Samantha M. Yeligar

Exhaled Breath Condensate: A Promising Source for Biomarkers of Lung Disease

Exhaled breath condensate (EBC) has been increasingly studied as a noninvasive research method for sampling the alveolar and airway space and is recognized as a promising source of biomarkers of lung diseases. Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines. Although EBC has great potential as a source of biomarkers in many lung diseases, the low concentrations of compounds within the EBC present challenges in sample collection and analysis. Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF. Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF. The aims of this paper are to address the characteristics of EBC; strategies to standardize EBC sample collection and review available analytical techniques for EBC analysis.

Ethanol-Induced Expression of ET-1 and ET-BR in Liver Sinusoidal Endothelial Cells and Human Endothelial Cells Involves Hypoxia-Inducible Factor-1α and MicroRNA-199

Chronic alcohol consumption leads to inflammation and cirrhosis of the liver. In this study, we observed that liver sinusoidal endothelial cells (LSEC) derived from ethanol-fed rats showed several fold increases in the mRNA expression of endothelin-1 (ET-1), hypoxia-inducible factor-1α (HIF-1α), and inflammatory cytochemokines compared with control rat LSEC. We also observed the same results in acute ethanol-treated LSEC from control rats and human dermal microvascular endothelial cells. Ethanol-mediated ET-1 expression involved NADPH oxidase and HIF-1α activation. Furthermore, ethanol increased the expression of the ET-1 cognate receptor ET-BR in Kupffer cells and THP-1 monocytic cells, which also involved HIF-1α activation. Promoter analysis and chromatin immunoprecipitation showed that hypoxia response element sites in the proximal promoter of ET-1 and ET-BR were required for the binding of HIF-1α to up-regulate their expression. We showed that microRNAs, miR-199 among several microRNAs, attenuated HIF-1α and ET-1 expression, while anti-miR-199 reversed the effects, suggesting that ethanol-induced miR-199 down-regulation may contribute to augmented HIF-1α and ET-1 expression. Our studies, for the first time to our knowledge, show that ethanol-mediated ET-1 and ET-BR expression involve HIF-1α, independent of hypoxia. Additionally, ethanol-induced ET-1 expression in rat LSEC is regulated by miR-199, while in human endothelial cells, ET-1 expression is regulated by miR-199 and miR-155, indicating that these microRNAs may function as novel negative regulators to control ET-1 transcription and, thus, homeostatic levels of ET-1 to maintain microcirculatory tone.

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.

Ethanol Augments RANTES/CCL5 Expression in Rat Liver Sinusoidal Endothelial Cells and Human Endothelial Cells via Activation of NF-κB, HIF-1α, and AP-1

Chronic alcohol consumption leads to liver inflammation and cirrhosis. Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. However, less is known about the molecular mechanisms for ethanol-induced RANTES up-regulation. In this study, we observed that liver sinusoidal endothelial cells derived from ethanol-fed rats (E-rLSECs) showed severalfold increases in RANTES and hypoxia-inducible factor 1α (HIF-1α) mRNAs compared with control rLSECs (C-rLSECs). Similar effects were seen in acute ethanol treatment of isolated rLSECs and human dermal microvascular endothelial cells. Ethanol-induced RANTES mRNA expression required ethanol metabolism, p38 MAPK, HIF-1α, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1α binding to wild-type hypoxia response elements (HREs; −31 to −9 bp) within the RANTES promoter in response to ethanol. RANTES promoter analysis showed that cis elements proximal to the transcription start site, HRE-1 (nt −22 to −19), HRE-2 (nt −32 to −29), and AP-1 (nt −250 to −244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1α binding to HRE-1. Additionally, promoter analysis revealed c-Jun, c-Jun/c-Fos, and JunD, but not JunB, bound to the AP-1 site of the RANTES promoter. Ethanol-mediated activation of NF-κB led to HIF-1α activation and concomitant RANTES expression. Plasma of ethanol-fed c-Junflox/flox-Mx-1-Cre mice showed attenuated levels of RANTES compared with ethanol-fed control mice, supporting the role of c-Jun in ethanol-induced RANTES expression. Our studies showed that ethanol-mediated RANTES/CCL5 expression occurs via HIF-1α activation independently of hypoxia. The identification of HIF-1α and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.

Alcoholism Causes Alveolar Macrophage Zinc Deficiency and Immune Dysfunction

RATIONALE Alcohol use disorders cause oxidative stress in the lower airways and increase susceptibility to pneumonia and lung injury. Currently, no therapeutic options exist to mitigate the pulmonary consequences of alcoholism. OBJECTIVES We recently determined in an animal model that alcohol ingestion impairs pulmonary zinc metabolism and causes alveolar macrophage immune dysfunction. The objective of this research is to determine the effects of alcoholism on zinc bioavailability and alveolar macrophage function in human subjects. METHODS We recruited otherwise healthy alcoholics (n = 17) and matched control subjects (n = 17) who underwent bronchoscopy for isolation of alveolar macrophages, which were analyzed for intracellular zinc, phagocytic function, and surface expression of granulocyte-macrophage colony-stimulating factor receptor; all three of these indices are decreased in experimental models. MEASUREMENTS AND MAIN RESULTS Alcoholic subjects had normal serum zinc, but significantly decreased alveolar macrophage intracellular zinc levels (adjusted means [SE], 718 [41] vs. 948 [25] RFU/cell; P < 0.0001); bacterial phagocytosis (adjusted means [SE], 1,027 [48] vs. 1,509 [76] RFU/cell; P < 0.0001); and expression of granulocyte-macrophage colony-stimulating factor receptor β subunit (adjusted means [SE], 1,471 [42] vs. 2,114 [35] RFU/cell; P < 0.0001]. Treating alveolar macrophages with zinc acetate and glutathione in vitro increased intracellular zinc levels and improved their phagocytic function. CONCLUSIONS These novel clinical findings provide evidence that alcohol abuse is associated with significant zinc deficiency and immune dysfunction within the alveolar space and suggest that dietary supplementation with zinc and glutathione precursors could enhance airway innate immunity and decrease the risk for pneumonia or lung injury in these vulnerable individuals.

Chronic-Alcohol-Abuse-Induced Oxidative Stress in the Development of Acute Respiratory Distress Syndrome

Chronic alcohol ingestion increases the risk of developing acute respiratory distress syndrome (ARDS), a severe form of acute lung injury, characterized by alveolar epithelial and endothelial barrier disruption and intense inflammation. Alcohol abuse is also associated with a higher incidence of sepsis or pneumonia resulting in a higher rate of admittance to intensive care, longer inpatient stays, higher healthcare costs, and a 2–4 times greater mortality rate. Chronic alcohol ingestion induced severe oxidative stress associated with increased ROS generation, depletion of the critical antioxidant glutathione (GSH), and oxidation of the thiol/disulfide redox potential in the alveolar epithelial lining fluid and exhaled breath condensate. Across intracellular and extracellular GSH pools in alveolar type II cells and alveolar macrophages, chronic alcohol ingestion consistently induced a 40–60 mV oxidation of GSH/GSSG suggesting that the redox potentials of different alveolar GSH pools are in equilibrium. Alcohol-induced GSH depletion or oxidation was associated with impaired functions of alveolar type II cells and alveolar macrophages but could be reversed by restoring GSH pools in the alveolar lining fluid. The aims of this paper are to address the mechanisms for alcohol-induced GSH depletion and oxidation and the subsequent effects in alveolar barrier integrity, modulation of the immune response, and apoptosis.

PPARγ Ligands Regulate NADPH Oxidase, eNOS, and Barrier Function in the Lung Following Chronic Alcohol Ingestion

BACKGROUND Chronic alcohol ingestion increases the incidence and severity of the acute respiratory distress syndrome (ARDS), where reactive species contribute to alveolar-capillary barrier dysfunction and noncardiogenic pulmonary edema. Previous studies demonstrated that chronic alcohol ingestion increased lung NADPH oxidase and endothelial nitric oxide synthase (eNOS) expression and that ligands for the peroxisome proliferator-activated receptor gamma (PPARγ) reduced NADPH oxidase expression. Therefore, we hypothesized that the PPARγ ligand, rosiglitazone, would attenuate alcohol-induced NADPH oxidase expression and pulmonary barrier dysfunction. METHODS C57Bl/6 mice were treated ± alcohol in drinking water (20% w/v) for 12 weeks. During the final week of alcohol treatment, mice were gavaged with rosiglitazone (10 mg/kg/d) or vehicle. Selected animals were treated twice with lipopolysaccharide (LPS, 2 mg/kg IP) prior to sacrifice. Pulmonary barrier dysfunction was estimated from protein content of bronchoalveolar lavage (BAL) fluid. RESULTS LPS treatment increased BAL protein in alcohol-fed but not control mice, and rosiglitazone attenuated LPS and alcohol-induced pulmonary barrier dysfunction. Alcohol- and LPS-induced increases in lung eNOS, Nox1, and Nox4 expression were attenuated by rosiglitazone. In vitro, alcohol (0.10% w/v) increased H(2)O(2) production, barrier dysfunction, eNOS, Nox1, and Nox4 expression in human umbilical vein endothelial cell (HUVEC) monolayers, effects also attenuated by rosiglitazone (10 μM). Alcohol-induced HUVEC barrier dysfunction was attenuated by inhibition of NOS or addition of the eNOS cofactor, tetrahydrobiopterin. CONCLUSIONS These results indicate that PPARγ activation reduced expression of eNOS, Nox1, Nox4, the production of reactive species, and barrier dysfunction caused by chronic alcohol ingestion and suggest that PPARγ represents a novel therapeutic target for strategies designed to reduce the risk of lung injury in patients with a history of chronic alcohol ingestion.

Alcohol and lung injury and immunity

Annually, excessive alcohol use accounts for more than

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer

220 billion in economic costs and 80,000 deaths, making excessive alcohol use the third leading lifestyle-related cause of death in the US. Patients with an alcohol-use disorder (AUD) also have an increased susceptibility to respiratory pathogens and lung injury, including a 2-4-fold increased risk of acute respiratory distress syndrome (ARDS). This review investigates some of the potential mechanisms by which alcohol causes lung injury and impairs lung immunity. In intoxicated individuals with burn injuries, activation of the gut-liver axis drives pulmonary inflammation, thereby negatively impacting morbidity and mortality. In the lung, the upper airway is the first checkpoint to fail in microbe clearance during alcohol-induced lung immune dysfunction. Brief and prolonged alcohol exposure drive different post-translational modifications of novel proteins that control cilia function. Proteomic approaches are needed to identify novel alcohol targets and post-translational modifications in airway cilia that are involved in alcohol-dependent signal transduction pathways. When the upper airway fails to clear inhaled pathogens, they enter the alveolar space where they are primarily cleared by alveolar macrophages (AM). With chronic alcohol ingestion, oxidative stress pathways in the AMs are stimulated, thereby impairing AM immune capacity and pathogen clearance. The epidemiology of pneumococcal pneumonia and AUDs is well established, as both increased predisposition and illness severity have been reported. AUD subjects have increased susceptibility to pneumococcal pneumonia infections, which may be due to the pro-inflammatory response of AMs, leading to increased oxidative stress.

Alcohol and inflammatory responses: Highlights of the 2015 Alcohol and Immunology Research Interest Group (AIRIG) meeting

Significance The present work establishes that the addition of the prosthetic group lipoic acid to catabolic enzymes is a dynamically regulated posttranslational modification that increases metabolic plasticity under hypoxia and in cancer cells. We show that that the polymerase-δ interacting protein 2 (Poldip2) is an oxygen-sensitive protein that regulates the lipoylation and activation of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additionally, our work reveals that mitochondrial peptidases participate in an integrated response needed for metabolic adaptation. This study positions Poldip2 as a key regulator of mitochondrial function and cell metabolism. Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α–mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.