Epigenetic regulation appears to be an important modifier of disease susceptibility. Environmental exposures are believed to induce these epigenetic changes. Previous studies have shown that altered methylation affects in vitro Th differentiation, raising the possibility that the risk for developing allergic sensitization or asthma also may be affected. We show for the first time that concomitant exposure to inhaled DEP and allergen can induce hypermethylation at select CpG sites in the IFN-γ promoter, and induce hypomethylation at one proximal CpG site in the IL-4 promoter in vivo. In addition, these patterns were associated with changes in IgE production, suggesting that altered methylation exerts molecular effects downstream.
In general, the methylation levels of CD4+
splenocytes at select CpG sites in the IFN-γ promoter averaged higher than observed in previous murine studies of CD4+
thymocytes (Jones and Chen, 2006
), especially following combined inhaled exposure to DEP and allergen. However, the levels appeared lower than measured from human CD4+
naïve T cells following in vitro
Th2 polarizing conditions, or isolated from adult atopics (White et al., 2002
), suggesting fundamental differences exist across species and cell types.
A growing body of in vitro
data has begun to reveal how these methylation patterns may modulate downstream molecular signaling pathways. For example, CpG−53
are conserved in rat, dog, chimpanzee, and human (Jones and Chen, 2006
; White et al., 2002
is located in the proximal AP1-binding site of the IFN-γ promoter and its methylation resulted in a change in factor binding (Young et al., 1994
). A subsequent study demonstrated that methylation of CpG−53
in the IFN-γ promoter significantly inhibited CREB and ATF2/c-Jun transcription factor binding to the CpG-containing AP1 site, augmenting proallergic Th2 polarization. Methylation of CpG−53
alone was sufficient to inhibit the IFN-γ promoter-driven reporter gene expression in a Th1 cell line (Jones and Chen, 2006
also interacted with AP-1–CREB DNA binding complexes (White et al., 2002
; Ye et al., 1994
In the present study, DEP and A. fumigatus
sensitization induced demethylation in only one of the studied CpG sites of the IL-4 promoter (i.e., CpG−408
), and not in any of the sites in the conserved element in the first intron (CIRE). However, demethylation along the IL-4 gene tends to progress in sequential order during Th2 differentiation from 5′ to 3′ (Ansel et al., 2006
). Specifically, demethylation during Th2 differentiation has been shown to begin at CpG−408
(Tykocinski et al., 2005
). Interestingly, CpG−408
has been identified as a putative transcription factor AP-2 site, although not associated with IL-4 gene transcription to date (Comb and Goodman, 1990
We recognize several limitations to our approach, including the possibility that DEP and/or A. fumigatus
exposure also could act via more distant elements in the IFN-γ or IL-4 genes or other genes. Additional airway studies (i.e., airway hyperreactivity) would have provided more information on the effects at local target tissues. BALB/c mice have been shown to be predisposed toward Th2 allergic responses in some experiments (Whitehead et al., 2003
), raising the possibility that the selection of mouse strain may have introduced biased. Finally, it may be difficult to distinguish the independent effects of DEP on CpG methylation from those associated with a general enhancement of Th2-mediated immune responses. Nonetheless, DEP repeatedly augmented the molecular changes observed following sensitization to A. fumigatus
alone (, ), suggesting a direct effect of DEP exposure on T cells.
In sum, chronically inhaled exposure to DEP-induced hypermethylation at several CpG sites of the IFN-γ gene promoter, and hypomethylation of CpG−408 in the proximal promoter of IL-4 gene in CD4+ T cells among mice sensitized to A. fumigatus. Hypermethylation of the IFN-γ gene promoter was associated with greater IgE production, and hypomethylation of the CpG−408 site in the proximal promoter of IL-4 gene was associated with reduced IgE production. This study is the first to demonstrate that inhaled environmental exposures implicated in asthma can alter methylation of Th genes in vivo. These results suggest that combined ambient allergen exposure and air pollution can modulate airway disease through epigenetic modification, supporting a new paradigm in asthma pathogenesis.