Regulation of macrophage activation by S-Nitrosothiols following ozone-induced lung injury.

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.

Farnesoid X receptor regulates lung macrophage activation and injury following nitrogen mustard exposure.

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.

Comprehensive dataset to assess morphological changes subsequent to bleomycin exposure.

Intratracheal bleomycin causes pulmonary injury, inflammation and fibrosis. The characteristic patchy nature of the injury makes analysis challenging. Histological assessment of lung injury is a useful tool to evaluate damage, however quantification is not standardized. We propose a multi-factorial approach to assess morphological changes subsequent to intratracheal bleomycin mediated lung injury. Lungs were inflation fixed with paraformaldehyde, sectioned and stained with hematoxylin and eosin. Whole slide images were scanned and ten 400x images were randomly chosen throughout the tissue for further analysis. Using ImageJ software, alveolar wall width was measured, nuclei were counted and airspace was quantified. Morphological changes were identified in mice instilled with bleomycin. This combination offers a robust measure of lung morphology especially in a heterogenous injury.

It's time to reconsider how we define health: Perspective from disability and chronic condition.

Our understanding of health has changed substantially since the World Health Organization initially defined health in 1948 as "a state of complete physical, mental and social and well-being and not merely the absence of disease or infirmity". These changes include reconceptualizing health on a continuum rather than as a static state, and adding existential health to physical, mental, and social well-being. Further, good health requires adaptation in coping with stress and is influenced by social, personal and environmental factors. Building on prior work, we propose a reconsidered 2020 definition: "Health is the dynamic balance of physical, mental, social, and existential well-being in adapting to conditions of life and the environment." Health is dynamic, continuous, multidimensional, distinct from function, and determined by balance and adaptation. This new definition has implications for research, policies, and practice, with particular relevance for health considered within a context of disability and chronic conditions.

Myeloid cell dynamics in bleomycin-induced pulmonary injury in mice; effects of anti-TNFα antibody.

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.

Assessment of mustard vesicant lung injury and anti-TNF-α efficacy in rodents using live-animal imaging.

Nitrogen mustard (NM) causes acute lung injury, which progresses to fibrosis. This is associated with a macrophage-dominant inflammatory response and the production of proinflammatory/profibrotic mediators, including tumor necrosis factor alpha (TNF-α). Herein, we refined magnetic resonance imaging (MRI) and computed tomography (CT) imaging methodologies to track the progression of NM-induced lung injury in rodents and assess the efficacy of anti-TNF-α antibody in mitigating toxicity. Anti-TNF-α antibody was administered to rats (15 mg/kg, every 8 days, intravenously) beginning 30 min after treatment with phosphate-buffered saline control or NM (0.125 mg/kg, intratracheally). Animals were imaged by MRI and CT prior to exposure and 1-28 days postexposure. Using MRI, we characterized acute lung injury and fibrosis by quantifying high-signal lung volume, which represents edema, inflammation, and tissue consolidation; these pathologies were found to persist for 28 days following NM exposure. CT scans were used to assess structural components of the lung and to register changes in tissue radiodensities. CT scans showed that in control animals, total lung volume increased with time. Treatment of rats with NM caused loss of lung volume; anti-TNF-α antibody mitigated this decrease. These studies demonstrate that MRI and CT can be used to monitor lung disease and the impact of therapeutic intervention.

Fatty acid nitroalkenes inhibit the inflammatory response to bleomycin-mediated lung injury.

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-NO2) 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-NO2 (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-NO2 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-NO2. 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-NO2 (24 ± 2.4%). Additionally, OA-NO2 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-NO2 (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-NO2 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-NO2 inhibits ITB-induced pro-inflammatory responses by modulating resident cell function.

Nutraceuticals as potential therapeutics for vesicant-induced pulmonary fibrosis.

Exposure to vesicants, including sulfur mustard and nitrogen mustard, causes damage to the epithelia of the respiratory tract and the lung. With time, this progresses to chronic disease, most notably, pulmonary fibrosis. The pathogenic process involves persistent inflammation and the release of cytotoxic oxidants, cytokines, chemokines, and profibrotic growth factors, which leads to the collapse of lung architecture, with fibrotic involution of the lung parenchyma. At present, there are no effective treatments available to combat this pathological process. Recently, much interest has focused on nutraceuticals, substances derived from plants, herbs, and fruits, that exert pleiotropic effects on inflammatory cells and parenchymal cells that may be useful in reducing fibrogenesis. Some promising results have been obtained with nutraceuticals in experimental animal models of inflammation-driven fibrosis. This review summarizes the current knowledge on the putative preventive/therapeutic efficacy of nutraceuticals in progressive pulmonary fibrosis, with a focus on their activity against inflammatory reactions and profibrotic cell differentiation.

Role of extracellular vesicles in cell-cell communication and inflammation following exposure to pulmonary toxicants.

Extracellular vesicles (EVs) have emerged as key regulators of cell-cell communication during inflammatory responses to lung injury induced by diverse pulmonary toxicants including cigarette smoke, air pollutants, hyperoxia, acids, and endotoxin. Many lung cell types, including epithelial cells and endothelial cells, as well as infiltrating macrophages generate EVs. EVs appear to function by transporting cargo to recipient cells that, in most instances, promote their inflammatory activity. Biologically active cargo transported by EVs include miRNAs, cytokines/chemokines, damage-associated molecular patterns (DAMPs), tissue factor (TF)s, and caspases. Findings that EVs are taken up by target cells such as macrophages, and that this leads to increased proinflammatory functioning provide support for their role in the development of pathologies associated with toxicant exposure. Understanding the nature of EVs responding to toxic exposures and their cargo may lead to the development of novel therapeutic approaches to mitigating lung injury.

Regulation of Macrophage Foam Cell Formation During Nitrogen Mustard (NM)-Induced Pulmonary Fibrosis by Lung Lipids.

Nitrogen mustard (NM) is a vesicant known to target the lung, causing acute injury which progresses to fibrosis. Evidence suggests that activated macrophages contribute to the pathologic response to NM. In these studies, we analyzed the role of lung lipids generated following NM exposure on macrophage activation and phenotype. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in a time-related increase in enlarged vacuolated macrophages in the lung. At 28 days postexposure, macrophages stained positively for Oil Red O, a marker of neutral lipids. This was correlated with an accumulation of oxidized phospholipids in lung macrophages and epithelial cells and increases in bronchoalveolar lavage fluid (BAL) phospholipids and cholesterol. RNA-sequencing and immunohistochemical analysis revealed that lipid handling pathways under the control of the transcription factors liver-X receptor (LXR), farnesoid-X receptor (FXR), peroxisome proliferator-activated receptor (PPAR)-É£, and sterol regulatory element-binding protein (SREBP) were significantly altered following NM exposure. Whereas at 1-3 days post NM, FXR and the downstream oxidized low-density lipoprotein receptor, Cd36, were increased, Lxr and the lipid efflux transporters, Abca1 and Abcg1, were reduced. Treatment of naïve lung macrophages with phospholipid and cholesterol enriched large aggregate fractions of BAL prepared 3 days after NM exposure resulted in upregulation of Nos2 and Ptgs2, markers of proinflammatory activation, whereas large aggregate fractions prepared 28 days post NM upregulated expression of the anti-inflammatory markers, Il10, Cd163, and Cx3cr1, and induced the formation of lipid-laden foamy macrophages. These data suggest that NM-induced alterations in lipid handling and metabolism drive macrophage foam cell formation, potentially contributing to the development of pulmonary fibrosis.