Transgenerational transmission of asthma risk after exposure to environmental particles during pregnancy

Abstract

Exposure to environmental particles during pregnancy increases asthma susceptibility of the offspring. We tested the hypothesis that this transmission continues to F2 and F3 generations and occurs via epigenetic mechanisms. We compared allergic susceptibility of three generations of BALB/c offspring after a single maternal exposure during pregnancy to diesel exhaust particles or concentrated urban air particles. After pregnant dams received intranasal instillations of particle suspensions or control, their F1, F2, and F3 offspring were tested in a low-dose ovalbumin protocol for sensitivity to allergic asthma. We found that the elevated susceptibility after maternal exposure to particles during pregnancy persists into F2 and, with lesser magnitude, into F3 generations. This was evident from elevated eosinophil counts in bronchoalveolar lavage (BAL) fluid, histopathological changes of allergic airway disease, and increased BAL levels of IL-5 and IL-13. We have previously shown that dendritic cells (DCs) can mediate transmission of risk upon adoptive transfer. Therefore, we used an enhanced reduced representation bisulfite sequencing protocol to quantify DNA methylation in DCs from each generation. Distinct methylation changes were identified in F1, F2, and F3 DCs. The subset of altered loci shared across the three generations were not linked to known allergy genes or pathways but included a number of genes linked to chromatin modification, suggesting potential interaction with other epigenetic mechanisms (e.g., histone modifications). The data indicate that pregnancy airway exposure to diesel exhaust particles (DEP) triggers a transgenerationally transmitted asthma susceptibility and suggests a mechanistic role for epigenetic alterations in DCs in this process.

Keywords: DNA methylation; asthma; epigenetics; transgenerational.

Fig. 1.

Schematic of the transgenerational model. F0 mice were exposed at embryonic day (E) E14–E15 to intranasal instillation of environmental particles; part of their F1 offspring was tested in the X1 low-dose allergen protocol to assess the transmission of asthma risk, while others remained naïve. These naïve females were then mated to normal males, and the study continued to F2, and then similarly to F3.

Fig. 2.

Bronchoalveolar lavage (BAL) eosinophil percentages (left) and counts (right) in the F1, F2, and F3 generation offspring of DEP-exposed (DEP) and PBS-exposed control (Neg Ctrl) mothers, as well as positive control asthmatic mice of the same age that received a dual intraperitoneal OVA sensitization protocol (Pos Ctrl). In F3, the four litters of the DEP group are shown separately to illustrate the variability in transmission of the phenotype. We tested multiple litters using from each litter n = 4–6 pups, for a total N behind each bar in the chart to be 6–10 (varies with birth rate and technical issues). *P < 0.05.

Fig. 3.

A: inflammatory lung histopathology scores in the F1, F2, and F3 generation offspring of DEP-exposed and PBS-exposed control (Neg Ctrl) mothers, as well as X2 OVA-sensitized age-matched positive control asthmatic mice (Pos Ctrl). B: BAL fluid cytokine levels via ELISA. n = 3 per litter. *P < 0.05, Mann-Whitney U-test one-tailed.

Fig. 4.

A: BAL eosinophil counts in the F1, F2, and F3 generation offspring of DEP-exposed in comparison to concentrated urban air particles (CAP)-exposed group, vs. PBS-exposed control (Neg Ctrl). B: lung histopathology scores in the CAP cohorts (F1, data not shown). Number of animals tested was 3–6 per litter, n = 5–10 per group. *P < 0.05.

Fig. 5.

Effect of decitabine treatment in F1 on transmission of the phenotype to F2 and F3 generations. A: BAL eosinophil counts from a representative experiment. n = 4–8 per group B: eosinophil counts from three experiments over two separate cohorts were normalized to negative control and pooled. n = 6–12 per group. *P < 0.05.

Fig. 6.

Significant differentially methylated regions (DMRs) in F1, F2, and F3 asthma-at-risk dendritic cells (DCs) vs. Control. In A, the overlap is based on the genomic coordinate of the DMR; this demonstrates how many CpGs were altered. B: the specific cytosines were mapped to a nearest annotated gene; we then tested how many genes were involved. There were multiple differentially methylated CpGs per gene name; hence, the numbers differ from A. C: direction of the methylation changes: number of loci with increased or decreased methylation relative in each generation relative to control is plotted. Columns F1+F2, F1+F2+F3 indicate loci that are significantly changed in the same direction in both or all of the indicated generations.

Fig. 7.

Network analysis of a list of genes with associated CpG methylation change within 10-kb distance of the transcript (n = 89) (A) and without the 10 kb filtration (n = 324) (B) seen in F1+F2+F3 generations; “direct links” algorithm. Notice minimal or no involvement in interacting pathways. Various symbols for the genes reflect whether the respective protein is membranous, nuclear, or cytoplasmic, and sometimes a functional designation; these are automatically generated by the software for illustrative purposes only.

Fig. 8.

A: network analysis of a list of genes with associated CpG methylation change within 10-kb distance of the transcript seen in F1 vs. control within the 10 kb cutoff (n = 1888). Notice a central cluster of genes with known direct interactions in biological pathways. B: central cluster (n = 941) expanded. C: three representative examples dissecting the network interactions within the cluster. The interaction lines illustrate whether the factors are richly or poorly interconnected into nodes.