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Diesel Exhaust Time-Dependently Modulates DNA Methylation in Peripheral Blood Mononuclear Cells from Exposed Volunteers: Results from a Controlled Human Crossover Study
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A7717 - Diesel Exhaust Time-Dependently Modulates DNA Methylation in Peripheral Blood Mononuclear Cells from Exposed Volunteers: Results from a Controlled Human Crossover Study
Author Block: C. F. Rider1, L. M. McEwan2, M. J. Jones2, R. L. Clifford3, M. S. Kobor2, C. Carlsten1; 1Respiratory Medicine, University of British Columbia, Vancouver, BC, Canada, 2Medical Genetics, University of British Columbia, Vancouver, BC, Canada, 3University of Nottingham, Nottingham, United Kingdom.
RATIONALE: Exposure to air pollution enhances the development and exacerbation of asthma. While this predominately occurs through increased lung inflammation, we sought to determine whether changes to DNA methylation in blood cells could contribute. We therefore investigated the pattern of DNA methylation in peripheral blood mononuclear cells (PBMCs) collected during a human exposure study.
HYPOTHESIS: PBMC DNA methylation of genes associated with asthma is modulated by diesel exhaust (DE) exposure, in a time-dependent manner.
METHODS: We performed a randomized, crossover, double-blinded, clinical exposure study in atopic volunteers with inhalation of filtered air (FA) or DE (standardized to 300µg/m3 of PM2.5), followed by the instillation of allergen and saline into contralateral lungs. Blood was collected before exposure and 4, 24 and 48 hours after, and PBMCs isolated. Genomic DNA was bisulfite converted and methylation at ~850,000 CpG sites was measured using Infinium MethylationEPIC arrays. Data were processed to remove technical variation associated with batch and probe design, as well as adjusted for cell type composition. Normalized DNA methylation data were analyzed using linear mixed effects models incorporating treatment (FA/DE), exposure order, time, age, smoking history, ethnicity, and sex and corrected for multiple testing using a q-value threshold of 0.25. Annotation data matched specific CpGs with related genes.
RESULTS: We observed significantly differentially methylated CpG sites between DE and FA after the exposure. Specifically, there were 0, 5 and 299 CpGs significantly affected by DE at 4, 24 and 48 hours, respectively. Genes related to these CpGs included TTC34, PLCB1, LSM2, B3GALT2, and CDC73 at 24 hours, while the most significant of the 231 associated genes at 48 hours were NTM, C1orf61, SYCP1, TTC34, and PLCB1. Overall analysis showed that DE exhaust exposure induced DNA hypomethylation, in line with our earlier data. Other genes of interest associated with modulated CpGs included the IL4 receptor (IL4R), the asthma-related gene ADAMTSL1, MAP3K1, eosinophil peroxidase (EPX), and BCL11B, which contributes to innate lymphoid cell 2 (ILC2) development. Some CpGs associated with genes that play roles in oxidative stress responses, including EPX, ENOX1, FDX1, LOXL2, PTGR2, SESN1 and TH were also modulated.
CONCLUSIONS: DNA methylation in PBMCs was significantly and time-dependently modulated by DE, relative to FA. These changes may affect the expression of genes important in asthma, by, for example, increasing IL4 receptor expression. Changes in DNA methylation in the blood following air pollution exposure may contribute to the development or worsening of asthma.
Author Block: C. F. Rider1, L. M. McEwan2, M. J. Jones2, R. L. Clifford3, M. S. Kobor2, C. Carlsten1; 1Respiratory Medicine, University of British Columbia, Vancouver, BC, Canada, 2Medical Genetics, University of British Columbia, Vancouver, BC, Canada, 3University of Nottingham, Nottingham, United Kingdom.
RATIONALE: Exposure to air pollution enhances the development and exacerbation of asthma. While this predominately occurs through increased lung inflammation, we sought to determine whether changes to DNA methylation in blood cells could contribute. We therefore investigated the pattern of DNA methylation in peripheral blood mononuclear cells (PBMCs) collected during a human exposure study.
HYPOTHESIS: PBMC DNA methylation of genes associated with asthma is modulated by diesel exhaust (DE) exposure, in a time-dependent manner.
METHODS: We performed a randomized, crossover, double-blinded, clinical exposure study in atopic volunteers with inhalation of filtered air (FA) or DE (standardized to 300µg/m3 of PM2.5), followed by the instillation of allergen and saline into contralateral lungs. Blood was collected before exposure and 4, 24 and 48 hours after, and PBMCs isolated. Genomic DNA was bisulfite converted and methylation at ~850,000 CpG sites was measured using Infinium MethylationEPIC arrays. Data were processed to remove technical variation associated with batch and probe design, as well as adjusted for cell type composition. Normalized DNA methylation data were analyzed using linear mixed effects models incorporating treatment (FA/DE), exposure order, time, age, smoking history, ethnicity, and sex and corrected for multiple testing using a q-value threshold of 0.25. Annotation data matched specific CpGs with related genes.
RESULTS: We observed significantly differentially methylated CpG sites between DE and FA after the exposure. Specifically, there were 0, 5 and 299 CpGs significantly affected by DE at 4, 24 and 48 hours, respectively. Genes related to these CpGs included TTC34, PLCB1, LSM2, B3GALT2, and CDC73 at 24 hours, while the most significant of the 231 associated genes at 48 hours were NTM, C1orf61, SYCP1, TTC34, and PLCB1. Overall analysis showed that DE exhaust exposure induced DNA hypomethylation, in line with our earlier data. Other genes of interest associated with modulated CpGs included the IL4 receptor (IL4R), the asthma-related gene ADAMTSL1, MAP3K1, eosinophil peroxidase (EPX), and BCL11B, which contributes to innate lymphoid cell 2 (ILC2) development. Some CpGs associated with genes that play roles in oxidative stress responses, including EPX, ENOX1, FDX1, LOXL2, PTGR2, SESN1 and TH were also modulated.
CONCLUSIONS: DNA methylation in PBMCs was significantly and time-dependently modulated by DE, relative to FA. These changes may affect the expression of genes important in asthma, by, for example, increasing IL4 receptor expression. Changes in DNA methylation in the blood following air pollution exposure may contribute to the development or worsening of asthma.
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