Ozone impairs endogenous compensatory responses in allergic asthma

Abstract

Asthma is a chronic inflammatory airway disease characterized by acute exacerbations triggered by inhaled allergens, respiratory infections, or air pollution. Ozone (O₃), a major component of air pollution, can damage the lung epithelium in healthy individuals. Despite this association, little is known about the effects of O₃ and its impact on chronic lung disease. Epidemiological data have demonstrated that elevations in ambient O₃ are associated with increased asthma exacerbations. To identify mechanisms by which O₃ exposure leads to asthma exacerbations, we developed a two-hit mouse model where mice were sensitized and challenged with three common allergens (dust mite, ragweed and Aspergillus fumigates, DRA) to induce allergic inflammation prior to exposure to O₃ (DRAO₃). Changes in lung physiology, inflammatory cells, and inflammation were measured. Exposure to O₃ following DRA significantly increased airway hyperreactivity (AHR), which was independent of TLR4. DRA exposure resulted in increased BAL eosinophilia while O₃ exposure resulted in neutrophilia. Additionally, O₃ exposure following DRA blunted anti-inflammatory and antioxidant responses. Finally, there were significantly less monocytes and innate lymphoid type 2 cells (ILC2s) in the dual challenged DRA-O₃ group suggesting that the lack of these immune cells may influence O₃-induced AHR in the setting of allergic inflammation. In summary, we developed a mouse model that mirrors some aspects of the clinical course of asthma exacerbations due to air pollution and identified that O₃ exposure in the asthmatic lung leads to impaired endogenous anti-inflammatory and antioxidant responses and alterations inflammatory cell populations.

Keywords: Allergic asthma; Eosinophils; Murine model; Ozone.

Figure 1:. DRA-O₃ exposure results in mixed eosinophilic and neutrophilic inflammation with increased lung injury.

WT male mice were either untreated (Control (Ctrl), n=10), challenged with DRA treated alone (DRA, n=10), challenged with O₃ alone (O₃, n=8), or challenged with both DRA and O₃ (DRA-O₃, n=9). (A) A schematic of the timing for the DRA sensitization and challenges as well as O₃ challenge is provided. Mouse image is from Biorender. DRA-O₃ mice were sensitized with DRA over 17 days to induce allergic inflammation, followed by O₃ exposure on day 18. BAL was collected after methacholine challenge and the total number of cells in the BAL fluid was measured (B). (C) Total protein in the BAL fluid was measured by BCA assay. Differential cell counts were performed to determine the percentage of (D) eosinophils, (E) neutrophils, and (F) macrophages. Error bars shown as mean ± SEM. *P <0.05, **P<0.001.

Figure 2:. Sequential exposure to DRA and O₃ induces histologic inflammation.

WT male mice were challenged with either DRA-O₃ (n=9), DRA (n=10), or O₃ (n=9), and whole lung tissue was collected and compared against lung tissue isolated from control mice (n=10). (A) Whole lung histologic sections were H&E-stained following methacholine challenge. These are representative images from each treatment group. Scale bar in 1x section is 2 mm. The insert box in 1x section represents the location that is shown in the subsequent 10x and 40x images. Scale bar is 200 μm for 10x and 50 μm for 40x. All lungs were examined by a veterinarian pathologist and DRA-treated groups had increased (B) perivascular interstitial inflammation, (C) peribronchial interstitial inflammation, (D) alveolar inflammation, and (E) alveolar macrophages compared to untreated control mice. Supplemental Table 1 contains grading and numerical explanations for each individual mouse. Error bars shown as mean ± SEM. *P <0.05, **P<0.01, ***P<0.001.

Figure 3:. DRA and DRA-O₃ exposure results in increased goblet cell hyperplasia

WT male mice (n=10) were challenged with either DRA-O₃ (n=9), DRA (n=10), or O₃ (n=9), and whole lung tissue was collected and compared against lungs isolated from control mice. (A) Whole lung histologic sections were PAS-stained following methacholine challenge. These are representative images from each treatment group. Scale bar in 1x section is 2 mm for Ctrl, DRA, O₃; and 3 mm for DRA-O₃. The insert box in 1x section represents the location that is shown in the subsequent 10x and 40x images. Scale bar is 200 μm for 10x and 50 μm for 40x. (B) Quantification of goblet cell hyperplasia was assessed on the H&E slides but data is shown here. mRNA levels of (C) Muc5b as well as mRNA (D) and protein (E) expression of Muc5ac was assessed in whole lung tissue. Error bars shown as mean ± SEM. *P <0.05, **P<0.01, ***P<0.001

Figure 4:. DRA-O₃ exposure increases airway hyperreactivity compared to either injury alone in a TLR4 independent fashion.

(A) WT mice were challenged with either untreated (control, n=10), DRA-O₃ (n=9), DRA (n=10), or O₃ (n=9) and then challenged with escalating doses of methacholine (Mch). Airway hyperreactivity, as assessed by measuring airway resistance (Rn) using a flexiVent small-animal ventilator. (B) WT (TLR4^+/+, n=8) or knockout (TLR4^−/−, n=7) were exposed to DRA and then O₃. Mice were challenged with escalating doses of methacholine. Error bars shown as mean ± SEM. *P <0.05.

Figure 5:. Ozone exposure following DRA challenge impairs endogenous anti-oxidant and anti-inflammatory responses.

WT male mice were untreated (n=10) or challenged with DRA-O₃ (n=9), DRA (n=10), or O₃ (n=9), and whole lung tissue was collected and compared against whole lung isolated from control mice. (A) IL-10 was assessed by ELISA. Expression of (B) GPX-1 and (C) GPX-2 was measured by qPCR. Error bars shown as mean ± SEM. *P <0.05, **P<0.01, ***P<0.001.

Figure 6:. Ozone exposure following DRA challenge impairs endogenous anti-oxidant and anti-inflammatory responses

WT male mice were either untreated (control, n=5) or challenged with DRA (n=5), O₃ (n=5) or DRA-O₃ (n=5) and the populations of immune cells were determined by flow cytometry. A) Combined UMAP multidimensional reduction of all Live CD45+ cells with specific cellular populations highlighted by flow gating strategies. (B) UMAP of individual treatment groups displayed in a heat map where increased cell numbers are shown in hot (red-yellow) colors and lower cell numbers are shown in cool (blue) colors. Total number of classical monocytes (B), inflammatory monocytes (C) or innate lymphoid type 2 cells (D) were measured in each group (n=5 mice/group). Error bars are shown as mean ± SEM. *P<0.05, **P<0.01