RESUMEN
Nitrogen mustard (NM) is a cytotoxic vesicant known to cause pulmonary injury that can progress to fibrosis. NM toxicity is associated with an influx of inflammatory macrophages in the lung. Farnesoid X receptor (FXR) is a nuclear receptor involved in bile acid and lipid homeostasis that has anti-inflammatory activity. In these studies, we analyzed the effects of FXR activation on lung injury, oxidative stress, and fibrosis induced by NM. Male Wistar rats were exposed to phosphate-buffered saline (vehicle control) or NM (0.125 mg/kg) by intratracheal Penncentury-MicroSprayer aerosolization; this was followed by treatment with the FXR synthetic agonist, obeticholic acid (OCA, 15 mg/kg), or vehicle control (0.13-0.18 g peanut butter) 2 hours later and then once per day, 5 days per week thereafter for 28 days. NM caused histopathological changes in the lung, including epithelial thickening, alveolar circularization, and pulmonary edema. Picrosirius red staining and lung hydroxyproline content were increased, indicative of fibrosis; foamy lipid-laden macrophages were also identified in the lung. This was associated with aberrations in pulmonary function, including increases in resistance and hysteresis. Following NM exposure, lung expression of HO-1 and iNOS, and the ratio of nitrates/nitrites in bronchoalveolar lavage fluid (BAL), markers of oxidative stress increased, along with BAL levels of inflammatory proteins, fibrinogen, and sRAGE. Administration of OCA attenuated NM-induced histopathology, oxidative stress, inflammation, and altered lung function. These findings demonstrate that FXR plays a role in limiting NM-induced lung injury and chronic disease, suggesting that activating FXR may represent an effective approach to limiting NM-induced toxicity. SIGNIFICANCE STATEMENT: In this study, the role of farnesoid-X-receptor (FXR) in mustard vesicant-induced pulmonary toxicity was analyzed using nitrogen mustard (NM) as a model. This study's findings that administration of obeticholic acid, an FXR agonist, to rats reduces NM-induced pulmonary injury, oxidative stress, and fibrosis provide novel mechanistic insights into vesicant toxicity, which may be useful in the development of efficacious therapeutics.
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Ácido Quenodesoxicólico/análogos & derivados , Lesión Pulmonar , Mecloretamina , Ratas , Masculino , Animales , Mecloretamina/toxicidad , Irritantes/efectos adversos , Ratas Wistar , Pulmón , Fibrosis , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/patología , Lesión Pulmonar/metabolismo , Estrés Oxidativo , LípidosRESUMEN
Nitrogen mustard (NM; mechlorethamine) is a cytotoxic vesicant known to cause acute lung injury which can progress to chronic disease. Due to the complex nature of NM injury, it has been difficult to analyze early responses of resident lung cells that initiate inflammation and disease progression. To investigate this, we developed a model of acute NM toxicity using murine precision cut lung slices (PCLS), which contain all resident lung cell populations. PCLS were exposed to NM (1-100 µM) for 0.5-3 h and analyzed 1 and 3 d later. NM caused a dose-dependent increase in cytotoxicity and a reduction in metabolic activity, as measured by LDH release and WST-1 activity, respectively. Optimal responses were observed with 50 µM NM after 1 h incubation and these conditions were used in further experiments. Analysis of PCLS bioenergetics using an Agilent Seahorse showed that NM impaired both glycolytic activity and mitochondrial respiration. This was associated with injury to the bronchial epithelium and a reduction in methacholine-induced airway contraction. NM was also found to cause DNA damage in bronchial epithelial cells in PCLS, as measured by expression of γ-H2AX, and to induce oxidative stress, which was evident by a reduction in glutathione levels and upregulation of the antioxidant enzyme catalase. Cleaved caspase-3 was also upregulated in airway smooth muscle cells indicating apoptotic cell death. Characterizing early events in NM toxicity is key in identifying therapeutic targets for the development of efficacious countermeasures.
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Pulmón , Mecloretamina , Animales , Mecloretamina/toxicidad , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo , Ratones , Daño del ADN , Ratones Endogámicos C57BL , Relación Dosis-Respuesta a Droga , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Sustancias para la Guerra Química/toxicidad , Glucólisis/efectos de los fármacos , Masculino , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patologíaRESUMEN
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.
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Acetilcisteína , Metabolismo Energético , Lesión Pulmonar , Mecloretamina , Estrés Oxidativo , Animales , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Mecloretamina/toxicidad , Masculino , Metabolismo Energético/efectos de los fármacos , Ratas , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Ratas Sprague-Dawley , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Sustancias para la Guerra Química/toxicidadRESUMEN
AIM: Chronotype reflects a circadian rhythmicity that regulates endothelial function. While the morning chronotype (MORN) usually has low cardiovascular disease risk, no study has examined insulin action on endothelial function between chronotypes. We hypothesized intermediate chronotypes (INT) would have lower vascular insulin sensitivity than morning chronotype (MORN). MATERIALS AND METHODS: Adults with obesity were classified per Morningness-Eveningness Questionnaire (MEQ) as either MORN (n = 27, 22 female, MEQ = 63.7 ± 4.7, 53.8 ± 6.7 years, 35.3 ± 4.9 kg/m2) or INT (n = 29, 23 female, MEQ = 48.8 ± 6.7, 56.6 ± 9.0 years, 35.7 ± 6.1 kg/m2). A 120 min euglycaemic-hyperinsulinaemic clamp (40 mU/m2/min, 90 mg/dl) was conducted to assess macrovascular insulin sensitivity via brachial artery flow-mediated dilation (%FMD; conduit artery), post-ischaemic flow velocity (resistance arteriole), as well as microvascular insulin sensitivity via contrast-enhanced ultrasound [e.g. microvascular blood volume (perfusion)]. Fasting plasma arginine and citrulline, as well as fasting and clamp-derived plasma endothelin-1 and nitrate/nitrite, were assessed as surrogates of vasoconstriction and nitric oxide-mediated vasodilation. Aerobic fitness (VO2max) and body composition (dual-energy X-ray absorptiometry) were also collected. RESULTS: MORN had a higher VO2max compared with INT (p < .01), although there was no difference in fat mass. While fasting FMD was similar between groups, insulin lowered FMD corrected to shear stress and microvascular blood volume in INT compared with MORN after co-varying for VO2max (both p ≤ .02). INT also had a lower fasting nitrate (p = .03) and arginine (p = .07). Higher MEQ correlated with elevated FMD (r = 0.33, p = .03) and lower post-ischaemic flow velocity (r = -0.33, p = .03) as well as shear rate (r = -0.36, p = .02) at 120 min. CONCLUSION: When measured during the morning, INT had a lower vascular insulin sensitivity than MORN. Additional work is needed to understand endothelial function differences among chronotypes to optimize cardiovascular disease risk reduction.
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Enfermedades Cardiovasculares , Resistencia a la Insulina , Adulto , Humanos , Femenino , Cronotipo , Nitratos , Obesidad , Arteria Braquial/fisiología , Insulina , Endotelio Vascular , Vasodilatación , ArgininaRESUMEN
E-cigarette consumption is under scrutiny by regulatory authorities due to concerns about product toxicity, lack of manufacturing standards, and increasing reports of e-cigarette- or vaping-associated acute lung injury. In vitro studies have demonstrated cytotoxicity, mitochondrial dysfunction, and oxidative stress induced by unflavored e-cigarette aerosols and flavoring additives. However, e-cigarette effects on the complex lung parenchyma remain unclear. Herein, the impact of e-cigarette condensates with or without menthol flavoring on functional, structural, and cellular responses was investigated using mouse precision cut lung slices (PCLS). PCLS were exposed to e-cigarette condensates prepared from aerosolized vehicle, nicotine, nicotine + menthol, and menthol e-fluids at doses from 50 to 500 mM. Doses were normalized to the glycerin content of vehicle. Video-microscopy of PCLS revealed impaired contractile responsiveness of airways to methacholine and dampened ciliary beating following exposure to menthol-containing condensates at concentrations greater than 300 mM. Following 500 mM menthol-containing condensate exposure, epithelial exfoliation in intrabronchial airways was identified in histological sections of PCLS. Measurement of lactate dehydrogenase release, mitochondrial water-soluble-tetrazolium salt-1 conversion, and glutathione content supported earlier findings of nicotine or nicotine + menthol e-cigarette-induced dose-dependent cytotoxicity and oxidative stress responses. Evaluation of PCLS metabolic activity revealed dose-related impairment of mitochondrial oxidative phosphorylation and glycolysis after exposure to menthol-containing condensates. Taken together, these data demonstrate prominent menthol-induced pulmonary toxicity and impairment of essential physiological functions in the lung, which warrants concerns about e-cigarette consumer safety and emphasizes the need for further investigations of molecular mechanisms of toxicity and menthol effects in an experimental model of disease.
Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Nicotina , Animales , Ratones , Nicotina/toxicidad , Mentol/toxicidad , Aerosoles y Gotitas Respiratorias , Pulmón , Aromatizantes/toxicidadRESUMEN
Exaggerated exercise blood pressure (BP) is linked to cardiovascular disease (CVD). Although evening chronotypes have greater CVD risk than morning (Morn) types, it is unknown if exercise BP differs in intermediate (Int) types. Adults with obesity were classified as either Morn [n = 23 (18 females), Morning-Eveningness Questionnaire (MEQ) = 63.96 ± 1.0, 54.74 ± 1.4 yr, 33.7 ± 0.6 kg/m2] or Int [n = 23 (19 females), MEQ = 51.36 ± 1.1, 55.96 ± 1.8 yr, 37.2 ± 1.2 kg/m2] chronotype per MEQ. A graded, incremental treadmill test to maximal aerobic capacity (VÌo2max) was conducted. Systolic (SBP) and diastolic (DBP) blood pressure and mean arterial pressure (MAP), rate pressure product (RPP), heart rate (HR), and rate of perceived intensity (RPE) were determined at baseline, 4 min, 6 min, and maximal stages. HR recovery (HRR; maximum postexercise) was determined at 1 and 2 min postexercise. Preexercise fasted aortic waveforms (applanation tonometry), plasma leptin, nitrate/nitrite (nitric oxide bioavailability), and body composition (dual X-ray, DXA) were also collected. Int had lower VÌo2max and plasma nitrate (both P ≤ 0.02) than Morn. No difference in preexercise BP, aortic waveforms, or body composition were noted between groups, although higher plasma leptin was seen in Int compared with Morn (P = 0.04). Although Int had higher brachial DBP and MAP across exercise stages (both P ≤ 0.05) and higher HR, RPE, and RPP at 6 min of exercise (all P ≤ 0.05), covarying for VÌo2max nullified the BP, but not HR or RPE, difference. HRR was greater in Morn independent of VÌo2max (P = 0.046). Fasted leptin correlated with HR at exercise stage 4 (r = 0.421, P = 0.041) and 6 min (r = 0.593, P = 0.002). This observational study suggests that Int has exaggerated BP and HR responses to exercise compared with Morn, although fitness abolished BP differences.NEW & NOTEWORTHY This study compares blood pressure and heart rate responses with graded, incremental exercise between morning and intermediate chronotype adults with obesity. Herein, blood pressure responses to exercise were elevated in intermediate compared with morning chronotype, although VÌo2max abolished this observation. However, heart rate responses to exercise were higher in intermediate vs. morning chronotypes independent of fitness. Collectively, this exercise hemodynamic response among intermediate chronotype may be related to reduced aerobic fitness, altered nitric oxide metabolism, and/or elevated aortic waveforms.
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Enfermedades Cardiovasculares , Prueba de Esfuerzo , Adulto , Femenino , Humanos , Presión Sanguínea/fisiología , Leptina , Frecuencia Cardíaca/fisiología , Cronotipo , Nitratos , Óxido Nítrico , Obesidad/diagnósticoRESUMEN
Macrophage efferocytosis of apoptotic neutrophils (PMNs) plays a key role in the resolution of inflammation. In these studies, we describe a novel flow cytometric method to assess efferocytosis of apoptotic PMNs. Resident alveolar macrophages and PMNs were collected from lungs of mice exposed to inhaled ozone (0.8 ppm, 3 h) followed by lipopolysaccharide (3 mg/kg, i.v.) to induce acute lung injury. PMNs were labeled with PKH26 or DilC18(5)-DS (D12730) cell membrane dye and then incubated with resident alveolar macrophages at a ratio of 5:1. After 90 min, macrophage efferocytosis was analyzed by flow cytometry and confirmed by confocal microscopy. Whereas alveolar macrophages incubated with D12730-labeled PMNs could readily be identified as efferocytotic or non-efferocytotic, this was not possible with PKH26 labeled PMNs due to confounding macrophage autofluorescence. Using D12730 labeled PMNs, subsets of resident alveolar macrophages were identified with varying capacities to perform efferocytosis, which may be linked to the activation state of these cells. Future applications of this method will be useful in assessing the role of efferocytosis in the resolution of inflammation in response to toxicant exposure.
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Macrófagos Alveolares , Neutrófilos , Ratones , Animales , Neutrófilos/metabolismo , Macrófagos Alveolares/metabolismo , Citometría de Flujo , Fagocitosis , Inflamación/metabolismo , ApoptosisRESUMEN
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/6J 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.
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Cloro , Lesión Pulmonar , Ratones , Masculino , Animales , Cloro/toxicidad , Ratones Endogámicos C57BL , Pulmón , Macrófagos , Líquido del Lavado Bronquioalveolar , Estrés Oxidativo , Metabolismo EnergéticoRESUMEN
Nitric oxide can interact with a wide range of proteins including many that are involved in metabolism. In this review we have summarized the effects of NO on glycolysis, fatty acid metabolism, the TCA cycle, and oxidative phosphorylation with reference to skeletal muscle. Low to moderate NO concentrations upregulate glucose and fatty acid oxidation, while higher NO concentrations shift cellular reliance toward a fully glycolytic phenotype. Moderate NO production directly inhibits pyruvate dehydrogenase activity, reducing glucose-derived carbon entry into the TCA cycle and subsequently increasing anaploretic reactions. NO directly inhibits aconitase activity, increasing reliance on glutamine for continued energy production. At higher or prolonged NO exposure, citrate accumulation can inhibit multiple ATP-producing pathways. Reduced TCA flux slows NADH/FADH entry into the ETC. NO can also inhibit the ETC directly, further limiting oxidative phosphorylation. Moderate NO production improves mitochondrial efficiency while improving O2 utilization increasing whole-body energy production. Long-term bioenergetic capacity may be increased because of NO-derived ROS, which participate in adaptive cellular redox signaling through AMPK, PCG1-α, HIF-1, and NF-κB. However, prolonged exposure or high concentrations of NO can result in membrane depolarization and opening of the MPT. In this way NO may serve as a biochemical rheostat matching energy supply with demand for optimal respiratory function.
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Metabolismo Energético , Óxido Nítrico , Metabolismo Energético/fisiología , Glucólisis/fisiología , Glucosa/metabolismo , Ácidos GrasosRESUMEN
Acute lung injury (ALI) is characterized by epithelial damage, barrier dysfunction, and pulmonary edema. Macrophage activation and failure to resolve play a role in ALI; thus, macrophage phenotype modulation is a rational target for therapeutic intervention. Large, lipid-laden macrophages have been observed in various injury models, including intratracheal bleomycin (ITB), suggesting that lipid storage may play a role in ALI severity. The endoplasmic reticulum-associated enzyme acyl coenzyme A acyltransferase-1 (Acat-1/Soat1) is highly expressed in macrophages, where it catalyzes the esterification of cholesterol, leading to intracellular lipid accumulation. We hypothesize that inhibition of Acat-1 will reduce macrophage activation and improve outcomes of lung injury in ITB. K-604, a selective inhibitor of Acat-1, was used to reduce cholesterol esterification and hence lipid accumulation in response to ITB. Male and female C57BL6/J mice (n = 16-21/group) were administered control, control + K-604, ITB, or ITB + K-604 on d0, control or K-604 on d3, and were sacrificed on day 7. ITB caused significant body weight loss and an increase in cholesterol accumulation in bronchoalveolar lavage cells. These changes were mitigated by Acat-1 inhibition. K-604 also significantly reduced ITB-induced alveolar thickening. Surfactant composition was normalized as indicated by a significant decrease in phospholipid: SP-B ratio in ITB+K-604 compared with ITB. K-604 administration preserved mature alveolar macrophages, decreased activation in response to ITB, and decreased the percentage mature and pro-fibrotic interstitial macrophages. These results show that inhibition of Acat-1 in the lung is associated with reduced inflammatory response to ITB-mediated lung injury. SIGNIFICANCE STATEMENT: Acyl coenzyme A acyltransferase-1 (Acat-1) is critical to lipid droplet formation, and thus inhibition of Acat-1 presents as a pharmacological target. Intratracheal administration of K-604, an Acat-1 inhibitor, reduces intracellular cholesterol ester accumulation in lung macrophages, attenuates inflammation and macrophage activation, and normalizes mediators of surface-active function after intratracheal bleomycin administration in a rodent model. The data presented within suggest that inhibition of Acat-1 in the lung improves acute lung injury outcomes.
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Lesión Pulmonar Aguda , Neumonía , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Acilcoenzima A , Aciltransferasas , Animales , Bencimidazoles , Bleomicina , Colesterol , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Esterol O-Aciltransferasa/genéticaRESUMEN
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.
RESUMEN
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.
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Lesión Pulmonar Aguda , Mecloretamina , Lesión Pulmonar Aguda/patología , Animales , Ciclooxigenasa 2/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Irritantes/toxicidad , Lípidos , Pulmón , Activación de Macrófagos , Masculino , Mecloretamina/toxicidad , RatonesRESUMEN
Activated macrophages have been implicated in lung injury and fibrosis induced by the cytotoxic alkylating agent, nitrogen mustard (NM). Herein, we determined if macrophage activation is associated with histone modifications and altered miRNA expression. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in increases in phosphorylation of H2A.X in lung macrophages at 1 d and 3 d post-exposure. This DNA damage response was accompanied by methylation of histone (H) 3 lysine (K) 4 and acetylation of H3K9, marks of transcriptional activation, and methylation of H3K36 and H3K9, marks associated with transcriptional repression. Increases in histone acetyl transferase and histone deacetylase were also observed in macrophages 1 d and 28 d post-NM exposure. PCR array analysis of miRNAs (miR)s involved in inflammation and fibrosis revealed unique and overlapping expression profiles in macrophages isolated 1, 3, 7, and 28 d post-NM. An IPA Core Analysis of predicted mRNA targets of differentially expressed miRNAs identified significant enrichment of Diseases and Functions related to cell cycle arrest, apoptosis, cell movement, cell adhesion, lipid metabolism, and inflammation 1 d and 28 d post NM. miRNA-mRNA interaction network analysis revealed highly connected miRNAs representing key upstream regulators of mRNAs involved in significantly enriched pathways including miR-34c-5p and miR-27a-3p at 1 d post NM and miR-125b-5p, miR-16-5p, miR-30c-5p, miR-19b-3p and miR-148b-3p at 28 d post NM. Collectively, these data show that NM promotes histone remodeling and alterations in miRNA expression linked to lung macrophage responses during inflammatory injury and fibrosis.
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Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Histonas/biosíntesis , Activación de Macrófagos/efectos de los fármacos , Mecloretamina/toxicidad , MicroARNs/biosíntesis , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Expresión Génica , Histonas/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Activación de Macrófagos/fisiología , Masculino , Ratones , MicroARNs/genética , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Pulmón/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Neumonía/prevención & control , Fibrosis Pulmonar/prevención & control , Inhibidores del Factor de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Bleomicina , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrosis , Pulmón/metabolismo , Pulmón/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Fenotipo , Fosfolípidos/metabolismo , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Alquenos/farmacología , Bleomicina , Ácidos Grasos/farmacología , Inflamación/prevención & control , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Animales , Líquido del Lavado Bronquioalveolar , Inflamación/inducido químicamente , Inflamación/patología , Antígenos Comunes de Leucocito/metabolismo , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosfolípidos/metabolismo , Pérdida de Peso/efectos de los fármacos , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/biosíntesis , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
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 (0.08â¯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+Ly6Chi 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+Ly6Clo anti-inflammatory/pro-fibrotic macrophages also increased in the lung after NM peaking at 14 d, a time coordinate with increases in TGFß 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.
Asunto(s)
Lesión Pulmonar Aguda/inducido químicamente , Sustancias para la Guerra Química/toxicidad , Pulmón/patología , Mecloretamina/toxicidad , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Animales , Modelos Animales de Enfermedad , Estudios de Factibilidad , Femenino , Fibrosis , Humanos , Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacosRESUMEN
RATIONALE: A primary goal of therapy for patients with peripheral artery disease (PAD) and intermittent claudication is increased ambulatory function. Supervised exercise rehabilitation was recently shown to confer superior walking benefits to pharmacological or surgical interventions. Increases in plasma inorganic nitrite, via oral nitrate, have been shown to increase exercise performance in both human and animal models, especially in hypoxic conditions. OBJECTIVE: To determine whether a 36-session exercise rehabilitation program while consuming oral inorganic nitrate (4.2 mmol concentrated beetroot juice) would produce superior benefits over exercise plus placebo in pain-free walking and markers of increased skeletal muscle perfusion in patients with PAD and intermittent claudication. METHODS AND RESULTS: This was a randomized, double-blind, per-protocol study design. After the 12-week protocol, claudication onset time on a maximal treadmill test increased by 59.2±57.3 s for the exercise plus placebo group (n=13) and by 180.3±46.6 s for the exercise plus beetroot juice group (n=11; P≤0.05). This produced a between treatment medium to large standardized effect size (Cohen d) of 0.62 (95% CI, -0.23 to +1.44). The data for 6-minute walk distance showed a similar pattern with increases of 24.6±12.1 and 53.3±19.6 m ( P≤0.05) in the exercise plus placebo and exercise plus beetroot juice groups, respectively. Measures of gastrocnemius perfusion, including ankle-brachial index, peak reactive hyperemic blood flow, and tissue deoxygenation characteristics, during exercise (assessed my near-infrared spectroscopy) all changed significantly for the exercise plus beetroot juice group with moderate-to-large effect sizes over exercise plus placebo changes. CONCLUSIONS: Although it is premature to speculate on overall clinical utility of a nitrate-based therapy for PAD, this early pilot study evidence is encouraging. Specifically, our data suggests that increasing plasma nitrite before exercise may allow PAD subjects to train with less pain, at higher workloads for longer durations at each training session, thereby maximizing the beneficial peripheral vascular and skeletal muscle adaptations. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01684930 and NCT01785524.
Asunto(s)
Beta vulgaris , Terapia por Ejercicio/métodos , Tolerancia al Ejercicio , Jugos de Frutas y Vegetales , Claudicación Intermitente/rehabilitación , Extremidad Inferior/irrigación sanguínea , Enfermedad Arterial Periférica/rehabilitación , Raíces de Plantas , Anciano , Biomarcadores/sangre , Método Doble Ciego , Femenino , Hemodinámica , Humanos , Claudicación Intermitente/sangre , Claudicación Intermitente/diagnóstico , Claudicación Intermitente/fisiopatología , Masculino , Persona de Mediana Edad , Nitritos/sangre , North Carolina , Enfermedad Arterial Periférica/sangre , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/fisiopatología , Proyectos Piloto , Recuperación de la Función , Flujo Sanguíneo Regional , Factores de Tiempo , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S-nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2, which is reduced 10-fold by SP-D OE-BAL. S-nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20, Cxcl1, and Vcam1, that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a, Nos2, Tnf, and Ptgs2, which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.
Asunto(s)
Macrófagos Alveolares/inmunología , Compuestos Nitrosos/metabolismo , Infecciones por Pneumocystis/genética , Procesamiento Proteico-Postraduccional , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Quimiocina CCL20/genética , Quimiocina CCL20/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Femenino , Inmunidad Innata , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Compuestos Nitrosos/inmunología , Fenotipo , Pneumocystis/crecimiento & desarrollo , Pneumocystis/patogenicidad , Infecciones por Pneumocystis/inmunología , Infecciones por Pneumocystis/metabolismo , Infecciones por Pneumocystis/microbiología , Proteína D Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/inmunología , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Pulmonary lymphangioleiomyomatosis (LAM) is a slow-progressing metastatic disease that is driven by mutations in the tumor suppressor tuberous sclerosis complex 1/2 (TSC1/2). Rapamycin inhibits LAM cell proliferation and is the only approved treatment, but it cannot cause the regression of existing lesions and can only stabilize the disease. However, in other cancers, immunotherapies such as checkpoint blockade against PD-1 and its ligand PD-L1 have shown promise in causing tumor regression and even curing some patients. Thus, we asked whether PD-L1 has a role in LAM progression. In vitro, PD-L1 expression in murine Tsc2-null cells is unaffected by mTOR inhibition with torin but can be upregulated by IFN-γ. Using immunohistochemistry and single-cell flow cytometry, we found increased PD-L1 expression both in human lung tissue from patients with LAM and in Tsc2-null lesions in a murine model of LAM. In this model, PD-L1 is highly expressed in the lung by antigen-presenting and stromal cells, and activated T cells expressing PD-1 infiltrate the affected lung. In vivo treatment with anti-PD-1 antibody significantly prolongs mouse survival in the model of LAM. Together, these data demonstrate that PD-1/PD-L1-mediated immunosuppression may occur in LAM, and suggest new opportunities for therapeutic targeting that may provide benefits beyond those of rapamycin.
Asunto(s)
Antígeno B7-H1/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Linfangioleiomiomatosis/metabolismo , Esclerosis Tuberosa/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/inmunología , Estudios de Casos y Controles , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/inmunología , Linfangioleiomiomatosis/patología , Ratones , Ratones Endogámicos C57BL , Esclerosis Tuberosa/tratamiento farmacológico , Esclerosis Tuberosa/inmunología , Esclerosis Tuberosa/patología , Regulación hacia ArribaRESUMEN
Both aging and chronic inflammation produce complex structural and biochemical alterations to the lung known to impact work of breathing. Mice deficient in surfactant protein D (Sftpd) develop progressive age-related lung pathology characterized by tissue destruction/remodeling, accumulation of foamy macrophages and alteration in surfactant composition. This study proposes to relate changes in tissue structure seen in normal aging and in chronic inflammation to altered lung mechanics using a computational model. Alterations in lung function in aging and Sftpd -/- mice have been inferred from fitting simple mechanical models to respiratory impedance data (Zrs), however interpretation has been confounded by the simultaneous presence of multiple coexisting pathophysiologic processes. In contrast to the inverse modeling approach, this study uses simulation from experimental measurements to recapitulate how aging and inflammation alter Zrs. Histologic and mechanical measurements were made in C57BL6/J mice and congenic Sftpd-/- mice at 8, 27 and 80 weeks of age (n = 8/group). An anatomic computational model based on published airway morphometry was developed and Zrs was simulated between 0.5 and 20 Hz. End expiratory pressure dependent changes in airway caliber and recruitment were estimated from mechanical measurements. Tissue elements were simulated using the constant phase model of viscoelasticity. Baseline elastance distribution was estimated in 8-week-old wild type mice, and stochastically varied for each condition based on experimentally measured alteration in elastic fiber composition, alveolar geometry and surfactant composition. Weighing reduction in model error against increasing model complexity allowed for identification of essential features underlying mechanical pathology and their contribution to Zrs. Using a maximum likelihood approach, alteration in lung recruitment and diminished elastic fiber density were shown predictive of mechanical alteration at airway opening, to a greater extent than overt acinar wall destruction. Model-predicted deficits in PEEP-dependent lung recruitment correlate with altered lung lining fluid composition independent of age or genotype.