Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Jan 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2022 Dec 8;459:116341. doi: 10.1016/j.taap.2022.116341

Ozone impairs endogenous compensatory responses in allergic asthma

Kevin Ho 1, David Weimar 1, Gina Torres-Matias 1,2, Hyunwook Lee 1, Saaleha Shamsi 1, Emily Shalosky 1, Michael Yaeger 1,2, Hannah Hartzler-Lovins 1,3, Katelyn Dunigan-Russell 1, Daria Jelic 1, Caymen M Novak 1, Kymberly M Gowdy 1, Joshua A Englert 1,*, Megan N Ballinger 1,*
PMCID: PMC9840700  NIHMSID: NIHMS1860358  PMID: 36502870

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 (DRA-O3). 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, ozone, murine model

Introduction

Asthma is a chronic inflammatory airway disease characterized by acute exacerbations in response to inhaled allergens or respiratory infections. Atopic asthma, distinguished from non-atopic asthma by eosinophilic inflammation, makes up approximately half of all asthma cases worldwide1. Asthma exacerbations are characterized by an acute worsening of lung function that in the most severe cases can be life-threatening. A common trigger for atopic asthma is air pollution, specifically increases in ambient O32. O3 is an air pollutant that induces pulmonary inflammation and oxidative stress. Epidemiologic studies have demonstrated an increase in asthma-related symptoms during times of high air pollution3.

Despite this epidemiological association, little is known about the underlying mechanisms by which O3 induces allergic asthma exacerbations. Early human studies of O3 exposure in healthy volunteers revealed increased peripheral blood and bronchoalveolar neutrophilia4–5, but the significance of this finding and whether patients with a preexisting lung disease (e.g. asthma) have a similar response is unknown. Murine models of acute O3 exposure have consistently demonstrated neutrophilic airway inflammation6–7, but its link to asthma exacerbation in the context of chronic allergic inflammation is still unclear. One of the major barriers to exploring the association between O3 and asthma is the lack of mouse models of allergic inflammation that can mirror the course of patients with atopic asthma who suffer from exacerbations due to air pollution. Existing asthma models are limited by the use of a single antigen for sensitization (leading to possible tolerance)8–9, short duration of allergen exposure, or the use of less clinically relevant antigens such as ovalbumin (OVA)10–11. Bronchoalveolar lavage results from these mouse models have varying degrees of lymphocytosis, neutrophilia, and eosinophilia, which differs from the expected eosinophilic and neutrophilic response based on prior human studies4–5.

Here, we present a novel two-hit murine model where mice were exposed to O3 following the induction of allergic inflammation with a mixture of clinically relevant environmental allergens12. We hypothesized that O3 exposure in this model would induce airway remodeling, goblet cell hyperplasia, inflammatory cell recruitment to the lungs, airway hyperreactivity (AHR), and the expression of anti-inflammatory cytokines and antioxidants compared to control mice or mice exposed to either allergens or O3 alone.

Material and Methods

Mice.

C57BL/6J (stock number: 000664) or TLR4−/− (stock number: 029015) male mice (8–10 weeks old) were purchased from The Jackson Laboratory (Bay Harbor, ME). Mice were randomly assigned to treatment groups. The animals were housed in specific pathogen-free conditions within The Ohio State University Animal Care Facility (Columbus, OH). All animal experiments were performed in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Institutional Animal Care and Use Committee at The Ohio State University.

Allergic inflammation model.

The DRA triple-allergen mixture was composed of extracts of 2.5 mg/ml of dust mite (Dermatophagoides farina), 25 mg/ml of ragweed (Ambrosia artenmisiifolia), and 2.5 mg/ml of A. fumigatus (Greer Laboratories) as previously described13–14. The DRA mixture contained 3624 EU/mL of endotoxin. Mice were anesthetized with ketamine-xylazine before all intranasal exposures. On days 0 and 5, mice were sensitized with 20 μl (200 μg) of the DRA allergen mixture. Mice were then challenged with 20 μl of DRA on days 12, 14 and 17.

O3 challenge.

Mice were exposed to room air (control) or O3 at 2 ppm for 3 hours in a custom-designed plexiglass chamber15. As described by Hatch et al.16, this was done to ensure O3-induced toxic effects in mice, which is comparable to an O3 concentration of 400 ppb for humans. Ozone was generated by directing 100% oxygen through an ultraviolet light generator that was mixed with room air inside the chamber. Temperature (24.9–25.5°C), humidity (68.6–75.0 RH), and ozone concentration were monitored continuously. For mice in the DRA-O3 group, the O3 challenge occurred on day 18. Mice exposed to O3 alone were challenged the day preceding the methacholine challenge.

Measurement of Airway Hyperreactivity.

For mice in the DRA-O3 group, airway measurements were taken 24 hours following O3 exposure. For the remaining mice, measurements were taken the day following the last exposure (DRA or O3). Mice were anesthetized with pentobarbital (100 mg/kg intraperitoneally), a tracheostomy tube was inserted, and then were paralyzed with pancuronium bromide (0.8 mL/kg intraperitoneally) before initiation of methacholine challenge. Mice were connected to a FlexiVent FX small-animal ventilator (SCIREQ) and increasing concentrations of methacholine were administered via an Aeroneb laboratory nebulizer (2.5 mg/ml, 5 mg/ml, 10 mg/ml, 25 mg/ml, and/or 50 mg/ml). The forced oscillatory technique was used to perform sequential quick-prime 3 maneuvers following two deep inflations to normalize volume history. Data obtained from these maneuvers were analyzed using the constant phase model to calculate the airway resistance (Rn) and other measurements of lung function using flexiWare software (version 8)17. The average of the top three Rn values at each methacholine dose were reported as the peak response to methacholine.

Bronchoalveolar Lavage (BAL) Cell Counts.

Lungs were lavaged with 1 mL of PBS and cells were counted using the Countess automated cell counter (Life Technologies). Cytospins were prepared and stained with HEMA 3 (Thermo Scientific). Following staining, differential cell counts were performed on 100 cells in a blinded fashion.

mRNA Extraction, cDNA synthesis, and qPCR.

mRNA was extracted from lung tissue homogenates by using a Direct-zol RNA miniprep Plus Kit (Zymo Research) according to the manufacturer’s instructions. cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). Gene expression was measured by quantitative PCR (qPCR) in a QuantStudio 3 Real-time PCR system using Fast PowerUp SyBR Green Master Mix (Applied Biosystems) unless otherwise noted. A list of forward and reverse primers used for qPCR are provided in Supplemental Table 1. TaqMan probe and primers were utilized to measure Muc5ac and Muc5b expression. Data were analyzed by the 2−ΔΔCt method with 18S expression as an endogenous control.

Measurement of cytokines.

Measurements of TSLP, IL-33, IL-1β and IL-10 were quantified in undiluted whole lung homogenates using ELISA kits (R&D systems) following manufacturer’s protocols. Muc5AC ELISA (Novus Biological) was performed according to the manufacturer’s instructions.

Flow cytometry cell preparation, cellular gating strategies and UMAP analysis.

Cell were isolated from whole lungs of mice by collagenase digestion as previous described18. Cells were incubated with Fc receptor block anti-mouse CD16/32 antibody (BD biosciences) followed by specific antibodies listed in Supplemental Table 2 Lung cells were then fixed in 2% paraformaldehyde and analyzed on a Cytek Aurora Spectral Flow Cytometry. The UMAP FlowJo Plugin was used to dimensionally reduce and visualize data sets. A total of 15,000 CD45+ live events from each sample were concatenated into a single FCS file, with the use of keywords to preserve the group identity. The UMAP algorithm was run on the single FCS file and the resulting plot is a 2-dimensional visual representative of all of the CD45+ live cells present in the various treatment groups. Each dot on the UMAP represents a cell and cells with similar expression profiles for the various markers used in the flow analysis will be groups closer together. The cellular populations highlighted in Figure 6A were identified by specific gating schemes for the whole lung digest are presented in Supplemental Figure 4 and 5 and based on those previously described by our group19 and Yu et al20.

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

Figure 6:

Open in a new tab

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

Lung Histology.

The upper right lobe was fixed in 10% formalin prior to processing and embedding in paraffin. H&E and PAS staining were conducted by the Comparative Pathology and Mouse Phenotyping Shared Resource at The Ohio State University. Whole lung images were obtained using Aperio ImageScope. Histopathologic analysis was performed by a board certified veterinary pathologist from Experimental Pathology Laboratories utilizing an established grading methodology to measure interstitial inflammation and goblet cell metaplasia21. Briefly the grades assigned to mucin were based on the number of goblet cells and their pattern of distribution in the main bronchus, proximal branches, preterminal bronchioles, and terminal bronchioles and the scoring was as follows: 1:represents no goblet cells; 2: scattered goblet cells in the main bronchus ± proximal branches; 3: increased number of goblet cells, confluent in some places; 4:same as 3 but goblet cell extending distally, and 5: same as 3 but extending distally into terminal bronchioles. The grading criteria for increased alveolar macrophages was as follows: 1: Alveolar macrophages with increased cytoplasm and up to 2 alveolar spaces per 1.5 mm2 field contain multiple macrophages; 2: alveolar macrophages with increased cytoplasm, alveolar spaces frequently contain multiple macrophage and multinucleated macrophages present; 3: 25–50% of alveolar spaces contain 1 or more macrophages; 4: >50% of alveolar spaces contain 1 or more macrophages. The grading criteria for alveolar inflammation was as follows: 1: <10% of alveoli contain mixed inflammatory cell infiltrates; 2: between 10–25% of alveoli contain mixed inflammatory cell infiltrates; 3: between 25–50% of alveoli contain mixed inflammatory cell infiltrates; 4: >50% of alveoli contain mixed inflammatory infiltrates. A summary incidence and severity of microscopic changes is located in Supplemental Table 3.

Statistics.

For laboratory-based experiments, statistical analysis was performed using Graphpad Prism 9. Data sets were tested for normality via a Shapiro-Wilk test. All data are presented as mean + SEM except where noted otherwise. Student’s t-tests were used to compare two groups with normally distributed data. A student’s t-test on log-transformed data was used to compare two groups with log-normally distributed data. Mann–Whitney tests were used to the compare ranks if the data were not normally distributed. For analysis of qPCR data, the statistical comparison was performed on log-transformed fold change data. For comparison among multiple groups, all data were normally or log-normally distributed and an ANOVA was performed on log-transformed or transformed data as appropriate. For experiments with two-independent variables, a two-way ANOVA was performed after testing data for normality as described. If a significance was determined by ANOVA, a Tukey post-hoc test for individual group comparisons was performed. A p-value of <0.05 was considered statistically significant. Outliers were identified using non-linear regression via the ROUT method with a Q threshold of 1%.

Results:

DRA-O3 asthma exacerbation model recapitulates the inflammatory characteristics of allergic inflammation and O3 exposure.

To create a background of allergic inflammation, mice were treated with DRA over a three-week period. Following DRA sensitization, mice were exposed to O3 to mimic the effects of this specific pollutant on asthmatics with allergic asthma (Figure 1A). There were four treatment groups: 1) control 2) O3 alone 3) DRA alone 4) DRA followed by O3 (DRA-O3). Total number of cells present in the airways were increased in mice with allergic inflammation but O3 exposure following DRA sensitization did not further increase BAL cell numbers (Figure 1B). BAL protein levels were measured as a marker of barrier permeability and were increased in DRA and O3 exposed groups (Figure 1C). Inflammatory cell populations within the BALs of the different treatment groups were assessed using cytospin slides. Groups exposed to DRA (DRA alone, DRA followed by O3) had significantly increased BAL eosinophils compared to control and O3 exposure alone (Figure 1D and Supplemental Figure 1A). BAL samples from groups treated with O3 (O3 alone, DRA followed by O3) had significantly higher BAL neutrophils compared to control and DRA exposure alone (Figure 1E and Supplemental Figure 1B). There was no significant difference in BAL eosinophilia between DRA alone compared to DRA-O3 exposure and no significant difference in BAL neutrophilia between O3 alone and DRA-O3. Control and O3 treatment alone had a higher percentage of macrophages than DRA-treated groups (Figure 1F) but there was no difference in the total number of macrophages (Supplemental Figure 1C). In summary, these data suggest that the combination of DRA sensitization and O3 exposure leads to a mixed inflammatory phenotype consisting of both eosinophilic and neutrophilic inflammation.

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

Figure 1:

Open in a new tab

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.

Sequential exposure to DRA and O3 induces inflammation within lung tissue.

Lung tissue was examined and assessed for different features of airway inflammation and remodeling using an established methodology21. A summary of the incidence and severity of microscopic changes in the lungs in found in Supplemental Table 3. Mice exposed to DRA (in combination with O3 or alone) had evidence of increased perivascular (Figure 2A–B) and peribronchiolar (Figure 2A,C) interstitial inflammation as well as alveolar inflammation (Figure 2A,D), which was characterized by a mixed inflammatory cell infiltrate. There were increased alveolar macrophages present in the alveolar spaces as well as increased cytoplasm and multinucleated macrophages present in the DRA and DRA-O3 groups compared to O3 or untreated (Figure 2E). O3 exposure alone at the dose and timepoint studied induced minimal histologic injury (Figure 2A). This analysis suggests that our 2-hit model induced histopathologic changes resembling the chronic airway inflammation found in human asthmatics.

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

Figure 2:

Open in a new tab

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.

O3 exposure in the setting of allergic inflammation does not alter goblet cell hyperplasia

Mucus overproduction can result in cough and airway obstruction during asthma exacerbations. Goblet cell hyperplasia is thought to be responsible for this excessive mucus production and secretion22. Examination of lung tissue following PAS staining revealed increased goblet cell hyperplasia in DRA-treated groups (Figure 3A). Exposure to O3 following DRA sensitization and challenge also resulted in increased goblet cell hyperplasia, but it was not significantly different than DRA alone (Figure 3B). Muc5ac and Muc5ab have been implicated in physiologic and pathophysiologic mucus secretion. There was no difference in lung tissue Muc5ac mRNA levels between all of the groups (Figure 3C). A single O3 exposure alone did not increase mRNA levels (Figure 3D) or protein expression (Figure 3E) of Muc5ac, but DRA-exposed groups did have significantly higher Muc5ac mRNA levels compared to controls (Figure 3D). There was a significant decrease in Muc5ac protein expression (Figure 3E) in the DRA-O3 group compared to the mice that received DRA alone. These data suggest that DRA exposure increases Muc5ac expression and goblet cell hyperplasia and that a single exposure to O3 does not significantly alter mucus production.

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

Figure 3:

Open in a new tab

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

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

Increased airway resistance is a physiologic hallmark of asthma exacerbations. To test for airway hyperreactivity (AHR) in our model, mice were given increasing doses of nebulized methacholine prior to measurements of airway resistance. As expected, mice exposed to each individual injury had increased airway resistance following methacholine challenge, but this was similar to the effect seen in control mice. DRA-O3 exposure led to a significant increase in AHR in response to nebulized methacholine compared to other treatment groups (Figure 4A). There were no significant differences in the tissue damping (G) or tissue elastase (H) across the groups (Supplemental Figure 2A and 2B). Previous data have demonstrated that O3-induced airway hyperreactivity and inflammation were dependent upon activation of the TLR4 signaling pathway23–24. To test whether changes in AHR and lung inflammation were TLR4 dependent in our 2-hit model, we subjected wild type (TLR4+/+) and TLR4 knockout (TLR4−/−) mice to O3 exposure following DRA sensitization and challenge. Both wild-type and TLR4 knockout mice had similar increases in airway resistance following methacholine challenge (Figure 4B). Next, we measured lung inflammatory cell recruitment and found similar levels of neutrophils, eosinophils, macrophages, and lymphocytes following DRA-O3 challenge (Supplemental Figure 3A and 3B). These data demonstrate that the DRA-O3 model recapitulates the increased airway resistance that is characteristic of asthma exacerbations and that this increase in AHR is not dependent on expression of TLR4.

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

Figure 4:

Open in a new tab

(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.

Sequential exposure to DRA-O3 alters expression of anti-oxidant enzymes and anti-inflammatory cytokines.

Production of Th2 cytokines has been shown to regulate the development of allergic asthma. TSLP, initially isolated from thymic stromal cell line, is a cytokine that has been implicated as a major factor in the development of asthma, especially Th2-related phenotypes. TSLP protein expression in the lung homogenate was significantly higher in mice treated with DRA alone compared to all other treatment groups, including DRA-O3 (Supplemental Figure 4A). Several different cytokines or chemokines have been implicated in eosinophil recruitment25–27. We measured protein expression of IL-33 and found it to be unexpectedly decreased in the combination DRA-O3 group (Supplemental Figure 4B). In contrast, there were no differences in expression of the pro-inflammatory cytokine IL-1β (Supplemental Figure 4C) between the DRA and DRA-O3 challenged groups.

Cells have various anti-inflammatory and anti-oxidant mediators to attenuate injury and respond to exogenous stresses. IL-10 is an anti-inflammatory cytokine that inhibits inflammation by regulating cytokine and chemokine expression from activated macrophages28. IL-10 protein expression was increased following DRA exposure alone, but O3 exposure following DRA sensitization impaired the compensatory increase in IL-10 (Figure 5A). A single O3 exposure alone did not alter IL-10 expression in lung tissue. Cellular antioxidants play a key role in protecting cells from injury following exposure to O3 or other types of oxidant injury. Specifically, glutathione peroxidases plays a protective role following O3 exposure or allergic inflammation29. In our model, DRA as well as DRA prior to O3 exposure decreased expression of glutathione peroxidase-1 (GPX-1, Figure 5A) and glutathione peroxidase-2 (GPX-2, Figure 5C) levels compared to control mice or O3 alone. These data suggest that allergic inflammation from DRA sensitization and challenge impairs the endogenous antioxidant response to O3. Similarly, O3 exposure in the setting of allergic inflammation impairs the compensatory increase in IL-10 induced by DRA alone. Together these data demonstrate the complex regulation of endogenous anti-oxidant and anti-inflammatory pathways in our 2-hit model.

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

Figure 5:

Open in a new tab

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.

O3 exposure in the setting of allergic inflammation impairs recruitment of monocytes and decreases type 2 innate lymphoid cells.

To interrogate the mechanisms by which O3 exposure following allergic inflammation impairs IL-10 release, different immune cell populations within the lung microenvironment were profiled via flow cytometry. Whole lung tissue was collected from the various treatment groups and collagenase digested to obtain a single cell suspension. Differences of lung leukocytes were distinguished using antibodies against various cell surface markers (Supplemental Table 2) and then specific cellular populations were identified using established gating strategies (Supplemental Figures 5A and 6A). To visualize the different immune cell populations within the lung, uniform manifold approximation and projection (UMAP) was used to dimensionally reduce and visualize the data sets (Figure 6A). Mice exposed to allergen challenge with DRA alone had significantly higher numbers of lung macrophages including tissue resident alveolar macrophages, monocyte-derived alveolar macrophages, and interstitial macrophages compared to control mice or mice exposed to O3 alone (Supplemental Figure 5B and Figure 6B). Mice exposed to DRA also had higher levels of monocytes including classical and inflammatory monocytes (Figure 6B–D) that are known to produce counter-regulatory cytokines such as IL-10. Interestingly, mice exposed to O3 following DRA challenge did not have a compensatory increase in these monocyte populations and had similar numbers of these cells compared to control mice. We also measured the number of other cells known to produce IL-10, such as T-cells, B-cells, and NK cells, and there was no significant differences in the absolute number of these cells between DRA and DRA-O3 treated mice (Supplemental Figure 6). Alternatively, mice exposed to DRA-O3 had decreased type 2 innate lymphoid cells (Figure 6E), which may be important given that a subset of these cells has been shown to produce IL-10 in response to allergic inflammation30. In summary, these data suggest that O3 may exacerbate airway hyperreactivity by decreasing immune cells that are responsible for the production of counter-regulatory cytokines during allergic inflammation.

Discussion:

Asthma, specifically allergic asthma, is a major health concern. Although new therapeutics have been developed for allergic phenotypes of asthma, few established murine models of allergic inflammation have focused on preventing asthma exacerbations triggered by air pollution. Clinically asthma exacerbations are characterized by increased symptoms (e.g. cough, wheezing, dyspnea) and impaired lung function. Experimentally asthma exacerbations are typically modeled by exposing laboratory animals to a known asthma trigger (e.g. environmental allergen, viral infection, etc) in the setting of pre-existing inflammation. This second hit typically leads to increased inflammation, impaired lung function, or both. To model how air pollution can lead to asthma exacerbations, we developed a novel model where mice with allergic inflammation due to sequential DRA exposure were exposed to ozone. Using this model, we found differences in the recruitment of inflammatory cells, airway hyperreactivity, and compensatory anti-oxidant and anti-inflammatory pathways. By exposing mice to O3 in the background of mixed eosinophilic and neutrophilic inflammation, we induced an acute worsening in airway hyperreactivity. This suggests that O3 has a deleterious effect on mice with allergic inflammation.

Other groups have reported two-hit allergic mouse models involving O3 exposure31–32, but there is inconsistency regarding the type of inflammation and how this correlates to human disease. Previous work has shown that a combination of common environmental allergens induced a mixed inflammatory phenotype, and this is important because eosinophilic inflammation is a pathophysiologic hallmark of atopic asthma in patients33 and these cells are not always present in murine models. Li et al.10 showed that O3 exposure following allergic inflammation induced by ovalbumin (OVA) led to increased mucus production, airway inflammation, and increased airway hyperreactivity. However, the BAL inflammatory profile consisted of lymphocytes instead of eosinophils. Last et al. studied the effects of a prolonged O3 exposure over several weeks following OVA sensitization and found a mixed inflammatory infiltrate that consisted of macrophages but did not examine airway hyperreactivity31. These findings provide insight into the effects of O3 on allergic inflammation but are limited by the use of OVA11. Our study examines the effects of O3 following intranasal sensitization and challenge with DRA, which is known to contain common antigenic triggers for asthmatics12. Our findings are consistent with the work of Keirstein et al who showed that mice with eosinophilic lung inflammation due Aspergillus exposure alone had increased airway hyperreactivity following subsequent O3 exposure8. These data suggest that O3 exposure in the setting of allergic inflammation can lead to impaired lung function which is a hallmark of asthma exacerbations. To our knowledge, we are the first to report a two-hit mouse model where mice were exposed to O3 following the induction of eosinophilic inflammation with DRA.

Signaling via a variety of different pathogen recognition receptors regulates expression of cytokines. Toll-like receptor (TLR) signaling pathways are involved in the initiation of inflammatory responses to a variety of pathogens, allergens and environmental exposures within the lungs. However, the role of these pathways can be difficult to investigate because both structural cells and immune cells express different receptors and expression of these receptors can change throughout disease progression. In addition, TLRs can exert dual roles during asthma exacerbations with some receptors causing tolerance or sensitization, while others promote inflammation, particularly early in life34. Our model demonstrates that the O3-induced exacerbation of DRA-induced allergic inflammation is independent of TLR4 signaling pathways (Figure 4B). Additionally, there was no difference in expression of TLR4-induced proinflammatory cytokines (i.e. IL-1β and IL-33) in our dual hit model (Supplement Figure 4C). Previous work has shown that other TLRs35–36 play a role in both asthma and O3 lung inflammation. In addition, recent work37 has shown that TLR4 heterodimerizes with other TLR receptors to modulate environmental lung injury. Additional work is needed to examine how TLR receptors other than TLR4 play a role in ozone induced asthma exacerbations.

There are several possible mechanisms to explain the physiologic and histopathologic findings observed in our DRA-O3 model. One potential mechanism of lung injury is that allergic inflammation from DRA exposure impairs endogenous anti-oxidant pathways that normally protect the lung when it is exposed to high levels of O3. Multiple studies have shown the importance of the anti-oxidant response in protecting the lung from oxidative stress, particularly O3-induced lung injury. Bao et al.29 showed that exposure to O3, OVA, or OVA followed by O3 increased levels of GPX compared to control. Previous data demonstrated that injurious exposures alone (O3 or OVA) or in combination (O3-OVA)28 caused elevated levels of anti-oxidant enzymes. In our model following O3 exposure alone, we observe significant increases in GPx-1 and GPx-2 (Figure 5B and 5C), but there was no increase in anti-oxidants in whole lung homogenates following DRA or DRA-O3. Similarly, IL-10, an anti-inflammatory cytokine, is critically involved in lung injury and asthma38–39. There was significantly elevated IL-10 in the lung homogenates after DRA exposure alone but not O3 group (Figure 5A). There was no difference in the amount of IL-10 in the DRA-O3 treated group. Together, this suggests that O3 in the setting of allergic inflammation inhibits endogenous cytoprotective responses.

Our study characterized the how O3 exposure in the setting of allergic inflammation altered the composition of immune cells in both the BAL and whole lung digests. To our knowledge, our study is the first to comprehensively profile immune cell populations within the lung after a 2-hit model using O3 exposure as a trigger. Although there was a significant increase in the number of T cells in the lungs of mice exposed to both DRA and DRA-O3 challenge (Supplemental Figure 6B), there was no difference in total CD4+ or CD8+ T cells between these two groups. Previous work has demonstrated different CD4+ T cell subsets regulate allergic inflammation, and additional studies are underway to determine if there are different Th cell subsets in the different treatment groups. Innate lymphoid type 2 cells (ILC2s) are involved in allergic inflammation as well as O3-induced eosinophilic inflammation and goblet cell hyperplasia40, however, ILC2s have not been examined in our current model. Our data demonstrates a significant decrease in ILC2 after DRA-O3 (Figure 6E). This is consistent with the fact that there were lower levels of IL-33, a known activator or ILC2s40, in mice subjected to our 2-hit model (Supplemental Figure 4B). Sex hormones regulate the number, function and phenotype of ILC2s, and C57Bl/6 male mice have less ILC2s compared to female mice41. Our study also found a significant increase in mast cells after DRA-O3 exposure. Mast cells are known to be localized within the smooth muscle bundles of asthmatic patients and play an important role in inflammation, bronchoconstriction and mucus secretion. In addition to degranulation, mast cells also produce a variety of cellular mediators42, such as TSLP, IL-33, IL-4, and IL-13, which were found in our 2-hit model and have an important role in the development of ozone-induced lung injury43. Additional work is in progress to determine the role of ILC2s and mast cells in this model.

As shown previously44, our work demonstrated that mice exposed to DRA alone had a significant increase in macrophages and monocytes in the lung (Figures 6C and 6D, Supplemental Figure 5B). The origin of various lung macrophage subpopulations within the lung microenvironment regulates allergic inflammation45, with circulating monocytes promoting allergic inflammation and resident myeloid cells dampening the immune response. Different subsets of monocytes regulate immune responses in the lung, and markers to distinguish these subpopulations vary according to species46. In humans, expression of the surface markers CD14, LPS binding protein, and CD16, an Fc-receptor, distinguished classical (CD14+CD16−), non-classical (CD14dimCD16+) and intermediate (CD14+CD16+) monocytes. In mice, other surface markers can distinguish classical/patrolling (CD11b+Ly6clo) verses inflammatory (CD11b+Ly6chi) monocytes. In murine studies, it has been shown that Ly6clo monocytes are present at baseline, and the presence of Ly6chi monocytes increases in the setting of injury or inflammation. There was a significant decrease in the expression of both the classical and inflammatory monocytes after DRA-O3 exposure. Although previous work demonstrated that O3 exposure resulted in novel macrophage subpopulations being recruited to the lung microenvironment47, 24 hrs after exposure there was no increase in macrophage or monocyte subpopulations within the lung tissue. Alternatively, exposure to the mixed allergens results in significant increases in all myeloid subpopulations within the lung. These studies suggest an important role for macrophages within the lung during environmental exacerbations of lung disease.

There are several limitations to this study. First, the concentration of O3 we used is several times higher than O3 levels during days with high air pollution, which may limit the clinical applicability of the study. However, 2 ppm for 3 hours is equivalent to a O3 concentration of 400 ppb in humans16 and has been used by other groups suggesting our results are generalizable. Studies are ongoing to currently investigate this model using a range of O3 exposures more in line with the NAAQS guidelines. Second, the ideal DRA concentration and administration timing is currently unknown. Because we used a single timepoint, allergic inflammation may not have peaked before O3 exposure and methacholine challenge. This is consistent with data from other groups that found peak neutrophil inflammation following O3 exposure occurred at 12 hrs; whereas the peak macrophage inflammation occurs at 48 hrs post O341,48. Despite this limitation, we were still able to observe significant histopathologic changes with our DRA protocol and increased AHR with O3 exposure following DRA sensitization. Third, we did not test for allergen -derived proteases and the effect this would have in the sensitization of mice in our triple antigen model. Previous work has demonstrated that proteases, such as serine proteases and metalloproteinases, can disrupt lung epithelial cell barriers driving the allergenicity and inflammatory responses and additional work is needed to determine their role in our triple antigen model49–50. Fourth, we used C57/BL6 mice, which are known to have more of a proinflammatory phenotype at baseline and are more resistant to type 2 airway inflammation. Performing these experiments in other wild-type strains that are more susceptible to allergic inflammation would provide additional information regarding this phenotype51. Finally, we performed all of our experiments with male mice as an initial first step. Other groups have described sex differences in mouse models of allergic inflammation41,52 and O3-induced lung injury53–55. Examining whether there are sex differences in our 2-hit model will be an important next step.

In summary, O3 is a common environmental trigger for asthma exacerbations, but the underlying mechanisms aggravating these underlying chronic lung disease is unclear. Using a novel, two-hit murine model, we demonstrated that O3 exposure following the induction of allergic inflammation resulted in increased AHR and induced histopathologic changes that mirror human allergic asthma exacerbations. O3 exposure in the setting of allergic inflammation impaired the endogenous release of IL-10 that accompanies DRA exposure alone. DRA exposure impaired the upregulation of anti-oxidant enzymes that normally occurs following O3 exposure. Future studies will be needed to determine the mechanisms responsible for these findings. Using models such as the one we describe may be help to identify novel therapeutic targets for asthmatic patients to prevent exacerbations in response to environmental pollutants.

Supplementary Material

1

Supplemental Figure 1: Total numbers of inflammatory subtypes within the airspace following challenge with DRA, O3, or DRA-O3 compared to control mice. WT male mice were either untreated (Control, n=10) or challenged with DRA (n=10), O3 (n=9) or DRA-O3 (n=9). BALs were performed and differential inflammatory subtypes were quantified by cytospin analysis. Absolute numbers of eosinophils (A), neutrophils (B), and macrophages (C) are shown in the number of cells/ml of BAL fluid returned. Error bars are shown as mean ± SEM. *P<0.05, ***P<0.001

Supplemental Figure 2: No increase in tissue dampening and elastance measurements in DRA, O3, or DRA-O3 challenged mice compared to control mice. WT male mice were either untreated (Control, n=10) or challenged with DRA (n=10), O3 (n=9) or DRA-O3 (n=9) and then challenged with escalating doses of methacholine (Mch). Elastance (H) and tissue dampening (G) were assessed using a flexiVent small-animal ventilator. Error bars are shown as mean ± SEM.

Supplemental Figure 3: No difference in total number of BAL cells or subtype of BALs in TLR4+/+ compared to TLR4−/− after DRA-O3 challenge. WT (TLR4+/+) or TLR4 knockout mice (TLR4−/−) were challenged with DRA and then O3. The total number of cells present in the BAL fluid was assessed by automatic cell counter (A) and cell differentials were determined by cytospin analysis (B). Error bars are shown as mean ± SEM.

Supplemental Figure 4: DRA-O3 exposure alters some Th-2 mediated cytokines and proinflammatory cytokines. Whole lung tissue was collected from WT mice unchallenged mice (n=10) as well as mice challenged with DRA-O3 (n=9), DRA (n=10), and O3 (n=9) and expression of cytokines was assessed. Expression of (A) TSLP, (B) IL-33, and (E) IL-1β was measured by ELISA. Error bars shown as mean ± SEM. *P <0.05. **P<0.01, ***P<0.001

Supplemental Figure 5: Flow gating scheme and absolute numbers of innate immune cells with the lung from the different groups. WT male mice were either untreated (control) or challenged with DRA, O3 or DRA-O3 and total lung cells were analyzed by flow cytometry. (A) The specific gating scheme for the innate immune cells are as shown. (B) Total number of each individual subpopulations is quantified. N=5 mice/group. Error bars are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001

Supplemental Figure 6: Flow gating scheme and absolute numbers of adaptive immune cells with the lung from the different groups. WT male mice were either untreated (control) or challenged with DRA, O3 or DRA-O3 and total lung cells were analyzed by flow cytometry. (A) The specific gating scheme for the adaptive immune cells are as shown. (B) Total number of each individual subpopulations is quantified. N=5 mice/group. Error bars are shown as mean ± SEM. *P<0.05, **P<0.01

Supplemental Table 1: List of primers used for qPCR

Supplemental Table 2: List of antibodies used for flow cytometry analysis

Supplemental Table 3: Histological grading of lung histology sections

Impact Statement:

These studies demonstrate that allergic asthma can be exacerbated by an environmental pollutant, such as ozone. Ozone-induced asthma exacerbation is due to an impaired endogenous anti-inflammatory and antioxidant response as well as a differential recruitment of immune cells into the lung

Highlights:

  • Ozone (O3) exposure in mice with allergic asthma increased airway hyperactivity

  • Increased airway resistance in O3 exposed asthmatic mice was independent of TLR4.

  • O3 exposure impaired endogenous anti-inflammatory and antioxidant pathways during asthma

Acknowledgements:

We would like to acknowledge Debra Tokarz for performing the pathology review. In addition, we would like to acknowledge that this work was performed in the Davis Heart and Lung Research Institute at The Ohio State Wexner College of Medicine.

Funding:

This was funded by The Ohio State University College of Medicine Office of Research Dean’s Discovery Program (MNB and JAE) and NIH R01ES028829 (KMG).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

COI: The authors have nothing to disclose

Credit author statement:

Conceptualization and Methodology: MNB, JAE, and KMG; Investigation: KH, DW, GTM, HL, SS, ES, MY, HHL, KDR, DJ, CMN; Data Curation: KH, DW and DJ; Writing original draft and review editing: KH, MNB ad JAE; Supervision, Project Management and Funding acquisition: MNB and JAE

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References:

  • 1.Knudsen TB, Thomsen SF, Nolte H et al. A population-based clinical study of allergic and non-allergic asthma. J Asthma. 2009. Feb;46(1):91–4 [DOI] [PubMed] [Google Scholar]
  • 2.Guarnieri M, Balmes JR. Outdoor air pollution and asthma. Lancet. 2014. May 3;383(9928):1581–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Khatri SB, Holguin FC, Ryan PB et al. Association of ambient ozone exposure with airway inflammation and allergy in adults with asthma. J Asthma. 2009;46(8):777–785. [PMC free article] [PubMed] [Google Scholar]
  • 4.Bosson JA, Blomberg A, Stenfors N et al. Peripheral blood neutrophilia as a biomarker of ozone-induced pulmonary inflammation. PLoS One. 2013;8(12):e81816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Schelegle ES, Siefkin AD, McDonald RJ. Time course of ozone-induced neutrophilia in normal humans. Am Rev Respir Dis. 1991. Jun;143(6):1353–8. [DOI] [PubMed] [Google Scholar]
  • 6.Che L, Jin Y, Zhang C et al. Ozone-induced IL-17A and neutrophilic airway inflammation is orchestrated by the caspase-1-IL-1 cascade. Sci Rep. 2016;6:18680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Johnston R, Mizgerd J, Shore S. CXCR2 is essential for maximal neutrophil recruitment and methacholine responsiveness after ozone exposure. Am J Physiol-Lung C. 2005; 288(1):L61–L67. [DOI] [PubMed] [Google Scholar]
  • 8.Kierstein S, Krytska K, Sharma S et al. Ozone inhalation induces exacerbation of eosinophilic airway inflammation and hyperresponsiveness in allergen-sensitized mice. Allergy. 2008. Apr; 63(4):438–46. [DOI] [PubMed] [Google Scholar]
  • 9.Goplen N, Karim MZ, Liang Q et al. Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma. J Allergy Clin Immunol. 2009;123(4):925–32.e11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Li J, Chen Y, Chen Q et al. Role of transient receptor potential cation channel subfamily V member 1 (TRPV1) on ozone-exacerbated allergic asthma in mice. Environ. Pollut 2019;247:586–594. [DOI] [PubMed] [Google Scholar]
  • 11.Kumar RK, Herbert C, Foster PS. Mouse models of acute exacerbations of allergic asthma. Respirology. 2016;21(5):842–9. [DOI] [PubMed] [Google Scholar]
  • 12.“Common Asthma Triggers.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 21 Aug. 2020, www.cdc.gov/asthma/triggers.html.
  • 13.Lee YG, Reader BF, Herman D et al. Sirtuin 2 enhances allergic asthmatic inflammation. JCI Insight. 2019. Feb 21;4(4):e124710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Park GY, Lee YG, Berdyshev E et al. Autotaxin production of lysophosphatidic acid mediates allergic asthmatic inflammation. Am J Respir Crit Care Med. 2013. Oct 15;188(8):928–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Howenstine M, Eigen H, Tepper R. Pulmonary function in infants after pertussis. J Pediatr. 1991. Apr;118(4 Pt 1):563–6. [DOI] [PubMed] [Google Scholar]
  • 16.Hatch GE, McKee J, Brown J et al. Biomarkers of Dose and Effect of Inhaled Ozone in Resting versus Exercising Human Subjects: Comparison with Resting Rats. Biomark Insights. 2013;8:53–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chung S, Lee YG, Karpurapu M et al. Depletion of microRNA-451 in response to allergen exposure accentuates asthmatic inflammation by regulating Sirtuin2. Am J Physiol Lung Cell Mol Physiol. 2020. May 1;318(5):L921–L930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ballinger MN, Paine R, Serezani CH et al. Role of granulocyte macrophage colony-stimulating factor during gram-negative lung infection with Pseudomonas aeruginosa. Am J Respir Cell Mol Biol. 2006. Jun;34(6):766–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Reader BF, Sethuraman S, Hay BR et al. IRAK-M Regulates Monocyte Trafficking to the Lungs in Response to Bleomycin Challenge. J Immunol. 2020. May 15;204(10):2661–2670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Yu YR, O’Koren EG, Hotten DF et al. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues. PLoS One. 2016. Mar 3;11(3):e0150606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Reece SW, Varikuti S, Kilburg-Basnyat B et al. Scavenger Receptor BI Attenuates IL-17A-dependent Neutrophilic Inflammation in Asthma. Am J Respir Cell Mol Biol. 2021. Mar 1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Curran DR, Cohn L. Advances in mucous cell metaplasia: a plug for mucus as a therapeutic focus in chronic airway disease. Am J Respir Cell Mol Biol. 2010;42(3):268–275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Williams AS, Leung SY, Nath P et al. Role of TLR2, TLR4, and MyD88 in murine ozone-induced airway hyperresponsiveness and neutrophilia. J Appl Physiol (1985). 2007. Oct;103(4):1189–95. [DOI] [PubMed] [Google Scholar]
  • 24.Li Z, Potts-Kant EN, Garantziotis S et al. Hyaluronan signaling during ozone-induced lung injury requires TLR4, MyD88, and TIRAP. PLoS One. 2011;6(11):e27137. [DOI] [PMC free article] [PubMed] [Google Scholar] [Research Misconduct Found]
  • 25.Schmid-Grendelmeier P, Altznauer F, Fischer B et al. Eosinophils express functional IL-13 in eosinophilic inflammatory diseases. J Immunol. 2002. Jul 15;169(2):1021–7. [DOI] [PubMed] [Google Scholar]
  • 26.Johnston LK, Bryce PJ. Understanding Interleukin 33 and Its Roles in Eosinophil Development. Front Med (Lausanne). 2017;4:51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yi S, Zhai J, Niu R et al. Eosinophil recruitment is dynamically regulated by interplay among lung dendritic cell subsets after allergen challenge. Nat Commun. 2018. Sep 24;9(1):3879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lang R, Patel D, Morris JJ et al. Shaping gene expression in activated and resting primary macrophages by IL-10. J Immunol. 2002. Sep 1;169(5):2253–63. [DOI] [PubMed] [Google Scholar]
  • 29.Bao A, Yang H, Ji J et al. Involvements of p38 MAPK and oxidative stress in the ozone-induced enhancement of AHR and pulmonary inflammation in an allergic asthma model. Respir Res. 2017;18(1):216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Seehus CR, Kadavallore A, Torre B et al. Alternative activation generates IL-10 producing type 2 innate lymphoid cells. Nat Commun. 2017. Dec 1;8(1):1900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Last JA, Ward R, Temple L et al. Ovalbumin-induced airway inflammation and fibrosis in mice also exposed to ozone. Inhal Toxicol. 2004. Jan;16(1):33–43. [DOI] [PubMed] [Google Scholar]
  • 32.North ML, Amatullah H, Khanna N et al. Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma. Respir Res. 2011. Feb 3;12(1):19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wardlaw AJ, Dunnette S, Gleich GJ et al. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Relationship to bronchial hyperreactivity. Am Rev Respir Dis. 1988. Jan;137(1):62–9. [DOI] [PubMed] [Google Scholar]
  • 34.Hammad H, Chieppa M, Perros F et al. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells. Nat Med. 2009. Apr;15(4):410–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Whitehead GS, Hussain S, Fannin R et al. TLR5 Activation Exacerbates Airway Inflammation in Asthma. Lung. 2020. Apr;198(2):289–298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Oakes JL, O’Connor BP, Warg LA et al. Ozone enhances pulmonary innate immune response to a Toll-like receptor-2 agonist. Am J Respir Cell Mol Biol. 2013. Jan;48(1):27–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Hussain S, Johnson CG, Sciurba J et al. TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury. Elife. 2020. Jan 28;9:e50458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Nie W, Fang Z, Li B et al. Interleukin-10 promoter polymorphisms and asthma risk: a meta-analysis. Cytokine. 2012. Dec;60(3):849–55. [DOI] [PubMed] [Google Scholar]
  • 39.Ogawa Y, Duru EA, Ameredes BT. Role of IL-10 in the resolution of airway inflammation. Curr Mol Med. 2008. Aug;8(5):437–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yang Q, Ge MQ, Kokalari B et al. Group 2 innate lymphoid cells mediate ozone-induced airway inflammation and hyperresponsiveness in mice. J Allergy Clin Immunol. 2016. Feb;137(2):571–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Yang Q, Ge MQ, Kokalari B et al. Group 2 innate lymphoid cells mediate ozone-induced airway inflammation and hyperresponsiveness in mice. J Allergy Clin Immunol. 2016. Feb;137(2):571–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Elieh Ali Komi D, Bjermer L. Mast Cell-Mediated Orchestration of the Immune Responses in Human Allergic Asthma: Current Insights. Clin Rev Allergy Immunol. 2019. Apr;56(2):234–247. [DOI] [PubMed] [Google Scholar]
  • 43.Kong X, Bennett WC, Jania CM et al. Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness. JCI Insight. 2021. Nov 8;6(21):e140207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Takeda M, Tanabe M, Ito W et al. Gender difference in allergic airway remodeling and immunoglobulin production in mouse model of asthma. Respirology. 2013. Jul;18(5):797–806. [DOI] [PubMed] [Google Scholar]
  • 45.Lee YG, Jeong JJ, Nyenhuis S et al. Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/CCl2, regulate airway inflammation and remodeling in allergic asthma. Am J Respir Cell Mol Biol. 2015. Jun;52(6):772–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Zasłona Z, Przybranowski S, Wilke C et al. Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma. J Immunol. 2014. Oct 15;193(8):4245–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yang J, Zhang L, Yu C et al. Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomark Res. 2014. Jan 7;2(1):1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Tighe RM, Li Z, Potts EN et al. Ozone inhalation promotes CX3CR1-dependent maturation of resident lung macrophages that limit oxidative stress and inflammation. J Immunol. 2011. Nov 1;187(9):4800–8. [DOI] [PMC free article] [PubMed] [Google Scholar] [Research Misconduct Found]
  • 49.Matsumura Y Role of Allergen Source-Derived Proteases in Sensitization via Airway Epithelial Cells. J Allergy (Cairo). 2012;2012:903659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Gaspar R, de Matos MR, Cortes L et al. Pollen Proteases Play Multiple Roles in Allergic Disorders. Int J Mol Sci. 2020. May 19;21(10):3578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Birukova A, Cyphert-Daly J, Cumming RI et al. Sex Modifies Acute Ozone-Mediated Airway Physiologic Responses. Toxicol Sci. 2019. Jun 1;169(2):499–510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Teufelberger AR, Van Nevel S, Hulpiau et al. Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33. Front Immunol. 2020. Sep 25;11:582044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Fuentes N, Nicoleau M, Cabello N et al. 17β-Estradiol affects lung function and inflammation following ozone exposure in a sex-specific manner. Am J Physiol Lung Cell Mol Physiol. 2019. Nov 1;317(5):L702–L716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Cabello N, Mishra V, Sinha U et al. Sex differences in the expression of lung inflammatory mediators in response to ozone. Am J Physiol Lung Cell Mol Physiol. 2015. Nov 15;309(10):L1150–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Fuentes N, Roy A, Mishra V et al. Sex-specific microRNA expression networks in an acute mouse model of ozone-induced lung inflammation. Biol Sex Differ. 2018. May 8;9(1):18. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure 1: Total numbers of inflammatory subtypes within the airspace following challenge with DRA, O3, or DRA-O3 compared to control mice. WT male mice were either untreated (Control, n=10) or challenged with DRA (n=10), O3 (n=9) or DRA-O3 (n=9). BALs were performed and differential inflammatory subtypes were quantified by cytospin analysis. Absolute numbers of eosinophils (A), neutrophils (B), and macrophages (C) are shown in the number of cells/ml of BAL fluid returned. Error bars are shown as mean ± SEM. *P<0.05, ***P<0.001

Supplemental Figure 2: No increase in tissue dampening and elastance measurements in DRA, O3, or DRA-O3 challenged mice compared to control mice. WT male mice were either untreated (Control, n=10) or challenged with DRA (n=10), O3 (n=9) or DRA-O3 (n=9) and then challenged with escalating doses of methacholine (Mch). Elastance (H) and tissue dampening (G) were assessed using a flexiVent small-animal ventilator. Error bars are shown as mean ± SEM.

Supplemental Figure 3: No difference in total number of BAL cells or subtype of BALs in TLR4+/+ compared to TLR4−/− after DRA-O3 challenge. WT (TLR4+/+) or TLR4 knockout mice (TLR4−/−) were challenged with DRA and then O3. The total number of cells present in the BAL fluid was assessed by automatic cell counter (A) and cell differentials were determined by cytospin analysis (B). Error bars are shown as mean ± SEM.

Supplemental Figure 4: DRA-O3 exposure alters some Th-2 mediated cytokines and proinflammatory cytokines. Whole lung tissue was collected from WT mice unchallenged mice (n=10) as well as mice challenged with DRA-O3 (n=9), DRA (n=10), and O3 (n=9) and expression of cytokines was assessed. Expression of (A) TSLP, (B) IL-33, and (E) IL-1β was measured by ELISA. Error bars shown as mean ± SEM. *P <0.05. **P<0.01, ***P<0.001

Supplemental Figure 5: Flow gating scheme and absolute numbers of innate immune cells with the lung from the different groups. WT male mice were either untreated (control) or challenged with DRA, O3 or DRA-O3 and total lung cells were analyzed by flow cytometry. (A) The specific gating scheme for the innate immune cells are as shown. (B) Total number of each individual subpopulations is quantified. N=5 mice/group. Error bars are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001

Supplemental Figure 6: Flow gating scheme and absolute numbers of adaptive immune cells with the lung from the different groups. WT male mice were either untreated (control) or challenged with DRA, O3 or DRA-O3 and total lung cells were analyzed by flow cytometry. (A) The specific gating scheme for the adaptive immune cells are as shown. (B) Total number of each individual subpopulations is quantified. N=5 mice/group. Error bars are shown as mean ± SEM. *P<0.05, **P<0.01

Supplemental Table 1: List of primers used for qPCR

Supplemental Table 2: List of antibodies used for flow cytometry analysis

Supplemental Table 3: Histological grading of lung histology sections

NIHMS1860358-supplement-1.pdf (1.2MB, pdf)

RESOURCES