Download Supplementary methods

Supplementary methods
Study participants and measurements
Adult CF patients (n=58; 35 males and 23 females; age range 23-63 years) represent a
cross-section of patients attending the regional Adult Cystic Fibrosis Service at The Alfred
Hospital (E1-E3). Pulmonary function was assessed by measuring forced expiratory volume
in 1 second (FEV1) and calculating the percentage predicted FEV1 (FEV1%) (E3). Height
and weight measurements were used to calculate BMI (in kilograms per metre squared;
kg/m2). Annual % change in weight was determined by assessing the change in weight over
the previous 3-4 years and standardizing for baseline weight and follow-up interval (mean
interval between baseline and follow-up measurements was 3.6 ± 0.4 years) (E1, E2).
Healthy controls (n=60; 29 males and 31 females) were mean age-matched (range 21-49
years) to the patient cohort (E4). Serum from CF participants was sampled on 1-3 occasions
over 6 months (E1) and sampled once for healthy controls (for CF participants the mean of
the 3 activin A and follistatin measurements is shown). The study was approved by The
Alfred Hospital Institutional Ethics Committee and written informed consent was obtained.
Recombinant follistatin
Follistatin 288 (FS288) was produced using the follistatin expressing plasmid
(pSV2HF288), a gift from Professor Shunichi Shimasaki (University of California, San
Diego, USA). The FS288 gene was amplified from pSV2HF288 by PCR and sub-cloned into
pAPEX3P vector. The pAPEX3P-FS288 plasmid was transfected into 293EBNA cells, and
puromycin-resistant cells expanded to form the stable 293EBNA FS cell line. FS288 was
purified from conditioned media of cultured 293EBNA FS cells by successive rounds of
chromatography through heparin-Sepharose affinity (5 ml Hi-Trap Heparin column, GE
Healthcare Bio-Sciences), size exclusion (Superdex 200 prep grade, Hi-load 16/60) and RPHPLC (Reversed Phase, OD-300, Aquapore ODS, C-18, 7 um, 300 A, 10 cm, 2.1 mm i.d.
Brownlee Cartridge Column; PerkinElmer) columns.
1
Follistatin luciferase bioassay
To assess the ability of recombinant follistatin to inhibit activin activity, HEK293T
cells, plated on poly-lysine-coated 24-well plates at a density of 150,000 cells per well, were
co-transfected with 50 ng A3-luc (a Smad2-responsive luciferase reporter), 25 ng FAST-2 (a
transcriptional co-activator), and 25 ng β-galactosidase (to normalise for transfection
efficiency). Transfections were performed under optimized conditions using lipofectamine
2000 transfection reagent (Invitrogen, Carlsbad, CA). 16 h post-transfection, cells were
treated with activin-A and increasing doses of recombinant human FS288 (rhFS288) for ~16
h. Cells were harvested in solubilization buffer (1% Triton X-100, 25 mM glycylglycine (pH
7.8), 15 mM MgSO4, 4 mM EGTA, and 1 mM dithiothreitol), and luciferase reporter activity
was measured (Victor2 Multilabel Counter; Perkin Elmer, Waltham, MA) and normalized
relative to -galactosidase activity.
Blood, BAL and tissue sampling and processing
Blood was collected from the inferior vena-cava of mice and serum separated (E5).
Bronchoalveolar lavage (BAL) fluid was collected from mice by cannulating the trachea and
lavaging the airways with 0.3 ml of 1% fetal calf serum in PBS, followed by three further
lavages of 0.2 ml (E5). BAL cells were collected by centrifugation at 350 g for 4 min, and
BAL fluid stored at –70°C for cytokine/chemokine analysis.
BAL cell cytospots were
Giemsa-stained (Merck, Darmstadt, Germany) and ≥ 200 cells identified by morphological
criteria. After BAL collection, the lung was perfusion-fixed via the trachea with fresh 2%
formalin at constant pressure (20 cm water) for 4-5 min. The lung was then removed and
placed in formalin prior to paraffin embedding.
2
REFERENCES
1. King SJ, Nyulasi IB, Bailey M, Kotsimbos T, Wilson JW. Loss of fat-free mass over four
years in adult cystic fibrosis is associated with high serum interleukin-6 levels but not tumour
necrosis factor-alpha. Clin Nutr 2014; 33: 150-155.
2. King SJ, Nyulasi IB, Strauss BJ, Kotsimbos T, Bailey M, Wilson JW. Fat-free mass
depletion in cystic fibrosis: associated with lung disease severity but poorly detected by body
mass index. Nutrition 2010; 26: 753-759.
3. King SJ, Topliss DJ, Kotsimbos T, Nyulasi IB, Bailey M, Ebeling PR, Wilson JW.
Reduced bone density in cystic fibrosis: DeltaF508 mutation is an independent risk factor.
Eur Respir J 2005; 25: 54-61.
4. de Kretser DM, Bensley JG, Pettila V, Linko R, Hedger MP, Hayward S, Allan CA,
McLachlan RI, Ludlow H, Phillips DJ. Serum activin A and B levels predict outcome in
patients with acute respiratory failure: a prospective cohort study. Crit Care 2013; 17: R263.
5. Hardy CL, Nguyen HA, Mohamud R, Yao J, Oh DY, Plebanski M, Loveland KL, Harrison
CA, Rolland JM, O'Hehir RE. The activin A antagonist follistatin inhibits asthmatic airway
remodelling. Thorax 2013; 68: 9-18.
3