Ozone Inhalation Provokes Glucocorticoid-Dependent and -Independent Effects on Inflammatory and Metabolic Pathways

Abstract

Growing evidence implicates air pollutants in adverse health effects beyond respiratory and cardiovascular disease, including metabolic impacts (diabetes, metabolic syndrome, obesity) and neurological/neurobehavioral outcomes (neurodegenerative disease, cognitive decline, perceived stress, depression, suicide). We have shown that inhalation of particulate matter or ozone activates the hypothalamic-pituitary-adrenal axis in rats and increases plasma levels of the glucocorticoid corticosterone. To investigate the role of corticosterone in mediating inflammatory and metabolic effects of pollutant exposure, in this study male Fischer-344 rats were administered the 11β-hydroxylase inhibitor metyrapone (0, 50, 150 mg/kg body weight) and exposed by nose-only inhalation for 4 h to air or 0.8 ppm ozone. Ozone inhalation provoked a 2-fold increase in plasma corticosterone, an effect blocked by metyrapone, but did not alter epinephrine levels. Inhibition of corticosterone production was associated with increased inflammatory signaling in the lungs and plasma in response to ozone, consistent with a role for glucocorticoids in limiting local and systemic inflammatory responses. Effects of ozone on insulin and glucagon, but not ghrelin or plasminogen activator inhibitor-1, were modified by metyrapone, revealing glucocorticoid-dependent and -independent effects on circulating metabolic and hemostatic factors. Several immunosuppressive and metabolic impacts of ozone in the lungs, heart, liver, kidney, and spleen were blocked by metyrapone and reproduced through exogenous administration of corticosterone (10 mg/kg body weight), demonstrating glucocorticoid-dependent effects in target tissues. Our results support involvement of endogenous glucocorticoids in ozone-induced inflammatory and metabolic effects, providing insight into potential biological mechanisms underlying health impacts and susceptibility.

Keywords: air pollution; glucocorticoid; hypothalamic–pituitary–adrenal (HPA) axis; inflammation; metabolic effects.; ozone; stress response; systemic effects.

FIG. 1.

.Effects of ozone and metyrapone on plasma corticosterone. Animals were treated with vehicle (Veh) or metyrapone (Met; 50, 150 mg/kg) and exposed for 4 h to clean air or 0.8 ppm ozone as described in the Materials and Methods. Corticosterone (Cort; 10 mg/kg) was administered to a separate set of animals and exposed to air, and naïve animals remained in their cages. Each bar represents mean ± SE (n = 5/group). OZONE × MET interaction, P < .001 (two-way ANOVA). Symbols above bars denote statistical significance of pairwise comparisons (P < .05, Holm-Sidak) as follows: (*) Air versus Ozone within vehicle; (a) 50 or 150 versus 0 mg/kg metyrapone within Ozone; (b) 150 versus 50 mg/kg metyrapone within Ozone

FIG. 2.

Inflammatory and antioxidant response in the lungs. Animals were administered metyrapone (Met; 50, 150 mg/kg) or vehicle (Veh), exposed to clean air or 0.8 ppm ozone for 4 h, and euthanized immediately after exposure. Corticosterone (Cort; 10 mg/kg) was administered to a separate set of animals and exposed to air, and naïve animals remained in their cages. Cytokines were recovered by bronchoalveolar lavage (BAL) and assessed by multiplex immunoassay, and gene expression was measured in lung tissue by real-time PCR. Data were assessed for significant factor interactions or main effects by two-way ANOVA, followed by Holm-Sidak pairwise comparison. Complete statistical analyses are presented in Supplementary Data. Each bar represents mean ± SE (n = 5/group). A, IL-17A. OZONE main effect, P = .013; MET main effect, P < .001. B, IL-6. OZONE × MET interaction, P < .001. C, CXCL1. OZONE × MET interaction, P = .01. D, CCL2. OZONE × MET interaction, P = .003. E, IL-6 mRNA. OZONE × MET interaction, P < .001. F, TNF mRNA. OZONE × MET interaction, P < .001. G, CCL2 mRNA. OZONE × MET interaction, P = .007. H, HMOX mRNA. OZONE × MET interaction, P = .027. I, MT-1 mRNA. OZONE × MET interaction, P < .001. J, MT-2 mRNA. OZONE main effect, P < .001; MET main effect, P < .001. Symbols above bars denote statistical significance of pairwise comparisons (P < .05, Holm-Sidak) as follows: (*) Air versus Ozone within each drug dose; (a) 50 or 150 versus 0 mg/kg metyrapone within Air or Ozone; (b) 150 versus 50 mg/kg metyrapone within Air or Ozone.

FIG. 3.

Effects of ozone and metyrapone on plasma cytokines. Cytokine levels were assessed in the plasma of animals administered vehicle (Veh) or metyrapone (Met; 50, 150 mg/kg) and exposed to air or 0.8 ppm ozone for 4 h. Corticosterone (Cort; 10 mg/kg) was administered to a separate set of animals and exposed to air, and naïve animals remained in their cages. Data were assessed for significant factor interactions or main effects by two-way ANOVA, followed by Holm-Sidak pairwise comparison. Complete statistical analyses are presented in Supplementary Data. Each bar represents mean ± SE (n = 5/group). A, IL-17A. OZONE main effect, P = .006; MET main effect, P < .001. B, M-CSF. OZONE main effect, P = .024; MET main effect, P = .018. C, IL-10. OZONE × MET interaction, P = .032; D, CXCL1. OZONE main effect, P = .002; MET main effect, P < .001. E, CCL2. OZONE main effect, P = .008; MET main effect, P < .001. Symbols above bars denote statistical significance of pairwise comparisons (P < .05, Holm-Sidak) as follows: (*) Air versus Ozone within each drug dose; (a) 50 or 150 versus 0 mg/kg metyrapone within Air or Ozone; (b) 150 versus 50 mg/kg metyrapone within Air or Ozone.

FIG. 4.

Tissue-level effects of ozone and metyrapone on the expression of inflammatory, metabolic, and antioxidant genes. The mRNA levels were assessed in tissues of animals exposed to air or 0.8 ppm ozone following administration of vehicle or metyrapone (Met; 50, 150 mg/kg). Values are expressed as mRNA fold-change relative to vehicle air control group ± SE (n = 5/group). Data were assessed for significant factor interactions or main effects by two-way ANOVA, followed by Holm-Sidak pairwise comparison. For simplicity, only significant Air versus Ozone pairwise comparisons are indicated (*P < .05). Complete statistical analyses are presented in Supplementary Data. A, Glucocorticoid-inducible leuzine zipper (GILZ). Lung: MET main effect, P < .001, OZONE main effect, P = .051. Heart: MET main effect, P < .001. Liver: MET main effect, P < .001. Kidney: MET × OZONE interaction, P = .020. Spleen: MET × OZONE interaction, P = .009. B, Hypoxia-inducible factor (HIF)-3α. Lung: MET × OZONE interaction, P < .001. Heart: MET main effect, P < .001, OZONE main effect, P < .001. Liver: MET main effect, P < .001, OZONE main effect, P = .016. Kidney: MET main effect, P < .001, OZONE main effect, P < .001. Spleen: MET × OZONE interaction, P = .01. C, Metallothionein (MT)-1. Lung: MET main effect, P < .001, OZONE main effect, P < .001. Heart: MET main effect, P < .001. Liver: MET main effect, P < .001, OZONE main effect, P < .001. Kidney: MET main effect, P < .001, OZONE main effect, P < .001. Spleen: MET main effect, P < .001, OZONE main effect, P < .001.

FIG. 5.

Comparison of transcriptional responses to ozone and exogenous corticosterone. The mRNA levels of immune response (glucocorticoid-inducible leucine zipper (GILZ), chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β, tumor necrosis factor (TNF)) and metabolic (hypoxia inducible factor (HIF)-3α, BCL2/adenovirus E1B 19 kDa-interacting protein (BNIP)-3; sterol regulatory element-binding protein (SREBP)-1) genes were assessed in the lung, heart, liver, kidney, and spleen of rats administered vehicle and exposed to 0.8 ppm ozone or administered corticosterone (10 mg/kg body weight) and exposed to air (n = 5/group). A log2 transformation was used to linearize data. Results were regressed and compared using Pearson correlations. FC, fold-change relative to vehicle air group.