Dataset DOI: 10.5061/dryad.3ffbg79zc

Description of the data and file structure

Organotypic BEC cultures were generated from 10 children with asthma and 6 healthy children from bronchial brushings obtained while donors were undergoing elective surgery, as previously described.¹² BEC donors were characterized at an initial research visit to confirm diagnosis and medical history and to record spirometry and fraction of exhaled nitric oxide measurements. Age ranged from 6.4-17.6 years in donors with asthma and 7.9-18.1 years in non-atopic, healthy donors; 40 % of the donors with asthma and 50 % of healthy donors were female. Fraction of exhaled nitric oxide (FeNO) was similar among the two groups (10.9 +/- 4.3 ppb vs 10.5 +/- 3.6 ppb). Fraction of exhaled volume in 1 second (FEV1 97.4 % +/- 11.3 vs 101.3 % +/- 21.5) was similar, while FEV1/FVC (forced vital capacity) was lower in asthma donors (0.81 +/- 0.05 vs 0.88 +/- 0.03). Among the donors with asthma, 30 % (n = 3) were positive for 3 or more aeroallergens on allergen-specific IgE testing, and 40 % (n = 4) had a history of severe exacerbation defined as needing systemic corticosteroids for ≥ 3 d and/or a hospitalization/emergency room visit for asthma requiring systemic corticosteroids.¹³ After differentiation to an organotypic, pseudostratified state, BEC cultures were infected with RV-16 at an MOI of 0.5 or stimulated with IL-13 at 10 ng/mL added every 48 hours to the media. RNA and DNA were harvested 10 days after RV16 infection and 7 days after IL-13 treatment.¹² Collected RNA was analyzed via RNA-sequencing for gene expression and DNA methylation was quantified in DNA using the Asthma & Allergy array.¹⁴ RNA was sequenced and aligned to the human genome (GRCh38, Ensembl 91) with quality control as previously described.^7,12 Genes were filtered to retain only protein coding genes nearest to the CpGs on the array, resulting in an expression dataset of 3,821 genes. DNA methylation probes were filtered to remove those located on sex chromosomes, those with SNPs within 3 bp and a minor allele frequency ≥ 5 % in Morin et al 2023, and those with < 90 % of samples having detection p-value < 0.01 as calculated by minfi.¹⁴ After quality control, 44,900 CpGs were included for analysis. Sample-specific quality was estimated using minfi and all samples were confirmed to be above a quality cutoff value of 10.5. Methylated and unmethylated channel intensities were quantile normalized separately and implemented in the ENMix R package, and methylation M-values (M) were calculated as M = log2 (Methylated + a) / (Unmethylated + a) with an offset value (a) of 100.¹⁵ Differential methylation was estimated using linear mixed effects contrasting RV-16 infection or IL-13 stimulation to the uninfected/unstimulated control (condition), with covariates accounting for age, sex, and DNA concentration as well nested random effects for donor and array. M value ~ condition + age + sex + log10 DNA concentration + (1|array) + (1|array: donor)

Differential gene expression was estimated using analogous linear mixed effects contrasts, with covariates accounting for age and sex, random effect for donor, and observation-level quality weights calculated using limma. Expression ~ condition + age + sex + (1|donor) + quality weights

Mixed effects models were estimated using lme4 implemented in kimma, and multiple hypothesis testing correction was performed within each analysis to adjust p-values using the Benjamini-Hochberg false discovery rate (FDR). We calculated the Pearson correlation of expression ✕ methylation.

References

12.       Doni Jayavelu N, Altman MC, Benson B, Dufort MJ, Vanderwall ER, Rich LM, White MP, Becker PM, Togias A, Jackson DJ, Debley JS. Type 2 inflammation reduces SARS-CoV-2 replication in the airway epithelium in allergic asthma through functional alteration of ciliated epithelial cells. J Allergy Clin Immunol. 2023 Jul;152(1):56–67. PMCID: PMC10052850

13.       Reddel HK, Taylor DR, Bateman ED, Boulet LP, Boushey HA, Busse WW, Casale TB, Chanez P, Enright PL, Gibson PG, de Jongste JC, Kerstjens HAM, Lazarus SC, Levy ML, O’Byrne PM, Partridge MR, Pavord ID, Sears MR, Sterk PJ, Stoloff SW, Sullivan SD, Szefler SJ, Thomas MD, Wenzel SE, American Thoracic Society/European Respiratory Society Task Force on Asthma Control and Exacerbations. An official American Thoracic Society/European Respiratory Society statement: asthma control and exacerbations: standardizing endpoints for clinical asthma trials and clinical practice. Am J Respir Crit Care Med. 2009 Jul 1;180(1):59–99. PMID: 19535666

14.       Morin A, Thompson EE, Helling BA, Shorey-Kendrick LE, Faber P, Gebretsadik T, Bacharier LB, Kattan M, O’Connor GT, Rivera-Spoljaric K, Wood RA, Barnes KC, Mathias RA, Altman MC, Hansen K, McEvoy CT, Spindel ER, Hartert T, Jackson DJ, Gern JE, McKennan CG, Ober C, program collaborators for Environmental Influences on Child Health Outcomes and Children’s Respiratory and Environmental Workgroup. A functional genomics pipeline to identify high-value asthma and allergy CpGs in the human methylome. J Allergy Clin Immunol. 2023 Jun;151(6):1609–1621. PMCID: PMC10859971

15.       Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, Hou L, Lin SM. Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics. 2010 Nov 30;11:587. PMCID: PMC3012676

Files and variables

File: supplement_DMP_DEG_cor_IL13.xlsx

Description: List of differential methylation and expression after 7 days stimulation with IL-13.

Variables

File: supplement_DMP_DEG_cor_RV16.xlsx

Description: List of differential methylation and expression 10 days after RV16 infection.

Variables

Human subjects data

All data collected following ethical and legal guidelines with IRB approval and de-identified to protect subject identity.