AJRCMB Articles in Press. Published on 13-April-2015 as 10.1165/rcmb.2014-0356OC

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Title

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In utero vitamin D deficiency increases airway smooth muscle mass and impairs lung

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function

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Authors

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1

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Hart, 2Graeme R Zosky

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Author contributions

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Conception and design of research: REF, PHH, SG, GRZ; Acquisition of data: REF, ACJ;

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Data analysis: REF, AB, AG. Interpretation of results: REF, AB; Drafting the manuscript for

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important intellectual content: REF; Revision of manuscript: REF, AB, SG, PHH, GZ;

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Approval of final version of manuscript: REF, AB, ACJ, AG, SG, PHH, GRZ.

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Affiliations

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1

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Australia;

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2

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Australia.

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Corresponding author

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Rachel E Foong

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Tel no.: +618 9489 7819

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Fax no.: +618 9489 7700

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Email: [email protected]

Rachel E Foong, 1Anthony Bosco, 1 Anya C Jones, 1Alex Gout, 1Shelley Gorman, 1Prue H

Telethon Kids Institute, the University of Western Australia, Perth, Western Australia,

School of Medicine, Faculty of Health Science, University of Tasmania, Hobart, Tasmania,

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Copyright © 2015 by the American Thoracic Society

AJRCMB Articles in Press. Published on 13-April-2015 as 10.1165/rcmb.2014-0356OC

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Running title

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In utero vitamin D deficiency causes lung deficits

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Funding Sources

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This study was funded by the National Health and Medical Research Council of Australia

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(1042235). RF is a recipient of an Australian Postgraduate Award, Stan and Jean Perron Top-

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Up Award and Asthma Foundation Western Australia Top-Up Award. AB is supported by a

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BrightSpark Foundation McCusker Fellowship. ACJ is a recipient of an Australian

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Postgraduate Award and a Top-Up Award from the University of Western Australia. SG is a

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recipient of the BrightSpark Foundation Fellowship.

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Copyright © 2015 by the American Thoracic Society

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AJRCMB Articles in Press. Published on 13-April-2015 as 10.1165/rcmb.2014-0356OC

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Abstract

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We have previously demonstrated increased airway smooth muscle (ASM) mass and airway

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hyperresponsiveness (AHR) in whole-life vitamin D-deficient female mice. In this study we

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aimed to uncover the molecular mechanisms contributing to altered lung structure and

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function. RNA was extracted from lung tissue of whole-life vitamin D-deficient and -replete

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female mice, and gene expression patterns were profiled by RNA-Seq. The data showed that

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genes

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morphogenesis, Wnt signalling, and inflammation were differentially expressed in vitamin D-

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deficient mice. Network analysis suggested that differentially expressed genes were

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connected by the hubs MMP9, NFKBIA, EGFR and EP300. Given our findings that

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developmental pathways may be altered, we investigated if the timing of vitamin D exposure

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(in utero versus postnatal) had an impact on lung health outcomes. Gene expression was

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measured in in utero or postnatal vitamin D-deficient mice, as well as whole-life vitamin D-

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deficient and -replete mice at 8-weeks of age. Baseline lung function, AHR and airway

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inflammation were measured and lungs fixed for lung structure assessment using

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stereological methods, and quantification of ASM mass. In utero vitamin D deficiency was

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sufficient to increase ASM mass, baseline airway resistance, and alter lung structure. There

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were increased neutrophils but decreased lymphocytes in bronchoalveolar lavage. Expression

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of inflammatory molecules S100A9 and S100A8 was mainly increased in postnatal vitamin

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D-deficient mice. These observations suggest that in utero vitamin D deficiency can alter

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lung structure and function, and increase inflammation, contributing to symptoms in chronic

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diseases such as asthma.

involved

in

embryonic

organ

development,

pattern

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Key words

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Copyright © 2015 by the American Thoracic Society

formation,

branching

AJRCMB Articles in Press. Published on 13-April-2015 as 10.1165/rcmb.2014-0356OC

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Airway remodeling, inflammation, RNA-Seq, mouse model, vitamin D

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Introduction

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Vitamin D deficiency is associated with chronic lung diseases such as asthma and chronic

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obstructive pulmonary disease (COPD) (1, 2). Asthma and COPD are both characterized by

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airway hyperresponsiveness (AHR) and airway remodeling (3), including an increase in

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airway smooth muscle (ASM) mass (4). We have recently demonstrated increased ASM mass

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in mice exposed to whole-life vitamin D deficiency (5). The increases in ASM were

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associated with gene expression levels of transforming growth factor (TGF)-β signalling

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molecules in vitamin D-deficient embryos at embryonic day 17.5, indicating that vitamin D

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deficiency may impinge on developmental pathways in early life. These observations support

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previous studies showing that low serum vitamin D levels are associated with increased ASM

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mass in children with severe asthma (6). Similarly, in vitro studies show that 1,25-

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hydroxyvitamin D (1,25(OH)2D3), the active metabolite of vitamin D, inhibits the

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proliferation of ASM cells (7), providing evidence that vitamin D has the capacity to

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modulate airway remodeling.

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Experimental animal studies suggest that vitamin D affects lung development. The

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maturation of fetal rat type II alveolar epithelial cells (AECs), which are important for the

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synthesis of surfactant and represent a key event in fetal lung development, is induced by

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1,25(OH)2D3 (8). Alveolar epithelial-mesenchymal interactions that are important for alveolar

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development are also influenced by 1,25(OH)2D3 (9). Furthermore, genetic studies show a

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high number of vitamin D regulated genes are represented in the developing human and

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murine lung transcriptome (10). One-third of these vitamin D regulated genes were

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differentially expressed in asthmatic children compared to non-asthmatic sibling controls

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Copyright © 2015 by the American Thoracic Society

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AJRCMB Articles in Press. Published on 13-April-2015 as 10.1165/rcmb.2014-0356OC

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suggesting that vitamin D deficiency during lung development may lead to asthma later in

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life.

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A recent study from our group investigating the effects of maternal vitamin D status on

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postnatal lung function outcomes found impaired lung function and increased asthma risk in

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children exposed to maternal vitamin D deficiency (11). This was consistent with our earlier

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work showing a causal association between early life vitamin D deficiency and lung structure

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and function in a mouse model (12). Based on our recent observation that whole-life vitamin

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D deficiency increases ASM mass, we sought to investigate pathways that might be altered

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by vitamin D deficiency in the adult lung using RNA sequencing (RNA-Seq). We focused on

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female mice because vitamin D deficiency induced physiological changes are only present in

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female mice (5). Moreover, the maternal effects of vitamin D deficiency on postnatal lung

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function are strongest in girls (11). Given the existing evidence supporting a link between

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early life vitamin D deficiency and lung health, we also investigated the relative impact of in

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utero and/or postnatal vitamin D deficiency on ASM, lung mechanics, AHR, inflammation

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and gene expression patterns to determine when lung structure and function are the most

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sensitive to the effects of vitamin D deficiency.

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Materials and Methods

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Mouse model

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A mouse model of vitamin D deficiency was established by dietary manipulation as described

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previously (5). To compare the effects of in utero and postnatal vitamin D deficiency, pups

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were cross-fostered to produce in utero vitamin D-deficient (VitD-|+) or postnatally vitamin

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D-deficient (VitD+|-), as well as whole-life vitamin D-deficient (VitD-|-) or whole-life

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vitamin D-replete (VitD+|+) mice. All studies were carried out on female mice when they

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were 8-weeks of age. All procedures were approved by the Telethon Kids Institute Animal 5

Copyright © 2015 by the American Thoracic Society

AJRCMB Articles in Press. Published on 13-April-2015 as 10.1165/rcmb.2014-0356OC

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Ethics Committee and conformed to National Health and Medical Research Council of

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Australia guidelines. Further details are provided in the online supplement.

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RNA-Seq protocol and data analysis

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Total RNA was extracted from lung tissue of whole-life vitamin D-deficient and -replete

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mice using TRIzol (Life Technologies, Victoria, Australia) followed by RNeasy (Qiagen,

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Victoria, Australia). Library preparation (Illumina TruSeq RNA Sample Prep Kit v2) and

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sequencing (50bp single-end using an Illumina HiSeq2000) was performed by the Australian

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Genome Research Facility Ltd. The raw sequencing results are available at the NCBI Short

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Read Archive under accession SRP046087. Further details regarding the RNA-Seq data

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analysis is provided in the online supplement.

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Reverse transcription quantitative PCR (RT-qPCR)

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A subset of differentially expressed genes identified in the RNA-Seq experiment was

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validated using RT-qPCR. Reverse transcription was carried out using the QuantiTect

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Reverse Transcription Kit (Qiagen). Real-time PCR primer assay sequences were obtained

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from Primerbank (13) and the primer list included in the online supplement. QuantiTect

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SYBR Green was used for qPCR on the ABI7900HT instrument. Relative standard curves

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were prepared from serially diluted PCR products (14).

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Lung function and methacholine challenge

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Baseline lung function was assessed using the low-frequency forced oscillation technique

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(15). After lung function measurements, mice were transferred to a flexiVent system

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(SCIREQ, Montreal, Canada) for assessment of responsiveness to methacholine (β-

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methacholine chloride, Sigma-Aldrich, Missouri, USA) as described previously (5). Further

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details are provided in the online supplement.

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Copyright © 2015 by the American Thoracic Society

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Differential cell counts

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Bronchoalveolar lavage (BAL) was collected from mice by washing 500 µL of sterile saline

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into the lungs 3 times. Total cell collected in the BAL were counted using a haemocytometer.

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The remaining cells were cytospun and stained with Leischman’s stain for differential cell

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counts.

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Histology and immunohistochemistry

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Lung structure was assessed in independent groups of mice following ATS/ERS guidelines

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(16). The right lung was used for lung stereology as described previously (12). Transverse

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sections from the left lung were stained with Masson’s Trichrome for measurement of ASM

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mass (5). Immunohistochemical staining was performed using primary antibodies for

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S100A9, S100A8, phosphorylated IκBα, phosphorylated EGFR and MMP9. Further details

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are provided in the online supplement.

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Statistical analyses

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Between group comparisons were made using t tests or 2-way ANOVAs with Holm-Sidak

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posthoc tests. Pearson’s correlation tests were performed to assess correlations between AHR

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and inflammatory parameters. Statistical analyses were performed using SigmaPlot software

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(SysStat Software, Illinois, USA) and Prism (GraphPad Software, California, USA). Data are

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shown as mean (SD). P values of

The effects of in utero vitamin D deficiency on airway smooth muscle mass and lung function.

We have previously demonstrated increased airway smooth muscle (ASM) mass and airway hyperresponsiveness in whole-life vitamin D-deficient female mice...
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