem
Biophys Res Commun.} 2016;478(3):1436-1441.

7. Millar NL, Gilchrist DS, Akbar M, et al. MicroRNA29a regulates
IL-33-mediated tissue remodelling in tendon disease. \emph{Nat
Commun.}2015;6:6774.

8. Kwon JJ, Factora TD, Dey S, Kota J. A Systematic Review of miR-29 in
Cancer. \emph{Mol Ther Oncolytics.} 2019;12:173-194.

9. Stoorvogel W. Functional transfer of microRNA by
exosomes.\emph{Blood.} 2012;119:646-648.

\textbf{Figure legends}

\textbf{FIGURE 1}

\textbf{miR-29s suppress cytokine-induced expression of sST2 and IFNAR1
in BEAS-2B cells}

A, Cytokine-induced soluble ST2 (sST2) release from BEAS-2B cells. A
human bronchial epithelial cell line, BEAS-2B cells, was stimulated with
the indicated concentrations of IL-4 and TNF-\selectlanguage{greek}α, \selectlanguage{english}alone or in combination,
for 48 h. The protein concentration of sST2 in each culture supernatant
was measured by ELISA. B, Schematic representation of the gene
structures of miR-29 family members. C, The effects of inhibition or
overexpression of miR-29s on sST2 release from BEAS-2B cells were
examined. Following 24-h transfection of 10 nM of miRNA for miR-29
inhibitors, mimics or a negative control, the cells were stimulated with
a combination of 100 ng/ml IL-4 and TNF-\selectlanguage{greek}α \selectlanguage{english}for 48 h. The protein
concentration of sST2 in each culture supernatant was measured by ELISA,
and the data are shown as the mean \selectlanguage{ngerman}± SD of triplicate samples.
*\emph{P}\textless{} .05 by Student's \emph{t} -test. D, Effects of
inhibition or overexpression of miR-29s on the expression of ST2 and
IFNAR1 proteins in BEAS-2B cells. BEAS-2B cells were treated as above in
C. Whole-cell lysates were subjected to Western blot analysis of the
expression of ST2, IFNAR1 and \selectlanguage{greek}β-\selectlanguage{english}actin (as a loading control). The data
are representative of two separate experiments. Chr.: chromosome; NC:
negative control; IFNAR1: IFN (\selectlanguage{greek}α, β \selectlanguage{english}and \selectlanguage{greek}ω\selectlanguage{english}) receptor 1; ACTB: \selectlanguage{greek}β-\selectlanguage{english}actin.

\textbf{FIGURE 2}

\textbf{Epithelial-derived miR-29s in exosomes}

BEAS-2B cells were stimulated with a combination of 100 ng/ml IL-4 and
TNF-\selectlanguage{greek}α \selectlanguage{english}for 48 h. Cell culture supernatants were collected, and the
exosomal fractions were extracted. A, The expression levels of miR-29a
and miR-29b in the exosomal fractions were measured by qPCR. The data
are shown as the mean \selectlanguage{ngerman}± SD of triplicate samples. Statistical analysis
was performed by Student's \emph{t} -test. *\emph{P} \textless{} .05. B,
The expression of CD81, a marker of exosomes, was analyzed by Western
blot analysis.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

