Childhood exposure to ambient polycyclic aromatic hydrocarbons is linked to epigenetic modifications and impaired systemic immunity in T cells

Abstract

Background: Evidence suggests that exposure to polycyclic aromatic hydrocarbons (PAHs) increases atopy; it is unclear how PAH exposure is linked to increased severity of atopic diseases.

Objective: We hypothesized that ambient PAH exposure is linked to impairment of immunity in atopic children (defined as children with asthma and/or allergic rhinitis) from Fresno, California, an area with elevated ambient PAHs.

Methods: We recruited 256 subjects from Fresno, CA. Ambient PAH concentrations (ng/m(3) ) were measured using a spatial-temporal regression model over multiple time periods. Asthma diagnosis was determined by current NHLBI criteria. Phenotyping and functional immune measurements were performed from isolated cells. For epigenetic measurements, DNA was isolated and pyrosequenced.

Results: We show that higher average PAH exposure was significantly associated with impaired Treg function and increased methylation in the forkhead box protein 3 (FOXP3) locus (P < 0.05), conditional on atopic status. These epigenetic modifications were significantly linked to differential protein expression of FOXP3 (P < 0.001). Methylation was associated with cellular functional changes, specifically Treg dysfunction, and an increase in total plasma IgE levels. Protein expression of IL-10 decreased and IFN-γ increased as the extent of PAH exposure increased. The strength of the associations generally increased as the time window for average PAH exposure increased from 24 hr to 1 year, suggesting more of a chronic response. Significant associations with chronic PAH exposure and immune outcomes were also observed in subjects with allergic rhinitis.

Conclusions and clinical relevance: Collectively, these results demonstrate that increased ambient PAH exposure is associated with impaired systemic immunity and epigenetic modifications in a key locus involved in atopy: FOXP3, with a higher impact on atopic children. The results suggest that increased atopic clinical symptoms in children could be linked to increased PAH exposure in air pollution.

Keywords: FOXP3; IFN-γ; T regulatory cells; Treg function; epigenetics; polycyclic aromatic hydrocarbons; total IgE.

Fig. 1

Ambient polycyclic aromatic hydrocarbons (PAH) exposure is associated with changes in Treg function. Linear regression analysis of ambient average PAH exposure at different time periods (in ng/m³) and Treg function. Treg function reported as % of suppressive function. (a, b) 24-hr mean PAH exposure (n = 41 asthmatic, n = 146 non-asthmatic), (c, d) 1-week mean PAH exposure (n = 38 asthmatic, n = 149 non-asthmatic), (e, f) 1-month mean PAH exposure (n = 44 asthmatic, n = 150 non-asthmatic), (g, h) 3-month mean PAH exposure (n = 47 asthmatic, n = 153 non-asthmatic), (i, j) 6-month mean PAH exposure (n = 61 asthmatic, n = 156 non-asthmatic) and (k,l) 1-year mean PAH exposure (n = 85 asthmatic, n = 171 non-asthmatic). Data points represent individuals. Red = asthmatic subjects, Blue = non-asthmatic subjects. Dotted lines represent 95% confidence intervals.

Fig. 2

Ambient polycyclic aromatic hydrocarbons (PAH) exposure is associated with changes in total IgE. Linear regression analysis of ambient average PAH exposure at different time periods (in ng/m³) and plasma total IgE levels. IgE levels reported as IU/mL. (a, b) 24-hr mean PAH exposure (n = 41 asthmatic, n = 146 non-asthmatic), (c, d) 1-week mean PAH exposure (n = 38 asthmatic, n = 149 non-asthmatic), (e, f) 1-month mean PAH exposure (n = 44 asthmatic, n = 150 non-asthmatic), (G, H) 3-month mean PAH exposure (n = 47 asthmatic, n = 153 non-asthmatic), (i, j) 6-month mean PAH exposure (n = 61 asthmatic, n = 156 non-asthmatic) and (k, l) 1-year mean PAH exposure (n = 85 asthmatic, n = 171 non-asthmatic). Data points represent individuals. Red = asthmatic subjects, Blue = non-asthmatic subjects. Dotted lines represent 95% confidence intervals.

Fig. 3

Ambient polycyclic aromatic hydrocarbons (PAH) exposure is associated with changes in FOXP3 methylation. Linear regression analysis of ambient average PAH exposure at different time periods (ng/m³) and FOXP3 methylation in CD4⁺CD25^hiCD127⁻ T cells. Methylation is reported as percent methylated CpG sites of 13 total examined CpG sites within the FOXP3 locus; the cut-off for positive methylation at each site was set at 70% of all sequencing reactions. (a, b) 24-hr mean PAH exposure (n = 41 asthmatic, n = 146 non-asthmatic), (c, d) 1-week mean PAH exposure (n = 38 asthmatic, n = 149 non-asthmatic), (e, f) 1-month mean PAH exposure (n = 44 asthmatic, n = 150 non-asthmatic), (g, h) 3-month mean PAH exposure (n = 47 asthmatic, n = 153 non-asthmatic), (i, j) 6-month mean PAH exposure (n = 61 asthmatic, n = 156 non-asthmatic) and (k,l) 1-year mean PAH exposure (n = 85 asthmatic, n = 171 non-asthmatic). Data points represent individuals. Red = asthmatic subjects, Blue = nonasthmatic subjects. Dotted lines represent 95% confidence intervals.

Fig. 4

Effect of 1 ng/m³ mean annual polycyclic aromatic hydrocarbons (PAH) exposure on changes in immune function in subjects with asthma and/or allergic rhinitis. Stock plots showing estimates and confidence intervals for changes in immune parameter units with a 1 ng/m³ increase in mean annual PAH concentration. (a) Stratified by asthma. Red triangles = asthmatic subjects (n = 85); Blue triangles = non-asthmatic subjects (n = 171). (b) Stratified by allergic rhinitis. Black diamonds = non-asthmatic subjects without rhinitis (n = 160); Red diamonds = non-asthmatic subjects with allergic rhinitis (n = 11). (c) Stratified by asthma and allergic rhinitis. Black squares = asthmatic subjects without rhinitis (n = 61); Blue squares = asthmatic subjects with allergic rhinitis (n = 24).

Fig. 5

Changes in immune parameters in a follow-up subset. Comparison (a–c) and correlation (d–f) of (a, b) % Treg function, (c, d) levels of total IgE and (e,f) FOXP3 methylation between initial and follow-up visit. The average time between visits was 252.1 + 13.4 days. (a–c) Data are presented as mean + standard error of mean (SEM). Black circles = non-asthmatic subjects (n = 19), Red circles = asthmatic subjects (n = 6). Paired t-test *, P < 0.05; (d–f) data are presented as correlation between initial and follow-up visit. Pearson’s correlation test, r², *P < 0.05.