University of Groningen Chronic mucus hypersecretion in COPD and asthma Tasena, Hataitip

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Chronic mucus hypersecretion in COPD and asthma

Tasena, Hataitip

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Tasena, H. (2019). Chronic mucus hypersecretion in COPD and asthma: Involvement of microRNAs and stromal cell-epithelium crosstalk. University of Groningen.

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531891-L-bw-Tasena 531891-L-bw-Tasena 531891-L-bw-Tasena 531891-L-bw-Tasena Processed on: 6-6-2019 Processed on: 6-6-2019 Processed on: 6-6-2019

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Chronic mucus hypersecretion in COPD and

asthma: Involvement of microRNAs and

stromal cell-epithelium crosstalk

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Groningen University Institute for Drug Exploration (GUIDE), the Groningen

Research Institute for Asthma and COPD (GRIAC), and U4 Ageing Lung

Consortium. They are financially supported by the University of Groningen,

Stichting Astma Bestrijding (SAB), and de Cock foundation.

Printing of this thesis was financially supported by the University of Groningen,

the Graduate school of Medical Sciences Groningen (GSMS), and Stichting

Astma Bestrijding (SAB).

ISBN (printed): 978-94-034-1704-2

ISBN (electronic): 978-94-034-1703-5

Cover design by Nick (fiverr.com/glitchfool)

Lay out by Abubakar Abdullahi Dubagari and Hataitip Tasena

Printed by Ipskamp Printing, Enschede

© 2019, Hataitip Tasena, The Netherlands

All rights reserved. No part of this thesis may be reproduced or transmitted

in any form or by any means without prior permission of the author or, when

appropriate, of the publisher of the publication.

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Involvement of microRNAs and

stromal cell-epithelium crosstalk

PhD thesis

to obtain the degree of PhD at the

University of Groningen

on the authority of the

Rector Magnificus prof. E. Sterken

and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on

Wednesday 3 July 2019 at 12.45 hours

by

Hataitip Tasena

born on 23 April 1989

in Chiang Rai, Thailand

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Prof. H.I. Heijink

Prof. W. Timens

Co-supervisors

Dr. C.A. Brandsma

Dr. M. van den Berge

Assessment Committee

Prof. K. Bracke

Prof. G.H. Koppelman

Prof. A. van den Berg

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Jennie Ong

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CHAPTER 1

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Chronic mucus hypersecretion (CMH) is a feature observed in various chronic respiratory diseases, particularly chronic obstructive pulmonary disease (COPD)1,2

and asthma3,4_{. While COPD is one of the top leading causes of death worldwide}

especially in elderly5_{, asthma is the most common chronic disease among children}6

and responsible for more than 400,000 deaths overall each year worldwide7_{. Both}

genetic and environmental factors contribute to COPD and asthma8–10_{, and cigarette}

smoke is widely recognized as a major cause of COPD10._{COPD and asthma patients}

may experience similar symptoms, e.g. breathing difficulty, chest tightness, wheezing, and cough; however, these symptoms are driven by different—though partly overlapping—mechanisms11_{. In COPD, exposure to air pollutants or irritant particles}

(e.g. cigarette or wood smoke) triggers inflammatory responses characterized by infiltration of neutrophils, macrophages, T lymphocytes and innate lymphoid cells, which in the long-term causes damage to the lung12_{. Abnormal tissue repair is a key}

pathophysiological feature of COPD which leads to small airway wall thickening and/or destruction of alveolar tissue (emphysema)13_{, resulting in airflow limitation}

that is not fully reversible and accelerated lung function decline13,14_{. In asthma, the}

most common type is allergen-induced asthma characterized by chronic eosinophilic airway inflammation, airway remodeling with increased smooth muscle mass, subepithelial fibrosis and goblet cell hyperplasia14,15_{. In patients with allergic asthma,}

allergen-specific type-2 T-helper cells drive the airway inflammatory response that gives rise to clinical symptoms such as wheezing, variable airflow limitation and airway hyperresponsiveness16_{. As both are highly heterogeneous diseases, COPD and}

asthma patients can be categorized into different subgroups based on their clinical features. One of the important features shared by both COPD and asthma patients is CMH.

CMH in COPD is often referred to as chronic bronchitis1,2_{. Here, it is associated}

with lower quality of life, an accelerated decline of lung function, more severe airflow obstruction and an increased risk of exacerbations and mortality17_{. In asthma,}

CMH is most prevalent in patients with severe asthma4_{and associated with acute}

exacerbations18_{. Mucus plugging in the airways is found in the vast majority of fatal}

asthma cases and may be an important, but underappreciated, cause of respiratory failure19_{. Although some anti-inflammatory drugs, bronchodilators and antibiotics}

have been reported to reduce mucus production or improve mucus clearance, the results of the studies were either inconsistent or not yet proven to be beneficial in humans20_{. The lack of effective CMH-targeted therapy, while it represents a high}

burden, reflects an urgent need to better understand mechanisms underlying CMH pathophysiology in order to find better treatment options.

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1 Factors contributing to chronic mucus hypersecretion

Patients with CMH suffer from chronic cough and sputum expectoration as a result of increased mucus accumulation in their airways, a process attributed to exaggerated mucin secretion and/or impaired mucus clearance21_{. Increased mucin secretion}

in COPD and asthmatic airways likely results from increased numbers of goblet cells22–24_{and enlargement of mucous glands}25,26_{, increased mucin synthesis}24,27–30

and/or increased degranulation of goblet cells31,32_{. Ineffective mucus clearance can}

be driven by defective cilia function33,34_{, changes in mucus composition}35_{, mucus}

dehydration36_{and/or higher viscoelasticity resulting from more mucin cross-linking}

or impaired mucin degradation37–39_{. All these factors are commonly used as markers}

for studying CMH, as summarized in figure 1.

Mucins are heavily glycosylated proteins responsible for mucus viscoelasticity40_.

MUC5AC and MUC5B are the most common mucins present in the respiratory tract and they are associated with both COPD29,30_{and asthma}27,28_{. When external pathogens}

or noxious particles are inhaled, the airway epithelium is the first line of defense protecting the lungs from these harmful stimuli. It is composed of various epithelial cell types including basal cells and more differentiated cells, e.g. club cells, goblet cells and ciliated cells41_{. Mucins are synthesized by goblet cells as a constituent}

of the airway epithelium and by mucous cells in submucosal glands21_{. After being}

synthesized, mucins are stored in intracellular granules before being released into the airway lumen. Here, secreted mucins are mixed with lipids, antimicrobial proteins, electrolytes, and water to form a mucus layer40_{, which can then be transported from}

distal to proximal airways by the rhythmic beating of cilia before entering the larynx and being expectorated by a sudden opening of vocal cords42_.

Mucus which is accumulated in the airways requires effective clearance, a process that is often impaired in patients with COPD or asthma33,34,43_{. As described}

above, one of the factors contributing to ineffective mucus clearance is ciliary dysfunction. Cilia on airway epithelium of COPD smokers are shorter, more vulnerable and show disorderly beating33_{, while lower cilia beating frequency has been observed}

in asthmatic airways34_{. In addition, smoking causes squamous metaplasia in the}

airways, that even further worsens mucus clearance. The type of secreted mucins may also influence mucociliary clearance. A recent study suggested that coating of secreted MUC5B with MUC5AC may provide anchoring activity that slows down mucus transport44_{. In asthma, impaired mucus transport has been proposed to be}

caused by tethering of MUC5AC to the epithelium rather than by cilia dysfunction35_.

Other studies suggest a negative impact of airway surface dehydration on mucus clearance36,45,46_{. Even without an increase of mucin synthesis and goblet cell number,}

dehydration of constitutively secreted mucus alone is sufficient to cause mucus plugging36_{. Apart from dehydration, impaired cleavage of mucins or extracellular}

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acute asthma, more cross-linking of mucin polymers is associated with reduced cough clearance, while mucin degradation is suppressed possibly due to inhibition of protease by excess plasma proteins39_{. These mechanisms can contribute to ineffective}

mucus clearance leading to CMH in asthma and COPD.

Figure 1. Factors contributing to CMH.

Molecular mechanisms involved in mucus production

Various well-known transcription factors play a crucial role in mucin synthesis/ secretion and cilia function. The first one is SAM pointed domain-containing ETS transcription factor (SPDEF). Expression of SPDEF is dependent on Signal transducer and activator of transcription 6 (STAT6)47_{. Goblet cells are absent in}

SPDEF-deficient mice, while SPDEF overexpression in club cells leads to goblet cell differentiation without proliferation, suggesting that club cells can function as a progenitor for goblet cells in these mice48_{. The second transcription factor is Forkhead}

box protein A2 (FOXA2), a winged helix protein known to play an important role in embryonic development. FOXA2-deleted mice developed goblet cell hyperplasia and increased MUC5AC synthesis in the airways49_{. Upon mucociliary differentiation}

of airway epithelial cells, SPDEF is upregulated while FOXA2 is downregulated50_,

suggesting their opposite roles in mucociliary development. Another transcription factor involved in epithelial differentiation is Forkhead box protein J1 (FOXJ1) which plays an essential role in epithelial polarization51_{and differentiation towards}

ciliated cells52_{and is often used as a marker to assess ciliated cell differentiation in}

vitro. These molecular processes may be regulated by pro-inflammatory cytokines, which are also known to play a role in CMH pathophysiology. IL-13, for instance, is an important T cell-derived mediator of type 2 inflammatory responses in allergic asthma53_{, which is also upregulated in COPD}54_{and is well known for its central}

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IL-1 family members, e.g. IL-1α and IL-1β, can promote mucus hypersecretion. Inhibition of IL-1 receptor 1 (IL-1R1) in βENaC-overexpressing (βENaC-Tg) mice, which develop spontaneous mucus plugging associated with cystic fibrosis/COPD-like phenotypes, alleviates airway mucus obstruction56_{. IL-1β acts on IL-1R1 and}

has been shown to stimulate MUC5AC and MUC5B expression in human airway epithelial cells via NF-κB activation57,58_.

Role of stromal cells in chronic mucus hypersecretion

Recent reports suggest that stromal cells, such as airway smooth muscle cells (ASMCs) and fibroblasts, play a crucial role in epithelial homeostasis and differentiation59–62_{and therefore may also be involved in CMH development. ASMCs}

respond to various pro-inflammatory cytokines, which are secreted by other cell types including epithelial cells, e.g. IL-1β and TGF-β63,64_{. Co-culturing tracheal epithelium}

with fibroblasts stimulates epithelial proliferation, mucin production and basement membrane formation in vitro65_{. It is still unknown which mechanisms mediate this}

crosstalk and whether these findings are relevant in the context of airway diseases. Our group previously observed that during fibroblast co-culture, epithelial-derived IL-1α increases CXCL8 and IL-6 production by fibroblasts66_{. Furthermore,}

we observed a significant association between a polymorphism in the Frizzled (FZD) 8 region and CMH67_{. FZD8 is a receptor for WNT growth factors, which}

are involved in lung development and regeneration as well as pro-inflammatory responses68_{. Lung fibroblasts responded to epithelial-derived stimuli (IL-1 and EGF)}

by upregulating FZD8 expression and this upregulation was more pronounced in fibroblasts from patients with CMH than those without CMH69_{. Fibroblasts derived}

from COPD patients with CMH, compared to ones without CMH, secreted higher levels of CXCL8 and IL-6 cytokines, which have been shown to promote MUC5AC production in differentiated epithelial cells67_{. Together, these findings suggest a}

potential involvement of fibroblasts in CMH development. How abnormalities in the complex interplay between structural cells in the airways lead to CMH and which gene networks are involved is currently unknown.

Role of microRNAs in chronic mucus hypersecretion and stromal

cell-epithelium crosstalk

As microRNAs (miRNA) play a key role in many cellular processes and have been implicated in a variety of diseases, it is likely that they also are involved in CMH pathogenesis. miRNAs are small non-coding RNA molecules that regulate messenger RNA (mRNA) expression at post-transcriptional level by targeting the 3’ untranslated region (3’ UTR) of their mRNA target, leading to mRNA degradation or translational inhibition70_{. It is predicted that miRNAs regulate the expression of over 60% of all}

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genes in mammalian genome71_{. Expression patterns of miRNAs in differentiated}

airway epithelial cells differ from those in basal cells72_{. Several miRNAs have been}

shown to be associated with asthma or COPD73_{. The expression of 7 miRNAs were}

higher and of 15 miRNAs were lower in bronchial brushings of asthma compared

to healthy controls74_{. IL-13 stimulation alters expression of several miRNAs in this}

list, including suppression of miR-34/449 family, consistent with lower expression

in asthma74_{. In addition, the expression of 5 miRNAs was higher and of 23 miRNAs,}

including miR-146a-5p, was lower in bronchial epithelium derived from

current-smokers than from never-current-smokers75_{. miRNAs may also be involved in the crosstalk}

between epithelial cells and fibroblasts. The induction of miR-146a-5p by pro-inflammatory cytokines was lower in primary lung fibroblasts from COPD patients

compared to those from healthy controls76_{. Interestingly, miR-146a-5p silencing}

promotes MUC5AC secretion by 16HBE cells77_.

Altogether, these findings led us to the hypothesis that abnormal stromal-epithelial crosstalk contributes to CMH pathophysiology and that miRNAs act as mediators of disturbed molecular mechanisms underlying abnormalities in mucociliary differentiation, pro-inflammatory responses, and stromal cell-epithelium crosstalk in CMH (figure 2).

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1 The scope of this thesis

We started this thesis with a comprehensive review discussing the role of miRNAs and exosomes in asthma pathogenesis (chapter 2). As to date no studies on

differential miRNA expression profiles in relation to CMH or chronic bronchitis have been reported, we applied an unbiased approach to identify novel candidate miRNAs potentially involved in CMH pathophysiology. Moreover, since most in vitro studies of CMH focused solely on airway epithelial cells, we developed co-culture models using patient-derived cells that take cell-cell interactions into consideration to shed new light on our current understanding of CMH regulatory mechanisms.

The aims of this thesis were to; 1) identify CMH-associated miRNAs and their associated biological pathways in COPD (Chapter 3) and asthma (Chapter 4)

using miRNA and mRNA expression profiles of patient-derived bronchial biopsies; 2) to investigate whether and how COPD patient-derived fibroblasts promote mucin secretion and mucociliary differentiation by airway epithelial cells from COPD patients with CMH using a long-term air-liquid interface (ALI) co-culture model (Chapter 5); 3) to assess the expression of selected CMH-associated miRNAs

identified in bronchial biopsies in COPD patient-derived airway fibroblasts and epithelial cells co-cultured at ALI (Chapter 6); 4) to elucidate the function of

miR-146a-5p in disturbed fibroblast-epithelial cell crosstalk in COPD using a submerged co-culture model of lung fibroblasts and airway epithelial cells from controls and COPD patients (Chapter 7), and 5) to compare gene expression profiles of asthma-

and control-derived ASMCs and to determine the subsequent role of a selected soluble factor on mucin production using ALI-cultured Calu-3 bronchial adenocarcinoma cells and primary airway epithelial cells (Chapter 8).

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75. Schembri, F. et al. MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc. Natl. Acad. Sci. U. S. A. 106, 2319–2324 (2009).

76. Sato, T. et al. Reduced miR-146a increases prostaglandin E₂in chronic obstructive pulmonary disease fibroblasts. Am. J. Respir. Crit. Care Med. 182, 1020–1029 (2010).

77. Zhong, T., Perelman, J. M., Kolosov, V. P. & Zhou, X. D. MiR-146a negatively regulates neutrophil elastase-induced MUC5AC secretion from 16HBE human bronchial epithelial cells. Mol. Cell. Biochem. 358, 249–255 (2011).

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CHAPTER 2

Role of microRNAs and exosomes in asthma

Curr Opin Pulm Med 2019: 25(1):87-93

Maarten van den Berge1

Hataitip Tasena2

1_{University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases,} Groningen, Box 30.001 NL-9700-RB, Groningen, The Netherlands.

2_{University of Groningen, University Medical Center Groningen, Department of Pathology and Medical} Biology, Groningen, Box 30.001 NL-9700-RB, Groningen, The Netherlands.

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ABSTRACT

Purpose of this review: Numerous signaling pathways and inflammatory responses

in cells and tissues are under microRNA (miRNA) control. In the present review, the role of miRNAs and exosomes in the pathogenesis of asthma will be discussed.

Recent findings: miRNAs differentially expressed with asthma, e.g. miRNA-34/449,

let-7, miRNA-19, miRNA-21, and miRNA-455, were identified in various cell types and tissues including epithelial cells, T cells, type 2 innate lymphoid cells, lung tissues and smooth muscles. Current data suggest the involvement of these miRNAs in epithelial differentiation, mucus production, airway remodeling, inflammation, etc. However, it is often difficult to predict which genes are targeted by a specific miRNA. We recently combined genome-wide miRNA analyses together with transcriptome in bronchial biopsies, in relation to chronic mucus hypersecretion, then performed a genome-wide miRNA-mRNA network analysis and identified the key miRNA regulators for chronic mucus hypersecretion.

Summary: There is now growing evidence suggesting that miRNAs play critically

important roles in asthma. Several asthma-associated miRNAs have already been identified. Although miRNAs are attractive targets for therapeutic intervention, a safe and effective delivery to target tissues and cells in humans remains a challenge.

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2 Introduction

Asthma is a commonly occurring inflammatory airways disease characterized by variable airflow obstruction in association with symptoms of wheeze and dyspnea. The asthmatic inflammatory process is characterized by a complex interplay of resident cells (i.e. epithelial and dendritic cells, fibroblasts, nerves, endothelial cells) and inflammatory cells (eosinophils, mast cells, neutrophils, macrophages, T-lymphocytes).

At present, physicians have limited choice in anti-inflammatory and bronchodilator treatments for asthma and there is no cure available for the disease. Better insight into the underlying mechanisms that drive asthma is needed as a first step toward discovering new druggable targets. microRNAs (miRNAs) are recognized to play an important role in asthma. miRNAs are small non-coding RNA transcripts (18-25 nucleotides) that are highly conserved across species. They are involved in the posttranscriptional regulation of gene expression. Once formed, miRNAs bind to Argonaute-2 (AGO2) proteins, which are part of the RNA-induced silencing complex (RISC). In the RISC complex, miRNAs induce cleavage of their target mRNAs or inhibit their translation. A single miRNA can target hundreds of genes and it is estimated that as many as 60% of mRNAs are controlled by miRNAs1_.

It is now known that numerous cellular responses are under miRNA control enabling miRNAs to regulate signaling pathways and inflammatory responses in tissues. Some miRNAs are secreted into exosomes, cell-derived nano-sized vesicles reported to be involved in various human diseases including asthma2, 3_{. In the present review, the}

role of miRNAs and exosomes in the pathogenesis of asthma will be discussed.

MicroRNAs and asthma

Several studies investigated miRNA expression levels both in vitro and in vivo in human and experimental models of asthma, a table with an overview can be found in reference 4_{. Although many studies have been performed in vitro and in animal}

models of asthma, studies on the role of miRNAs in humans are scarce. Williams et al compared the expression of 227 miRNAs in bronchial biopsies between eight corticosteroid-naïve patients with mild allergic asthma and eight healthy controls 5_.

No differences were found which may have been due to the fact that mild asthma patients were included or to the cellular heterogeneity within bronchial biopsies which may have masked differences in miRNA expression within specific cell types. Another possible explanation may simply be that the study was underpowered for an unbiased approach analyzing 227 miRNAs as only 16 subjects were included. Solberg et al compared the miRNA expression profile in isolated epithelial cells derived from bronchial brushings between 16 steroid-naïve patients with asthma and 12 healthy controls using microarrays 6_{. They identified 22 differentially expressed miRNAs}

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family are downregulated in asthma. They validated their findings in air-liquid interface cultured epithelial cells demonstrating that exposure to interleukin(IL)13 represses miRNA-34/449 levels. This effect persisted after treating the cells with corticosteroids. These findings are of interest as miRNA-449 has previously been found to regulate differentiation of airway ciliated cells promoting centriole multiplication and multiciliogenesis, in part by targeting NOTCH1 mRNA7_{. It has}

been shown in animal models that an increase in NOTCH contributes to the airway mucous metaplasia frequently observed in asthma8_{. Taken together, the IL13-induced}

reduction in miRNA-34/449 in the bronchial epithelium, as observed by Solberg et al, may contribute to the alterations in epithelial differentiation that are often seen in asthma.

Other miRNAs found to be downregulated in epithelial cells derived from steroid-naïve patients with asthma were members of the 7 family (7a-5p, let-7c, let-7f-5p, let-7g-5p, and let-7i-5p)6_{. Several studies have shown the let-7 family}

to be important in asthma. Polikepahad et al demonstrated that IL13 is a direct target of let-7 using a luciferase reporter system and inhibition of let-7a significantly upregulated IL13 expression in T cells 9, 10._{In ovalbumin(OVA)-challenged murine}

model, expression of several let-7 family members decreased in allergic inflammatory lungs compared to healthy lungs 11_{. Intranasal delivery of a let-7 mimic improved}

airway hyperresponsiveness and mucus production and decreased inflammatory cell infiltration in this experimental model of asthma. These findings suggest that decreased levels of let-7 increases type 2 inflammation thus contributing to a more severe asthma. However, this could not be confirmed in a subsequent study by Polikepahad et al where an anti-let-7 locked nucleic acid (LNA) antagomir did not worsen the asthmatic inflammatory process in their OVA-challenged murine model, but contrastingly reduced inflammatory cell counts in bronchoalveolar lavage fluid and decreased levels of IL4, IL5 and IL13 9_{. Possible explanations for the discrepant}

findings between the studies could be different dose regimens or route of administration of the anti-let-7 antagomir used or the fact that the antagomir used by Polikepahad et al only inhibited four members of the let-7 family. In a recent study by Kim et al, exposure of human airway smooth muscle cells to β2-agonists increased let-7f by 2-3-fold together with a ~90% decrease in β2-receptors, while inhibition of let-7f reduced this downregulation by 50%12_{. These findings suggest that inhibition of let-7}

may render airway smooth muscle more sensitive to bronchodilator effects of β2-agonists. In a study in COPD, we recently found increased let-7 in bronchial biopsies to be associated with chronic mucus hypersecretion (CMH)13_{. Whether this is also}

the case in asthma is currently under investigation. One miRNA can target multiple mRNAs, validation of its target genes is challenging since many miRNA-mRNA interactions are still unknown14_{. Several algorithms have been proposed to predict}

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different algorithms and the false positive rates are high15_{. Importantly, we analyzed}

both miRNA and mRNA data available from the same bronchial biopsies derived from COPD patients13_{, which allowed us to create miRNA-mRNA co-expression networks}

to directly evaluate the miRNA-mRNA interactions. This approach enabled us to identify the let-7 family with its CMH-associated targets including EDN1, NKD1, PDGFB, COL4A1, and COL4A2 as key important regulators of CMH in COPD.

In a recent study, Martinez-Nunez et al performed miRNA sequencing (miRNA-seq) in cultured bronchial epithelial cells derived from 8 severe asthma patients and 5 healthy controls. To determine the genome-wide relationship between differentially expressed miRNAs and their mRNA targets, they performed subcellular fractionation and RNA-seq (frac-seq) in the same samples allowing detection of both cytoplasmic mRNA as well as polyribosome-bound mRNA transcripts16_{. A total of 21}

miRNAs were differentially expressed between severe asthma and healthy controls. Importantly, these miRNAs were found to preferentially target polyribosome-bound rather than cytoplasmic mRNA transcripts, suggesting a higher impact on translating mRNAs. Amongst the most differentially expressed miRNAs in the study by Martinez-Nunez was miRNA-19. This is in agreement with the findings of Haj-Salem et al who showed miRNA-19a to be specifically upregulated in cultured bronchial epithelial cells derived from patients with severe asthma compared to those with mild asthma and healthy controls17_{. Follow-up functional studies revealed that}

upregulation of miRNA-19a leads to an increased epithelial cell proliferation rate by targeting TGFB2 mRNA, thus possibly contributing to airway remodeling in severe asthma. In another study, miRNA-19, part of the 17~92 cluster (also including miRNA-17, miRNA-18, and miRNA-92), was found to play an important role in T cells regulating Th2 cell differentiation18_{. The miRNA was upregulated in CD3+}

CD4+ T cells sorted from bronchoalveolar lavage fluid (BAL) fluid from asthma patients compared to healthy controls, independent of the use of steroids. In follow-up experiments, it was demonstrated that CD4 cells lacking the miRNA-17~92 cluster produced fewer Th2 cytokines like IL4 and IL13, whereas this cytokine defect could be completely restored after transfection with a miRNA-19a or miRNA-19b mimic, but not after transfection with a mimic for other members of the 17~92 cluster. In a recent study, miRNAs of the miRNA-17~92 cluster were also found to be critically important for normal type 2 innate lymphoid cell (ILC2) survival, another source of type 2 cytokines; and similar as in T cells, miRNA-19 was particularly involved in IL13 production19_.

Little is known about how miRNAs contribute to airway hyperresponsiveness and remodeling which are important features of asthma and COPD. Chiba et al reported that miR-133a inhibition in in vitro cultured human bronchial smooth muscle cells leads to an upregulation of RhoA, a key protein regulating contractility of smooth muscles20_{. Recently, we investigated how miRNAs contribute to airway remodeling,}

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an important feature in asthma and COPD. To this end, we investigated how TGF-β influences miRNA expression in fibroblasts and how this affects their function 21_.

After exposure to TGF-β, miRNA-21 and miRNA-455 expression increased in cultured human lung fibroblasts. We then used the AGO2 ImmunoPrecipitation-gene Chip approach to identify miRNA targets on a large scale. With this approach the AGO2 protein is directly immunoprecipitated. Subsequently, the AGO2-associated mRNA transcripts were analyzed by microarray, to investigate the miRNA targetome (the whole miRNA-regulated gene set) in fibroblasts either or not exposed to TGF-β. We found that the predicted miRNA-455 and miRNA-21 targets present in the miRNA targetome of unstimulated and TGF-β-stimulated human lung fibroblasts are significantly enriched for TGF-β associated signaling processes. These findings show that there exists a cross-talk between the TGF-β pathway and the miRNAs, i.e. miRNA-445 and miRNA-21. In animal models of asthma, miRNA-21 has been shown to be upregulated in the airway wall22, 23_{. Using the OVA-model, Lu et al found}

that miRNA-21-deficient mice had reduced levels of eosinophilic inflammation and IL4 in their lungs and BAL fluid22, 23_{. A plausible implication of miRNA-455 was}

reported in a study by Garbacki et al who observed miR-455 upregulation in the lungs of allergen-exposed mice.24_{Taken together, accumulating evidence points}

towards a potentially important role of several miRNAs in airway inflammation and remodeling in asthma, including miRNA-34/449, let-7, miRNA-19, miRNA-21, and miRNA-455, as summarized in figure 1.

Exosomes

Exosomes are small (10-150 nm) membrane-bound vesicles. They can be released by e.g. inflammatory cells upon activation and are thought to play a crucial role in intercellular signaling and communication25_{. Genetic material, proteins, and lipid}

mediators can be packaged in exosomes and transferred to cells both locally and distally. Exosomes are found in a large variety of body fluids including blood, urine, BAL fluid, exhaled breath condensate, saliva and nasal lavage fluid. Exosomes also contain miRNAs and it has been shown that miRNAs packaged in exosomes can contribute to inflammation in many diseases including asthma. Sinha et al were able to demonstrate that exosomes are present in exhaled breath condensate from patients with asthma and healthy controls and contain the majority of the 634 miRNAs detectable in exhaled breath condensate26_{. Levänen et al demonstrated the}

presence of miRNAs in exosomes isolated from BAL fluid from 10 asthmatics and 10 healthy controls3_{. Despite the fact that patients with mild intermittent asthma}

were included, as many as 24 exosomal miRNAs were found to be differentially expressed, including let-7 which was downregulated in asthma. A total of 22 out of the 24 exosomal miRNAs in BAL fluid were previously also found to be altered in the airway epithelium of patients with asthma in the study by Solberg et al, with the

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same direction of effect6_{. Thus, exosomes can carry and transport miRNAs making}

intercellular communication possible and may play important roles in asthma.

Figure 1. Key miRNAs and their potential roles in asthma. The figure summarizes miRNAs of which

expression is associated with asthma, cell types in which the differential expression was determined, and how the miRNAs possibly play a role in asthma. AHR is airway hyperresponsiveness; ILC2 is type 2 innate lymphoid cells.

MicroRNA-based treatment of asthma

There is now increasing evidence that specific miRNAs play potentially important roles in asthma and they can be either inhibited or overexpressed. Therefore, miRNA-based therapeutics are an attractive area of investigation.

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inhibitors or mimics in animal models of asthma, safe and effective delivery to target tissues or cells in humans remains a challenge. The latter is important as miRNA targeting can affect multiple genes and therefore ‘off-target’ side-effects should be prevented as much as possible. A commonly used method to inhibit the function of a specific miRNA is to deliver oligonucleotides with a complementary sequence that bind and inactivate the miRNA. To prevent degradation of these oligonucleotides by RNases, chemical modifications can be used, including LNAs and addition of 2’-O-methyl groups.27_{Further, addition of cholesterol tags may help to facilitate}

their uptake into target cells28_{. An alternative to chemically modified antisense}

oligonucleotides may be the introduction of a so-called ‘microRNA sponge’-encoding transgene into the cell, the expressed transcript being an engineered RNA molecule with multiple complementary binding sites to the target miRNA. Conversely, overexpression of miRNAs can be achieved with synthetic miRNAs that mimic natural miRNAs. These miRNA mimics often require liposomes, lipoprotein-based carriers, or nanoparticles as vehicles for their delivery29, 30_{. Currently,}

miRNA-based therapeutics are not (yet) in development for asthma, but there are successful examples that modulation of miRNAs can be treatment strategy. The best example so far is miravirsen, the world’s first miRNA therapeutic, which is a short LNA for miR-122 currently in phase II clinical trials for the treatment of hepatitis C virus (HCV) infection31_{. Other examples of miRNA therapeutics in clinical trials include}

antimiR-103/107, antimiR-155, miR-29 mimic, miR-16 mimic, and miR-34 mimic29_.

There are also several other miRNA therapeutics currently being investigated in preclinical models29_.

Conclusions

There is now increasing evidence that miRNAs play potentially important roles in asthma. Several miRNAs have already been identified including miRNA-34/449, let-7, miRNA-19, miRNA-21, and miRNA-455. A limitation of studies performed so far is that they were mostly done in in vitro and in animal models of asthma and evidence in humans remains limited. Adequately powered studies leading to a better insight in the role of miRNAs in relevant human cells and tissues are now needed. miRNAs can play a central role in the regulation of inflammatory responses as one miRNA can target multiple genes. However, once a relevant miRNA has been identified, it is often difficult to predict which genes/pathways are actually regulated by that specific miRNA as target prediction software is far from perfect. Therefore, we have previously combined genome-wide miRNA analyses with transcriptome data from the same sample, in this case bronchial biopsies, in relation to chronic mucus hypersecretion. Using this approach, we examined direct and indirect miRNA-mRNA associations responsible for CMH. In addition, we were able to perform a genome-wide miRNA-mRNA network analysis to identify the key miRNA regulators (figure 2). Another

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approach to identify miRNA target genes is to perform AGO2-Immunoprecipation

in cultured cells, i.e. with and without overexpression or inhibition of a miRNA of interest. With this technique, all genes regulated by miRNAs are captured and can then further be investigated using microarray or RNA-sequencing to identify the targetome of the miRNA of interest. The central role of miRNAs in regulation of inflammatory processes makes them attractive targets for therapeutic intervention and there have been several successful examples of treatments with miRNA inhibitors or mimics in animal models of asthma. Although the safe and effective delivery to target tissues and cells in humans remains a challenge, improved technologies are emerging to facilitate the development of potential miRNA-based treatments in several indications including asthma.

Figure 2. miRNA-mRNA co-expression network showing the key regulatory miRNA and their correlated predicted target genes associated with chronic mucus hypersecretion in COPD. This

analysis identified amongst other, the let-7 family with its CMH-associated targets including EDN1, NKD1, PDGFB, COL4A1, and COL4A2 as key important regulators. Red diamonds represent miRNAs higher expressed with CMH; blue diamonds represent miRNAs lower expressed with CMH; green circles represent predicted target genes negatively correlated with that miRNA; black circles represent predicted target genes negatively correlated with that miRNA whose expression was also associated with CMH. Line width correlates to degree of significance of the miRNA-mRNA correlation. Figure reproduced with permission from Tasena et al.13

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3. Levanen B, Bhakta NR, Torregrosa Paredes P, Barbeau R, Hiltbrunner S, Pollack JL, Skold CM, Svartengren M, Grunewald J, Gabrielsson S, Eklund A, Larsson BM, Woodruff PG, Erle DJ, Wheelock AM. Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients. J Allergy Clin Immunol 2013; 131: 894-903.

4. Svitich OA, Sobolev VV, Gankovskaya LV, Zhigalkina PV, Zverev VV. The role of regulatory RNAs (miRNAs) in asthma. Allergol Immunopathol (Madr) 2018; 46: 201-205.

5. Williams AE, Larner-Svensson H, Perry MM, Campbell GA, Herrick SE, Adcock IM, Erjefalt JS, Chung KF, Lindsay MA. MicroRNA expression profiling in mild asthmatic human airways and effect of corticosteroid therapy. PLoS One 2009; 4: e5889.

6. Solberg OD, Ostrin EJ, Love MI, Peng JC, Bhakta NR, Hou L, Nguyen C, Solon M, Nguyen C, Barczak AJ, Zlock LT, Blagev DP, Finkbeiner WE, Ansel KM, Arron JR, Erle DJ, Woodruff PG. Airway epithelial miRNA expression is altered in asthma. Am J Respir Crit Care Med 2012; 186: 965-974.

7. Marcet B, Chevalier B, Luxardi G, Coraux C, Zaragosi LE, Cibois M, Robbe-Sermesant K, Jolly T, Cardinaud B, Moreilhon C, Giovannini-Chami L, Nawrocki-Raby B, Birembaut P, Waldmann R, Kodjabachian L, Barbry P. Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway. Nat Cell Biol 2011; 13: 693-699.

8. Guseh JS, Bores SA, Stanger BZ, Zhou Q, Anderson WJ, Melton DA, Rajagopal J. Notch signaling promotes airway mucous metaplasia and inhibits alveolar development. Development 2009; 136: 1751-1759.

9. Polikepahad S, Knight JM, Naghavi AO, Oplt T, Creighton CJ, Shaw C, Benham AL, Kim J, Soibam B, Harris RA, Coarfa C, Zariff A, Milosavljevic A, Batts LM, Kheradmand F, Gunaratne PH, Corry DB. Proinflammatory role for let-7 microRNAS in experimental asthma. J Biol Chem 2010; 285: 30139-30149.

10. Orom UA, Kauppinen S, Lund AH. LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 2006; 372: 137-141.

11. Kumar M, Ahmad T, Sharma A, Mabalirajan U, Kulshreshtha A, Agrawal A, Ghosh B. Let-7 microRNA-mediated regulation of IL-13 and allergic airway inflammation. J Allergy Clin Immunol 2011; 128: 1077-1085 e1071-1010.

12. Kim D, Cho S, Woo JA, Liggett SB. A CREB-mediated increase in miRNA let-7f during prolonged beta-agonist exposure: a novel mechanism of beta2-adrenergic receptor down-regulation in airway smooth muscle. FASEB J 2018; 32: 3680-3688.

13. Tasena H, Faiz A, Timens W, Noordhoek J, Hylkema MN, Gosens R, Hiemstra PS, Spira A, Postma DS, Tew GW, Grimbaldeston MA, van den Berge M, Heijink IH, Brandsma CA. microRNA-mRNA regulatory networks underlying chronic mucus hypersecretion in COPD. Eur Respir J 2018.

14. Tan LP, Seinen E, Duns G, de Jong D, Sibon OC, Poppema S, Kroesen BJ, Kok K, van den Berg A. A high throughput experimental approach to identify miRNA targets in human cells. Nucleic Acids Res 2009; 37: e137.

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15. Sethupathy P, Megraw M, Hatzigeorgiou AG. A guide through present computational approaches for the identification of mammalian microRNA targets. Nat Methods 2006; 3: 881-886.

16. Martinez-Nunez RT, Rupani H, Plate M, Niranjan M, Chambers RC, Howarth PH, Sanchez-Elsner T. Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq. J Immunol 2018; 201: 251-263.

17. Haj-Salem I, Fakhfakh R, Berube JC, Jacques E, Plante S, Simard MJ, Bosse Y, Chakir J. MicroRNA-19a enhances proliferation of bronchial epithelial cells by targeting TGFbetaR2 gene in severe asthma. Allergy 2015; 70: 212-219.

18. Simpson LJ, Patel S, Bhakta NR, Choy DF, Brightbill HD, Ren X, Wang Y, Pua HH, Baumjohann D, Montoya MM, Panduro M, Remedios KA, Huang X, Fahy JV, Arron JR, Woodruff PG, Ansel KM. A microRNA upregulated in asthma airway T cells promotes TH2 cytokine production. Nat Immunol 2014; 15: 1162-1170. 19. Singh PB, Pua HH, Happ HC, Schneider C, von Moltke J, Locksley RM, Baumjohann D, Ansel KM. MicroRNA

regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation. J Exp Med 2017; 214: 3627-3643.

20. Chiba Y, Tanabe M, Goto K, Sakai H, Misawa M. Down-regulation of miR-133a contributes to up-regulation of Rhoa in bronchial smooth muscle cells. Am J Respir Crit Care Med 2009; 180: 713-719.

21. Ong J, Timens W, Rajendran V, Algra A, Spira A, Lenburg ME, Campbell JD, van den Berge M, Postma DS, van den Berg A, Kluiver J, Brandsma CA. Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts. PLoS One 2017; 12: e0183815. 22. Lu TX, Hartner J, Lim EJ, Fabry V, Mingler MK, Cole ET, Orkin SH, Aronow BJ, Rothenberg ME.

MicroRNA-21 limits in vivo immune response-mediated activation of the IL-12/IFN-gamma pathway, Th1 polarization, and the severity of delayed-type hypersensitivity. J Immunol 2011; 187: 3362-3373.

23. Mattes J, Collison A, Plank M, Phipps S, Foster PS. Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease. Proc Natl Acad Sci U S A 2009; 106: 18704-18709.

24. Garbacki N, Di Valentin E, Huynh-Thu VA, Geurts P, Irrthum A, Crahay C, Arnould T, Deroanne C, Piette J, Cataldo D, Colige A. MicroRNAs profiling in murine models of acute and chronic asthma: a relationship with mRNAs targets. PLoS One 2011; 6: e16509.

25. Sastre B, Canas JA, Rodrigo-Munoz JM, Del Pozo V. Novel Modulators of Asthma and Allergy: Exosomes and MicroRNAs. Front Immunol 2017; 8: 826.

26. Sinha A, Yadav AK, Chakraborty S, Kabra SK, Lodha R, Kumar M, Kulshreshtha A, Sethi T, Pandey R, Malik G, Laddha S, Mukhopadhyay A, Dash D, Ghosh B, Agrawal A. Exosome-enclosed microRNAs in exhaled breath hold potential for biomarker discovery in patients with pulmonary diseases. J Allergy Clin Immunol 2013; 132: 219-222.

27. Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M. Silencing of microRNAs

in vivo with ‘antagomirs’. Nature 2005; 438: 685-689.

28. Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z, Zamore PD. Target RNA-directed trimming and tailing of small silencing RNAs. Science 2010; 328: 1534-1539.

29. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov 2017; 16: 203-222.

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30. Kreth S, Hubner M, Hinske LC. MicroRNAs as Clinical Biomarkers and Therapeutic Tools in Perioperative Medicine. Anesth Analg 2018; 126: 670-681.

31. Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol 2012; 199: 407-412. *Describes discovery of first miRNA-based treatment for a human disease.

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CHAPTER 3

MicroRNA-mRNA regulatory networks underlying

chronic mucus hypersecretion in COPD

Eur Respir J 2018: 52(3): 1701556

Hataitip Tasena1,2_,_{Alen Faiz}1,2,3_,_{Wim Timens}1,2_,_{Jacobien Noordhoek}1,2,3_,

Machteld N Hylkema1,2_,_{Reinoud Gosens}2,4_,_{Pieter S Hiemstra}5_,

Avrum Spira6_,_{Dirkje S Postma}2,3_,_{Gaik W Tew}7_,

Michele A Grimbaldeston8_{, Maarten van den Berge}2,3_,

*Irene H Heijink1,2,3 _{and *Corry-Anke Brandsma}1,2

(*both authors contributed equally)

1_{University of Groningen, University Medical Centre Groningen, Department of Pathology and Medical} Biology, Groningen, the Netherlands.

2_{University of Groningen, University Medical Centre Groningen, Groningen Research nstitute for Asthma} and COPD, Groningen, the Netherlands.

3_{University of Groningen, University Medical Centre Groningen, Department of Pulmonary Diseases,} Groningen, the Netherlands.

4_{University of Groningen, Department of Molecular Pharmacology, Groningen, the therlands.} 5_{Leiden University Medical Centre, Department of Pulmonology, Leiden, The Netherlands.}

6_{Boston University Medical Centre, Department of Medicine, Division of Computational Biomedicine,} Boston, Massachusetts, USA.

7_{Research & Early Development, Genentech Inc, South San Francisco, California, USA.} 8_{OMNI-Biomarker Development, Genentech Inc, South San Francisco, California, USA.}

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ABSTRACT

Chronic mucus hypersecretion (CMH) is a common feature in COPD and associated with worse prognosis and quality of life. This study aimed to identify microRNA (miRNA)-mRNA regulatory networks underlying CMH.

miRNA and mRNA expression profiles in bronchial biopsies from 63 COPD patients were associated with CMH using linear regression. Potential mRNA targets of each CMH-associated miRNA were identified using Pearson correlations. GSEA and STRING analyses were used to identify key genes and pathways.

Twenty miRNAs and 539 mRNAs were differentially expressed with CMH in COPD. The expression of 10 miRNAs was significantly correlated with the expression of one or more mRNAs. Of these, miR-134-5p, miR146a-5p and the let-7 family had the highest representation of CMH-associated mRNAs among their negatively correlated predicted targets. KRAS and EDN1 were identified as key regulators of CMH and were negatively correlated predicted targets of miR-134-5p and the let-7a/ d/f-5p, respectively. GSEA suggested involvement of MUC5AC-related genes and several other relevant gene sets in CMH. The lower expression of miR-134-5p was confirmed in primary airway fibroblasts from COPD patients with CMH.

We identified miR-134-5p, miR-146a-5p and let-7 family, along with their potential target genes including KRAS and EDN1, as potential key miRNA-mRNA networks regulating CMH in COPD.

University of Groningen Chronic mucus hypersecretion in COPD and asthma Tasena, Hataitip

Chronic mucus hypersecretion in COPD and asthma

Tasena, Hataitip

Chronic mucus hypersecretion in COPD and

asthma: Involvement of microRNAs and

stromal cell-epithelium crosstalk

Groningen University Institute for Drug Exploration (GUIDE), the Groningen

Research Institute for Asthma and COPD (GRIAC), and U4 Ageing Lung

Consortium. They are financially supported by the University of Groningen,

Stichting Astma Bestrijding (SAB), and de Cock foundation.

Printing of this thesis was financially supported by the University of Groningen,

the Graduate school of Medical Sciences Groningen (GSMS), and Stichting

Astma Bestrijding (SAB).

ISBN (printed): 978-94-034-1704-2

ISBN (electronic): 978-94-034-1703-5

Cover design by Nick (fiverr.com/glitchfool)

Lay out by Abubakar Abdullahi Dubagari and Hataitip Tasena

Printed by Ipskamp Printing, Enschede

© 2019, Hataitip Tasena, The Netherlands

All rights reserved. No part of this thesis may be reproduced or transmitted

in any form or by any means without prior permission of the author or, when

appropriate, of the publisher of the publication.

Involvement of microRNAs and

stromal cell-epithelium crosstalk

PhD thesis

to obtain the degree of PhD at the

University of Groningen

on the authority of the

Rector Magnificus prof. E. Sterken

and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on

Wednesday 3 July 2019 at 12.45 hours

by

Hataitip Tasena

born on 23 April 1989

in Chiang Rai, Thailand

Prof. H.I. Heijink

Prof. W. Timens

Co-supervisors

Dr. C.A. Brandsma

Dr. M. van den Berge

Assessment Committee

Prof. K. Bracke

Prof. G.H. Koppelman

Prof. A. van den Berg

Jennie Ong

CONTENTS

CHAPTER 1

General introduction

9

CHAPTER 2

Role of microRNAs and exosomes in asthma

21

Opin Pulm Med 2019: 25(1):87-93

CHAPTER 3

MicroRNA-mRNA regulatory networks underlying 33

chronic mucus hypersecretion in COPD

Eur Respir J 2018: 52(3): 1701556

CHAPTER 4

MiR-31-5p: a shared regulator of chronic

71

mucus hypersecretion in asthma and chronic

obstructive pulmonary disease

CHAPTER 5

Airway mucus secretion in COPD-derived

87

airway epithelial cells is promoted by

fibroblast-epithelium crosstalk

CHAPTER 6

Involvement of miRNAs associated with chronic

109

mucus hypersecretion in fibroblast-epithelium

crosstalk in COPD

CHAPTER 7

MiR-146a-5p plays an essential role in the aberrant 119

epithelial-fibroblast crosstalk in COPD

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CHAPTER 8

Profiling of healthy and asthmatic airway smooth