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SUPPLEMENTARY INFORMATION TITLE: Sputum gene expression reveals dysregulation of mast cells and basophils in eosinophilic COPDAppendix S1- MethodsCOPD participantsParticipants required a doctor-confirmed diagnosis of stable COPD with incompletely reversible airflow limitation (based on post-bronchodilator forced expiratory volume in one second [FEV1] % predicted values <80% and forced expiratory ratio [FER] <0.70). Stable disease was defined as an absence of recent respiratory infection, acute exacerbations or change in maintenance therapy in four weeks prior to recruitment. Participants were excluded if they were currently smoking, pregnant, breastfeeding, or had a primary diagnosis of another respiratory disease (ie bronchiectasis, pulmonary fibrosis, active tuberculosis or lung cancer). Exacerbation definitionAn exacerbation was defined as symptoms, either increased or new in onset, including cough, sputum, wheezing, dyspnea, or chest tightness for ≥3 days requiring antibiotics or systemic steroids.1, 2 A severe exacerbation was defined as a hospitalization and/or emergency department visit, as per the 2019 GOLD guidelines.3Sputum processing, RNA extraction, cDNA synthesis and qPCRSputum was induced and processed as described previously.4 Sputum portions were selected and dispersed using dithiothreitol. Sputum cytospins were stained with May Grunwald Giemsa and a differential cell count of 400 non-squamous cells was performed. Sputum RNA Extraction and RT-qPCR has been previously described.5-7 Sputum RNA was extracted using an automated process (Qiacube with RNeasy Mini Kit components, Qiagen, Hilden, Germany). Extracted RNA was quantified using the Quant-iT RiboGreen RNA Quantitation Assay Kit (Molecular Probes Inc, Invitrogen, Eugene, Ore). DNase treatment was applied to 10 μL neat RNA samples or 10 μL RNA and RNase/DNase free water samples (calculated based on RNA concentration [ng/μL]), as per manufacturer’s instructions (DNase I, Amplification Grade, Invitrogen, California, USA) and reverse transcribed to cDNA, as per manufacturer’s instructions (high capacity cDNA Reverse Transcription Kit, Applied Biosystems). cDNA was used to detect gene expression using standard TaqMan probe-based qPCR methods (Applied Biosystems). The expression of eight target genes (CPA3 (Cat# Hs00157019_m1), TPSAB1/TPSB2 (Cat# Hs02576518_gH), KIT (Cat# Hs00174029_m1), GATA2 (Cat# Hs00231119_m1), SOCS2 (Cat# Hs00919620_m1), ENO2 (Cat# Hs00157360_m1), GPR56/ADGRG1 (Cat# Hs00938474_m1), HDC (Cat# Hs00157914_m1)) were measured using real-time PCR (Life Technologies) and normalized to the B-actin (Cat# 4326315E) housekeeping gene. Samples were measured at either a 1/10 or 1/2 cDNA dilution. To determine cDNA dilution, an initial measure of 18S (Cat# 4319413E) and B-actin was performed at a 1/10 dilution. Samples expressing a cycle threshold (Ct) value of >20 for 18S and >30 for B-actin were repeated at a 1/2 dilution. If 18S and B-actin Ct values remained above threshold at a 1/2 dilution, the sample was excluded. Statistical analysis was performed on ΔCt (gene expression) and 2-ΔCT (relative mRNA abundance) values, with ΔCt calculated as the change in Ct between the target gene and B-actin. Of note, a greater ΔCt value corresponds to lower relative target gene expression, while a greater 2-ΔCt value corresponds to higher relative target gene binatorial gene metric calculationMC/basophil genes were analyzed in a combinatorial gene metric.8 To generate the gene metric, ΔCt values of eight individual MC/basophil signature genes were standardized (centered and scaled) and a row mean across standardized gene values was calculated for each participant. ReferencesE1.Albert RK, Connett J, Bailey WC, et al. Azithromycin for Prevention of Exacerbations of COPD. N Engl J Med. 2011;365(8):689-698.E2.Niewoehner DE, Rice K, Cote C, et al. Prevention of exacerbations of chronic obstructive pulmonary disease with tiotropium, a once-daily inhaled anticholinergic bronchodilator: a randomized trial. Ann Intern Med. 2005;143(5):317-326.E3.Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease 2019 Report . Published 2019. Accessed October 23, 2020.E4. Gibson PG, Wlodarczyk JW, Hensley MJ, et al. Epidemiological Association of Airway Inflammation with Asthma Symptoms and Airway Hyperresponsiveness in Childhood. Am J of Respir Crit Care Med. 1998;158(1):36-41.E5. Baines KJ, Simpson JL, Wood LG, Scott RJ, Gibson PG. Transcriptional phenotypes of asthma defined by gene expression profiling of induced sputum samples. J Allergy Clin Immunol. 2011;127(1):153-160. e151-159.E6. Baines KJ, Simpson JL, Wood LG, Scott RJ, Gibson PG. Transcriptional phenotypes of asthma defined by gene expression profiling of induced sputum samples. J Allergy Clin Immunol. 2011;127(1):153-160. e151-159.E7. Berthon BS, Gibson PG, Wood LG, MacDonald-Wicks LK, Baines KJ. A sputum gene expression signature predicts oral corticosteroid response in asthma. Eur Respir J. 2017;49(6):1700180. E8. Peters MC, Mekonnen ZK, Yuan S, Bhakta NR, Woodruff PG, Fahy JV. Measures of gene expression in sputum cells can identify TH2-high and TH2-low subtypes of asthma. J Allergy Clin Immunol. 2014;133(2):Appendix S2: Supplementary Figures and LegendFigure S1: Relative mRNA abundance of A) TPSB2, B) CPA3, C) KIT, D) GATA2, E) SOCS2, F) ENO2, G) GPR56, H) HDC, and I) combinatorial MC/basophil gene metric between COPD airway inflammatory phenotypes and control participants. Bars represent median, with error bars representing Q1, Q3. Relative mRNA abundance compared between groups are expressed as 2-ΔCt relative to the housekeeping gene β-actin. Combinatorial gene metric based on ΔCt gene expression values. # p<0.005 compared to control participants; ^ p<0.005 versus neutrophilic participants (N-COPD); * p<0.005 compared to paucigranulocytic participants (PG-COPD). Control participants (n=17); N-COPD (n=34); Eosinophilic-COPD [E-COPD] (n=31); Mixed granulocytic COPD [MG-COPD] (n=13); PG-COPD (n=18). ................
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