Lung injury and Oxidative Stress Induced by Inhaled Chlorine in Mice is Associated with Proinflammatory Activation of Macrophages and Altered Bioenergetics

Abstract

Chlorine (Cl₂) gas is a highly toxic and oxidizing irritant that causes life-threatening lung injuries. Herein, we investigated the impact of Cl₂-induced injury and oxidative stress on lung macrophage phenotype and function. Spontaneously breathing male C57BL/6 mice were exposed to air or Cl₂ (300 ppm, 25 min) in a whole-body exposure chamber. Bronchoalveolar lavage (BAL) fluid and cells, and lung tissue were collected 24 h later and analyzed for markers of injury, oxidative stress and macrophage activation. Exposure of mice to Cl₂ resulted in increases in numbers of BAL cells and levels of IgM, total protein, and fibrinogen, indicating alveolar epithelial barrier dysfunction and inflammation. BAL levels of inflammatory proteins including surfactant protein (SP)-D, soluble receptor for glycation end product (sRAGE) and matrix metalloproteinase (MMP)-9 were also increased. Cl₂ inhalation resulted in upregulation of phospho-histone H2A.X, a marker of double-strand DNA breaks in the bronchiolar epithelium and alveolar cells; oxidative stress proteins, heme oxygenase (HO)-1 and catalase were also upregulated. Flow cytometric analysis of BAL cells revealed increases in proinflammatory macrophages following Cl₂ exposure, whereas numbers of resident and antiinflammatory macrophages were not altered. This was associated with increases in numbers of macrophages expressing cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), markers of proinflammatory activation, with no effect on mannose receptor (MR) or Ym-1 expression, markers of antiinflammatory activation. Metabolic analysis of lung cells showed increases in glycolytic activity following Cl₂ exposure in line with proinflammatory macrophage activation. Mechanistic understanding of Cl₂-induced injury will be useful in the identification of efficacious countermeasures for mitigating morbidity and mortality of this highly toxic gas.

INTRODUCTION

Chlorine (Cl₂) is a highly reactive gas which causes severe pulmonary injury. It was used as a chemical weapon on a large-scale during World War I and the Iraq-Iran war in the 1980’s, and more recently in 2014 and 2018, during the Syrian civil war (Zellner and Eyer, 2020). There have also been several incidences of accidental exposure to Cl₂ (Govier and Coulson, 2018). Cl₂ exposure doses and duration vary depending on the proximity to the source and amount of Cl₂ present in the local environment of its release. In humans, chlorine concentrations above 400 ppm have been reported to be fatal over 30 min duration (White and Martin 2010). Respiratory complications in individuals exposed to Cl₂ are of major concern as these account for most morbidity and mortality (Mackie et al., 2014). Common symptoms of acute exposure to Cl₂ include cough, choking, irritation and burning of the upper respiratory tract, sore throat, and dyspnea, which occur immediately or within 24 h. These are associated with pulmonary edema and pneumonitis. Long term pathologies include bronchitis, emphysema, airway obstruction, reactive airway disease and fibrosis (Zellner and Eyer, 2020; Achanta and Jordt, 2021).

Inhaled Cl₂ readily dissolves in airway lining fluid forming hydrochloric acid (HCl) and hypochlorous acid (HOCl). While HCl is neutralized by bicarbonates within the airway lining fluid, HOCl and Cl₂ can react with the respiratory tract mucosa causing prolonged or delayed toxicological effects (Yadav et al., 2010). Cl₂ also rapidly oxidizes membrane lipids and causes DNA damage and cytotoxicity within epithelial and endothelial cells in the airways leading to epithelial barrier dysfunction, alterations in pulmonary surfactants, and activation of inflammatory cascades (Massa et al., 2014; Achanta and Jordt, 2021).

Inflammatory macrophages are known to play a role in acute injury and disease pathogenesis induced by diverse pulmonary toxicants (Laskin et al., 2019; Stegelmeier et al., 2019). These cells become activated by inflammatory signals they encounter in the lung and release mediators that contribute to both initiation and resolution of the inflammatory response. This activity is mediated by distinct subsets of macrophages broadly classified as proinflammatory M1 and antiinflammatory/proresolution M2. Whereas M1 macrophages produce proinflammatory/cytotoxic mediators, M2 macrophages release mediators which promote inflammation resolution and tissue repair. It appears that the outcome of the response to tissue injury depends on the balance between the M1 and M2 subsets (Wynn and Vannella, 2016; Laskin et al., 2019). Previous studies have shown that the acute toxicity of Cl₂ is associated with increases in expression of M1 (interleukin [IL]-1β, nitric oxide synthase [iNOS], CCL2 and cyclooxygenase [COX]-2) and M2 (arginase [Arg]-1, mannose receptor [MR] and resistin like-alpha [RETNLA]) markers in lung macrophages suggesting that both subsets accumulate in the tissue (Martin et al., 2003; Massa et al., 2014; Musah et al., 2019). In the present studies we analyzed this directly using techniques in flow cytometry, immunohistochemistry, and cellular metabolism. We found that the majority of macrophages accumulating in the lungs after Cl₂ are functionally and metabolically activated towards a proinflammatory M1 phenotype. These findings suggest that targeting proinflammatory macrophages, and mediators they release in response to Cl₂ exposure may represent an efficacious approach for mitigating pulmonary toxicity.

MATERIALS AND METHODS

Animals and treatments

Specific pathogen-free C57BL/6J male mice (10–12 wk) were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in an AALAC approved animal facility in filter top microisolation cages and provided food and water ad libitum. Animals received humane care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Mice were exposed to Cl₂ gas (Airgas, Piscataway, NJ) in a whole-body exposure chamber (CH Technologies, Westwood, NJ) under a chemical safely fume hood in a designated room following Rutgers University Environmental Health and Safety guidelines. Mice were placed in the chamber and the chamber equilibrated with 300 ppm Cl₂ gas for 5 min at a flow rate of 3 L/min. This was followed by exposure of mice for 25 min. The exposure chamber was ventilated with air for 7 min prior to removing the animals. Cl₂ flow rate entering and exiting the exposure chamber was monitored continuously using a digital flow meter. Chamber exhaust was passed through a flask containing 800 mL of sodium bicarbonate (500 mM) and then exhausted into the fume hood. Air exposed mice served as controls. No mortality was observed in mice up to 24 h post exposure.

Bronchoalveolar lavage (BAL), tissue and cell collection

Animals were euthanized 24 h after exposure by i.p. injection of xylazine (30 mg/kg; Bayer, Shawnee, KS) and ketamine (135 mg/kg; Henry Schein Animal Health, Dublin, OH). BAL was collected by slowly instilling and withdrawing 1 ml of ice-cold phosphate buffer saline (PBS), pH 6.0 into the lung in situ three times through a 20-gauge cannula placed in the trachea. BAL fluid was centrifuged (300 × g, 8 min, 4 °C), supernatants collected, aliquoted, and stored at −80 °C. Cell pellets were resuspended in 250 μl PBS and viable cells enumerated on a hemocytometer using trypan blue dye exclusion.

For differential analysis, cytospins were prepared (10⁴ BAL cells/slide), fixed and stained with Hema-3 Stat Pack (Fisher Scientific, Pittsburgh, PA). A total of 300 cells/slide were analyzed. In air-treated control mice, BAL cells were comprised of 100% macrophages; after Cl₂ exposure, 64.8 ± 6.0% were macrophages, 33.8 ± 6.2% neutrophils, and 1.4 ± 0.3% eosinophils (mean ± SE, n = 6–8 slides/treatment group). A BCA protein assay kit (Pierce Biotechnologies, Rockford, IL) was used to assess protein levels in cell-free BAL with bovine serum albumin as the standard. BAL IgM levels were quantified by enzyme-linked immunosorbent assay (Bethyl Laboratories, Montgomery, TX). For histological evaluation, the left lung lobe was inflated with 3% paraformaldehyde, excised, and submerged in paraformaldehyde overnight. The tissue was then rinsed and transferred to 50% ethanol and then embedded in paraffin.

A separate set of animals was used for lung cell isolation. Following BAL collection, the lung was perfused in situ via the right ventricle with 5 ml of ice-cold PBS. The entire lung was then removed and instilled five times with 1 ml Hank’s balanced salt solution (HBSS) while gently massaging the tissue. Lavage fluid was centrifuged (300 × g, 8 min, 4 °C), the cell pellet resuspended in 1 ml HBSS and combined with the first BAL lavage cell suspension collected as described above. The cells were then washed three times with HBSS containing 2% fetal bovine serum, and viable cells enumerated using trypan blue dye exclusion.

For analysis of extracellular vesicles (Evs), cell-free BAL was centrifuged (600 × g, 8 min, 4 °C) and 15 μl of supernatant transferred to tubes containing 215 μl PBS and 50 μl of counting beads (3 μm; 10⁴ beads/ml; Spherotech, Lake Forest, IL). Samples were incubated for 30 min at 4°C in the dark and then analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) equipped with 405 nm, 488 nm, and 638 nm lasers using a 120 sec acquisition protocol. The upper and lower range of the Ev gate was set based on side scatter distributions of 0.3, 0.5, and 0.9 μm mega mix beads (Biocytex, Marseille, France), and 0.1 μm 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC] liposomes (T&T scientific, Knoxville, TN) (Supplementary Fig. 1). Evs were quantified as previously described (McVey et al., 2016; Cointe et al., 2017) with some modifications, and calculated as: Ev events × number of beads added/bead events × sample volume. To assess the origin of the Evs, BAL (10 μl) was incubated with 2 μl anti-mouse CD31-AF647 (Biolegend, San Diego, CA), or CD9-PerCP-Vio700 or CD326-PEVio-770C (Miltenyi Biotech, Gaithersburg, MD) antibodies for 45 min. Tetramethylindocarbocyanine (3 μl, 500 mg/ml; Invitrogen, Carlsbad, CA) was then added. After an additional 15 min incubation, 600 μl of PBS was added, the samples were then centrifuged (108,000 × g, 60 min, 4 °C) and the pellets resuspended in 600 μl PBS and analyzed on a CytoFLEX. Data were analyzed using Kaluza software (version 1.2; Beckman Coulter).

Flow cytometry

Lung cells were resuspended in 100 μl of staining buffer (PBS containing 2% fetal calf serum and 0.02% sodium azide) and incubated with anti-mouse CD16/CD32 (1:100; Biolegend, San Diego, CA) for 10 min at 4°C to block nonspecific binding. The cells were then incubated with FITC-conjugated anti-mouse CD11b (1:100, Biolegend), PE-conjugated anti-mouse Ly6C (1:100, Biolegend), BV-421 conjugated anti-mouse CD11c (1:100, Biolegend), AF 700-conjugated anti-mouse CD45 (1:100, Biolegend) and AF 647-conjugated anti-mouse Ly6G (1:100, Biolegend) antibodies for 30 min at 4°C followed by 30 min incubation with eFluor 780-conjugated fixable viability dye (1:100; eBioscience, San Diego, CA). Cells were then washed with PBS, fixed in 3% paraformaldehyde, and analyzed using a Beckman Coulter Gallios flow cytometer (Brea, CA). Data were analyzed using Beckman Coulter Kaluza software (version 1.2) as previously described (Sunil et al., 2015).

Bioenergetic measurements

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe96 Analyzer (Agilent Technologies, Inc., Santa Clara, CA). Cells were resuspended in HBSS and inoculated into 96 well plates (2 × 10⁵ cells/well). After 30 min at RT, supernatants were gently removed, the cells washed, refed with freshly prepared assay media (Agilent XF Base Media supplemented with 2 mM glutamine), and then incubated for 1 h at 37°C in the absence of CO₂. OCR and ECAR were assayed before and after sequential additions of glucose (25 mM), oligomycin (2 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 μM), and rotenone (0.5 μM) / antimycin A (0.5 μM) (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. For calculation of bioenergetic parameters, data were normalized to protein content.

Immunohistochemistry

Tissue sections (5 μm) were deparaffinized and endogenous peroxidase quenched using 3% hydrogen peroxide diluted in methanol. Antigen retrieval was performed by warming the specimens for 20 – 30 min in 10 mM sodium citrate buffer (pH 6.0). To block nonspecific binding, sections were incubated for 2 h at room temperature (RT) in Tris-buffer containing 5–100% horse, goat or rabbit serum. Sections were then incubated overnight at 4°C in a humidified chamber with anti-MR (1:400), anti-heme oxygenase (HO)-1 (1:1000), anti-COX-2 (1:1500), anti-iNOS (1:200), anti-Ym-1 (1:300) or anti-phospho-histone H2A.X (γH2A.X; 1:400) antibodies (Supplementary Table 1) or appropriate IgG controls, rinsed and then incubated with biotinylated secondary antibody (Vectastain Elite ABC kit, Vector Labs, Burlingame, CA) for 30 min at room temperature. For catalase immunostaining, an M.O.M. Immunodetection kit was used (Vector Labs). Sections were incubated with anti-catalase antibody (1:250) at room temperature for 30 min, rinsed and incubated with biotinylated anti-mouse IgG reagent for 10 min. Binding was visualized using an avidin-biotinylated enzyme complex with 3, 3’-diaminobenzidine (DAB) as the substrate. Random sections from the left lobe of 6 mice/treatment group were evaluated for each antibody (1 section/antibody/animal) and images acquired at high resolution using Olympus light microscope and DP controlled software (Olympus Corporation, Center Valley, PA). Tissue sections were scanned using an Olympus VS120 Virtual Microscopy System and viewed using OlyVIA version 2.6 software (Center Valley, PA). Positively stained alveolar macrophages were enumerated in 15 – 20 random fields/lung lobe by light microscopy. The samples were selected randomly and assessed blindly.

Western blot analysis

Equal volumes of cell free native BAL samples or BAL samples reduced with dithiothreitol and denatured, were fractionated on 3 – 8% Tris-Acetate or 4 – 12% Novex Bis-Tris gels (Invitrogen, San Diego, CA), respectively. Proteins were then transferred to nitrocellulose membranes and non-specific binding blocked by incubation of the blots with 10% non-fat dry milk in Tris-buffered saline (20 mM Tris Base, 137 mM sodium chloride, pH 7.6)/0.5% Tween-20) for 1 h at RT. The blots were incubated overnight at 4°C with antibody (Supplementary Table 1) against soluble receptor for advanced glycation end product (sRAGE; 1:1,000), surfactant protein (SP)-D (1:10,000), fibrinogen (1:1,000) or matrix metalloproteinase (MMP)-9 (1:1,000) in Tris-buffered saline/0.5% Tween-20/1% non-fat dry milk. After washing 3 times, the blots were incubated for 1 h at RT with HRP-conjugated secondary antibody (1:5,000) diluted in Tris-buffered saline/0.5% Tween-20/1% non-fat dry milk. Immunoreactive bands were visualized using an ECL detection system (GE Healthcare Biosciences, Piscataway, NJ).

Statistical analysis

An unpaired t-test was used to analyze data utilizing GraphPad Prism V6.01 (GraphPad Software Inc., La Jolla, CA). A p-value ≤ 0.05 was considered statistically significant.

RESULTS

Inhaled Cl₂ causes an accumulation of activated proinflammatory lung macrophages in the lung

In initial studies, we used flow cytometry to assess the phenotype of inflammatory cells accumulating in the lung lining fluid in response to Cl₂. In these experiments, BAL cells were analyzed for expression of CD11b followed by Ly6G, Ly6C and CD11c, as previously described (Sunil et al., 2015) (Supplementary Fig. 2). Cl₂ exposure resulted in a 6-fold increase in mature CD11b⁺Ly6G⁻Ly6C⁺CD11c⁺ and a 367-fold increase in immature CD11b⁺Ly6G⁻Ly6C⁺CD11c⁻ proinflammatory macrophages in the lung (Fig. 1). Increases in CD11b⁺Ly6G⁺Ly6C⁻ granulocytes (643-fold) and CD11b⁺Ly6G⁻Ly6C⁻CD11c⁻ immature antiinflammatory macrophages (3-fold) were also observed following Cl₂ exposure. In contrast, Cl₂ exposure had no effect on the numbers of CD11b⁺Ly6G⁻Ly6C⁻CD11c⁺ mature antiinflammatory macrophages or CD11b⁻Ly6G⁻Ly6C⁻CD11c⁺ resident macrophages (Fig. 1). To determine if lung macrophages are activated following Cl₂ exposure, we analyzed expression of proinflammatory (iNOS, COX-2) and antiinflammatory (MR, Ym-1) proteins in histologic sections. Following exposure of mice to Cl₂, numbers of macrophages expressing iNOS and COX-2 increased. In contrast, there were no effects on numbers of macrophages expressing MR or Ym-1 (Fig. 2 and Table 1).

Figure 1. Effects of Cl₂ exposure on lung cell phenotype.

Lung cells collected by BAL + massage 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) were stained with antibodies to CD11b, Ly6G, Ly6C, and CD11c or appropriate isotype controls, and analyzed by flow cytometry. MP, macrophages. Bars, mean ± SE (n = 5 mice/treatment group). ^aSignificantly different (p ≤ 0.05) from air treated mice.

Figure 2. Effects of Cl₂ exposure on macrophage pro- and anti-inflammatory protein expression.

Lung sections, prepared 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) were stained with antibody to COX-2, iNOS, MR or Ym-1. Binding was visualized using a Vectastain kit. Original magnification, 600x. Representative sections from 6 mice/treatment group are shown.

Table 1:

Effect of chlorine (CI₂) exposure on the numbers of alveolar macrophages expressing inflammatory and oxidative stress markers.

	CTL	CI2
COX-2	4.6 ± 1.5	9.7 ± 3.5
iNOS	1.1 ± 0.5	2.2 ± 0.4
MR	4.0 ± 0.5	5.0 ± 0.6
Ym-1	7.8 ± 0.5	9.0 ± 1.0
HO-1	3.6 ± 0.2	5.2 ± 0.6a
Catalase	0.6 ± 0.2	2.8 ± 0.6a

Sections of the left lobe were prepared 24 h after exposure of mice to air control (CTL) or CI₂. Numbers of alveolar macrophages staining positively for cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), mannose receptor (MR), Ym-1, heme oxygenase (HO)-1, or catalase were enumerated in 20 random fields/tissue section (n = 6 mice/treatment group). Data are mean ± SE of positively stained cells per field. Data were analyzed using an unpaired t test.

^aSignificantly (p ≤ 0.05) different from CTL.

In further studies we analyzed intracellular energy metabolism in lung cells, which is linked to phenotypic activation (Viola et al., 2019). Changes in ECAR in response to glucose, oligomycin and FCCP were used to estimate glycolytic function in the presence of glutamine. In parallel, characteristics of mitochondrial oxidative phosphorylation were assessed by measuring OCR in response to oligomycin, FCCP, and rotenone/antimycin A. Cells from Cl₂ exposed mice displayed an overall increase in ECAR; most notable were significant increases in basal (1.5-fold) and maximal glycolytic acidification (1.7-fold); Cl₂ exposure was also associated with increases in non-mitochondrial oxygen consumption (1.2-fold) and maximal respiration (1.2-fold) (Fig. 3).

Figure 3. Effects of Cl₂ exposure on macrophage bioenergetics.

Lung cells collected by BAL + massage 24 h after exposure of mice to air (CTL) or chlorine (Cl₂), were plated for 30 min, washed and then treated sequentially with glucose, oligomycin (Oligo.), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin (Rot/Ant). Extracellular acidification rate (ECAR, left upper panel) and oxygen consumption rate (OCR, right upper panel) were analyzed using an XFe96 Agilent Seahorse. Basal glycolytic acidification and maximal glycolytic acidification were calculated from ECAR values; non-mitochondrial oxygen consumption, basal respiration, and maximal respiration were calculated from OCR values. Bars, mean ± SE (n = 5 mice/treatment group). ^aSignificantly (p ≤ 0.05) different from CTL.

Cl₂-induced proinflammatory activation of lung macrophages is associated with lung injury and oxidative stress

We next assessed whether inflammatory macrophage activation in the lung following Cl₂ exposure is linked to lung injury and oxidative stress. Treatment of mice with Cl₂ resulted in the appearance of phosphorylated histone H2A.X (γH2A.X) in epithelial cells in proximal and distal bronchioles, and in lung macrophages and epithelial cells in alveolar regions of the lung, consistent with DNA damage (Fig. 4). Increases in BAL cell (3.6-fold), protein (1.4-fold) and IgM (3.3-fold) levels were also noted after Cl₂ exposure indicating inflammation and alveolar epithelial and endothelial cell injury (Fig. 5). This is supported by our findings that BAL levels of both native fibrinogen and α, β and γ fibrinogen chains increased (1.7-fold) after Cl₂ exposure (Fig. 6). Markers of inflammation including SP-D (1.8-fold), sRAGE (3.9-fold) and MMP-9 (13.6-fold) were also increased in BAL fluid from mice treated with Cl₂ (Fig. 6).

Figure 4. Effects of Cl₂ exposure on expression of γpH2A.X.

Lung sections, prepared 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) were stained with antibody to pH2A.X. Binding was visualized using a Vectastain kit. Original magnification, 200x (proximal bronchioles, top panels); 200x (distal bronchioles, middle panels); 400x (alveoli, bottom panels). Representative sections from 6 mice/treatment group are shown.

Figure 5. Effects of Cl₂ exposure on BAL protein, cell and IgM content.

BAL collected 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) was analyzed for protein (upper panel), cell content (middle panel) and IgM (lower panel). Bars, mean ± SE (n = 10 – 16 mice/treatment group). ^aSignificantly (p ≤ 0.05) different from CTL.

Figure 6. Effects of Cl₂ exposure on lung injury and inflammation.

BAL collected 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) was analyzed for levels of native and reduced fibrinogen, surfactant protein (SP)-D, soluble receptor for advanced glycation end product (sRAGE), and matrix metalloproteinase (MMP)-9 by western blotting. Representative blots from 5 – 10 mice/treatment group are shown.

Cl₂ exposure is known to cause oxidative stress, which has been implicated in lung injury (Wigenstam et al., 2015; Achanta and Jordt, 2021). In line with this, we found that the antioxidant enzymes HO-1 and catalase were upregulated in bronchiolar epithelial cells in response to Cl₂; numbers of enlarged macrophages expressing HO-1 and catalase were also increased (Fig. 7 and Table 1). Conversely, minimal staining was observed in the alveolar epithelium.

Figure 7. Effects of Cl₂ exposure on markers of oxidative stress.

Lung sections prepared 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) were stained with antibody to HO-1 or catalase. Binding was visualized using a Vectastain kit. Original magnification, 600x (alveolar regions, upper panels); 200x (bronchiolar regions, lower panels) for each protein. Representative sections from 6 mice/treatment group are shown.

In response to injury and oxidative stress, cells release Evs, which are important in cell - cell communication (Nieri et al., 2016). Evs were detected in BAL from control mice; these were released by CD9⁺ myeloid cells, CD31⁺ endothelial cells and CD326⁺ epithelial cells. Cl₂ administration resulted in a significant increase (2-fold) in total Evs in BAL (Fig. 8). This was correlated with increases in myeloid (2-fold) and endothelial derived (4.5-fold) Evs, with no effect on epithelial cell derived Evs.

Figure 8. Effects of Cl₂ exposure on extracellular vesicles.

BAL collected 24 h after exposure of mice to air (CTL) or chlorine (Cl₂) was analyzed for total extracellular vesicles (Evs), CD9⁺ Evs, CD31⁺ Evs and CD326⁺ Evs as described in Materials and Methods. Bars, mean ± SE (n = 5 mice/treatment group). ^aSignificantly (p ≤ 0.05) different from CTL.

DISCUSSION

Cl₂ gas is a potent oxidizing agent that causes severe injury to the airways characterized by damage to the bronchiolar epithelium and alterations in lung function (Demnati et al., 1995; Song et al., 2011; Jonasson et al., 2013; Balakrishna et al., 2014). This is associated with an accumulation of inflammatory cells in the lung (Cheng et al., 2021; Lee et al., 2021). In the present studies, we characterized inflammatory cells in the lung following Cl₂ exposure with the long-term goal of analyzing their role in pulmonary toxicity.

Following Cl₂ exposure, two subsets of CD11b⁺ inflammatory cells increased in the lung: proinflammatory macrophages and granulocytes. Conversely, antiinflammatory macrophages were not altered. We also found that proinflammatory, but not antiinflammatory macrophages were functionally activated after Cl₂ exposure, as evidenced by increased expression of iNOS and COX-2, with no effect on Ym-1 or MR. These findings are consistent with impaired resolution of inflammation by antiinflammatory M2 macrophages, which we speculate contributes to tissue injury and oxidative stress (Zhao et al., 2022). iNOS and COX-2 mediate the production of proinflammatory/cytotoxic reactive nitrogen species and prostaglandins, respectively (Laskin et al., 2011). Similar increases in iNOS and COX-2 levels have previously been described in the lung following Cl₂ exposure (Martin et al., 2003; Massa et al., 2014). The fact that corticosteroids and iNOS inhibitors abrogate acute lung injury and inflammation provide support for a role of proinflammatory macrophages and mediators such as eicosanoids and reactive nitrogen species in Cl₂ toxicity (Demnati et al., 1998; Martin et al., 2003; Chen et al., 2013; Hoyle et al., 2016). Of note, numbers of resident macrophages were unchanged after Cl₂ exposure. These findings suggest that these cells do not play a major role in innate immune responses to inhaled Cl₂.

Proinflammatory activation of lung macrophages was confirmed by assessing their metabolic profile. Macrophages that promote inflammation require a higher rate of energy supply and hence tend to rely on glycolysis, while the activity of antiinflammatory macrophages is characterized by fatty acid oxidation and oxidative phosphorylation as the energy demand rate is relatively lower (Freemerman et al., 2014; Jha et al., 2015; Viola et al., 2019; Woods et al., 2020). Increases in glycolysis and a reduction in the tricarboxylic acid (TCA) cycle coupled with attenuated mitochondrial oxidative phosphorylation leads to the generation of TCA intermediates such as citrate, succinate, and reactive oxygen and nitrogen species which are important in propagating inflammation (El Kasmi and Stenmark, 2015). Our findings of significant increases in glycolytic activity in lung cells following Cl₂ inhalation are consistent with proinflammatory activation (El Kasmi and Stenmark, 2015; Lavrich et al., 2018). Differences were also observed in non-mitochondrial oxygen consumption and maximal respiration following Cl₂ exposure which may reflect the activity of inflammatory granulocytes. These findings indicate that Cl₂ induces a complex metabolic pattern that involves upregulation of both glycolysis and oxidative phosphorylation. Previous studies have shown that monocytes exhibit stimuli-specific divergent metabolic responses and activation of both glycolysis and oxidative phosphorylation in response to microbial stimuli or TLR2-specific activation (Lachmandas et al., 2016). Moreover, under conditions of glucose deprivation, monocytes depend on oxidative phosphorylation to meet the increased energy demand during microbial stimulation (Raulien et al., 2017). Whether the metabolic response of lung macrophages to inhaled Cl₂ is also stimulus specific or whether the oxidative changes are a result of granulocyte activation remains to be investigated.

In accord with earlier studies (Leustik et al., 2008; Tian et al., 2008; Hoyle et al., 2010; Wigenstam et al., 2015), treatment of mice with Cl₂ caused disruption in the alveolar epithelial barrier, as reflected by increases in BAL cell, protein and IgM content; this was associated with upregulation of HO-1 and catalase in bronchiolar and alveolar epithelial cells and in lung macrophages indicating oxidative stress. Increases in antioxidants including HO-1, glutathione peroxidase 2, and superoxide dismutase have been described in the lungs following Cl₂ exposure (Leustik et al., 2008; Tuck et al., 2008; Yadav et al., 2010; McGovern et al., 2011). Oxidative stress is thought to be a consequence of the formation of HOCl and its breakdown products HCl and oxygen, produced by the reaction of Cl₂ with airway lining fluid (Tuck et al., 2008). Reactive oxygen and nitrogen species generated by inflammatory macrophages may also contribute to this response (Roberts et al., 2009). Oxidative stress is known to cause DNA damage (Risom et al., 2005). Following Cl₂ exposure, we detected increases in expression of γH2A.X, a marker of double-strand DNA damage (Ismail and Hendzel, 2008), in bronchiolar epithelial cells, as well as in alveolar macrophages and epithelial cells. Additional studies are required to determine if this is due to Cl₂ induced oxidative stress or direct damage to DNA by HOCl or its breakdown products (Masuda et al., 2001). Pulmonary toxicants including radiation, nitrogen mustard and chromium have been reported to induce γH2A.X in bronchiolar and alveolar cells (Xie et al., 2005; Malaviya et al., 2015; Venosa et al., 2021). Our findings that γH2A.X is evident in alveolar macrophages and epithelial cells following Cl₂ exposure, together with increases in oxidative stress markers, demonstrate that these cells are targeted by Cl₂.

Fibrinogen is an acute phase glycoprotein released by the liver in response to tissue damage and inflammation (Luyendyk et al., 2019). Its presence in the airways is considered to be a biomarker of acute lung injury (Kulkarni et al., 2022). Following lung injury, fibrinogen is converted to fibrin which suppresses the activity of pulmonary surfactants and increases vascular smooth muscle contractility. This results in atelectasis and reduced lung functioning (Wagers et al., 2004; Wygrecka et al., 2008; Wigenstam et al., 2015). Fibrinogen interacts with the Mac-1 (CD11b/CD18, CR3, αMβ2) on monocytes/macrophages resulting in NF-κB activation and production of proinflammatory proteins (Mosesson, 2005; Davalos and Akassoglou, 2012). Our findings that both native fibrinogen and α, β and γ degradation products of fibrinogen are present in BAL are in accord with reports of increased fibrillar content in alveoli and serum following Cl₂ exposure (Jonasson et al., 2013; Wigenstam et al., 2015). HOCl has been shown to inhibit blood clot retraction and fibrinolysis (Misztal et al., 2019). This may contribute to coagulation abnormalities and the development of lung fibrosis after Cl₂ exposure (Yildirim et al., 2004; Wigenstam et al., 2016).

SP-D is a pulmonary collectin that functions to dampen macrophage inflammatory responses (Sorensen, 2018). Increased levels of SP-D in BAL is an indicator of lung inflammation (Gaunsbaek et al., 2013; Kulkarni et al., 2022). Consistent with Cl₂-induced inflammation, levels of SP-D in BAL were increased. Analogous increases in BAL SP-D levels have been reported after exposure of rodents to bleomycin, cigarette smoke, sulfur mustard and lipopolysaccharide, pulmonary toxicants known to induce a robust inflammatory response (Winkler et al., 2011; Gaunsbaek et al., 2013; Malaviya et al., 2020). Reactive oxygen and nitrogen species generated by inflammatory cells and HOCl cause S-nitrosylation of SP-D (Matalon et al., 2009; Massa et al., 2014; Guo et al., 2019). This changes the function of SP-D from antiinflammatory to proinflammatory (Guo et al., 2008; Atochina-Vasserman, 2012). Studies are in progress to determine if SP-D structure is modified after Cl₂ exposure and if this contributes to pulmonary inflammation.

Increases in BAL levels of the proinflammatory proteins sRAGE and MMP-9 were also detected after exposure of mice to Cl₂. RAGE is a membrane receptor expressed on alveolar macrophages and epithelial cells that promotes proinflammatory signaling (Hudson and Lippman, 2018; Sanders et al., 2019). It is cleaved to sRAGE by proteinases, including MMP-9 generated at sites of tissue injury (Oczypok et al., 2017). RAGE is a key mediator of pulmonary inflammatory responses and an important contributor to acute lung injury in animals and humans (Kulkarni et al., 2022). Our findings that Cl₂-induced increases in BAL levels of sRAGE are correlated with alveolar epithelial cell barrier dysfunction, and BAL levels of inflammatory proteins are in line with previous studies in rodent models of lung injury induced by mustards, lipopolysaccharide, and cigarette smoke (Zhang et al., 2009; Kodavanti et al., 2011; Wang et al., 2018; Malaviya et al., 2020; Wang et al., 2020). Binding of RAGE to ligands such as HMGB1 initiates intracellular signaling leading to the sustained activation of NF-κB and transcription of proinflammatory genes including iNOS and MMP-9, as well as RAGE itself (Hergrueter et al., 2011; Stogsdill et al., 2013; Sanders et al., 2019). Studies are ongoing to determine if this contributes to amplification of inflammatory signaling cascades following Cl₂ exposure and tissue injury. MMP-9 has been reported to play a central role in pulmonary inflammation and disease pathogenesis following acute lung injury. This is based on findings that levels of MMP-9 rise in the lung and/or BAL following exposure to pulmonary toxicants such as ozone, bleomycin, and mustard vesicants and that this directly correlates with structural damage and altered pulmonary function (Chakrabarti and Patel, 2005; Kim et al., 2009; Francis et al., 2017; Malaviya et al., 2020). We postulate that MMP-9 is similarly involved in the toxicity of inhaled Cl₂. In this context, we previously reported that acute Cl₂ gas exposure produces progressive alterations in surfactant composition, a response accompanied by mechanical respiratory dysfunction (Massa et al., 2014).

Evs are lipid vesicles released from injured or stressed cells that play a role in cell-cell communication (Andres et al., 2020; Mohan et al., 2020). Evs transport cargo consisting of transcription factors, mRNAs, noncoding regulatory RNAs, lipids, and proteins to recipient cells and thus regulate their response (Andres et al., 2020). Exposure of mice to Cl₂ resulted in a significant increase in total Evs in BAL. Assessment of the cellular origin of the Evs showed that the majority of the Evs in BAL from air exposed mice were from epithelial cells (CD326⁺), myeloid cells (CD9⁺) and endothelial cells (CD31⁺). Treatment of mice with Cl₂ resulted in significant increases in myeloid cell- and endothelial cell-derived Evs, with no effect on epithelial-derived Evs. We speculate that Evs are released from myeloid and endothelial cells as a mechanism to signal repair of the damaged epithelium. Further studies characterizing the cargo carried by the Evs are required to determine if they contribute to propagating and/or resolving Cl₂-induced inflammation.

The present studies demonstrate that inhalation of toxic doses of Cl₂ is associated with an accumulation of activated proinflammatory macrophages in the lung. These cells have been implicated in lung injury and oxidative stress induced by diverse pulmonary toxicants (Laskin et al., 2019). The fact that proinflammatory macrophage accumulation is correlated with lung injury, oxidative stress and increased inflammatory protein levels suggests that these cells may also contribute to the pathogenic response to Cl₂. Importantly, numbers of resident macrophages and antiinflammatory macrophages, which are important in suppressing inflammation and promoting wound repair were unchanged after Cl₂ exposure. These findings suggest that impaired resolution of inflammation and wound repair may also contribute to toxicity. Further research is needed to assess the precise role of macrophage subsets in acute Cl₂ toxicity; this will aid in identifying efficacious countermeasures for mitigating morbidity and mortality of this highly toxic gas.

Highlights.

• Acute chlorine (Cl₂) gas inhalation causes lung injury and oxidative stress.

• Macrophages responding to Cl₂ exhibit a proinflammatory phenotype.

• Levels of antiinflammatory macrophages are not affected by Cl₂ exposure.