Grandmaternal allergen sensitization reprograms epigenetic and airway responses to allergen in second generation offspring

SUPPLEMENTARY FIGURE LEGEND

Supplemental Figure 1. Grandmaternal allergen exposure regulates differentially methylated cytosines in airway epithelium of second-generation offspring at baseline and after allergen sensitization. Grandmaternal HDM exposure alone resulted in 1,390 DMCs in airway epithelium of F2 offspring (F0 HDM•F2 Base vs F0 Veh•F2 Base). F2 HDM sensitization alone resulted in 1,467 DMCs (F0Veh•F2Sens vs F0 Veh•F2 Base). In contrast, HDM sensitization in F2 mice from HDM-exposed founders resulted in 3,756 total DMCs (F0 HDM•F2 Sens vs F0 HDM•F2 Base). N=8 total for each group (n=4 males and n=4 females).

Supplementary Figure 2. Differential methylation is linked to altered gene expression in airway epithelium of second-generation offspring. A subset of F2 airway epithelial genes identified during differentially-methylated pathway analyses were analyzed by real-time RT-PCR to assess for expression. Grandmaternal HDM exposure significantly increased Smad3 expression at baseline and potentiated its response after F2 HDM sensitization. Paxillin expression was also increased after HDM sensitization compared to non-sensitized F2 descendants from HDM exposed founders, although this difference did not meet statistical significance (p = 0.06). No differences were observed between Ets3, Cdh3, or Cacna1d expression between groups. *p < 0.05. Delta CT values analyzed with two-way ANOVA and Tukey’s post-test. N=8 total for each group (n=4 males and n=4 females), except for F0 HDM•F2 Base, n=5.

Supplementary Figure 3. Grandmaternal allergen exposure regulates vagal ganglia cytosine methylation in second-generation offspring at baseline and after allergen sensitization. Grandmaternal HDM exposure alone resulted in 901 DMCs (F0 HDM•F2 Base vs F0 Veh•F2 Base). F2 HDM sensitization alone resulted in 1,467 DMCs (F0Veh•F2Sens vs F0 Veh•F2 Base). In contrast, HDM sensitization in F2 mice from HDM-exposed maternal founders resulted in 1,787 total DMCs (F0 HDM•F2 Sens vs F0 HDM•F2 Base). N=8 total for each group (n=4 males and n=4 females).

Supplementary Figure 4. Epigenetics changes after grandmaternal allergen exposure in second-generation offspring at baseline and after allergen sensitization are tissue-specific. Unique genes are differentially methylated in vagal ganglia neurons compared to airway epithelium after grandmaternal HDM exposure (15 shared genes), and after HDM sensitization in F2 mice from Vehicle-exposed maternal founders (only 9 shared genes) and HDM-exposed maternal founders (only 41 shared genes). N=8 total for each group (n=4 males and n=4 females).

Supplementary Figure 5. Gene ontology analysis of biological process enrichment in vagal ganglia from second-generation offspring.

Gene ontology analysis of DMCs in vagal ganglia of F2 mice identified enriched biological processes (individual small circles), which were then grouped based on functional annotation using Cytoscape Enrichment Map (larger, labeled, black circles). Each small circle represents an individual biological process and size is proportional to number of involved genes, while connecting edges represent shared genes between individual biological processes. (A) Gene ontology analysis showing effects of HDM sensitization on biological process enrichment in F2 offspring from vehicle-exposed F0 mice (F0 Veh•F2 Sens vs F0 VEH•F2 Base). (B) Gene ontology analysis showing potentiating effects of F0 grandmaternal HDM exposure on biological process enrichment in F2 mice after HDM sensitization (F0 HDM•F2 Sens vs F0 HDM•F2 Base). N=8 total for each group (n=4 males and n=4 females).

Supplemental Figure 6. Quantification of airway sensory nerve density using 3D confocal microscopy. Airway epithelial substance P-positive sensory nerves were analyzed in whole mount specimens using immunofluorescence and 3-dimensional confocal microscopy. Total nerve length and the number of branch points were quantified from nerve models generated based on substance P-positive voxels. Top) Representative images of substance P-expressing nerves (purple, top left) and nerve modeling (white filaments, top right). Bottom) Nerve length (left) and the number of branch points (right) were similar between untreated F2 mice from HDM exposed grandmothers and vehicle exposed grandmothers. N=6 both groups (n=3 males and n=3 females).

SUPPLEMENTARY DATA FILES

Lebold&Cook_DMC_Gene List.xlsx. 

Lebold&Cook_DMR Gene List.xlsx.

Differentially methylated cytosines (DMC) and regions (DMR) source data files.