Our findings demonstrate that in utero exposure to a diet rich in methyl donors can enhance the severity of allergic airway disease in the offspring of mice and that this predisposition appears to be partially transmitted to subsequent generations. Moreover, methylation studies identified specific genes, including Runx3, that are under epigenetic control and that may play a fundamental role in the development and severity of this condition. Since diets rich in methyl donors did not significantly affect the airway disease phenotype of mice when exposed during either lactation or adulthood, there appears to be a period of vulnerability that is limited to gestation. This study provides what we believe is the first in vivo evidence that in utero diet can have an impact on CpG methylation and transgenerational inheritance of allergic airway disease.
Pregnancy is associated with a strong skewing of the immune system toward the Th2 cytokine profile, which enables survival of the fetus (19
). Postnatal persistence of Th2 polarization would favor the future development of allergic diseases, such as asthma. Consistent with this logic, failure to suppress genes that negatively regulate Th2 and favor normal maturation of the immune system could lead to enhanced severity of allergic airway disease in adults. Our observation that exposure to methyl donors was associated with altered lymphocyte development and function suggests that suppression of regulatory genes might contribute to enhanced allergic airway disease observed in animals exposed to dietary methyl donors. Previous in vitro studies indicate that DNA methylation can regulate T lymphocyte differentiation to either Th1 or Th2 lineages (9
). In vitro, hypomethylation enhances the expression of STAT4 and IFN-γ (11
) in CD4+
T cells and induces FoxP3 expression (14
), skewing toward a Th1 response and increasing the population of Treg cells. We provide in vivo evidence that in utero diet modifies differentiation of splenic T lymphocytes to express either CD4 or CD8. Interestingly, we identify enhanced CpG methylation and mRNA expression of Runx3
, a gene known to regulate CD4+
T lymphocyte development (21
) by silencing CD4+
expression. The reduced level of Runx3
corresponds well with an increased CD4+
is a tumor suppressor gene (25
) as well as a suppressor of dendritic cell maturation (26
), and it can regulate T lymphocyte development (21
). Recent studies also suggest that Runx3
expression can be attenuated through increased DNA methylation (28
). Importantly, Runx3-deficient mice spontaneously develop an asthma-like phenotype (30
). Recent findings indicate that Runx3 cooperates with T-bet in the silencing of IL-4 in the Th1 cell (31
). A reduced Runx3 level might lead to enhanced transcription of IL-4 skewing toward Th2 differentiation as observed in anti-CD3+
lymphocytes. In aggregate, our findings further support the importance of Runx3
as an epigenetically regulated candidate gene in allergic airway disease. Our study indicates that maternal dietary intake is an important early exposure that can directly influence CpG methylation, gene expression, and T lymphocyte development.
Consistent with an epigenetic mechanism that regulates the severity of allergic asthma in this model, we observed transgenerational inheritance of this phenotype. Previous work indicates that maternal diet consumption results in alterations in coat color in the agouti mouse as a result of differential CpG methylation (7
). Additionally, epigenetic modifications can be transgenerationally inherited through persistence of epigenetic changes in the germ line (6
). Our observation of partial transmission of disease risk suggests that the epigenetic effects on allergic airway disease are indeed reduced but persist into the F2 generation. Recently, dietary folic acid intake has been associated with CpG methylation in humans, which can be transmitted across generations (34
). In addition to dietary exposures, it is likely that other environmental exposures could have an impact on epigenetic transmission of complex disease. For example, it is known that tobacco smoke can modify gene expression by DNA hypermethylation (35
). Our discovery that the severity of allergic airway disease caused by in utero exposure to methyl donors can be passed through generations is consistent with an epigenetic mechanism of transmission. However, these transgeneration affects should be interpreted with caution, since the F2 generation germ line could have been exposed during gestation of the F1 generation (36
). In aggregate, these observations support the complexity of heritable diseases and raise the possibility that epigenetic modification could contribute to the limited success of both linkage and association studies in complex heritable diseases such as asthma.
These observations suggest that the increased prevalence of allergic asthma in humans may in part be related to increased perinatal dietary supplementation with methyl donors. Given the importance of folate supplementation in preventing congenital abnormalities (3
) and the differences in murine and human biology, we need to carefully evaluate the potential for adverse consequences of dietary supplementation during pregnancy before considering modifications in the current recommendations. However, our research suggests that too much dietary supplementation, especially with methyl donors during pregnancy, may have unexpected biological and pathophysiological consequences. Understanding the complex interactions between environmental exposures and genetic vulnerability will provide insight into future interventions for individuals at risk for the development of allergic asthma.