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

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1 1 Supplemental Information 2 Title 3 4 In utero vitamin D deficiency increases airway smooth muscle mass and impairs lung function 5 Authors Rachel E Foong, 1 Anthony Bosco, 1 Anya C Jones, 1 Alex Gout, 1 Shelley Gorman, 1 Prue H Hart, 2 Graeme R Zosky 8 Affiliations Telethon Kids Institute, the University of Western Australia, Perth, Western Australia, Australia; School of Medicine, Faculty of Health Science, University of Tasmania, Hobart, Tasmania, Australia. 13 Materials and Methods 14 Mouse model Three-week-old female BALB/c mice (Animal Resource Centre, Murdoch, Australia) were fed vitamin D 3 -deficient or -replete diets containing 0 or 2280 IU vitamin D 3, respectively (Specialty Feeds, Glen Forrest, Western Australia) as previously described (1). Deficient diets were supplemented with 2% (vs 1%) calcium to maintain serum calcium in vitamin D- deficient mice which has previously been shown to result in no differences in serum calcium levels between groups (2). Mice were housed in a room with a 12-hour ultraviolet B-free light/dark cycle. At 8-weeks of age, female mice were mated with vitamin D-replete male BALB/c mice. To compare the effects of in utero and postnatal vitamin D deficiency, vitamin

2 D-deficient pups were fostered to vitamin D-replete dams and vice versa. This produced mice that were vitamin D-deficient in utero (VitD- +) or postnatally vitamin D-deficient (VitD+ -). Pups were also fostered to different vitamin D-deficient dams, as well as -replete dams, as controls. These were the whole-life vitamin D-deficient (VitD- -) or whole life vitamin D- replete (VitD+ +) groups. Vitamin D deficiency was confirmed by measuring serum 25(OH)D using IDS OCTIEIA ELISA kits (Immunodiagnostic Systems Ltd, Boston Business Park, UK). Serum 25(OH)D levels in VitD- - and VitD+ - mice (14.6 [0.76] nmol/l) were significantly lower than VitD+ + and VitD- + mice (70.0 [12.1] nmol/l). Studies were carried out on female mice when they were 8-weeks of age. Bronchoalveolar lavage and lung tissue for gene expression studies were collected from euthanized mice upon completion of lung function assessment, while lungs fixed for structural assessment were obtained from a separate group of mice. All procedures were approved by the Telethon Kids Institute Animal Ethics Committee and conformed to National Health and Medical Research Council of Australia guidelines. 15 RNA-Seq protocol and data analysis Lung tissue samples from adult whole-life vitamin D-deficient and -replete female mice (n = 6 per group) were stabilized in RNAlater (Sigma-Aldrich, New South Wales, Australia). The samples were disrupted in TRIzol (Life Technologies, Victoria, Australia) employing a TissueRuptor rotor-stator homogenizer (Qiagen, Victoria, Australia). Total RNA was extracted from TRIzol followed by purification on RNeasy Minelute columns (Qiagen). RNA purity was assessed on the NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The total RNA samples were sent to the Australian Genome Research Facility for library preparation and sequencing. Library preparation was based on the TruSeq RNA Sample Preparation Kit v2 (Illumina, Victoria, E2

3 Australia), and the samples were sequenced on the HiSeq2000 instrument (50 bp single end, ~25 million reads per sample). The raw sequencing results are available at the NCBI Short Read Archive under accession SRP Sequencing reads were aligned to the mouse reference genome (mm10) using Subread and summarized at the gene-level using feature Counts (3). Differentially expressed genes were identified using an empirical Bayes approach and exact tests based on the negative binomial distribution using edger packages (Bioconductor) (4). Enriched biological functions among differentially expressed genes were identified using GeneTerm Linker (5). A molecular interaction network was constructed using prior knowledge from the Ingenuity Systems Knowledge Base (IPA) (Ingenuity Systems, California, USA) (6) Lung function assessment Mice were anaesthetized by intraperitoneal injection with 40 mg/ml ketamine and 2 mg/ml xylazine at a dose of 0.01 ml/g body weight. Two-thirds of the dose was administered initially to induce surgical anaesthesia prior to tracheostomy and cannulation with a polyethylene endotracheal tube (internal diameter = cm, length = 1.0 cm). Mice were connected to a small animal ventilator (HSE-Harvard MiniVent; Harvard Apparatus, Holliston, MA, USA) within a whole-body plethysmograph. Mice were ventilated at 400 breaths/min with a tidal volume of 10 ml/kg and 2 cmh 2 O positive end-expiratory pressure and the remaining anaesthetic given Baseline lung function was assessed using a modification of low-frequency forced oscillation technique (LFOT) (7). An oscillatory signal containing 9 frequencies ranging from 4 to 38 Hz was generated by a speaker and delivered to the endotracheal cannula via a wavetube of known impedance. A four-parameter model with constant phase tissue impedance was then E3

4 fitted to the respiratory impedance spectrum (Zrs) for calculation of the Newtonian resistance (R aw, which primarily reflects airway resistance in the mouse due to the low chest wall impedance), tissue damping (G, the parameter describing the viscous frequency dependent response in resistance which is thought to reflect resistance of the peripheral airways where airflow occurs primarily by diffusion) and tissue elastance (H, the elastic properties of the lung parenchyma). 7 Lung and airway histology After euthanasia, lungs were inflation-fixed via tracheal instillation with 4% formaldehyde solution at a pressure of 10 cmh 2 O. The left lung was separated from the right lung at the main bronchus prior to tissue processing for embedding in paraffin. The right lung was randomly orientated (8) and 5-µm sections were taken at 1000 µm intervals throughout the right lung starting at a random point. The sections were then stained with haematoxylin and eosin prior to stereological measurements for lung structure measurements using the NewCast stereology software (Visiopharm, Denmark). Lung volume was calculated using the Cavalieri method (9) while point counts were used to measure volume of parenchyma (V p ), alveolar septa (V s ), air in the major airways (V a ) and surface area density of alveoli (S v ). The left lung was embedded with the transverse section face down in order to obtain cross sections of the first generation airway. Five µm sections were taken at 500 µm intervals throughout the entire lung and stained with Masson s Trichrome for visualization of the ASM layer. We have previously verified using immunohistochemical staining for α-smooth muscle actin that Masson s Trichrome stains for structural smooth muscle elements (1). The crosssectional area of the ASM and basement membrane perimeter were measured using the NewCast system. 24 Immunohistochemistry E4

5 Immunohistochemical staining was performed using the following primary antibodies: S100A9 (1:200 dilution; Abcam, Cambridge, MA, USA), S100A8 (MRP8; 1:200 dilution; Abcam, Cambridge, MA, USA), phosphorylated IκBα (p-iκbα; 1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated EGFR (p-egfr; 1:200 dilution; Cell Signaling, Danvers, MA, USA) References 1. Foong RE, Shaw NC, Berry LJ, Hart PH, Gorman S, Zosky GR. Vitamin D deficiency causes airway hyperresponsiveness, increases airway smooth muscle mass, and reduces TGF-beta expression in the lungs of female BALB/c mice. Physiological reports 2014; 2: e Zosky GR, Berry LJ, Elliot JG, James AL, Gorman S, Hart PH. Vitamin D Deficiency Causes Deficits in Lung Function and Alters Lung Structure. Am J Respir Crit Care Med 2011; 183: Liao Y, Smyth GK, Shi W. featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014; 30: Robinson MD, McCarthy DJ, Smyth GK. edger: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010; 26: Fontanillo C, Nogales-Cadenas R, Pascual-Montano A, De Las Rivas J. Functional Analysis beyond Enrichment: Non-Redundant Reciprocal Linkage of Genes and Biological Terms. PLoS One 2011; 6: e Bosco A, Ehteshami S, Panyala S, Martinez FD. Interferon regulatory factor 7 is a major hub connecting interferon-mediated responses in virus-induced asthma exacerbations in vivo. J Allergy Clin Immunol 2012; 129: E5

6 Zosky GR, Janosi TZ, Adamicza Á, Bozanich EM, Cannizzaro V, Larcombe AN, Turner DJ, Sly PD, Hantos Z. The bimodal quasi-static and dynamic elastance of the murine lung. Am J Physiol 2008; 105: Nyengaard JR, Gundersen HJG. Sampling for stereology in lungs. Eur Respir Rev 2006; 15: Yan X, Polo Carbayo JJ, Weibel ER, Hsia CCW. Variation of lung volume after fixation when measured by immersion or Cavalieri method. Am J Physiol Lung Cell Mol Physiol 2003; 284: L242-L Tables 11 Table S1. Primer list for RT-qPCR validation Primer S100A9 forward S100A9 reverse S100A8 forward S100A8 reverse MMP8 forward MMP8 reverse MMP9 forward MMP9 reverse EGFR forward EGFR reverse NFKBIA forward Sequence 5 -ATACTCTAGGAAGGAAGGACACC-3 5 -TCCATGATGTCATTTATGAGGGC-3 5 -AAATCACCATGCCCTCTACAAG-3 5 -CCCACTTTTATCACCATCGCAA-3 5 -TGGTGATTTCTTGCTAACCCC-3 5 -TACACTCCAGACGTGAAAAGC-3 5 -GGACCCGAAGCGGACATTG-3 5 -CGTCGTCGAAATGGGCATCT-3 5 -GCCATCTGGGCCAAAGATACC-3 5 -GTCTTCGCATGAATAGGCCAAT-3 5 -TGAAGGACGAGGAGTACGAGC-3 E6

7 NFKBIA reverse EP300 forward EP300 reverse MUC5AC forward MUC5AC reverse RPL37 forward RPL37 reverse 5 -TGCAGGAACGAGTCTCCGT-3 5 -CTTCAGACAAGTCTTGGCATAGT-3 5 -CCGACTCCCATGTTGAGGTTT CAGGACTCTCTGAAATCGTACCA-3 5 -GAAGGCTCGTACCACAGGG-3 5 -ACGAAGGGAACGTCATCCTTT-3 5 -TGGCACTCCAGTTATACTTCCT Table S2. List for differentially regulated genes identified by edger analysis Gene Log fold change Log counts per million *P Value False discovery rate S100a E E-13 S100a E E-11 Cd E E-10 Cdkl E E-07 Foxo E E-06 Plcb E E-06 Mir E E-06 Trim E E-06 F13a E E-05 Adap E E-05 Slc7a E E-05 Krt E E-05 Gm E E-05 Deptor E E-05 Sned E E-05 Ccdc E E-05 Ago E E-05 Rora E E-05 Cbl E E-05 Csf3r E E-05 Gan E Slc40a E Ppp1r12b E Dst E Ly E Scand E Lmbrd E Rn18s E E7

8 Zc3h12c E Pkd E Rn45s E Mmp E Dennd4c E Zbtb E Pon E Ttll E Hmbox E Siglec E Mxd4 Ddi E E Mmp E Slit E Ralgapa E Lpin E Zfp E Bicc E Atf E Ccdc E Hoxb E Nebl E Pglyrp E Sntb E Mmp E Abca8b E Klf E Ppp1r9a E Ern E Mgat E Lars E Hipk E Gatad2b E Elk E D830031N03Rik E Col27a E Atp7b E Uhmk E Lrrk E Kif13b E Fam184a E Rapgef E Serpina3g E Plxna E Nbeal E Lrp E E8

9 Mertk E Tulp E Pdpr E Cdk E Vps13c E Mrc E Atp6v0d E Retnlg E Btbd7 Mical E E Shroom E F E Itgax E Emilin E Dpy19l E Dmxl E Vps13d E Rgcc E Glg E Dock E Zfp E Baz1a E Zbtb E Hivep E Atf E Cacna1c E Glt8d E Pfkfb E Cxcr E Foxa E Hhex E Zhx E Wdfy E Fzd E Iigp E Ckb E Gm E Pnrc E Trrap E Arid5b E Gramd E Ltf E Mll E Tsc22d E Cep85l E Cyp4f E E9

10 Abcg E Cers E Tbxas E Gm E Lpl E Rev E Pds5a Rnf E E Bdp E Ighg2b E Ttc E Ms4a8a E Ear E Mib E Fam43a E Scarb E Ash1l E Olr E Atp1a E Pbld D10Bwg1379e Nbea Fmo F H2afj Gadd45a Gatm Tmem Itga Slc24a Slc26a Slfn Sik Rgs Chi3l Abca8a Ankrd Crebrf B05Rik Nrarp Gbp Helz Vps13a Mgp Fnip Trip E10

11 E24Rik AU Ctsk Ccdc85b Gbp10 Ptar Sema5a Fkbp Xrn Serpina3f Itpr Phf Atad Zbed Greb AA Phlda Rorc Klf Jund Nup Lphn F630028O10Rik Eif3f Wdfy Serpina3h Ier I14Rik Strn Pltp AI Mapk Lyl Zfp Kcnn Steap Chchd Dync1h Igkj Mki Efcab4b Gm Gm S1pr Prex E11

12 Adamtsl2 Fat Tnxb Dnajb Ccl Cys A4galt Serpina3i Lpar Wnt7a Alms Cpeb Hes C20Rik Exph C230081A13Rik Rnd Srrm Ubr Atp10d Ubr Gm Fras Cd300lb Pde4c Ahctf Tnks Arid Mdm Slc10a Pde3a Robo Psd Art Ep Med13l Snhg Klhl Chad Dync2h Nfxl Abhd Prrc2c Des Cep Alox E12

13 Bmpr Gpr Per A630007B06Rik C1galt Aak Xdh Gt(ROSA)26Sor Usp Wdr Flrt A530099J19Rik Glul Dcdc2a Ighg2c M11Rik Axl Mob1b Rab3c Gbp Foxn Atp2b Tppp Sorl Tbc1d Egfr Atp5g Ppm1l Pelo Klhl Bpifb Clec7a Dock Mbd Tspan Cx3cr Snai Rprml Il18bp Tgtp Mrps Nipbl Gm Sema7a Wee Zfp E13

14 Phf Hoxb Nr2c Fam178a Gramd1b Lilra Ahnak Igtp Cd Sec14l Slc43a Gata Dopey Gtf2a Synrg Agfg Ttll Gbp Rplp Gm Creb Ankrd Map3k Ccdc Tmod Phlpp Gbp C530008M17Rik Gbp P16Rik Csf2rb Fam210a Pak Med Prr15l Fam129a Gbp Zfp Bcar Tubb4b Peli Trem Maff Plekhf Pcnt Nlrc E14

15 1 Apc Hnrnpa Mdn Plcb Havcr Epb Lrch Fry Pi4k2b Pcdhac Gbp Rhobtb Oasl Abca Cdc42ep Utrn Chd Shisa Myof Cnot Sf3a Huwe Ctss Gm Ppp1r3c Shroom Chd Nfkbia Gbp Rab * P - value adjusted for multiple testing 2 3 Figure legends Figure S1. Gene and protein expression of EGFR (A), MMP9 (C), MMP8 (E) and EP300 (F) in lungs from whole-life vitamin D-replete (VitD+ +), postnatal vitamin D-deficient (VitD+ -), vitamin D-deficient in utero (VitD- +) and whole life vitamin D-deficient (VitD- -) mice. Gene expression data are expressed as fold changes obtained by normalizing candidate genes to the ribosomal protein L37 (RPL37) reference gene. Representative images of lung sections immunohistochemically stained for the protein products of EGFR (phosphorylated-egfr;b) and MMP9 (D) are shown (Scale 100 E15

16 1 2 µm). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < Data are represented as mean (SD), n = 10/group Figure S2. Lung volume (V L ; A), volume of parenchyma (V p ; B), volume of air in major airways (V a ; C) and surface area of the alveoli (S v ; D) measured by stereology from fixed lungs of whole-life vitamin D-replete mice (VitD+ +), postnatal vitamin D-deficient (VitD+ - ), vitamin D-deficient in utero (VitD- +) and whole life vitamin D-deficient female mice (VitD- -). *indicates p < Data are represented as mean (SD), n = 7-11/group Figure S3. Correlation of neutrophil cell counts with airway hyperresponsiveness, presented as the percentage increase from saline at the highest dose of methacholine exposure (A), MMP8 gene expression (B) and MMP9 gene expression (C). E16

17 Figure S1. Gene and protein expression of EGFR (A), MMP9 (C), MMP8 (E) and EP300 (F) in lungs from whole-life vitamin D-replete (VitD+ +), postnatal vitamin D-deficient (VitD+ -), vitamin D-deficient in utero (VitD- +) and whole life vitamin D-deficient (VitD- -) mice. Gene expression data are expressed as fold changes obtained by normalizing candidate genes to the ribosomal protein L37 (RPL37) reference gene. Representative images of lung sections immunohistochemically stained for the protein products of EGFR (phosphorylated-egfr;b) and MMP9 (D) are shown (Scale 100 µm). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < Data are represented as mean (SD), n = 10/group. 173x156mm (300 x 300 DPI)

18 Figure S2. Lung volume (VL; A), volume of parenchyma (Vp; B), volume of air in major airways (Va; C) and surface area of the alveoli (Sv; D) measured by stereology from fixed lungs of whole-life vitamin D-replete mice (VitD+ +), postnatal vitamin D-deficient (VitD+ -), vitamin D-deficient in utero (VitD- +) and whole life vitamin D-deficient female mice (VitD- -). *indicates p < Data are represented as mean (SD), n = 7-11/group. 160x113mm (300 x 300 DPI)

19 Figure S3. Correlation of neutrophil cell counts with airway hyperresponsiveness, presented as the percentage increase from saline at the highest dose of methacholine exposure (A), MMP8 gene expression (B) and MMP9 gene expression (C). 83x145mm (300 x 300 DPI)