Alcohol potentiates RSV-mediated injury to ciliated airway epithelium.

Alcohol impairs resolution of respiratory viral infections. Numerous immune response pathways are altered in response to alcohol misuse, including alcohol-induced ciliary dysfunction in the lung. We hypothesized that mucociliary clearance-mediated innate immunity to respiratory syncytial virus (RSV) would be compromised by alcohol exposure. Cilia were assayed using Sisson-Ammons Video Analysis by quantitating the average number of motile points in multiple whole field measurements of mouse tracheal epithelial cells grown on an air-liquid interface. Pretreatment with ethanol alone (100 mM for 24 hours) had no effect on the number of motile cilia. A single dose (TCID50 1 × 105) of RSV resulted in a significant (p < 0.05) decrease in motile cilia after 2 days. Ethanol pretreatment significantly (p < 0.05) potentiated RSV-induced cilia loss by 2 days. Combined RSV and ethanol treatment led to a sustained activation-induced auto-downregulation of PKC epsilon (PKCε). Ethanol-induced enhancement of ciliated cell detachment was confirmed by dynein ELISA and LDH activity from the supernates. RSV-induced cilia loss was evident until 7 days, when RSV-only infected cells demonstrated no significant cilia loss vs. control cells. However, cells pretreated with ethanol showed significant cilia loss until 10 days post-RSV infection. To address the functional significance of ethanol-enhanced cilia detachment, mice fed alcohol ad libitum (20% for 12 weeks) were infected once with RSV, and clearance was measured by plaque-forming assay from lung homogenates for up to 7 days. After 3 days, RSV plaque formation was no longer detected from the lungs of control mice, while significant (p < 0.01) RSV plaque-forming units were detected at 7 days in alcohol-fed mice. Alcohol-fed mice demonstrated enhanced cilia loss and delayed cilia recovery from tracheal measurements in wild-type C57BL/6 mice, but not PKCε KO mice. These data suggest that alcohol worsens RSV-mediated injury to ciliated epithelium in a PKCε-dependent manner.

RSV-specific anti-viral immunity is disrupted by chronic ethanol consumption.

Alcohol-use disorders (AUD) persist in the United States and are heavily associated with an increased susceptibility to respiratory viral infections. Respiratory syncytial virus (RSV) in particular has received attention as a viral pathogen commonly detected in children and immune-compromised populations (elderly, asthmatics), yet more recently was recognized as an important viral pathogen in young adults. Our study evaluated the exacerbation of RSV-associated illness in mice that chronically consumed alcohol for 6 weeks prior to infection. Prior studies showed that lung viral titers remained elevated in these animals, leading to a hypothesis that T-cell activation and immune specificity were deficient in controlling viral spread and replication in the lungs. Herein, we confirm a reduction in RSV-specific IFNγ production by CD8 T cells and a depolarization of Th1 (CD4+IFNγ+) and Th2 (CD4+IL-4+) T cells at day 5 after RSV infection. Furthermore, over the course of viral infection (day 1 to day 7 after RSV infection), we detected a delayed influx of neutrophils, monocytes/macrophages, and lymphocytes into the lungs. Taken together, the data show that both the early and late adaptive immunity to RSV infection are altered by chronic ethanol consumption. Future studies will determine the interactions between the innate and adaptive immune systems to delineate therapeutic targets for individuals with AUD often hospitalized by respiratory infection.

Reactive Oxygen Species/Hypoxia-Inducible Factor-1α/Platelet-Derived Growth Factor-BB Autocrine Loop Contributes to Cocaine-Mediated Alveolar Epithelial Barrier Damage.

Abuse of psychostimulants, such as cocaine, has been shown to be closely associated with complications of the lung, such as pulmonary hypertension, edema, increased inflammation, and infection. However, the mechanism by which cocaine mediates impairment of alveolar epithelial barrier integrity that underlies various pulmonary complications has not been well determined. Herein, we investigate the role of cocaine in disrupting the alveolar epithelial barrier function and the associated signaling cascade. Using the combinatorial electric cell-substrate impedance sensing and FITC-dextran permeability assays, we demonstrated cocaine-mediated disruption of the alveolar epithelial barrier, as evidenced by increased epithelial monolayer permeability with a concomitant loss of the tight junction protein zonula occludens-1 (Zo-1) in both mouse primary alveolar epithelial cells and the alveolar epithelial cell line, L2 cells. To dissect the signaling pathways involved in this process, we demonstrated that cocaine-mediated induction of permeability factors, platelet-derived growth factor (PDGF-BB) and vascular endothelial growth factor, involved reactive oxygen species (ROS)-dependent induction of hypoxia-inducible factor (HIF)-1α. Interestingly, we demonstrated that ROS-dependent induction of another transcription factor, nuclear factor erythroid-2-related factor-2, that did not play a role in cocaine-mediated barrier dysfunction. Importantly, this study identifies, for the first time, that ROS/HIF-1α/PDGF-BB autocrine loop contributes to cocaine-mediated barrier disruption via amplification of oxidative stress and downstream signaling. Corroboration of these cell culture findings in vivo demonstrated increased permeability of the alveolar epithelial barrier, loss of expression of Zo-1, and a concomitantly increased expression of both HIF-1α and PDGF-BB. Pharmacological blocking of HIF-1α significantly abrogated cocaine-mediated loss of Zo-1. Understanding the mechanism(s) by which cocaine mediates barrier dysfunction could provide insights into the development of potential therapeutic targets for cocaine-mediated pulmonary hypertension.

Alcohol's Effects on Lung Health and Immunity.

It has long been known that people with alcohol use disorder (AUD) not only may develop physical dependence but also may experience devastating long-term health problems. The most common and identifiable alcohol-associated health problems include liver cirrhosis, pancreatitis, cardiomyopathies, neuropathies, and dementia. However, the lung also is adversely affected by alcohol abuse, a fact often overlooked by clinicians and the public. Individuals with AUD are more likely to develop pneumonia, tuberculosis (TB), respiratory syncytial virus (RSV) infection, and acute respiratory distress syndrome (ARDS). Increased susceptibility to these and other pulmonary infections is caused by impaired immune responses in people with AUD. The key immune cells involved in combating pulmonary conditions such as pneumonia, TB, RSV infection, and ARDS are neutrophils, lymphocytes, alveolar macrophages, and the cells responsible for innate immune responses. Researchers are only now beginning to understand how alcohol affects these cells and how these effects contribute to the pathophysiology of pulmonary diseases in people with AUD.

Peripherally cross-linking the shell of core-shell polymer micelles decreases premature release of physically loaded combretastatin A4 in whole blood and increases its mean residence time and subsequent potency against primary murine breast tumors after IV administration.

PURPOSE: Determine the feasibility and potential benefit of peripherally cross-linking the shell of core-shell polymer micelles on the premature release of physically loaded hydrophobic drug in whole blood and subsequent potency against solid tumors. METHODS: Individual Pluronic F127 polymer micelles (F127 PM) peripherally cross-linked with ethylenediamine at 76% of total PEO blocks (X-F127 PM) were physically loaded with combretastatin A4 (CA4) by the solid dispersion method and compared to CA4 physically loaded in uncross-linked F127 PM, CA4 in DMSO in vitro, or water-soluble CA4 phosphate (CA4P) in vivo. RESULTS: X-F127 PM had similar CA4 loading and aqueous solubility as F127 PM up to 10 mg CA4 / mL at 22.9 wt% and did not aggregate in PBS or 90% (v/v) human serum at 37°C for at least 24 h. In contrast, X-F127 PM decreased the unbound fraction of CA4 in whole blood (fu) and increased the mean plasma residence time and subsequent potency of CA4 against the vascular function and growth of primary murine 4T1 breast tumors over CA4 in F127 PM and water-soluble CA4P after IV administration. CONCLUSIONS: Given that decreasing the fu is an indication of decreased drug release, peripherally cross-linking the shell of core-shell polymer micelles may be a simple approach to decrease premature release of physically loaded hydrophobic drug in the blood and increase subsequent potency in solid tumors.

Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells.

Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3-7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3 to 7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.

Dietary antioxidants prevent alcohol-induced ciliary dysfunction.

Previously we have shown that chronic alcohol intake causes alcohol-induced ciliary dysfunction (AICD), leading to non-responsive airway cilia. AICD likely occurs through the downregulation of nitric oxide (NO) and cyclic nucleotide-dependent kinases, protein kinase G (PKG) and protein kinase A (PKA). Studies by others have shown that dietary supplementation with the antioxidants N-acetylcysteine (NAC) and procysteine prevent other alcohol-induced lung complications. This led us to hypothesize that dietary supplementation with NAC or procysteine prevents AICD. To test this hypothesis, C57BL/6 mice drank an alcohol/water solution (20% w/v) ad libitum for 6 weeks and were concurrently fed dietary supplements of either NAC or procysteine. Ciliary beat frequency (CBF) was measured in mice tracheas, and PKG/PKA responsiveness to ß-agonists and NOx levels were measured from bronchoalveolar lavage (BAL) fluid. Long-term alcohol drinking reduced CBF, PKG and PKA responsiveness to ß-agonists, and lung NOx levels in BAL fluid. In contrast, alcohol-drinking mice fed NAC or procysteine sustained ciliary function and PKG and PKA responsiveness to ß-agonists. However, BAL NO levels remained low despite antioxidant supplementation. We also determined that removal of alcohol from the drinking water for as little as 1 week restored ciliary function, but not PKG and PKA responsiveness to ß-agonists. We conclude that dietary supplementation with NAC or procysteine protects against AICD. In addition, alcohol removal for 1 week restores cilia function independent of PKG and PKA activity. Our findings provide a rationale for the use of antioxidants to prevent damage to airway mucociliary functions in chronic alcohol-drinking individuals.

Proteomic analysis of bovine axonemes exposed to acute alcohol: role of endothelial nitric oxide synthase and heat shock protein 90 in cilia stimulation.

BACKGROUND: Cilia are finger-like motor-driven organelles, which propel inhaled particles and mucus from the lung and airways. We have previously shown that brief alcohol exposure stimulates ciliary motility through an endothelial nitric oxide synthase (eNOS)-dependent pathway localized in the ciliary metabolon. However, the signaling molecules of the ciliary metabolon involved in alcohol-triggered ciliary beat frequency (CBF) stimulation upstream of eNOS activation remain unknown. METHODS: We hypothesized that brief alcohol exposure alters threonine and serine phosphorylation of proteins involved in stimulating CBF. Two-dimensional electrophoresis indicated both increases and decreases in the serine and threonine phosphorylation states of several proteins. One of the proteins identified was heat shock protein 90 (HSP90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP90 has been shown to associate with eNOS in lung tissue, we hypothesized that HSP90 is a key component in alcohol-triggered eNOS activation and that these 2 proteins co-localize within the ciliary metabolon. RESULTS: Immunofluorescence experiments demonstrate that eNOS and HSP90 co-localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP90 chaperone functions, prevented eNOS-HSP90 association and prevented the translocation of eNOS from the ciliary metabolon to the axoneme. Functional cilia motility studies revealed that geldanamycin blocked alcohol-stimulated ciliary motility in bovine bronchial epithelial cells and mouse tracheal rings. CONCLUSIONS: On the basis of the HSP90 localization with eNOS, alcohol activation of HSP90 phosphorylation, and geldanamycin's ability to inhibit HSP90-eNOS association, prevent eNOS translocation to the axoneme, and block alcohol-stimulated ciliary motility, we conclude that alcohol-induced cilia stimulation occurs through the increased association of HSP90 with eNOS. These data help further elucidate the mechanism through which brief alcohol exposure stimulates CBF.

Alcohol increases the permeability of airway epithelial tight junctions in Beas-2B and NHBE cells.

BACKGROUND: Tight junctions form a continuous belt-like structure between cells and act to regulate paracellular signaling. Protein kinase C (PKC) has been shown to regulate tight junction assembly and disassembly and is activated by alcohol. Previous research has shown that alcohol increases the permeability of tight junctions in lung alveolar cells. However, little is known about alcohol's effect on tight junctions in epithelium of the conducting airways. We hypothesized that long-term alcohol exposure reduces zonula occluden-1 (ZO-1) and claudin-1 localization at the cell membrane and increases permeability through a PKC-dependent mechanism. METHODS: To test this hypothesis, we exposed normal human bronchial epithelial (NHBE) cells, cells from a human bronchial epithelial transformed cell line (Beas-2B), and Beas-2B expressing a PKCα dominant negative (DN) to alcohol (20, 50, and 100 mM) for up to 48 hours. Immunofluorescence was used to assess changes in ZO-1, claudin-1, claudin-5, and claudin-7 localization. Electric cell-substrate impedance sensing was used to measure the permeability of tight junctions between monolayers of NHBE, Beas-2B, and DN cells. RESULTS: Alcohol increased tight junction permeability in a concentration-dependent manner and decreased ZO-1, claudin-1, claudin-5, and claudin-7 localization at the cell membrane. To determine a possible signaling mechanism, we measured the activity of PKC isoforms (alpha, delta, epsilon, and zeta). PKCα activity significantly increased in Beas-2B cells from 1 to 6 hours of 100 mM alcohol exposure, while PKCζ activity significantly decreased at 1 hour and increased at 3 hours. Inhibiting PKCα with Gö-6976 prevented the alcohol-induced protein changes in both ZO-1 and claudin-1 at the cell membrane. PKCα DN Beas-2B cells were resistant to alcohol-induced protein alterations. CONCLUSIONS: These results suggest that alcohol disrupts ZO-1, claudin-1, claudin-5, and claudin-7 through the activation of PKCα, leading to an alcohol-induced "leakiness" in bronchial epithelial cells. Such alcohol-induced airway-leak state likely contributes to the impaired airway host defenses associated with acute and chronic alcohol ingestion.

Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function.

Exposure to cigarette smoke is associated with airway epithelial mucus cell hyperplasia and a decrease in cilia and ciliated cells. Few models have addressed the long-term effects of chronic cigarette smoke exposure on ciliated epithelial cells. Our previous in vitro studies showed that cigarette smoke decreases ciliary beat frequency (CBF) via the activation of protein kinase C (PKC). We hypothesized that chronic cigarette smoke exposure in an in vivo model would decrease airway epithelial cell ciliary beating in a PKC-dependent manner. We exposed C57BL/6 mice to whole-body cigarette smoke 2 hours/day, 5 days/week for up to 1 year. Tracheal epithelial cell CBF and the number of motile cells were measured after necropsy in cut tracheal rings, using high-speed digital video microscopy. Tracheal epithelial PKC was assayed according to direct kinase activity. At 6 weeks and 3 months of smoke exposure, the baseline CBF was slightly elevated (~1 Hz) versus control mice, with no change in ß-agonist-stimulated CBF between control mice and cigarette smoke-exposed mice. By 6 months of smoke exposure, the baseline CBF was significantly decreased (2-3 Hz) versus control mice, and a ß-agonist failed to stimulate increased CBF. The loss of ß-agonist-increased CBF continued at 9 months and 12 months of smoke exposure, and the baseline CBF was significantly decreased to less than one third of the control rate. In addition to CBF, ciliated cell numbers significantly decreased in response to smoke over time, with a significant loss of tracheal ciliated cells occurring between 6 and 12 months. In parallel with the slowing of CBF, significant PKC activation from cytosol to the membrane of tracheal epithelial cells was detected in mice exposed to smoke for 6-12 months.