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Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chlorine (Cl 2 ) gas is a highly toxic and oxidizing irritant that causes life-threatening lung injuries. Herein, we investigated the impact of Cl 2 -induced injury and oxidative stress on lung macrophage phenotype and function. Spontaneously breathing male C57BL/6J mice were exposed to air or Cl 2 (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 Cl 2 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. Cl 2 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 Cl 2 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 Cl 2 exposure in line with proinflammatory macrophage activation. Mechanistic understanding of Cl 2 -induced injury will be useful in the identification of efficacious countermeasures for mitigating morbidity and mortality of this highly toxic gas., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Debra L Laskin reports financial support was provided by National Institutes of Health., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Acute exposure to ozone causes oxidative stress, characterized by increases in nitric oxide (NO) and other reactive nitrogen species in the lung. NO has been shown to modify thiols generating S-nitrosothiols (SNOs); this results in altered protein function. In macrophages this can lead to changes in inflammatory activity which impact the resolution of inflammation. As SNO formation is dependent on the redox state of both the NO donor and the recipient thiol, the local microenvironment plays a key role in its regulation. This dictates not only the chemical feasibility of SNO formation but also mechanisms by which they may form. In these studies, we compared the ability of the SNO donors, ethyl nitrite (ENO), which targets both hydrophobic and hydrophilic thiols, SNO-propanamide (SNOPPM) which targets hydrophobic thiols, and S-nitroso-N-acetylcysteine. (SNAC) which targets hydrophilic thiols. to modify macrophage activation following ozone exposure. Mice were treated with air or ozone (0.8 ppm, 3 h) followed 1 h later by intranasal administration of ENO, SNOPPM or SNAC (1-500 μM) or appropriate controls. Mice were euthanized 48 h later. Each of the SNO donors reduced ozone-induced inflammation and modified the phenotype of macrophages both within the lung lining fluid and the tissue. ENO and SNOPPM were more effective than SNAC. These findings suggest that the hydrophobic SNO thiol pool targeted by SNOPPM and ENO plays a major role in regulating macrophage phenotype following ozone induced injury., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Debra Laskin reports financial support was provided by National Institutes of Health. Andrew Gow reports financial support was provided by National Institutes of Health. Sheryse Taylor reports financial support was provided by National Institutes of Health. Alexa Murray reports financial support was provided by National Institutes of Health. Elena Abramova reports financial support was provided by National Institutes of Health. Changjiang Guo reports financial support was provided by National Institutes of Health. Dr. Laskin, a co-author of the paper, is a guest editor for the journal, (Copyright © 2022. Published by Elsevier Inc.)

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause acute lung injury which progresses to fibrosis. Alveolar Type II cells are primarily responsible for surfactant production; they also play a key role in lung repair following injury. Herein, we assessed the effects of NM on Type II cell activity. Male Wistar rats were administered NM (0.125 mg/kg) or PBS control intratracheally. Type II cells, lung tissue and BAL were collected 3 d later. NM exposure resulted in double strand DNA breaks in Type II cells, as assessed by expression of γH2AX; this was associated with decreased expression of the DNA repair protein, PARP1. Expression of HO-1 was upregulated and nitrotyrosine residues were noted in Type II cells after NM exposure indicating oxidative stress. NM also caused alterations in Type II cell energy metabolism; thus, both glycolysis and oxidative phosphorylation were reduced; there was also a shift from a reliance on oxidative phosphorylation to glycolysis for ATP production. This was associated with increased expression of pro-apoptotic proteins activated caspase-3 and -9, and decreases in survival proteins, β-catenin, Nur77, HMGB1 and SOCS2. Intracellular signaling molecules important in Type II cell activity including PI3K, Akt2, phospho-p38 MAPK and phospho-ERK were reduced after NM exposure. This was correlated with dysregulation of surfactant protein production and impaired pulmonary functioning. These data demonstrate that Type II cells are targets of NM-induced DNA damage and oxidative stress. Impaired functioning of these cells may contribute to pulmonary toxicity caused by mustards., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Debra Laskin reports financial support was provided by National Institutes of Health. She is an Associate Editor of Toxicology and Applied Pharmacology, (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause acute lung injury which progresses to fibrosis; this is associated with a sequential accumulation of pro- and anti-inflammatory macrophages in the lung which have been implicated in NM toxicity. Farnesoid X receptor (FXR) is a nuclear receptor involved in regulating lipid homeostasis and inflammation. In these studies, we analyzed the role of FXR in inflammatory macrophage activation, lung injury and oxidative stress following NM exposure. Wild-type (WT) and FXR -/- mice were treated intratracheally with PBS (control) or NM (0.08 mg/kg). Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 3, 14 and 28 d later. NM caused progressive histopathologic alterations in the lung including inflammatory cell infiltration and alveolar wall thickening and increases in protein and cells in BAL; oxidative stress was also noted, as reflected by upregulation of heme oxygenase-1. These changes were more prominent in male FXR -/- mice. Flow cytometric analysis revealed that loss of FXR resulted in increases in proinflammatory macrophages at 3 d post NM; this correlated with upregulation of COX-2 and ARL11, markers of macrophage activation. Markers of anti-inflammatory macrophage activation, CD163 and STAT6, were also upregulated after NM; this response was exacerbated in FXR -/- mice at 14 d post-NM. These findings demonstrate that FXR plays a role in limiting macrophage inflammatory responses important in lung injury and oxidative stress. Maintaining or enhancing FXR function may represent a useful strategy in the development of countermeasures to treat mustard lung toxicity., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2022. Published by Elsevier Inc.)

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15-30 min later by anti-TNFα antibody (15mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bleomycin is a cancer therapeutic known to cause lung injury which progresses to fibrosis. Evidence suggests that macrophages contribute to this pathological response. Tumor necrosis factor (TNF)α is a macrophage-derived pro-inflammatory cytokine implicated in lung injury. Herein, we investigated the role of TNFα in macrophage responses to bleomycin. Treatment of mice with bleomycin (3 U/kg, i.t.) caused histopathological changes in the lung within 3 d which culminated in fibrosis at 21 d. This was accompanied by an early (3-7 d) influx of CD11b + and iNOS + macrophages into the lung, and Arg-1 + macrophages at 21 d. At this time, epithelial cell dysfunction, defined by increases in total phospholipids and SP-B was evident. Treatment of mice with anti-TNFα antibody (7.5 mg/kg, i.v.) beginning 15-30 min after bleomycin, and every 5 d thereafter reduced the number and size of fibrotic foci and restored epithelial cell function. Flow cytometric analysis of F4/80 + alveolar macrophages (AM) isolated by bronchoalveolar lavage and interstitial macrophages (IM) by tissue digestion identified resident (CD11b - CD11c + ) and immature infiltrating (CD11b + CD11c - ) AM, and mature (CD11b + CD11c + ) and immature (CD11b + CD11c - ) IM subsets in bleomycin treated mice. Greater numbers of mature (CD11c + ) infiltrating (CD11b + ) AM expressing the anti-inflammatory marker, mannose receptor (CD206) were observed at 21 d when compared to 7 d post bleomycin. Mature proinflammatory (Ly6C + ) IM were greater at 7 d relative to 21 d. These cells transitioned into mature anti-inflammatory/pro-fibrotic (CD206 + ) IM between 7 and 21 d. Anti-TNFα antibody heightened the number of CD11b + AM in the lung without altering their activation state. Conversely, it reduced the abundance of mature proinflammatory (Ly6C + ) IM in the tissue at 7 d and immature pro-fibrotic IM at 21 d. Taken together, these data suggest that TNFα inhibition has beneficial effects in bleomycin induced injury, restoring epithelial function and reducing numbers of profibrotic IM and the extent of pulmonary fibrosis., (Copyright © 2021. Published by Elsevier Inc.)

Fatty acid nitroalkenes are reversibly-reactive electrophiles, endogenously detectable at nM concentrations, displaying anti-inflammatory actions. Nitroalkenes like 9- or 10-nitro-octadec-9-enoic acid (e.g. nitro-oleic acid, OA-NO 2 ) pleiotropically suppress cardiovascular inflammatory responses, with pulmonary responses less well defined. C57BL/6 J male mice were intratracheally administered bleomycin (3 U/kg, ITB), to induce pulmonary inflammation and acute injury, or saline and were treated with 50 μL OA-NO 2 (50 μg) or vehicle in the same instillation and 72 h post-exposure to assess anti-inflammatory properties. Bronchoalveolar lavage (BAL) and lung tissue were collected 7d later. ITB mice lost body weight, with OA-NO 2 mitigating this loss (-2.3 ± 0.94 vs -0.4 ± 0.83 g). Histology revealed ITB induced cellular infiltration, proteinaceous debris deposition, and tissue injury, all significantly reduced by OA-NO 2 . Flow cytometry analysis of BAL demonstrated loss of Siglec F + /F4/80 + /CD45 + alveolar macrophages with ITB (89 ± 3.5 vs 30 ± 3.7%). Analysis of CD11b/CD11c expressing cells showed ITB-induced non-resident macrophage infiltration (4 ± 2.3 vs 43 ± 2.4%) was decreased by OA-NO 2 (24 ± 2.4%). Additionally, OA-NO 2 attenuated increases in mature, activated interstitial macrophages (23 ± 4.8 vs. 43 ± 5.4%) in lung tissue digests. Flow analysis of CD31 - /CD45 - /Sca-1 + mesenchymal cells revealed ITB increased CD44 + populations (1 ± 0.4 vs 4 ± 0.4MFI), significantly reduced by OA-NO 2 (3 ± 0.4MFI). Single cell analysis of mesenchymal cells by western blotting showed profibrotic ZEB1 protein expression induced by ITB. Lung digest CD45 + cells revealed ITB increased HMGB1 + cells, with OA-NO 2 suppressing this response. Inhibition of HMGB1 expression correlated with increased basal phospholipid production and SP-B expression in the lung lining. These findings indicate OA-NO 2 inhibits ITB-induced pro-inflammatory responses by modulating resident cell function., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause acute lung injury which progresses to fibrosis. Herein, we developed a murine model of NM-induced pulmonary toxicity with the goal of assessing inflammatory mechanisms of injury. C57BL/6J mice were euthanized 1-28 d following intratracheal exposure to NM (0.08 mg/kg) or PBS control. NM caused progressive alveolar epithelial thickening, perivascular inflammation, bronchiolar epithelial hyperplasia, interstitial fibroplasia and fibrosis, peaking 14 d post exposure. Enlarged foamy macrophages were also observed in the lung 14 d post NM, along with increased numbers of microparticles in bronchoalveolar lavage fluid (BAL). Following NM exposure, rapid and prolonged increases in BAL cells, protein, total phospholipids and surfactant protein (SP)-D were also detected. Flow cytometric analysis showed that CD11b + Ly6G - F4/80 + Ly6C hi proinflammatory macrophages accumulated in the lung after NM, peaking at 3 d. This was associated with macrophage expression of HMGB1 and TNFα in histologic sections. CD11b + Ly6G - F4/80 + Ly6C lo anti-inflammatory/pro-fibrotic macrophages also increased in the lung after NM peaking at 14 d, a time coordinate with increases in TGFβ expression and fibrosis. NM exposure also resulted in alterations in pulmonary mechanics including increases in tissue elastance and decreases in compliance and static compliance, most prominently at 14 d. These findings demonstrate that NM induces structural and inflammatory changes in the lung that correlate with aberrations in pulmonary function. This mouse model will be useful for mechanistic studies of mustard lung injury and for assessing potential countermeasures., Competing Interests: Declaration of Competing Interest 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., (Published by Elsevier Inc.)

Acute Cl2 exposure following industrial accidents or military/terrorist activity causes pulmonary injury and severe acute respiratory distress. Prior studies suggest that antioxidant depletion is important in producing dysfunction, however a pathophysiologic mechanism has not been elucidated. We propose that acute Cl2 inhalation leads to oxidative modification of lung lining fluid, producing surfactant inactivation, inflammation and mechanical respiratory dysfunction at the organ level. C57BL/6J mice underwent whole-body exposure to an effective 60ppm-hour Cl2 dose, and were euthanized 3, 24 and 48h later. Whereas pulmonary architecture and endothelial barrier function were preserved, transient neutrophilia, peaking at 24h, was noted. Increased expression of ARG1, CCL2, RETLNA, IL-1b, and PTGS2 genes was observed in bronchoalveolar lavage (BAL) cells with peak change in all genes at 24h. Cl2 exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO3(-) or NO2(-). Expression of the alternative macrophage activation markers, Relm-α and mannose receptor was increased in alveolar macrophages and pulmonary epithelium. Capillary surfactometry demonstrated impaired surfactant function, and altered BAL phospholipid and surfactant protein content following exposure. Organ level respiratory function was assessed by forced oscillation technique at 5 end expiratory pressures. Cl2 exposure had no significant effect on either airway or tissue resistance. Pulmonary elastance was elevated with time following exposure and demonstrated PEEP refractory derecruitment at 48h, despite waning inflammation. These data support a role for surfactant inactivation as a physiologic mechanism underlying respiratory dysfunction following Cl2 inhalation., (Copyright © 2014 Elsevier Inc. All rights reserved.)