Maternal allergen exposure reprograms the developmental lung transcriptome in atopic and normoresponsive rat pups

Abstract

The "fetal origins hypothesis" argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.

Fig. 1.

Ovalbumin (OVA) exposure protocol. Adult female rats were sensitized to OVA 14 days before mating. Control rats received saline (SAL). Pregnant rats sensitized to OVA were challenged with OVA for 30 min, 4 times during the pregnancy. Control rats received SAL. Pups were collected at postnatal day 1 (PN1), 7, or 14. E1, embryonic day 1.

Fig. 2.

Lung resistance is elevated in Brown Norway (BN) and Lewis pups following maternal allergen exposure. Airway resistance is significantly increased after OVA exposure in Lewis pups (top) after 12.5 mg of methacholine (MCh) (*P < 0.05, n = 4 SAL, n = 15 OVA), in BN (bottom) after 50 mg/ml of MCh (*P < 0.05, n = 5 SAL, n = 16 OVA). All measurements were taken at PN14.

Fig. 3.

Inflammatory cell infiltrates in bronchoalveolar lavage (BAL) fluid isolated from rat pups at PN1, 7, and 14. The percentage (A) and total number of inflammatory cell infiltrates (B) were assessed in BAL fluid isolated from rat pups at PN1, 7, and 14 (n > 4 all groups, except Lewis SAL and BN OVA PN14, n = 3). A: percentage of neutrophils was elevated in OVA-exposed Lewis pups at PN7 and 14 and BN pups at PN1 and 7. Following maternal OVA exposure, BN pups display higher neutrophil levels than Lewis pups at PN1. Eosinophil percentage was significantly increased in BN pups after maternal OVA exposure at PN7 and 14 and was higher than in Lewis pups at all 3 time points. B: OVA exposure was associated with an increase in the total number of neutrophils from BAL fluid in Lewis pups at PN14 and in BN pups at PN1 and 7. The total number of eosinophils was elevated in OVA-exposed BN pups at PN1 and 7 and was significantly higher than that observed in Lewis pups at PN1 (*P < 0.05 OVA vs. SAL; #P < 0.05 vs. Lewis rat).

Fig. 4.

A: Serum IgE levels are increased in Lewis and BN pups after maternal OVA exposure. OVA exposure is associated with increased serum IgE in BN pups at PN14 (n = 4, *P < 0.05 OVA vs. SAL, #P < 0.05 vs. Lewis). B: BAL levels of IL-1β, TNF-α, and GRO/KC are elevated in OVA-exposed BN pups at PN14. BAL levels of a panel of inflammatory serum cytokines including IFN-γ, IL-1β, IL-4, IL-13, KC/GRO, and TNF-α were assessed. OVA-exposed BN but not Lewis pups had elevated levels of IL-1β, TNF-α, and KC/GRO (n = 4, *P < 0.05).

Fig. 5.

Principal component analysis of whole lung samples in 22,523-gene probe expression space. 2 replicate samples were selected per condition: 2 strains × 3 ages × 2 exposures. The principal components 1–3 (PC1–3) coordinate centroids are plotted for each condition. Samples are marked Strain.Age. Connecting lines are colored gray for SAL and black for OVA. PC1 appears to correspond to developmental age, PC2 to strain, and PC3 to a combination of age and strain.

Fig. 6.

Gene expression trajectories perturbed by maternal OVA exposure. A: 2 hypothetical genes illustrating the use of linear correlation between developmental time series expression profiles to identify developmental expression affected by maternal SAL vs. OVA exposures. The SAL vs. OVA correlation value is inversely proportional to effect of OVA exposure. The expression time series for gene A is well correlated in SAL vs. OVA and poorly correlated for gene B. The expression of gene B during lung development is potentially affected by OVA exposure. If OVA exposure causes a uniform basal shift in gene expression from SAL exposure as in gene A, the OVA-SAL correlation is 1, and therefore gene A is not considered to be affected by OVA exposure when correlation is used to measure similarity. B: 3,693 reproducible probes (correlation between 1st and 2nd replicate profiles > 0.8) and the histogram of their OVA vs. SAL correlation values for each rat strain. The dashed vertical line marks the threshold for OVA effect (OVA vs. SAL correlation < −0.1), and we count the number of probes below/above this threshold. The light gray solid vertical line marks the median of OVA vs. SAL correlation values for the strain. C: of the 3,693 reproducible probe from B, 1,889 probes have loading magnitudes that are in the top 5% in PC1–3, which correspond to age-strain variation, and 799 of these are affected by OVA exposure, OVA vs. SAL correlation < −0.1 for either strain. D: for gene ontology enrichment analyses, we relaxed the criteria in C. For determining whether a probe is affected by OVA exposure, we identified 5,724 reproducible probes correlation >0.7 between 1st and 2nd replicate profiles. Of these, 3,669 probes have loading magnitudes that are in the top 10% in PC1–3, which correspond to age-strain variation, and 1,806 of these are affected by OVA exposure, OVA vs. SAL correlation < 0.0 for either rat strain.

Fig. 7.

Select strain-specific gene probes whose PN1, 7, and 14 lung expression profiles were affected by maternal OVA exposure as reported by Illumina Rat microarray (12). Plots of the means ± SD of quantile normalized logarithm base 2 expression signal of gene probes in 6 conditions (3 ages × 2 exposures, duplicate measurements per condition). Gray and black represents SAL and OVA exposures, respectively.

Fig. 8.

Validation of strain-specific perturbation of trajectories of respiratory gene expression in OVA-exposed pups by qRT-PCR. mRNA expression in whole lung tissue isolated from OVA or SAL exposed BN (A) and Lewis (B) pups was assessed by quantitative real-time PCR at PN1, 7, and 14. (n = 4, *P < 0.05, all genes BN and Lewis).

Fig. 9.

Genes perturbed by OVA exposure are also differentially expressed in lymphoblastoid cell lines from asthmatic patients. Maternal OVA exposure affects genes in BN (n = 36) and Lewis (n = 61) rat strains that are also differentially expressed in lymphoblastoid cell lines of asthmatic vs. nonasthmatic human sibling pairs in Moffatt et al. 2007 (21). The contingency tables show the number of overlapping genes between the rat and human data. The significance of overlap was assessed using the Fisher exact test. The odds ratio represents the fold enrichment or strength of the overlap.