Asthma discordance in twins is linked to epigenetic modifications of T cells

Abstract

T cells mediate the inflammatory responses observed in asthma among genetically susceptible individuals and have been suspected to be prone to epigenetic regulation. However, these relationships are not well established from past clinical studies that have had limited capacity to control for the effects of variable genetic predisposition and early environmental exposures. Relying on a cohort of monozygotic twins discordant for asthma we sought to determine if epigenetic modifications in T cells were associated with current asthma and explored whether such modifications were associated with second hand smoke exposures. Our study was conducted in a monozygotic twin cohort of adult twin pairs (n = 21) all discordant for asthma. Regulatory T cell (Treg) and effector T cell (Teff) subsets were assessed for levels of cellular function, protein expression, gene expression and CpG methylation within Forkhead box P3 (FOXP3) and interferon gamma-γ (IFNγ) loci. Comparisons by asthma and current report of exposure to second hand smoke were made. Treg from asthmatic discordant twins demonstrated decreased FOXP3 protein expression and impaired Treg function that was associated with increased levels of CpG methylation within the FOXP3 locus when compared to their non-asthmatic twin partner. In parallel, Teff from discordant asthmatic twins demonstrated increased methylation of the IFNγ locus, decreased IFNγ expression and reduced Teff function when compared to Teff from the non-asthmatic twin. Finally, report of current exposure to second hand smoke was associated with modifications in both Treg and Teff at the transcriptional level among asthmatics. The results of the current study provide evidence for differential function of T cell subsets in monozygotic twins discordant for asthma that are regulated by changes in DNA methylation. Our preliminary data suggest exposure to second hand smoke may augment the modified T cell responses associated with asthma.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Differential T cell function between asthmatic and non-asthmatic twins.

Function of purified Treg (A) and purified Teff (B) from non-asthmatic twins (n = 21, white bars) and their asthmatic (n = 21, black bars) twin partner was assessed via 3H-thiymidine incorporation. A) Suppressive function of purified CD4+CD25+ Treg to CD4+ CD25neg Teff is presented as % function. B) Proliferation of purified CD4+ Teff to tetanus antigen. Data are presented as mean ± SD, * p<.05.

Figure 2. Reduced expression of proteins involved in Teff and Treg function during asthma.

A) FOXP3 gene expression and B) FOXP3 protein was assessed from purified Treg from asthmatic and non-asthmatic twin pairs. In C) IFNγ gene expression and D) IFNγ protein levels were assessed from purified Teff cells from asthmatic and non-asthmatic twin pairs. Gene expression was determined by QT-PCR and is shown as relative fold expression of candidate genes to expression of the housekeeping gene β-glucuronidase. Protein levels were determined by intracellular flow cytometry and shown as mean fluorescence intensity (MFI). Data points represent individuals and twin pairs are connected via lines, * p<.001.>

Figure 3. Epigenetic modification of FOXP3 and IFNγ during asthma.

Treg were purified from the PB (A, n = 21) or bronchoalveolar lavage (BAL) (B, n = 4) of MZT pairs discordant for asthma and methylation of 13 CpG sites within the FOXP3 locus was quantified. A CpG site was considered methylated after exhibiting methylation at that CpG site in ≥70% of the pyrosequencing reactions. Linear regression and Spearman’s correlation analysis of C) FOXP3 protein and % of methylated CpG sites within FOXP3(% calculated as number of methylated CpG sites divided by total number of CpG sites in the FOXP3 locus) or D) FOXP3 transcript (fold expression) and % of methylated CpG sites within FOXP3. E) Teff were purified from asthmatic and non-asthmatic MZT pairs (n = 21) and methylation of 6 CpG sites within the IFNγ locus was quantified. Data points represent individuals and twin pairs are connected via lines, r =  correlation coefficient.

Figure 4. Reduction of FOXP3 and IFNγ is augmented with recent SHS exposure.

Peripheral blood Treg or Teff (CD4+CD25neg) were purified from twin pairs discordant for asthma and concordant for no SHS exposure (n = 15) and discordant for both asthma and SHS exposure (n = 6). In A and B, Treg were assessed for FOXP3 transcript (A) and FOXP3 protein (B). In C and D, Teff were assessed for IFNγ transcript (C) and protein (D). Gene expression was determined by QT-PCR and is shown as relative fold expression of candidate genes to expression of the housekeeping gene β-glucuronidase. Protein levels were determined by intracellular flow cytometry and shown as mean fluorescence intensity (MFI). Data points (circles) represent individuals and individual twin pairs are connected via a line, p<.05 for non-asthmatic vs. asthmatic or shs all groups.>

Figure 5. Increased epigenetic modification of T cells with current SHS exposure during asthma.

Peripheral blood CD4+CD25+ Treg or CD4+CD25neg Teff were purified from twin pairs discordant for asthma and concordant for no SHS exposure (n = 15) and discordant for both asthma and SHS exposure (n = 6). Genomic DNA was sequenced for methylation of CpG sites within A) FOXP3 or B) IFNγ. *p<.05 n.s.: not significant.>

Figure 6. Decreased immune function in asthmatics with SHS.

Immune parameters from MZT pairs discordant for asthma (n = 15 non-asthmatic (Twin A) and asthmatic (Twin B)) or MZT pairs discordant for both asthma and SHS (n = 6 non-asthmatic (Twin A) and asthmatic + SHS (Twin B) were assessed. A) Teff proliferation to tetanus antigen. B) Tetanus Ig titers. C) Total serum IgE titers. Data are presented as mean ± SD, *p<.05.>