Rachel L. Clifford, PhD, Meaghan J. Jones, PhD, Julia L

Inhalation of diesel exhaust and allergen alters human bronchial epithelium DNA methylation
Rachel L. Clifford, PhD, Meaghan J. Jones, PhD, Julia L. MacIsaac, PhD, Lisa M. McEwen, BSc, Sarah J. Goodman, BSc, Sara Mostafavi, PhD, Michael S. Kobor, PhD, Chris Carlsten, MD Journal of Allergy and Clinical Immunology Volume 139, Issue 1, Pages (January 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Study design (A) and explanation of sample naming according to group status (B). Individuals in group I were first exposed to FA; those in group II were first exposed to DE. Postexposure, allergen and saline were instilled; 48 hours later, bronchial brushings were taken for epithelial cell DNA isolation. Four weeks later, the process was repeated with opposing exposures. Samples 1 to 4 associated with group I and samples 5 to 8 associated with group II. LLL, Left lower lobe; RLL, right lower lobe. Journal of Allergy and Clinical Immunology , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 CpG methylation was significantly affected by allergen in lung previously exposed to DE. A, P value distribution for the effect of allergen in lung with prior DE exposure (7.FAA vs 1.FAS). Dashed line indicates distribution expected by chance. B, Plot of 43,485 variable CpG probes. Red = q values < 0.05; green = q value < 0.05 and Δβ of >0.1. C, Beta values of probes significantly different between control (1.FAS) and 7.FAA, with Δβ of >0.1 (75 total probes). Each vertical band is a study subject. Blue: high methylation; yellow: low methylation; D and E, Reduction/increase in DNA methylation (Δβ), relative to 1.FAS, among 70/5 hits following the intermediate DE exposure. Points are individual CpG sites and lines indicate mean ± SEM. Journal of Allergy and Clinical Immunology , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Characteristics of the CpGs affected by allergen in lung previously exposed to DE. A, CpG island type for all analysis probes and the 75 top hits.29 B, Relationship of Δβ and distance to the transcriptional start site. Red = q value < 0.05 and Δβ of >0.1. C, Individual CpG site plots of the 8 CpG hit sites associated with the TBX3 gene. Whiskers represent the minimum and maximum of all the data. HC, High CpG density; IC, intermediate CpG density; ICshore, intermediate CpG density overlapping with a high-density region; LC, low CpG density. Journal of Allergy and Clinical Immunology , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 CpG methylation was significantly affected by DE in lung previously exposed to allergen. A, P value distribution for the effect of DE in lung with prior allergen exposure (4.DES Vs 1.FAS). Dashed line indicates distribution expected by chance. B, Plot of 43,485 variable CpG probes. Red = q value < 0.05; green = q value < 0.05 and Δβ of >0.1. C, Heatmap of β values of probes that were significantly different between control (1.FAS) and 7.DES (DE with prior allergen) with a Δβ of >0.1 (548 probes total). Each vertical band is a study subject. Blue: high methylation; yellow: low methylation; D and E, Reduction/increase in DNA methylation (Δβ), relative to 1.FAS, among 528/20 hits following the intermediate allergen exposure. Points are individual CpG sites and lines are mean ± SEM. Journal of Allergy and Clinical Immunology , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Characteristics of the CpGs affected by DE in lung previously exposed to allergen. A, CpG island type for all analysis probes and the 548 top hits. B, Relationship of Δβ and distance to the transcriptional start site. Red = q value < 0.05 and Δβ of >0.1; C-F, Individual CpG site plots of the CpG sites associated with 4 HOX genes (Fig 5, C) HOXA3, (Fig 5, D) HOXA4, (Fig 5, E) HOXB1, and (Fig 5, F) HOXB3. Whiskers represent the minimum and maximum of all the data. HC, High CpG density; IC, intermediate CpG density; ICshore, intermediate CpG density overlapping with a high-density region; LC, low CpG density. Journal of Allergy and Clinical Immunology , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Rachel L. Clifford, PhD, Meaghan J. Jones, PhD, Julia L

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