Altered amino acid profile in patients with SARS-CoV-2 infection.

Low plasma arginine bioavailability has been implicated in endothelial dysfunction and immune dysregulation. The role of arginine in COVID-19 is unknown, but could contribute to cellular damage if low. Our objective was to determine arginine bioavailability in adults and children with COVID-19 vs. healthy controls. We hypothesized that arginine bioavailability would be low in patients with COVID-19 and multisystem inflammatory syndrome in children (MIS-C). We conducted a prospective observational study of three patient cohorts; arginine bioavailability was determined in asymptomatic healthy controls, adults hospitalized with COVID-19, and hospitalized children/adolescents <21 y old with COVID-19, MIS-C, or asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection identified on admission screen. Mean patient plasma amino acids were compared to controls using the Student's t test. Arginine-to-ornithine ratio, a biomarker of arginase activity, and global arginine bioavailability ratio (GABR, arginine/[ornithine+citrulline]) were assessed in all three groups. A total of 80 patients were included (28 controls, 32 adults with COVID-19, and 20 pediatric patients with COVID-19/MIS-C). Mean plasma arginine and arginine bioavailability ratios were lower among adult and pediatric patients with COVID-19/MIS-C compared to controls. There was no difference between arginine bioavailability in children with COVID-19 vs. MIS-C. Adults and children with COVID-19 and MIS-C in our cohort had low arginine bioavailability compared to healthy adult controls. This may contribute to immune dysregulation and endothelial dysfunction in COVID-19. Low arginine-to-ornithine ratio in patients with COVID-19 or MIS-C suggests an elevation of arginase activity. Further study is merited to explore the role of arginine dysregulation in COVID-19.

Pioglitazone Reverses Alcohol-Induced Alveolar Macrophage Phagocytic Dysfunction.

Alcohol use disorders (AUD) increase susceptibility to respiratory infections by 2- to 4-fold in part because of impaired alveolar macrophage (AM) immune function. Alcohol causes AM oxidative stress, diminishing AM phagocytic capacity and clearance of microbes from the alveolar space. Alcohol increases AM NADPH oxidases (Noxes), primary sources of AM oxidative stress, and reduces peroxisome proliferator-activated receptor γ (PPARγ) expression, a critical regulator of AM immune function. To investigate the underlying mechanisms of these alcohol-induced AM derangements, we hypothesized that alcohol stimulates CCAAT/enhancer-binding protein ß (C/EBPß) to suppress Nox-related microRNAs (miRs), thereby enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. Furthermore, we postulated that pharmacologic PPARγ activation with pioglitazone would inhibit C/EBPß and attenuate alcohol-induced AM dysfunction. AM isolated from human AUD subjects or otherwise healthy control subjects were examined. Compared with control AM, alcohol activated AM C/EBPß, decreased Nox1-related miR-1264 and Nox2-related miR-107, and increased Nox1, Nox2, and Nox4 expression and activity. These alcohol-induced AM derangements were abrogated by inhibition of C/EBPß, overexpression of miR-1264 or miR-107, or pioglitazone treatment. These findings define novel molecular mechanisms of alcohol-induced AM dysfunction mediated by C/EBPß and Nox-related miRs that are amenable to therapeutic targeting with PPARγ ligands. These results demonstrate that PPARγ ligands provide a novel and rapidly translatable strategy to mitigate susceptibility to respiratory infections and related morbidity in individuals with AUD.

Increasing nitric oxide bioavailability fails to improve collateral vessel formation in humanized sickle cell mice.

Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5-7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5-7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4-5), increased necrosis score (P < 0.05, n = 4-5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4-5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.

Impact of arginine therapy on mitochondrial function in children with sickle cell disease during vaso-occlusive pain.

Altered mitochondrial function occurs in sickle cell disease (SCD), due in part to low nitric oxide (NO) bioavailability. Arginine, the substrate for NO production, becomes acutely deficient in SCD patients with vaso-occlusive pain episodes (VOE). To determine if arginine improves mitochondrial function, 12 children with SCD-VOE (13.6 ± 3 years; 67% male; 75% hemoglobin-SS) were randomized to 1 of 3 arginine doses: (1) 100 mg/kg IV 3 times/day (TID); (2) loading dose (200 mg/kg) then 100 mg/kg TID; or (3) loading dose (200 mg/kg) followed by continuous infusion (300 mg/kg per day) until discharge. Platelet-rich plasma mitochondrial activity, protein expression, and protein-carbonyls were measured from emergency department (ED) presentation vs discharge. All VOE subjects at ED presentation had significantly decreased complex-V activity compared to a steady-state cohort. Notably, complex-V activity was increased at discharge in subjects from all 3 arginine-dosing schemes; greatest increase occurred with a loading dose (P < .001). Although complex-IV and citrate synthase activities were similar in VOE platelets vs steady state, enzyme activities were significantly increased in VOE subjects after arginine-loading dose treatment. Arginine also decreased protein-carbonyl levels across all treatment doses (P < .01), suggesting a decrease in oxidative stress. Arginine therapy increases mitochondrial activity and reduces oxidative stress in children with SCD/VOE. This trial was registered at www.clinicaltrials.gov as #NCT02536170.

TGF-ß1 Suppresses the Type I IFN Response and Induces Mitochondrial Dysfunction in Alveolar Macrophages.

TGF-ß1 is a pleiotropic cytokine with an established role in fibrosis; however, the immunosuppressive effects of TGF-ß1 are less characterized. Elevated levels of TGF-ß1 are found in patients with acute and chronic lung diseases, and the underlying disease processes are exacerbated by respiratory viral infections. The alveolar macrophage is the first line of cellular defense against respiratory viral infections, and its response to infections is dependent on environmental cues. Using the mouse alveolar macrophage line, MH-S, and human CD14+ monocyte-derived macrophages, we examined the effects of TGF-ß1 on the type I IFN antiviral response, macrophage polarization, and mitochondrial bioenergetics following a challenge with human respiratory syncytial virus (RSV). Our results showed that TGF-ß1 treatment of macrophages decreased the antiviral and proinflammatory response, and suppressed basal, maximal, spare mitochondrial respiration, and mitochondrial ATP production. Challenge with RSV following TGF-ß1 treatment further exacerbated mitochondrial dysfunction. The TGF-ß1 and TGF-ß1+RSV-treated macrophages had a higher frequency of apoptosis and diminished phagocytic capacity, potentially through mitochondrial stress. Disruption of TGF-ß1 signaling or rescue of mitochondrial respiration may be novel therapeutically targetable pathways to improve macrophage function and prevent secondary bacterial infections that complicate viral respiratory infections.

Targeting HIF-1α in combination with PPARα activation and postnatal factors promotes the metabolic maturation of human induced pluripotent stem cell-derived cardiomyocytes.

Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca2+ transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.

Ethyl linolenate is elevated in meconium of very-low-birth-weight neonates exposed to alcohol in utero.

BACKGROUND: The health implications of in utero alcohol exposure have been difficult to study in very-low-birth-weight newborns (VLBW) because of an inability to identify maternal alcohol exposure. Fatty acid ethyl esters (FAEEs) are elevated in meconium of alcohol-exposed term newborns. We hypothesized that meconium FAEEs would be similarly elevated in alcohol-exposed VLBW premature newborns. METHODS: In a retrospective cohort study of 64 VLBW neonates, newborns were classified into Non-Exposed, Any Exposure, or Weekly Exposure groups based on an in-depth structured maternal interview. Meconium FAEE concentrations were quantified via gas chromatography mass spectrometry. RESULTS: Alcohol exposure during Trimester 1 (Any Exposure) occurred in ~30% of the pregnancies, while 11% of the subjects reported drinking ≥ 1 drink/week (Weekly Exposure). Meconium ethyl linolenate was higher in Any Exposure (P = 0.01) and Weekly Exposure groups (P = 0.005) compared to the Non-Exposed VLBW group. There was a significant positive correlation between Trimester 1 drinking amounts and the concentration of meconium ethyl linolenate (P = 0.005). Adjusted receiver operating characteristic (ROC) curves evaluating ethyl linolenate to identify alcohol-exposed VLBW newborns generated areas under the curve of 88% with sensitivities of 86-89% and specificities of 83-88%. CONCLUSION: Despite prematurity, meconium FAEEs hold promise to identify the alcohol-exposed VLBW newborn.

In utero alcohol effects on foetal, neonatal and childhood lung disease.

Maternal alcohol use during pregnancy exposes both premature and term newborns to the toxicity of alcohol and its metabolites. Foetal alcohol exposure adversely effects the lung. In contrast to the adult "alcoholic lung" phenotype, an inability to identify the newborn exposed to alcohol in utero has limited our understanding of its effect on adverse pulmonary outcomes. This paper will review advances in biomarker development of in utero alcohol exposure. We will highlight the current understanding of in utero alcohol's toxicity to the developing lung and immune defense. Finally, we will present recent clinical evidence describing foetal alcohol's association with adverse pulmonary outcomes including bronchopulmonary dysplasia, viral infections such as respiratory syncytial virus and allergic asthma/atopy. With research to define alcohol's effect on the lung and translational studies accurately identifying the exposed offspring, the full extent of alcohol's effects on clinical respiratory outcomes of the newborn or child can be determined.

Peroxisome Proliferator-Activated Receptor γ Regulates Chronic Alcohol-Induced Alveolar Macrophage Dysfunction.

Peroxisome proliferator-activated receptor (PPAR) γ is critical for alveolar macrophage (AM) function. Chronic alcohol abuse causes AM phagocytic dysfunction and susceptibility to respiratory infections by stimulating nicotinamide adenine dinucleotide oxidases (Nox), transforming growth factor-ß1, and oxidative stress in the AM. Because PPARγ inhibits Nox expression, we hypothesized that alcohol reduces PPARγ, stimulating AM dysfunction. AMs were examined from: (1) patients with alcoholism or control patients; (2) a mouse model of chronic ethanol consumption; (3) PPARγ knockout mice; or (4) MH-S cells exposed to ethanol in vitro. Alcohol reduced AM PPARγ levels and increased Nox1, -2, and -4, transforming growth factor-ß1, oxidative stress, and phagocytic dysfunction. Genetic loss of PPARγ recapitulated, whereas stimulating PPARγ activity attenuated alcohol-mediated alterations in gene expression and phagocytic function, supporting the importance of PPARγ in alcohol-induced AM derangements. Similarly, PPARγ activation in vivo reduced alcohol-mediated impairments in lung bacterial clearance. Alcohol increased levels of microRNA-130a/-301a, which bind to the PPARγ 3' untranslated region to reduce PPARγ expression. MicroRNA-130a/-301a inhibition attenuated alcohol-mediated PPARγ reductions and derangements in AM gene expression and function. Alcohol-induced Toll-like receptor 4 endocytosis was reversed by PPARγ activation. These findings demonstrate that targeting PPARγ provides a novel therapeutic approach for mitigating alcohol-induced AM derangements and susceptibility to lung infection.

The Relationship Between Airway Antioxidant Levels, Alcohol Use Disorders, and Cigarette Smoking.

BACKGROUND: Alcohol use disorders (AUDs) and cigarette smoking are associated with pulmonary oxidative stress, likely related to antioxidant depletion. Pulmonary oxidative stress may adversely affect innate immunity, leading to increased pneumonia susceptibility and severity, including development of the acute respiratory distress syndrome. In people with AUDs, most of whom smoke, antioxidant therapy can potentially restore immune cell function and attenuate pneumonia development. Challenges to human investigations of antioxidant therapies include an inability to identify pulmonary oxidative stress noninvasively and the optimal route to deliver pulmonary antioxidants. We sought to determine whether bronchoalveolar lavage (BAL) measures of thiol antioxidants from a 50-ml upper airway aliquot approximated those in the alveolar space and to determine whether AUDs and/or smoking affected these relationships. METHODS: Healthy human subjects with and without AUDs, including smokers and nonsmokers, underwent BAL. Samples obtained after the first 50-ml normal saline aliquot were analyzed as representing bronchial airways; subsequent 50-ml aliquots were analyzed as representative of the alveolar space. Reduced and oxidized (GSSG) glutathione, cysteine (Cys), and its oxidized species, cystine, along with mixed disulfides (MDs) were quantified using high-performance liquid chromatography. The percent of total thiols present in their oxidized forms, and thiol redox potentials, were calculated. RESULTS: Positive correlations between upper and lower BAL fluid thiol species were observed that were most robust for GSSG (ρ = 0.85), Cys (ρ = 0.83), and MDs (ρ = 0.69), but poor for thiol redox potential measures. In contrast to nonsmokers (either with or without AUDs), in subjects with AUDs who smoked, upper BAL fluid %GSSG, Cys, and MD measures were relatively increased compared to lower. CONCLUSIONS: A small volume BAL procedure may be suitable to assess intrapulmonary oxidative stress related to thiol depletion. Factors including AUDs and smoking may disproportionately increase upper airways oxidative stress that could be relevant for therapeutic interventions.

Cysteine oxidation impairs systemic glucocorticoid responsiveness in children with difficult-to-treat asthma.

BACKGROUND: The mechanisms underlying glucocorticoid responsiveness are largely unknown. Although redox regulation of the glucocorticoid receptor (GR) has been reported, it has not been studied in asthmatic patients. OBJECTIVE: We characterized systemic cysteine oxidation and its association with inflammatory and clinical features in healthy children and children with difficult-to-treat asthma. We hypothesized that cysteine oxidation would be associated with increased markers of oxidative stress and inflammation, increased features of asthma severity, decreased clinically defined glucocorticoid responsiveness, and impaired GR function. METHODS: PBMCs were collected from healthy children (n = 16) and children with asthma (n = 118) aged 6 to 17 years. Children with difficult-to-treat asthma underwent glucocorticoid responsiveness testing with intramuscular triamcinolone. Cysteine, cystine, and inflammatory chemokines and reactive oxygen species generation were quantified, and expression and activity of the GR were assessed. RESULTS: Cysteine oxidation was present in children with difficult-to-treat asthma and accompanied by increased reactive oxygen species generation and increased CCL3 and CXCL1 mRNA expression. Children with the greatest extent of cysteine oxidation had more features of asthma severity, including poorer symptom control, greater medication use, and less glucocorticoid responsiveness despite inhaled glucocorticoid therapy. Cysteine oxidation also modified the GR protein by decreasing available sulfhydryl groups and decreasing nuclear GR expression and activity. CONCLUSIONS: A highly oxidized cysteine redox state promotes a posttranslational modification of the GR that might inhibit its function. Given that cysteine oxidation is prevalent in children with difficult-to-treat asthma, the cysteine redox state might represent a potential therapeutic target for restoration of glucocorticoid responsiveness in this population.

Receptor for advanced glycation end-products regulates lung fluid balance via protein kinase C-gp91(phox) signaling to epithelial sodium channels.

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 µg versus 2.48 ± 0.53 µg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

The relative balance of GM-CSF and TGF-ß1 regulates lung epithelial barrier function.

Lung barrier dysfunction is a cardinal feature of the acute respiratory distress syndrome (ARDS). Alcohol abuse, which increases the risk of ARDS two- to fourfold, induces transforming growth factor (TGF)-ß1, which increases epithelial permeability and impairs granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent barrier integrity in experimental models. We hypothesized that the relative balance of GM-CSF and TGF-ß1 signaling regulates lung epithelial barrier function. GM-CSF and TGF-ß1 were tested separately and simultaneously for their effects on lung epithelial cell barrier function in vitro. TGF-ß1 alone caused an ∼ 25% decrease in transepithelial resistance (TER), increased paracellular flux, and was associated with projections perpendicular to tight junctions ("spikes") containing claudin-18 that colocalized with F-actin. In contrast, GM-CSF treatment induced an ∼ 20% increase in TER, decreased paracellular flux, and showed decreased colocalization of spike-associated claudin-18 with F-actin. When simultaneously administered to lung epithelial cells, GM-CSF antagonized the effects of TGF-ß1 on epithelial barrier function in cultured cells. Given this, GM-CSF and TGF-ß1 levels were measured in bronchoalveolar lavage (BAL) fluid from patients with ventilator-associated pneumonia and correlated with markers for pulmonary edema and patient outcome. In patient BAL fluid, protein markers of lung barrier dysfunction, serum α2-macroglobulin, and IgM levels were increased at lower ratios of GM-CSF/TGF-ß1. Critically, patients who survived had significantly higher GM-CSF/TGF-ß1 ratios than nonsurviving patients. This study provides experimental and clinical evidence that the relative balance between GM-CSF and TGF-ß1 signaling is a key regulator of lung epithelial barrier function. The GM-CSF/TGF-ß1 ratio in BAL fluid may provide a concentration-independent biomarker that can predict patient outcomes in ARDS.

Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells.

Amiloride-sensitive epithelial Na(+) channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (Eh) would alter cell signaling and have an effect on ENaC open probability (Po). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG Eh became less negative (n = 21; P < 0.05). Treatment of 400 µM GSSG to the cell bath significantly decreased ENaC Po from 0.39 ± 0.06 to 0.13 ± 0.05 (n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 µM GSSG reduced ENaC Po from 0.32 ± 0.08 to 0.17 ± 0.05 (n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC Po. Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione Eh is an important determinant of alveolar fluid clearance in vivo.

Fatty acid ethyl esters disrupt neonatal alveolar macrophage mitochondria and derange cellular functioning.

BACKGROUND: Chronic alcohol exposure alters the function of alveolar macrophages (AM), impairing immune defenses in both adult and neonatal lungs. Fatty acid ethyl esters (FAEEs) are biological markers of prenatal alcohol exposure in newborns. FAEEs contribute to alcohol-induced mitochondrial (MT) damage in multiple organs. We hypothesized that in utero ethanol exposure would increase FAEEs in the neonatal lung and that direct exposure of neonatal AM to FAEEs would contribute to MT injury and cellular dysfunction. METHODS: FAEEs were measured in neonatal guinea pig lungs after ± in utero ethanol exposure via gas chromatography/mass spectrometry. The NR8383 cell line and freshly isolated neonatal guinea pig AM were exposed to ethyl oleate (EO) in vitro. MT membrane potential, MT reactive oxygen species generation (mROS), phagocytosis, and apoptosis were evaluated after exposure to EO ± the MT-specific antioxidant mito-TEMPO (mitoT) or ± the pan-caspase inhibitor Z-VAD-FMK. Whole lung FAEEs were compared using the Mann-Whitney U-test. Cellular results were analyzed using 1-way analysis of variance, followed by the Student-Newman-Keuls Method for post hoc comparisons. RESULTS: In utero ethanol significantly increased ethyl linoleate and the combinations of ethyl oleate + linoleate + linolenate (OLL), and OLL + stearate in the neonatal lung. In vitro EO caused significant MT dysfunction in both NR8383 and primary neonatal AM, as indicated by increased mROS and loss of MT membrane potential. Impaired phagocytosis and apoptosis were significantly increased in both the cell line and primary AM after EO exposure. MitoT conferred significant but only partial protection against EO-induced MT injury, as did caspase inhibition with Z-VAD-FMK. CONCLUSIONS: In utero ethanol exposure increased FAEEs in the neonatal guinea pig lung. Direct exposure to the FAEE EO significantly contributed to AM dysfunction, in part via oxidant injury to the MT and in part via secondary apoptosis.

Zinc insufficiency mediates ethanol-induced alveolar macrophage dysfunction in the pregnant female mouse.

AIMS: (a) Establish the minimum number of weeks of chronic ethanol ingestion needed to perturb zinc homeostasis, (b) Examine intracellular zinc status in the alveolar macrophages (AMs) when ethanol ingestion is combined with pregnancy, (c) Investigate whether in vitro zinc treatment reverses the effects of ethanol ingestion on the AM. METHODS: C57BL/6 female mice were fed a liquid diet (±25% ethanol-derived calories) during preconception and pregnancy. The control group was pair-fed to the ethanol group. In the isolated AMs, we measured intracellular AM zinc levels, zinc transporter expression, alternative activation and phagocytic index. Zinc acetate was added to some cells prior to analysis. RESULTS: Intracellular zinc levels in the AM decreased within 3 weeks of ethanol ingestion. After ethanol ingestion prior to and during pregnancy, zinc transporter expression and intracellular zinc levels were decreased in the AMs when compared with controls. Bacterial clearance was decreased because the AMs were alternatively activated. In vitro additions of zinc reversed these effects of ethanol. CONCLUSION: Ethanol ingestion prior to and during pregnancy perturbed AM zinc balance resulting in impaired bacterial clearance, but these effects were ameliorated by in vitro zinc treatments.

Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 µM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.

Chronic ethanol exposure alters the lung proteome and leads to mitochondrial dysfunction in alveolar type 2 cells.

The lungs can undergo irreversible damage from chronic alcohol consumption. Herein, we developed an animal model predisposed for edematous lung injury following chronic ingestion of alcohol to better understand the etiology of alcohol-related disorders. Using animal modeling, alongside high-throughput proteomic and microarray assays, we identified changes in lung protein and transcript in mice and rats, respectively, following chronic alcohol ingestion or a caloric control diet. Liquid chromatography-mass spectrometry identified several mitochondrial-related proteins in which the expression was upregulated following long-term alcohol ingestion in mice. Consistent with these observations, rat gene chip microarray analysis of alveolar cells obtained from animals maintained on a Lieber-DeCarli liquid alcohol diet confirmed significant changes in mitochondrial-related transcripts in the alcohol lung. Transmission electron microscopy revealed significant changes in the mitochondrial architecture in alcohol mice, particularly following lipopolysaccharide exposure. Chronic alcohol ingestion was also shown to worsen mitochondrial respiration, mitochondrial membrane polarization, and NAD(+)-to-NADH ratios in alveolar type 2 cells. In summary, our studies show causal connection between chronic alcohol ingestion and mitochondrial dysfunction, albeit the specific role of each of the mitochondrial-related proteins and transcripts identified in our study requires additional study.

Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition.

Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% (P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% (P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25-50 µM) activated endogenous transforming growth factor-ß1 (TGF-ß1), as evident by H2DCFDA immunofluorescence and ELISA (P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-ß1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-ß1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold (P < 0.05) and that this effect was prevented by intraperitoneal TGF-ß1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-ß1 signaling.

Exhaled breath condensate in intubated neonates--a window into the lung's glutathione status.

BACKGROUND: Analysis of exhaled breath condensates (EBC) is a non-invasive technique to evaluate biomarkers such as antioxidants in the pediatric population, but limited data exists of its use in intubated patients, particularly newborns. Currently, tracheal aspirate (TA) serves as the gold standard collection modality in critically ill newborns, but this method remains invasive. We tested the hypothesis that glutathione status would positively correlate between EBC and TA collections in intubated newborns in the Newborn Intensive Care Unit (NICU). We also hypothesized that these measurements would be associated with alveolar macrophage (AM) glutathione status in the newborn lung. METHODS: Reduced glutathione (rGSH), glutathione disulfide (GSSG), and total GSH (rGSH + (2 X GSSG)) were measured in sequential EBC and TA samples from 26 intubated newborns via high performance liquid chromatography (HPLC). Additionally, AM glutathione was evaluated via immunofluorescence. Pearson's correlation coefficient and associated 95% confidence intervals were used to quantify the associations between raw and urea-corrected concentrations in EBC and TA samples and AM staining. Statistical significance was defined as p ≤ 0.05 using two-tailed tests. The sample size was projected to allow for a correlation coefficient of 0.5, with 0.8 power and alpha of 0.05. RESULTS: EBC was obtainable from intubated newborns without adverse clinical events. EBC samples demonstrated moderate to strong positive correlations with TA samples in terms of rGSH, GSSG and total GSH. Positive correlations between the two sampling sites were observed in both raw and urea-corrected concentrations of rGSH, GSSG and total GSH. AM glutathione staining moderately correlated with GSSG and total GSH status in both the TA and EBC. CONCLUSIONS: GSH status in EBC samples of intubated newborns significantly correlated with the GSH status of the TA sample and was reflective of cellular GSH status in this cohort of neonatal patients. Non-invasive EBC sampling of intubated newborns holds promise for monitoring antioxidant status such as GSH in the premature lung. Further studies are necessary to evaluate the potential relationships between EBC biomarkers in the intubated premature newborn and respiratory morbidities.