Frank L. Harris

Ethanol Induces Oxidative Stress in Alveolar Macrophages via Upregulation of NADPH Oxidases

Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8–10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20–35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients’ bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2′,7′-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.

Fetal alcohol exposure impairs alveolar macrophage function via decreased glutathione availability.

Immature function of the alveolar macrophage increases the risk of pulmonary infections in premature newborns. In utero alcohol increases fetal systemic oxidative stress. Because the premature lung is deficient in glutathione (GSH), we hypothesized that chronic in utero alcohol (ethanol) exposure exacerbates the oxidative stress within the developing lung, thereby impairing alveolar macrophage function. Additionally, we evaluated the effects of in vivo and in vitro GSH availability on ethanol-exposed macrophage function. Using a guinea pig model of chronic in utero ethanol exposure, fetal epithelial lining fluid (ELF) and alveolar macrophage GSH were decreased with increased markers of oxidative stress. Ethanol-exposed macrophage exhibited impaired phagocytosis and increased apoptosis compared with gestational control. When the GSH precursor S-adenosyl-methionine (SAM) was added to the maternal drinking water containing ethanol, fetal ELF and macrophage GSH were maintained and ELF oxidative stress diminished. In vivo maternal SAM therapy maintained macrophage phagocytosis and decreased apoptosis. In vitro GSH supplements also improved phagocytosis and viability in both premature and ethanol-exposed macrophage. This suggested that in utero ethanol impaired premature macrophage function and viability via decreased GSH availability. Furthermore, GSH supplementation during and after ethanol exposure improved fetal macrophage function and viability. These results add a new dimension to the detrimental effects of fetal alcohol exposure on the developing alveolar macrophage, raising the possibility of GSH therapy to augment premature alveolar macrophage function.

In Utero Ethanol Exposure Impairs Defenses Against Experimental Group B Streptococcus in the Term Guinea Pig Lung

BACKGROUND The effects of fetal alcohol exposure on the risks of neonatal lung injury and infection remain under investigation. The resident alveolar macrophage (AM) is the first line of immune defense against pulmonary infections. In utero ethanol (ETOH) exposure deranges the function of both premature and term guinea pig AM. We hypothesized that fetal ETOH exposure would increase the risk of pulmonary infection in vivo. METHODS We developed a novel in vivo model of group B Streptococcus (GBS) pneumonia using our established guinea pig model of fetal ETOH exposure. Timed-pregnant guinea pigs were pair fed +/-ETOH and some were supplemented with the glutathione (GSH) precursor S-adenosyl-methionine (SAM-e). Term pups were given GBS intratracheally while some were pretreated with inhaled GSH prior to the experimental GBS. Neonatal lung and whole blood were evaluated for GBS while isolated AM were evaluated using fluorescent microscopy for GBS phagocytosis. RESULTS Ethanol-exposed pups demonstrated increased lung infection and sepsis while AM phagocytosis of GBS was deficient compared with control. When SAM-e was added to the maternal diet containing ETOH, neonatal lung and systemic infection from GBS was attenuated and AM phagocytosis was improved. Inhaled GSH therapy prior to GBS similarly protected the ETOH-exposed pup from lung and systemic infection. CONCLUSIONS In utero ETOH exposure impaired the neonatal lungs defense against experimental GBS, while maintaining GSH availability protected the ETOH-exposed lung. This study suggested that fetal alcohol exposure deranges the neonatal lungs defense against bacterial infection, and support further investigations into the potential therapeutic role for exogenous GSH to augment neonatal AM function.

Alcohol induced mitochondrial oxidative stress and alveolar macrophage dysfunction.

An alcohol use disorder increases the risk of invasive and antimicrobial resistant community-acquired pneumonia and tuberculosis. Since the alveolar macrophage (AM) orchestrates the immune response in the alveolar space, understanding the underlying mechanisms by which alcohol suppresses AM phagocytosis is critical to improving clinical outcomes. In the alveolar space, chronic alcohol ingestion causes severe oxidative stress and depletes antioxidants which are critical for AM function. The mitochondrion is important in maintaining cellular redox balance and providing the ATP critical for phagocytosis. The focus of this study was to understand how alcohol triggers mitochondrial reactive oxygen species (ROS), stimulates cellular oxidative stress, and induces AM dysfunction. The current study also investigated the capacity of the mitochondrial targeted antioxidant, mitoTEMPOL (mitoT), in modulating mitochondrial oxidative stress, and AM dysfunction. Using in vitro ethanol exposure and AMs from ethanol-fed mice, ethanol promoted mitochondrial dysfunction including increased mitochondrial ROS, decreased mitochondrial membrane potential, and decreased ATP. Treatment with mitoT reversed these effects. Ethanol-induced decreases in phagocytosis and cell viability were also attenuated with mitoT. Therefore, antioxidants targeted to the mitochondria have the potential to ameliorate ethanol-induced mitochondrial oxidative stress and subsequent decreases in AM phagocytosis and cell viability.

Ascorbate deficiency and oxidative stress in the alveolar type II cell

The objective of this study was to determine the impact of limited ascorbate (Asc) availability on type II cell sensitivity to oxidant stress. Guinea pigs were fed diets with or without Asc for 18 days, and type II cells were isolated. Although lung Asc was decreased by 90% in deficient animals (scorbutic), type II cell Asc was decreased by 50%. Upon treatment with 250 microM H2O2, the necrotic injury was twofold greater in scorbutic cells compared with control cells. With 100 microM H2O2 treatment, apoptotic injury was twofold greater in scorbutic cells compared with control cells. Although there was less necrotic injury in cells exposed to 95% O2, the scorbutic cells were more sensitive than control cells. Asc pretreatment protected against necrosis and apoptosis. The Asc analog isoascorbate provided partial protection and suggested that part of the protection was not chemical detoxification but was Asc specific. We conclude that limited Asc availability resulted in a functional type II cell but a cell more sensitive to oxidant-induced injury.The objective of this study was to determine the impact of limited ascorbate (Asc) availability on type II cell sensitivity to oxidant stress. Guinea pigs were fed diets with or without Asc for 18 days, and type II cells were isolated. Although lung Asc was decreased by 90% in deficient animals (scorbutic), type II cell Asc was decreased by 50%. Upon treatment with 250 μM H2O2, the necrotic injury was twofold greater in scorbutic cells compared with control cells. With 100 μM H2O2treatment, apoptotic injury was twofold greater in scorbutic cells compared with control cells. Although there was less necrotic injury in cells exposed to 95% O2, the scorbutic cells were more sensitive than control cells. Asc pretreatment protected against necrosis and apoptosis. The Asc analog isoascorbate provided partial protection and suggested that part of the protection was not chemical detoxification but was Asc specific. We conclude that limited Asc availability resulted in a functional type II cell but a cell more sensitive to oxidant-induced injury.

Alcohol induces mitochondrial redox imbalance in alveolar macrophages.

Alcohol abuse suppresses the immune responses of alveolar macrophages (AMs) and increases the risk of a respiratory infection via chronic oxidative stress and depletion of critical antioxidants within alveolar cells and the alveolar lining fluid. Although alcohol-induced mitochondrial oxidative stress has been demonstrated, the oxidation of the mitochondrial thioredoxin redox circuit in response to alcohol has not been examined. In vitro ethanol exposure of a mouse AM cell line and AMs from ethanol-fed mice demonstrated NADPH depletion concomitant with oxidation of mitochondrial glutathione and oxidation of the thioredoxin redox circuit system including thioredoxin 2 (Trx2) and thioredoxin 2 reductase (Trx2R). Mitochondrial peroxiredoxins (Prdxs), which are critical for the reduction of the thioredoxin circuit, were irreversibly hyperoxidized to an inactive form. Ethanol also decreased the mRNAs for Trx2, Trx2R, Prdx3, and Prdx5 plus the mitochondrial thiol-disulfide proteins glutaredoxin 2, glutathione reductase, and glutathione peroxidase 2. Thus, the mitochondrial thioredoxin circuit was highly oxidized by ethanol, thereby compromising the mitochondrial antioxidant capacity and ability to detoxify mitochondrial reactive oxygen species. Oxidation of the mitochondrial thioredoxin redox circuit would further compromise the transient oxidation of thiol groups within specific proteins, the basis of redox signaling, and the processes by which cells respond to oxidants. Impaired mitochondria can then jeopardize cellular function of AMs, such as phagocytosis, which may explain the increased risk of respiratory infection in subjects with an alcohol use disorder.

Elevated plasma and lung endothelial selectin levels in patients with acute respiratory distress syndrome and a history of chronic alcohol abuse.

ObjectiveActivation of endothelial cells is a critical step in the pathogenesis of acute respiratory distress syndrome (ARDS). Soluble endothelial selectin (sE-selectin), an endothelial cell–specific molecule that mediates leukocyte-endothelial cell adhesion has never been measured in the bronchoalveolar lavage fluid of critically ill patients. Based on the effects of alcohol on endothelial cell and alveolar-capillary barrier function, we hypothesized that chronic alcohol exposure may be associated with increased sE-selectin in ARDS patients. DesignProspective observational cohort study. SettingMedical and surgical intensive care units; an inpatient alcohol detoxification unit within university-affiliated hospitals. PatientsA total of 20 ARDS patients (50% with chronic alcohol abuse); seven individuals with a history of chronic alcohol abuse. InterventionPatients underwent bronchoalveolar lavage within 72 hrs of ARDS diagnosis. Individuals with a history of chronic alcohol abuse underwent bronchoalveolar lavage within 7 days of their last alcoholic beverage. A history of chronic alcohol abuse was determined by a Short Michigan Alcohol Screening Test score of ≥3 or a history of an alcohol-related diagnosis. sE-selectin was measured in plasma and bronchoalveolar lavage. Measurements and Main ResultsNeither severity of illness nor at-risk diagnosis differed by alcohol history. sE-selectin levels in the plasma of ARDS patients who abused alcohol chronically were significantly elevated compared with nonalcoholic ARDS patients (181 ng/mL [56–328] vs. 32 ng/mL [14–55], p < .01). The bronchoalveolar lavage sE-selectin levels from the patients with ARDS and alcohol abuse were also significantly elevated compared with nonalcoholic ARDS patients (1.51 ng/mL [1.09–3.11] vs. 0.69 ng/mL [0.33–0.94], p < .002) and were higher than those measured in individuals with a history of chronic alcohol abuse but without ARDS (0.46 ng/mL [0.12–2.75], p = .15). ConclusionsARDS patients who chronically abuse alcohol have elevated concentrations of sE-selectin in both the plasma and epithelial lining fluid consistent with altered endothelial cell and alveolar-capillary barrier function.

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.

Placental Fatty Acid ethyl esters are elevated with maternal alcohol use in pregnancies complicated by prematurity.

The accumulation of fatty acid ethyl esters (FAEEs) in meconium of term newborns has been described as one potential biomarker of maternal alcohol use during pregnancy. FAEEs accumulate in multiple alcohol-exposed fetal tissues and in the placenta. Limited research has focused on the identification of the premature newborn exposed to alcohol in utero. We hypothesized that maternal alcohol use occurs in a significant proportion of premature deliveries and that this exposure can be detected as elevated placental FAEEs. The goals of this study were to 1) determine the prevalence of maternal alcohol use in the premature newborn and 2) investigate whether placental FAEEs could identify those newborns with fetal alcohol exposure. This prospective observational study evaluated 80 placentas from 80 women after premature delivery. Subjects were interviewed for alcohol intake and placental FAEEs were quantified via GC/MS. Receiver Operator Characteristic (ROC) Curves were generated to evaluate the ability of placental FAEEs to predict maternal drinking during pregnancy. Adjusted ROC curves were generated to adjust for gestational age, maternal smoking, and illicit drug use. 30% of the subjects admitted to drinking alcohol during pregnancy and approximately 14% answered questions indicative of problem drinking (designated AUDIT+). The specific FAEEs ethyl stearate and linoleate, as well as combinations of oleate + linoleate + linolenate (OLL) and of OLL + stearate, were significantly (p<0.05) elevated in placentas from AUDIT+ pregnancies. Adjusted ROC Curves generated areas under the curve ranging from 88–93% with negative predictive values of 97% for AUDIT+ pregnancies. We conclude that nearly one third of premature pregnancies were alcohol-exposed, and that elevated placental FAEEs hold great promise to accurately determine maternal alcohol use, particularly heavy use, in pregnancies complicated by premature delivery.

Metabolic Consequences of Chronic Alcohol Abuse in Non-Smokers: A Pilot Study

An alcohol use disorder (AUD) is associated with an increased susceptibility to respiratory infection and injury and, upon hospitalization, higher mortality rates. Studies in model systems show effects of alcohol on mitochondrial function, lipid metabolism and antioxidant systems. The present study applied high-resolution metabolomics to test for these changes in bronchoalveolar lavage fluid (BALF) of subjects with an AUD. Smokers were excluded to avoid confounding effects and compliance was verified by cotinine measurements. Statistically significant metabolic features, differentially expressed by control and AUD subjects, were identified by statistical and bioinformatic methods. The results show that fatty acid and acylcarnitine concentrations were increased in AUD subjects, consistent with perturbed mitochondrial and lipid metabolism. Decreased concentrations of methyl-donor compounds suggest altered one-carbon metabolism and oxidative stress. An accumulation of peptides suggests proteolytic activity, which could reflect altered epithelial barrier function. Two metabolites of possible microbial origin suggest subclinical bacterial infection. Furthermore, increased diacetylspermine suggests additional metabolic perturbations, which could contribute to dysregulated alveolar macrophage function and vulnerability to infection. Together, the results show an extended metabolic consequence of AUD in the bronchoalveolar space.