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. 2024 May 15;200(2):299–311. doi: 10.1093/toxsci/kfae062

Effects of ozone exposure on lung injury, inflammation, and oxidative stress in a murine model of nonpneumonic endotoxemia

Jared Radbel 1,✉, Jaclynn A Meshanni 2, Kinal N Vayas 3, Oahn Le-Hoang 4, Elena Abramova 5, Peihong Zhou 6, Laurie B Joseph 7, Jeffrey D Laskin 8, Andrew J Gow 9, Debra L Laskin 10
PMCID: PMC11285192  PMID: 38749002

Abstract

Recent studies have identified exposure to environmental levels of ozone as a risk factor for the development of acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI) that can develop in humans with sepsis. The aim of this study was to develop a murine model of ALI to mechanistically explore the impact of ozone exposure on ARDS development. Mice were exposed to ozone (0.8 ppm, 3 h) or air control followed 24 h later by intravenous administration of 3 mg/kg lipopolysaccharide (LPS) or PBS. Exposure of mice to ozone + LPS caused alveolar hyperplasia; increased BAL levels of albumin, IgM, phospholipids, and proinflammatory mediators including surfactant protein D and soluble receptor for advanced glycation end products were also detected in BAL, along with markers of oxidative and nitrosative stress. Administration of ozone + LPS resulted in an increase in neutrophils and anti-inflammatory macrophages in the lung, with no effects on proinflammatory macrophages. Conversely, the numbers of resident alveolar macrophages decreased after ozone + LPS; however, expression of Nos2, Arg1, Cxcl1, Cxcl2, Ccl2 by these cells increased, indicating that they are activated. These findings demonstrate that ozone sensitizes the lung to respond to endotoxin, resulting in ALI, oxidative stress, and exacerbated pulmonary inflammation, and provide support for the epidemiologic association between ozone exposure and ARDS incidence.

Keywords: ozone, acute lung injury, inflammation, sepsis, oxidative stress

Acute respiratory distress syndrome (ARDS) is a severe clinical correlate of acute lung injury (ALI). In-hospital mortality from ARDS is approximately 40% and the incidence of ARDS has greatly increased due to COVID-19 (Bellani et al., 2016; Tzotzos et al., 2020). ARDS can develop as a consequence of either direct (ie, pneumonia) or indirect (ie, nonpneumonic sepsis, trauma) stress to the lung (Thompson et al., 2017). Recent studies have identified exposure to ozone, a ubiquitous urban air pollutant, and pulmonary toxicant, as a risk factor for the development of ARDS from indirect lung stressors (Reilly et al., 2019, 2023; Ware et al., 2016). Of particular concern is that these large independent epidemiological studies directly linked ozone levels below EPA standards to the increased ARDS risk (Reilly et al., 2019, 2023; Ware et al., 2016a). Mechanisms underlying the contribution of ozone exposure to the development of ARDS have not been established.

Lipopolysaccharide (LPS) is the biologically active component of bacterial-derived endotoxin. Depending on the route of LPS exposure, it has been used to model pneumonia (inhalation or intratracheal administration) or nonpneumonic sepsis-induced (intravenous administration/endotoxemia) ALI (Matute-Bello et al., 2008). Although the effects of ozone exposure on ALI induced directly by inhaled LPS, mimicking pneumonia-induced ARDS have been explored, its effects on intravenous administered LPS, a model of nonpneumonic sepsis-induced ARDS and indirect ALI, have not been analyzed and this represents the focus of the present studies (Duda et al., 2021; Hollingsworth et al., 2007).

ARDS is pathologically characterized by structural alterations in the alveoli, alveolar inflammation, and alveolar-capillary barrier dysfunction (Thompson et al., 2017). This is associated with oxidative and nitrosative stress (Fink, 2002; Matthay et al., 2019). The present studies demonstrate that pre-exposure of mice to inhaled ozone exacerbates lung inflammation caused by intravenous administration of LPS and induces ALI, alveolar-capillary barrier dysfunction, and oxidative/nitrosative stress. These findings provide new insights into the relationship between exposure to ozone, sepsis, and susceptibility to ARDS.

Materials and methods

 

 

Animals, exposures, and sample collection

C57Bl6/J mice (11–13 week, male, The Jackson Laboratories, Bar Harbor, ME) were exposed to air or ozone (0.8 ppm, 3 h) in a plexiglass chamber as described previously (Carnino et al., 2022). This exposure protocol was selected to overcome the inherent reduced sensitivity of mice to ozone when compared with humans due to differences in respiratory tract structure and predominant breathing methods (nose vs mouth); in this context, previous studies have demonstrated that exposure of rodents to 0.8 ppm ozone for 3 h is equivalent to exposure of humans to 0.2 ppm for 2 h while exercising (Hatch et al., 2013, 1994). Ozone was generated from oxygen gas via an ultraviolet light ozone generator (Gilmont Instruments, Barrington, Illinois) and mixed with air. Concentrations inside the chamber were monitored using a Photometric ozone analyzer (model 202, 2B Technologies, Inc., Boulder, Colorado). Twenty-four hours after exposure, mice were anesthetized with isoflurane and administered phosphate-buffered saline (PBS) control or intravenous LPS (3 mg/kg, E. coli O128: B12, Sigma-Aldrich). This dose and strain of LPS were utilized as earlier studies showed that they consistently induced endotoxemia, neutrophilia, and lung injury in mice (Everhart et al., 2006; Fink, 2014; Sunil et al., 2007). After an additional 24 h, mice were euthanized by intraperitoneal injection of xylazine (30 mg/kg) and ketamine (135 mg/kg). Our post-exposure analysis time was based on pilot studies that identified significant neutrophil accumulation in the lung 24 h after LPS administration, and earlier work showing alveolar-capillary barrier dysfunction 48 h after ozone exposure (Francis et al., 2017). The lung was perfused in situ through the right ventricle with 5 ml of ice-cold saline. Bronchoalveolar lavage fluid (BAL) was collected by slowly instilling and withdrawing 1 ml of ice-cold PBS into the lung 3 times through a 20-gauge cannula inserted into the trachea. BAL was centrifuged and cell-free supernatants aliquoted and stored at −80°C until analysis. The lung was then removed and lavaged 5 times with 1 ml cold perfusion medium (25 mM HEPES and 4.4 mM NaHCO3 in HBSS, pH 7.3) while gently massaging the tissue (Sunil et al., 2015). Lavage fluid was centrifuged (300 × g, 8 min, 4°C), cell pellets resuspended in HBSS +2% v/v fetal bovine serum and combined with the first BAL cell pellet. Cells were treated with red blood cell lysis buffer (1000 μl, Sigma), washed 3 times and viable cells enumerated on a hemocytometer using trypan blue dye exclusion.

For histopathology, lungs were inflated through the trachea with PBS containing 3% paraformaldehyde at 25 cm H2O. After overnight incubation at 4°C, lungs were transferred to 50% ethanol and stored at room temperature. Histologic sections (4 µm) were prepared and stained with hematoxylin and eosin or analyzed by immunohistochemistry as previously described or by in situ hybridization using Biotechne RNAscope® as previously described (Sunil et al., 2015; Wang et al., 2012). The extent of histological changes (terminal bronchiolar/alveolar hyperplasia and alveolar tissue: airspace ratio) and neutrophilia were quantified blindly using QuPath software (Bankhead et al., 2017) (Supplementary Figure 1). Histological changes were confirmed by a board-certified veterinary pathologist (M. Goedken, DVM, PhD, Rutgers Pathology Services).

Cell-free BAL analysis

BAL levels of IgM and albumin were quantified by ELISA (IgM, Bethyl Laboratory Inc, Montgomery, Texas; albumin, Thermo Fischer, Waltham, Massachusetts) and analyzed in duplicate or triplicate at 480 nm (IgM) or 562 nm (albumin) on a V max MAXline microplate reader (Molecular Devices, Sunnydale, California). Levels of SP-D, S-nitrosylated SP-D (SNO-SP-D), and soluble receptors for advanced glycation end products (sRAGE) in BAL were assessed by Western blotting. Briefly, equal volumes of BAL supernatants were separated on 4%–12% Bis-Tris gradient reducing/denaturing gels (Invitrogen, San Diego, California); proteins were then transferred to polyvinylidene difluoride membranes (Atochina-Vasserman et al., 2011). Nonspecific binding was blocked by incubation of the membranes with 10% milk in T-TBS (0.5% Tween 20 in Tris-buffered saline) for 1 h at room temperature. This was followed by overnight incubation at 4°C with rabbit anti-mouse polyclonal SP-D antibody (DU117, 1:10 000; a gift from Amy Pastva, Duke University, North Carolina) or rabbit anti-mouse RAGE antibody (catalog no. ab65965; 1:2000; Abcam, Cambridge, Massachusetts). Blots were washed and incubated for 1 h at room temperature with HRP-conjugated secondary antibody (1:5000, 1% nonfat milk in T-TBS; Bio-Rad, Hercules, California). Bands were visualized using an ECL Prime detection system (GE Health Care, Piscataway, New Jersey). SNO-SP-D was analyzed using a biotin switch assay followed by western blotting as described previously (Guo et al., 2019; Jaffrey and Snyder, 2001). In brief, free protein thiols in equal volumes of cell-free BAL were blocked, and new thiols generated using sodium ascorbate decomposition of S-N bonds; this was followed linking new thiols to biotin. Biotin proteins were then precipitated with streptavidin-agarose beads and analyzed by western blotting (Guo et al., 2019; Jaffrey and Snyder, 2001).

For analysis of total phospholipids, BAL fluid was separated into hydrophobic large aggregate and hydrophilic small aggregate fractions by ultracentrifugation (20 000 s × g, 1 h, 4°C). Phospholipids were quantified in large aggregate fractions by measuring inorganic phosphate content using a V max MAXline microplate reader (Atochina et al., 2004). Total NOx and acetone precipitated organic NOx (NOx-modified protein and lipid) levels were assayed by vanadium chloride reduction using the Sievers 280i Nitric Oxide Analyzer (General Electric, Niskayuna, New York) as per the manufacturer’s instructions (Atochina-Vasserman et al., 2007).

Flow cytometric analysis

BAL cells were resuspended in 100 μl of staining buffer (PBS, 2% v/v fetal bovine serum and 0.02% v/v sodium azide) and incubated for 10 min at 4°C with anti-mouse CD16/32 (1:100; Biolegend, San Diego, California, clone 93) to block nonspecific binding, followed by FITC-conjugated anti-mouse CD11b (1:100; Biolegend, clone M1/70), PE-CF594 conjugated anti-mouse Ly6C (1:100; BD Biosciences, San Jose, California, clone AL-21), PerCP-Cy5.5-conjugated anti-mouse Siglec F (1:100, BD Biosciences, clone E50-2440), PE/Cy7-conjugated anti-mouse F4/80 (1:100, Biolegend, clone BM8), AF647-conjugated anti-mouse Ly6G (1:100; Biolegend, clone1A8), AF 700-conjugated anti-mouse CD45 (1:100, Biolegend, clone 30-F11), and Brilliant Violet 421-conjugated anti-mouse Cd11c (1:100, Biolegend, clone N418) antibodies for 30 min, and then with eFluor 780-conjugated fixable viability dye (1:1000; eBioscience, San Diego, California) for an additional 30 min at 4°C. Cells were then washed with staining buffer, fixed with 3% paraformaldehyde, and analyzed on a Beckman Coulter Gallios flow cytometer (Brea, California) using the following lasers/filters/voltages (V): forward scatter/0 V, side scatter/0 V; 488/525 ± 20 nm/320V (CD11b); 488/620 ± 30 nm/345V (Ly6C); 488/695 ± 30 nm/410V (Siglec F); 488/755/440 V (F4/80); 633/660 ± 20 nm/400V (Ly6G); 633/725 ± 20 nm/370V (CD45); 633/755/380 V (Viability); 405/450 ± 50 nm/320V (CD11c). Lung cell populations were identified as described previously and shown in Supplementary Figure 2 (Sunil et al., 2015). Data were analyzed using Beckman Coulter Kaluza version 2.1 software.

Reverse transcription qPCR

BAL cells were magnetically separated into CD11b+ (inflammatory) and CD11b− (resident) cells using an EasySep Direct Cell Isolation kit (Stemcell Technologies, Cambridge, Massachusetts). Total RNA was extracted from CD11b− resident macrophages using an RNeasy Mini kit (Qiagen, Valencia, California). mRNA was reverse transcribed using a cDNA Reverse Transcription Kit (VWR, Radnor, Pennsylvania). RT-qPCR was performed using TaqMan PCR master mix (ThermoFisher) on a QuantStudio 6 Flex Real-Time PCR system (ThermoFisher). Ct values were measured and analyzed using the 2–ΔΔCt method to quantify fold change in mRNA expression; Actb (beta actin) was used as a control housekeeping gene. The following TaqMan primers were used: Actb (Mm00607939_s1), Arg1 (Mm00475988_s1), Ccl2 (Mm00441243_g1), Cxcl1 (Mm00433859_m1), Cxcl2 (Mm00436450_m1), and Nos2 (Mm00440502_m1).

Measurement of lung tissue glutathione and hydrogen peroxide

Lung homogenates were analyzed for oxidized (GSSG) and reduced (GSH) glutathione using a fluorescence Detection Kit (Invitrogen, Waltham, Massachusetts). Hydrogen peroxide (H2O2) was analyzed in S9 fractions of lung homogenates using an Amplex Red assay as described previously (Mishin et al., 2020).

Statistical analyses

All experiments were repeated at least 3 times (n = 4 mice/treatment group/experiment). Data greater than 1.5 × the interquartile range were excluded. Continuous data were analyzed for normality and then by 2-way ANOVA followed by a post hoc Tukey’s test. Semi-quantitative western blots, histology, and qPCR data or continuous data that did not fit a normal distribution were analyzed nonparametrically using Kruskal–Wallis and post hoc Dunn tests or Mann–Whitney test where appropriate. A p value ≤ .05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software (Version 9.5.0).

Results

 

Effects of exposure to ozone and LPS on lung histopathology and markers of injury, inflammation, and dyslipidemia in BAL

Exposure of mice to ozone + PBS, but not air + LPS, resulted in epithelial hyperplasia in the terminal bronchiolar-alveolar junction; LPS had no effect on ozone-induced histopathology (Figure 1 and Table 1). To quantify the extent of alveolar parenchymal thickening, we calculated the ratio of alveolar parenchyma to alveolar space in peribronchiolar and non-peribronchiolar regions of the lung. Consistent with epithelial hyperplasia, the parenchyma/space ratios in peribronchiolar areas of the lung were elevated in ozone-exposed mice when compared with air controls; LPS had no effect on this response (Table 1). No differences in the alveolar parenchyma/space ratios were observed in nonperibronchiolar areas of the lung in any of the treatment groups. Exposure of mice to ozone + LPS resulted in a significant increase in BAL levels of IgM and albumin, relative to the ozone + PBS or air + LPS exposure groups, demonstrating impaired alveolar-capillary barrier function (Figure 2). Significant increases in BAL levels of total phospholipids were also observed in mice exposed to air + LPS compared with air + PBS, with greater levels in ozone + LPS relative to air + LPS exposed mice. In contrast, total BAL cell counts decreased following exposure of mice to ozone + LPS, when compared with air + PBS, but not ozone + PBS or air + LPS.

Figure 1.

Figure 1.

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Effects of ozone and LPS on terminal bronchiolar-alveolar hyperplasia. Lung sections, prepared from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, were stained with H&E. Images were acquired using the Olympus VS120 Virtual Microscopy system and visualized with QuPath software. Fifteen random 30× fields of bronchiolar-alveolar junctions were examined for the presence of bronchiolar-alveolar epithelial hyperplasia. One representative field from 7 mice/treatment group is shown. Arrows indicate areas of alveolar parenchymal thickening. Abbreviations: A, alveolar space; TB terminal bronchiole. Original magnification, 10× (center panels), 40× (peripheral panels).

Table 1.

Effects of ozone and LPS on lung histopathology.

Air + PBS Air + LPS Ozone + PBS Ozone + LPS

  • Terminal bronchiolar/alveolar Epithelial hyperplasia (%)

 1 ± 3 0 ± 0 67 ± 19a,b 71 ± 16a,b

  • Alveolar tissue: airspace ratio (peribronchiolar)

 0.30 ± 0.03 0.33 ± 0.11 0.45 ± 0.16 0.51 ± 0.11a,b

  • Alveolar tissue: airspace ratio (nonperibronchiolar)

 0.29 ± 0.03 0.32 ± 0.08 0.29 ± 0.08 0.34 ± 0.05
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Lung sections, prepared from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, were stained with H&E. Images were acquired using the VS120 Virtual Microscopy system and visualized with QuPath software. Fifteen random 30× fields of bronchiolar-alveolar junctions were examined for the presence of bronchiolar-alveolar epithelial hyperplasia. Two 60× sections (1 peri-bronchiolar and 1 nonperibronchiolar) were examined in each random 30× field and the alveolar tissue: airspace ratio quantified.

a

Significantly different (p ≤ .05) from air + PBS.

b

Significantly different from air + LPS.

Figure 2.

Figure 2.

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Effects of ozone and LPS on alveolar-capillary barrier function, cells and lung phospholipids. BAL, collected from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, was centrifuged and cell-free supernatants analyzed for IgM, albumin and phospholipid content. Top left panel: IgM was measured by ELISA (n = 8–11 mice/treatment group). Top right panel: albumin was measured by ELISA (n = 13–15 mice/treatment group). Bottom left panel: Total phospholipids were quantified in large aggregate fractions of cell-free BAL as described in Materials and Methods (n = 5–12 mice/treatment group). Bottom right panel: Cells were enumerated by trypan blue dye exclusion using a hemocytometer (n = 11–12 mice/treatment group). Bars, mean ± SD; *Significant difference (p ≤ .05) between groups as indicated.

sRAGE and SP-D are markers of inflammation linked to ARDS (García-Laorden et al., 2017; Sorensen, 2018). Following administration of ozone + LPS, BAL levels of sRAGE and SP-D increased; SP-D levels also increased after exposure of mice to ozone + PBS (Figure 3). The combined effects of ozone + LPS were greater than ozone + PBS.

Figure 3.

Figure 3.

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Effects of ozone and LPS on inflammatory proteins in BAL. Equal volumes of cell-free BAL, prepared from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, were analyzed by western blotting for sRAGE (Left panel) and SP-D (Right panels). Bands were visualized using an ECL Prime detection system (GE Health Care). Blots are representative of 4 experiments; each lane is one animal. Band densities were quantified by ImageJ, normalized to air + PBS controls, and presented as fold change relative to control. Bars (sRAGE), mean ± SD (n = 10–12 mice/treatment group). Bars (SP-D), mean ± SD (n = 13–15 mice/treatment group). *Significant difference (p ≤ .05) between groups as indicated. Complete Western blots are shown in Supplementary Figure 3.

Effects of ozone and LPS on oxidative and nitrosative stress in the lung

Treatment of mice with ozone + LPS, but not ozone + PBS or air + LPS, resulted in a significant increase in the production of H2O2 in the lung (Figure 4). An increase in the ratio of oxidized (GSSG) to reduced (GSH) glutathione in the tissue and upregulation of heme oxygenase 1 (HO-1) expression in lung macrophages was also noted in ozone + LPS treated mice, with no evidence of increases in these oxidative stress markers in the other treatment groups (Figs. 4 and 5). Ozone + LPS and air + LPS also caused nitrosative stress in the lung; thus, levels of total NOx were elevated in BAL from these mice (Figure 6). In contrast, only ozone + LPS exposure resulted in increases in BAL levels of organic NOx (NOx-modified protein + lipid). Further analysis showed that NOx-mediated protein modifications in ozone + LPS-exposed mice included S-nitrosylation of SP-D (Figure 6).

Figure 4.

Figure 4.

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Effects of ozone and LPS on lung oxidative stress. Tissue was collected from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, and immediately frozen at −80°C. Left panel: Lung homogenates were separated into microsomal and cytosolic (S9) fractions and analyzed for hydrogen peroxide (H2O2) using an Amplex Red assay. Right panel: Lung homogenates were analyzed for oxidized (GSSG) and reduced (GSH) glutathione by ELISA. Bars, mean ± SD (n = 5–6 mice/treatment group). *Significant difference (p ≤ .05) between groups as indicated.

Figure 5.

Figure 5.

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Effects of ozone and LPS on HO-1 expression. Lung sections, prepared from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, were immunostained with antibody to HO-1 (StressGen Biotechnologies; 1:250) or rabbit anti-mouse IgG antibody control. Binding was visualized using a 3,3′-Diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories). One representative section from 3 to 4 mice/treatment group is shown (original magnification, 40×). Positively staining AMs were enumerated from 20 random fields (20× magnification/lung). Inset: mean ± SD (n = 3–4). aSignificantly different (p ≤ .05) from air + PBS. Lower magnifications of lung histology are shown in Supplementary Figure 4.

Figure 6.

Figure 6.

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Effects of ozone and LPS on markers of nitrosative stress. BAL was collected from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, centrifuged and cell-free supernatants analyzed for NOx levels by vanadium chloride reduction (upper left panel). Bars, mean ± SD, n = 7–12 mice/treatment group. Equal volumes of BAL were acetone precipitated to enrich for protein and lipid (organic fraction) and analyzed for organic NOx levels (lower left panel). Bars, mean ± SD, n = 4–5 mice/treatment group. A biotin switch assay followed by Western blotting was used to detect SNO-SP-D in BAL (right panels). Blots are representative of 2 experiments; each lane is 1 mouse. Band densities were quantified by ImageJ, normalized to air + PBS controls and presented as fold change. Bars, mean + SD (n = 5–6 mice/treatment group). *Significant difference (p ≤ .05) between groups as indicated. Complete Western blots are shown in Supplementary Figure 3.

Effects of exposure to ozone and LPS on infiltrating myeloid cells and resident alveolar macrophages (AMs)

Treatment of mice with ozone + LPS resulted in a significant increase in neutrophils in BAL, when compared with mice treated with air + PBS, ozone + PBS, or air + LPS (Figure 7). Increased numbers of neutrophils were also identified in the alveolar interstitium, and alveolar space of mice treated with LPS when compared with mice treated with PBS; this was not altered by ozone exposure (Figure 8). Greater numbers of neutrophils were noted in the alveolar space of mice exposed to ozone + LPS relative to air + LPS consistent with our flow cytometric results. The percentage of mature anti-inflammatory macrophages in BAL was also greater in ozone + LPS-treated mice relative to air + PBS or air + LPS-treated mice. Conversely, ozone had no effect on LPS induced increases in immature anti- or pro-inflammatory macrophages. Mature proinflammatory macrophages were not detectable in any of the treatment groups.

Figure 7.

Figure 7.

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Effects of ozone and LPS on lung inflammatory cells. Cells collected from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS by BAL + lung massage were immunostained with antibodies to CD45, CD11b, Siglec F, CD11c, Ly6G, F4/80, and Ly6C and analyzed by flow cytometry. PMNs were defined as CD45+CD11b+Siglec F-Ly6G+Cd11c−, immature proinflammatory macrophages as CD11b+ Siglec F−Ly6G−Cd11c−F4/80+Ly6Chi, mature anti-inflammatory macrophages as CD11b+ Siglec F−Ly6G−Cd11c+F4/80+Ly6Clo, immature anti-inflammatory macrophages as CD11b+ Siglec F−Ly6G−Cd11c-F4/80+Ly6Clo. Mature proinflammatory macrophages (CD11b+ Siglec F−Ly6G−Cd11c+F4/80+Ly6Chi cells) were not detected. Data are representative of at least 3 experiments and presented as % CD45+ cells (leukocytes). Bars, mean ± SD (n = 11–14 mice/treatment group). *Significant difference (p ≤ .05) between groups as indicated. MP, macrophage.

Figure 8.

Figure 8.

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Effects of ozone and LPS on neutrophil infiltration into the lungs. Sections, prepared from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS, were stained with H&E. Images were acquired using the VS120 Virtual Microscopy system and visualized with QuPath software. Neutrophils were enumerated in ten random 40× fields from 5 lobes (2 fields/lobe) in the interstitial (I) and alveolar (A) space. One representative field from 7 mice/treatment group is shown. Arrows indicate alveolar neutrophils; arrowheads indicate interstitial neutrophils. Original magnification, 40×. aSignificantly different (p ≤ .05) from air + PBS. bSignificantly different from air + LPS. cSignificantly different from ozone + PBS.

In further studies, we analyzed the effects of ozone and LPS on resident AMs. A significant decrease in resident AMs was observed in mice exposed to ozone + LPS, relative to air + PBS, with no change after ozone + PBS or air + LPS administration (Figure 9). Despite the reduction in resident AM number after ozone + LPS exposure, their expression of proinflammatory (Nos2) and anti-inflammatory (Arg1) activation markers increased, when compared with the other treatment groups (Figure 9). Resident AM activation in ozone + LPS exposed mice was confirmed in situ in histologic sections by analyzing Nos2 and Arg1 mRNA expression in Siglec F+ cells (Figure 10). Neutrophil and monocyte chemokine gene expression including Cxcl1, Ccl2, and Cxcl2 were also upregulated in resident AMs from ozone + LPS-exposed mice relative to the other exposure groups (Figure 9).

Figure 9.

Figure 9.

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Effects of ozone and LPS on resident alveolar macrophages (AMs). Lung cells from mice exposed to air + PBS, air + LPS, ozone + PBS, or ozone + LPS were immunostained with antibodies to CD45, CD11b, Siglec F, CD11c, Ly6G, F4/80, and Ly6C and analyzed by flow cytometry. Upper left panel: Resident AMs were identified as CD45+CD11b−Siglec F+. Data are presented as % CD45+ cells (n = 11–14 mice/treatment group). Separate groups of lung cells were immunostained with metallic bead-conjugated antibodies to CD11b; CD11b− AMs were collected by magnetic separation and analyzed by qPCR using the 2–ΔΔCt method with Actb as a control housekeeping gene. Arg1 (n = 5–6 mice/group), Cxcl1 (n = 5–6 mice/group), Cxcl2 (n = 5–6 mice/group), and Ccl2 (n = 3 mice/group); data are presented as fold change relative to air + PBS controls; Nos2 (n = 6–7 mice/group); data are presented as fold change relative to air+ LPS-treated mice, as no expression was detected in the air + PBS and ozone + PBS controls. Ct values above 36 were considered undetectable. Bars, mean ± SE. *Significant difference (p ≤ .05) between groups as indicated. ND, not detected.

Figure 10.

Figure 10.

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Nos2 and Arg1 expression in resident alveolar macrophages (AMs). Histological sections were prepared from mice exposed to ozone + LPS. Tissue was hybridized using probes to Arg1 or Nos2 mRNA and then immunostained with antibody to Siglec F (Abcam). Nos2 and Arg1 mRNA were visualized using the RNAScope® assay (Bio-Techne). Siglec F binding was visualized using a DAB peroxidase substrate kit. One representative section from 2 mice is shown. Original magnification, 40×; brown staining, Siglec F; red dots, Arg1 (upper panel) or Nos2 (lower panel). Insets: Enlarged Siglec F and mRNA-positive macrophages (100×). Lower magnifications are shown in Supplementary Figure 5.

Discussion

In these studies, we describe a model of ALI caused by exposure of mice to inhaled ozone followed by intravenous LPS, which replicates key features of ARDS in humans induced by ozone exposure and the indirect lung stressor, sepsis. Using this murine model, we demonstrate that ozone primes the lung for an exacerbated inflammatory response to intravenous LPS. Moreover, exposure to ozone and LPS causes damage to the alveolar epithelium and alveolar-capillary barrier dysfunction, as well as oxidative and nitrosative stress, consistent with ARDS in humans. These findings support the notion that environmental exposure to oxidant air pollutants such as ozone may contribute to the development of ARDS (Reilly et al., 2019, 2023; Ware et al., 2016a). Further studies using this model will be valuable for identifying mechanistic pathways underlying these effects.

Following exposure of mice to ozone, but not air, histologic evidence of early ALI, characterized by bronchiolar alveolar junction epithelial hyperplasia was observed. Although the percentage of the lung with hyperplasia and the degree of alveolar thickening were similar in mice exposed to ozone + PBS and ozone + LPS, exacerbated alveolar-capillary barrier dysfunction, measured by BAL levels of IgM and albumin, was greater after the combined ozone and LPS exposure. Previous experimental models of ARDS induced by intravenous administration of LPS reported predominant effects on the alveolar-capillary endothelium with only mild changes in alveolar capillary permeability (Matute-Bello et al., 2008). Findings of significant increases in both of these markers of injury in our model are more in line with pathology observed in human ARDS (Kulkarni et al., 2022; Matute-Bello et al., 2011; Thompson et al., 2017). The exacerbated alveolar-capillary barrier dysfunction in mice exposed to ozone + LPS when compared with air + LPS is similar to models of ozone + inhaled LPS exposure (Hollingsworth et al., 2007; Li et al., 2010). We also found that BAL levels of phospholipids, the main component of pulmonary surfactant, were increased in ozone + LPS-treated mice relative to the other exposure groups, consistent with alveolar epithelial type II cell dysfunction (Venosa et al., 2021). Although elevated BAL phospholipid levels were also observed in mice exposed to air + LPS, this was significantly greater after ozone + LPS exposure. These data support the notion that ozone exposure exacerbates LPS-induced dyslipidemia. Soluble RAGE and SP-D are markers of inflammation and lung epithelial cell injury in humans with ARDS (García-Laorden et al., 2017; Su et al., 2009; Uchida et al., 2006). Levels of these proteins were significantly elevated in BAL from mice exposed to ozone + LPS compared with the other treatment groups. Taken together, these data demonstrate that combined exposure to ozone and LPS induces changes in the lung consistent with pathologic alterations observed in ARDS.

Patients with ARDS exhibit a loss of oxidant buffering capacity and increased pulmonary levels of reactive oxygen species (ROS) indicating oxidative stress (Wilson et al., 2001). Oxidative stress is known to cause membrane lipid peroxidation which leads to epithelial and endothelial cell damage (Fink, 2002; Wilson et al., 2001). GSH is a major scavenger of ROS, playing an important role in protection against oxidative stress (Fink, 2002; Mytilineou et al., 2002; Sentellas et al., 2014). In line with oxidative stress, we observed a significant increase in the ratio of GSSG to GSH in the lungs of mice treated with ozone + LPS; H2O2 production also increased. We speculate that this contributes to alveolar-capillary barrier dysfunction in these mice. Further evidence of oxidative stress in ozone + LPS-treated mice includes our findings that HO-1 was upregulated in AMs, a characteristic response of cells to protect against intracellular ROS (Campbell et al., 2021; MacGarvey et al., 2012). Of note, despite increases in HO-1 and infiltration of anti-inflammatory macrophages into the lung lining fluid, ALI persisted after ozone + LPS administration. These findings suggest that ALI in these mice is due, at least in part, to an inability to effectively limit oxidative stress.

Reactive nitrogen species (RNS) also contribute to the pathogenesis of ALI and ARDS, both directly by inducing cytotoxicity and indirectly by modulating inflammatory cell and protein function (Sittipunt et al., 2001; Taylor et al., 2022; Wilson et al., 2001). In accord with this, we detected increased levels of NOx and NOx-modified proteins in BAL from mice with ALI caused by exposure to ozone + LPS. Although exposure to air + LPS also resulted in increased BAL NOx levels, increases in RNS-modified proteins were greater in mice exposed to ozone + LPS. This may be a consequence of increased production of ROS which interact with nitric oxide to generate higher oxides of nitrogen, such as peroxynitrite (Fink, 2002; Taylor et al., 2022). One NOx-modified protein that we identified in BAL is SNO-SP-D. In healthy lungs, SP-D binds to SIRP-1α on the surface of AMs helping maintain their anti-inflammatory activity (Guo et al., 2008; Hussell and Bell, 2014). In contrast, SNO-SP-D binds to CD91/calreticulin on macrophages, leading to proinflammatory activation (Guo et al., 2008). These data suggest that RNS may contribute to resident AM activation via posttranscriptional modification of SP-D.

Alveolar neutrophilia is a hallmark of ARDS and is considered a marker of ALI in animal models (Matute-Bello et al., 2011; Thompson et al., 2017). Consistent with ARDS in humans, we observed significantly greater numbers of neutrophils in BAL and in the alveolar space in histologic sections from mice exposed to ozone + LPS relative to LPS alone. Previously we showed that approximately 80% of lung neutrophils isolated from mice treated with ozone + LPS are undergoing apoptosis, suggesting activation (Radbel et al., 2023; Mayadas et al., 2014). Earlier studies using models of intravenous LPS-induced ARDS reported that neutrophils were mainly localized in the alveolar capillaries with relatively smaller numbers in the airspaces (Matute-Bello et al., 2008). Our data suggest that injury caused by ozone promotes migration of neutrophils from the capillaries into the airspace after endotoxin exposure and that these cells contribute to ALI, contrasting earlier reports of reduced BAL levels of neutrophils following exposure of mice to ozone + inhaled LPS (Hollingsworth et al., 2007). We speculate that neutrophil infiltration into the alveolar space in our model is due, at least in part, to AM production of chemokines and alveolar-capillary barrier dysfunction in ozone + LPS-exposed mice. This is supported by prior studies showing that these chemokines activate neutrophils and induce their migration across the capillary endothelium into the lungs of rodents (Kolaczkowska and Kubes, 2013; Maus et al., 2003). Our observation of greater infiltration of neutrophils into the lung and increases in inflammatory proteins including sRAGE and SP-D in BAL suggest that inhalation of ozone primes the lung to respond to intravenous endotoxin resulting in exacerbated inflammation.

Resident AMs are the major innate immune cell population in the lung (Bissonnette et al., 2020). Under homeostatic conditions, these cells function to dampen inflammatory responses protecting the lung against excessive responses to xenobiotics (Hussell and Bell, 2014). However, in the setting of infection and/or lung damage, evidence suggests that resident AMs can become activated and exhibit proinflammatory activity (Hussell and Bell, 2014; Tao et al., 2023). Following exposure of mice to ozone + LPS, a decrease in BAL cells was observed; this was due to a reduction in resident AMs. Importantly, remaining resident AM recovered from the lung were found to be activated as measured by increased expression of proinflammatory markers including Nos2, Cxcl1, Cxcl2, and Ccl2. Although in air + LPS-exposed mice, resident AM Nos2 was also upregulated, this was significantly less than in ozone + LPS-exposed mice. Interestingly, expression of the anti-inflammatory gene Arg1 was also upregulated in resident AM from ozone + LPS-exposed mice. These findings support the notion that resident AMs participate in both proinflammatory and anti-inflammatory responses to tissue injury (Mould et al., 2017). A similar reduction in resident AMs and concomitant activation of surviving cells and release of chemotactic factors has been described in models of inhaled ozone + inhaled LPS (Hollingsworth et al., 2007).

Increased numbers of mature anti-inflammatory macrophages were also identified in BAL from mice exposed to ozone + LPS. In contrast, only relatively small increases in immature anti- and proinflammatory macrophages were detected in LPS-exposed mice with no effects of ozone. These findings indicate that like ARDS in humans, ALI in mice following ozone + LPS exposure is mainly mediated by inflammatory neutrophils. Earlier reports suggest that anti-inflammatory macrophages accumulating in the injured lungs of mice are important in the resolution of inflammation (Watanabe et al., 2019). Studies are ongoing to more precisely elucidate the role of these cells in our ozone + LPS ALI model including their contribution to efferocytosis of apoptotic neutrophils, a key step in inflammation resolution (Radbel et al., 2023).

There are some limitations to our findings that need to be considered. Earlier studies reported that administration of LPS prior to ozone exposure protects against ozone-induced injury in rodents demonstrating that the order of exposures is important in the pathophysiologic response (Peavy and Fairchild, 1987; Pendino et al., 1996). However, this was not addressed in our studies as our goal was to model the human epidemiologic data in which humans are exposed to ozone prior to developing sepsis (Reilly et al., 2023). Additionally, we did not examine tissue for alveolar edema or physiologic dysfunction, which are additional elements of ARDS. Also important will be to assess sex differences in the development of ALI as our studies were only performed with male mice (Cabello et al., 2015; Yaeger et al., 2021).

Based on our findings a 2-hit model for the development of ARDS following ozone exposure is proposed (Figure 11). Accordingly, ozone inhalation damages the alveolar epithelium, whereas LPS activates resident AMs to release chemokines, which attract circulating neutrophils into the airspace. ROS and RNS released following ozone + LPS exposure further damages epithelial cells and disrupts the alveolar-capillary barrier further activating resident AMs. These cells release additional chemokines and inflammatory mediators, exacerbating neutrophil recruitment and ALI. Further mechanistic studies exploring ozone-induced ALI and ARDS in sepsis are essential as both ozone levels and the burden of infectious diseases are expected to rise as a consequence of climate change (Chen et al., 2018; Kurane, 2010).

Figure 11.

Figure 11.

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Two-hit model of ozone + LPS-induced ARDS. Ozone inhalation results in damage to the alveolar epithelium (bottom left panel) while endotoxemia activates resident AMs which release chemokines, such as Cxcl2, attracting circulating neutrophils (PMNs) into the airspace (top right panel). ROS and RNS generated following ozone + LPS exposure further damage epithelial cells, disrupt the alveolar-capillary barrier, and activate resident AMs. Activated AMs release chemokines and inflammatory mediators exacerbating PMN recruitment and ALI. Created with BioRender.com.

Supplementary Material

kfae062_Supplementary_Data
kfae062_supplementary_data.docx (6.8MB, docx)

Acknowledgments

The authors acknowledge the contributions of the following individuals: Changjiang Guo, MD and Gregory Pappas, PhD for assistance in measuring BAL NOx; Michael Goedkin DVM, PhD, DACVP for help characterizing histopathologic changes in the lung; and Renuka Rajagopal, MD for assistance in processing samples for qPCR.

Contributor Information

Jared Radbel, Department of Medicine, Division of Pulmonary and Critical Care Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901, USA.

Jaclynn A Meshanni, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Kinal N Vayas, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Oahn Le-Hoang, Department of Medicine, Division of Pulmonary and Critical Care Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901, USA.

Elena Abramova, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Peihong Zhou, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Laurie B Joseph, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Jeffrey D Laskin, Department of Environmental and Occupational Health and Justice, School of Public Health, Rutgers University, Piscataway, New Jersey 08854, USA.

Andrew J Gow, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Debra L Laskin, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.

Supplementary data

Supplementary data are available at Toxicological Sciences online.

Funding

This work was supported by the National Institutes of Health (ES031678, ES004738, ES033698, ES005022, S10OD026876); and the Society of Toxicology Donald Gardner Award.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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