Fig 1.
Variation in AHR among 101 inbred strains of HMDP.
A) Timeline of the sensitization and exposure protocol that was designed to sufficiently induce AHR across 101 HMDP strains of different genetic backgrounds and varying susceptibilities to DEP. Mice (n = 4–8 per strain) were first sensitized on day 0 through a 100μl intraperitoneal injection containing 200μg DEP and 25μg HDM + 2.25mg Alum, as an adjuvant. On days 7–10, mice were separated into two groups and placed in insulated chambers daily for 20mins supplied with ambient air and saturated with either aerosolized PBS (control; n = 2-4/strain) or 200μg DEP (exposed; n = 2-4/strain). To avoid batch effects, the same collection of DEP and lot of HDM was used for all strains and all exposures were carried out at the same time in the mornings. On day 11, airway hyperreactivity was measured by invasive plethysmography. Lung resistance at baseline and in response to increasing concentrations of methacholine is shown for each strain under control PBS exposure (B) and DEP-exposed conditions (C). Each colored line represents the maximum lung resistance of each strain (as an average of 2–4 mice) at each methacholine dose. D) Circular bar-graph shows variation in lung resistance among HMDP mice strains under PBS control (light blue bars) and DEP exposed conditions (dark blue bars) at a methacholine dose of 10mg/ml. Each bar represents the mean value of 2–4 mice per group per strain.
Fig 2.
Results of a GWAS for AHR in the HMDP under PBS-control exposure.
A) The Manhattan plot shows lung resistance under control PBS exposure is significantly associated with two loci on chromosomes 2 and 19. The GWAS analysis included 196,591 SNPs, whose genomic positions are shown along the x-axis with their corresponding -log10 p-values indicated by the y-axis. The genome-wide thresholds for significant (p = 4.1x10-6) and suggestive (p = 4.1x10-4) evidence of association are indicated by the horizontal red and blue lines, respectively. B) Lung resistance at a methacholine dose of 2.5mg/ml is lower among strains carrying the minor T allele of the peak SNP (rs51547574) on chromosome 19 compared to strains carrying the C allele. Dots represent the average of 2–4 mice per strain. C) Regional plot for the chromosome 19 locus shows that rs51547574 is located directly over Il33 (red box) although SNPs in strong LD encompass other genes as well.
Table 1.
Effect Sizes and significance levels at varying doses of methacholine for loci identified for AHR on chromosomes 19 and 3.
Fig 3.
Results of a GxE GWAS for AHR in the HMDP in response to DEP exposure.
A) The Manhattan plot shows that lung resistance at a methacholine dose of 10mg/ml is significantly associated with a locus on chromosome 3. The GWAS analysis included 203,074 SNPs, whose genomic positions are shown along the x-axis with their corresponding -log10 p-values indicated by the y-axis. The mean difference (delta, Δ) in lung resistance between DEP-exposed mice and PBS-exposed controls were calculated for each strain and used in a linear mixed-model GWAS analysis with normal inverse transformed traits as implemented in the program pylmm. Genome-wide thresholds for significant (p = 4.1x10-6) and suggestive (p = 4.1x10-4) evidence of association are indicated by the horizontal red and blue lines, respectively. B) The Δ lung resistance at a methacholine dose of 10mg/ml is greater among strains carrying the minor C allele of the peak SNP (rs30880385) on chromosome 3 compared to strains carrying the T allele. Dots represent mean strain difference in Δ lung resistance between DEP-exposed mice and PBS-exposed controls. C) Regional plot for chromosome 3 locus shows that rs30880385 is located directly over Dapp1 (red box) although SNPs in strong LD encompass other genes as well.
Fig 4.
Tissue expression pattern of DAPP1 and relationship to inflammatory genes in humans.
A) Levels of DAPP1 mRNA, expressed as transcripts per million reads (TPM), are highest in mucosal and immune system-related tissues, including lung (shown in red). Data were obtained from the Genotype-Tissue Expression (GTEx) project portal. B) DAPP1 expression in lung and spleen is strongly and positively correlated with several pro-inflammatory cytokines relevant to asthma, including IL1A, IL7, IL12A, IL17A, IL23A, and IL33. The color-range in the illustration reflects the strength of the Spearman correlation coefficients (r) and the size of the circles reflects the significance levels of the p-values.
Fig 5.
Functional validation of Dapp1 as a DEP-responsive gene.
A and D) Lung resistance after inhalation exposure to DEP and HDM is not increased in Dapp1-/- mice compared to WT animals. DEP/HDM exposure induced eosinophilia (B and E) and neutrophilia (C and F) to the same extent in BAL fluid from Dapp1-/- and WT mice. Mice were first sensitized on day 0 through a 100μl intraperitoneal injection containing 200μg DEP and 25μg HDM + 2.25mg Alum, as an adjuvant. On days 7–10, mice were placed in insulated chambers daily for 20mins supplied with ambient air and saturated with either aerosolized PBS or both 200μg DEP and 25μg HDM. On day 11, airway hyperreactivity was measured by invasive plethysmography, followed by collection of BAL. Cell counts in BAL fluid were determined by flow cytometry. WT control animals were either female C57BL/6J mice purchased from the Jackson Laboratories (A-C) or WT littermates of both sexes generated through an intercross between Dapp1+/- heterozygote mice (D-F). For both experiments, n = 4–8 mice in each group. Data are shown as mean ± SE. ****p<0.0001; ***p<0.001; **p<0.005; *p<0.05.