In utero supplementation with methyl donors enhances allergic airway disease in mice

Abstract

Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.

Figure 1. Gestational exposure to dietary methyl donors regulates severity of allergic asthma.

Adult C57BL/6J progeny exposed to HMD in utero demonstrate enhanced allergic inflammation when compared with those exposed to LMD. C57BL/6J breeding pairs were fed HMD, RD (NIH-31), or LMD beginning 2 weeks prior to mating and during pregnancy. Experimental diets were continued until the time of weaning, when F1 progeny were placed on RD (NIH-31). At 6 to 10 weeks of age, F1 mice were immunized and subjected to an OVA challenge. In utero exposure to HMD was associated with enhanced (A) airway responsiveness to methacholine (n = 5–6; *P < 0.05) (HMD, squares; LMD, diamonds), (B) concentration of lung lavage eosinophils (n = 31–63; HMD, white bars; LMD, black bars), (C) percentage of lung lavage eosinophils (n = 31–63), (D) IL-13 levels in whole-lung lavage fluid (n = 15–26), (E) total serum IgE (n = 15–26; *P < 0.05, HMD versus LMD), (F) OVA-specific IgE (n = 15–26; ^†P = 0.05, HMD versus LMD), and (G) OVA-specific IgG1 (n = 15–26; *P < 0.05, HMD versus LMD). Avg, average.

Figure 2. Gestational exposure to methyl donors enhances allergic airway disease in mice.

H&E staining of lung sections obtained from mice subjected in utero to HMD or LMD diet, then immunized and subjected to an OVA challenge. Low magnification of lungs from HMD (A) and LMD (B) mice. Higher magnification of small airways from HMD (C) and LMD (D) mice. Venule with perivascular cuffing from a dam fed HMD (E) or LMD (F) during gestation. Arrow shows multinucleated giant cells. Arteriole with perivascular cuffing from a dam fed HMD (G) and LMD (H) during gestation. Arrows identify margination of inflammatory cells. Original magnification, ×4 (A and B); ×40 (C–H).

Figure 3. Transgenerational inheritance of diet-associated risk of allergic asthma.

Male F1 progeny were mated with naive females and maintained on RD. F2 progeny were immunized and subjected to an OVA challenge at 6–10 weeks of age. The F2 progeny were kept on RD. F2 adults (HMD, n = 37; LMD, n = 68) demonstrated nonsignificant differences in the (A) concentration of lung lavage eosinophils (P = 0.099), significant differences in the (B) percentage of lung lavage eosinophils (*P = 0.007), (C) and significant differences in serum IgE (^†P < 0.001).

Figure 4. Diet modifies production of cytokines and chemokines in splenocytes.

Spleens from mice gestated on either HMD or LMD were harvested following immunization and challenge with OVA. To determine functional consequences of perinatal diet, splenocytes were minced, passed through a 50-μm cell strainer, and isolated by density gradient centrifugation with Histopaque 1083 as described in Methods. (A) CD4⁺/CD8⁺ ratios in the spleen were determined by flow cytometry (n = 5; *P < 0.05, HMD versus LMD). To determine the functional implications of respective diets on splenocytes, 1 × 10⁶ cells were exposed to saline or 4 μg OVA in vitro for 72 hours. Supernatants were collected and evaluated for cytokines/chemokines by Bio-Plex profiling. (B) Splenocytes derived from animals exposed to HMD demonstrate enhanced production of KC, CCL4, and CCL5 (^†P < 0.01, HMD versus LMD). (C) Next, we harvested splenocytes from mice with gestational exposure to HMD or LMD without immunization and challenge with OVA and isolated CD4⁺ lymphocytes by negative selection. These cells were challenged with antibodies to CD3⁺CD28⁺ (1 μg/ml each) for 48 hours. CD4⁺ cells from HMD mice demonstrate enhanced levels of IL-4 and no difference in IFN-γ.

Figure 5. DNA methylation affects mRNA and protein expression.

Relative mRNA expression was measured by real-time PCR using SYBR-Green as described in Methods. (A) Comparison between HMD/high responders (n = 8) and LMD/low responders (n = 3) in challenged C57BL/6 mice in lung tissue. P = 0.30 (Runx3); P = 0.10 (Nfact1); *P = 0.02 (Jak2); P = 0.20 (Rcor3); P = 0.08 (Ube2j1). (B) Runx3 protein expression was determined in lung tissue from individual F1 mice exposed in utero to either an HMD (n = 5) or an LMD (n = 3). Mouse Runx3 protein was detected in total lung cell extracts by a polyclonal antibody to human AML2^–/Runx3. ^†P < 0.05, HMD versus LMD. (C) Comparison of gene expression from splenocytes obtained from F1 mice treated in utero with a high (n = 5) or low (n = 6) methylation diet and subsequently sensitized and subjected to an OVA challenge. ^‡P = 0.01 (Nfact1); ^§P = 0.03 (Runx3); P = 0.06 (Jak2). (D) In vitro effect of a demethylating agent on gene expression from splenocytes. Splenocytes were obtained from F1 mice treated in utero with an HMD following sensitization and challenge with OVA. 1 × 10⁶ cells were exposed to 4 μg OVA in vitro for 72 hours and either pulsed daily with saline or 1.0 μM of azacytidine (Aza). ^¶P = 0.003 (Runx3); P = 0.008 (Nfact1); ^#P = 0.08 (Jak2).