A mouse model links asthma susceptibility to prenatal exposure to diesel exhaust

Abstract

Background: Most asthma begins in the first years of life. This early onset cannot be attributed merely to genetic factors because the prevalence of asthma is increasing. Epidemiologic studies have indicated roles for prenatal and early childhood exposures, including exposure to diesel exhaust. However, little is known about the mechanisms. This is largely due to a paucity of animal models.

Objective: We aimed to develop a mouse model of asthma susceptibility through prenatal exposure to diesel exhaust.

Methods: Pregnant C57BL/6 female mice were given repeated intranasal applications of diesel exhaust particles (DEPs) or PBS. Offspring underwent suboptimal immunization and challenge with ovalbumin (OVA) or received PBS. Pups were examined for features of asthma; lung and liver tissues were analyzed for transcription of DEP-regulated genes.

Results: Offspring of mice exposed to DEPs were hypersensitive to OVA, as indicated by airway inflammation and hyperresponsiveness, increased serum OVA-specific IgE levels, and increased pulmonary and systemic TH2 and TH17 cytokine levels. These cytokines were primarily produced by natural killer (NK) cells. Antibody-mediated depletion of NK cells prevented airway inflammation. Asthma susceptibility was associated with increased transcription of genes known to be specifically regulated by the aryl hydrocarbon receptor and oxidative stress. Features of asthma were either marginal or absent in OVA-treated pups of PBS-exposed mice.

Conclusion: We created a mouse model that linked maternal exposure to DEPs with asthma susceptibility in offspring. Development of asthma was dependent on NK cells and associated with increased transcription from aryl hydrocarbon receptor- and oxidative stress-regulated genes.

Keywords: IL-13; IL-17; IL-5; Prenatal exposure; aryl hydrocarbon receptor; asthma; diesel exhaust particles; mouse model; natural killer cells.

Figure 1. Experimental protocol

Timed pregnant C57BL/6 mice were given intranasal applications (IN) of either DEP or PBS on indicated gestation days (GD). Offspring were given intraperitoneal injections (IP) of PBS or a mixture of OVA and alum in PBS and then intranasal applications of OVA or PBS on indicated postnatal days (PND). These mice were then analyzed 3 days after the final IN application.

Figure 2. Airway inflammation and resistance

Experiments included PBS-PBS (prenatal and postnatal PBS), PBS-OVA (prenatal PBS, postnatal OVA), DEP-PBS (prenatal DEP, postnatal PBS), DEP-OVA (prenatal DEP, postnatal OVA) mice. A, B, peribronchial inflammation (histologic analysis), mean values (± standard error of the mean - SEM) from 3–6 mice/group; C–G, mean BAL cell counts (± SEM) from 10–21 mice/group; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; H, total lung resistance, mean values (± SEM) from 9–18 mice/group; ####, DEP-OVA vs. PBS-PBS; */****, DEP-OVA vs. PBS-OVA; ^/^^^^, DEP-OVA vs. DEP-PBS; $, PBS-OVA vs. PBS-PBS.

Figure 3. Expression of cytokines in lung

PBS-PBS, PBS-OVA, and DEP-OVA mice were analyzed for relative expression of cytokine transcripts in the lung tissue (A; mean values (± SEM) of 6–13 mice/group) and absolute level of cytokine proteins in BALF (B; mean values (± SEM) of 4–6 mice/group). Levels of each cytokine transcript were normalized to 18S rRNA and expressed as fold change in expression relative to the PBS-PBS group.

Figure 4. OVA-specific IgE in serum

Sera from PBS-PBS, PBS-OVA, DEP-PBS, and DEP-OVA mice were analyzed for concentration of OVA-specific IgE; mean values (± SEM) shown from 20–27 mice/group

Figure 5. Cytokine expression in splenocyte populations

OVA-stimulated and immunostained splenocytes (CD3, CD4, NK1.1, NKp46 and a cytokine) from PBS-PBS, PBS-OVA, and DEP-OVA mice were analyzed by flow cytometry. CD4 T cells, NK and NKT cells were defined as CD3+CD4+NK1.1−NKp46−, CD3−NK1.1+NKp46+ and CD3+NK1.1+ cells, respectively. A–C, Cytokine+ splenocytes expressed as a percentage of all splenocytes. D–F, Percentages of cytokine+ cells attributable to individual cell populations. G–I, Cytokine+ NK cells expressed as a percentage of all NK cells; mean values (± SEM) shown from 6 mice/group

Figure 6. Effect of NK cell depletion

Pregnant mice were exposed to DEP and offspring were immunized and challenged with OVA (see Fig 1). Anti-NK1.1 or isotype control IgG were injected 72 hrs, 48 hrs, and 24 hrs before the first OVA challenge. A–B, Peribronchial inflammation (histologic analysis); C–G, BAL cell numbers; mean values (± SEM) shown from 8 mice/group

Figure 7. Expression of AhR signature transcripts

Lungs (A) and livers (B) from PBS-PBS, PBS-OVA, DEP-PBS, and DEP-OVA mice were analyzed for relative levels of AhRR, Cyp1a1, and Cyp1b1 mRNA as in Fig 3; mean values (± SEM) shown from 6–13 mice/group

Figure 8. Levels of oxidative stress-regulated transcripts

Lungs (A) and livers (B) from PBS-PBS, PBS-OVA, and DEP-OVA mice were analyzed for relative levels of Hmox1 and Nrf2 mRNAs (as in Fig 3); mean values shown from 6–13 mice/group