Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function.

Alcohol consumption with concurrent cigarette smoking produces malondialdehyde acetaldehyde (MAA)-adducted lung proteins. Lung surfactant protein D (SPD) supports innate immunity via bacterial aggregation and lysis, as well as by enhancing macrophage-binding and phagocytosis. MAA-adducted SPD (SPD-MAA) has negative effects on lung cilia beating, macrophage function, and epithelial cell injury repair. Because changes in SPD multimer structure are known to impact SPD function, we hypothesized that MAA-adduction changes both SPD structure and function. Purified human SPD and SPD-MAA (1 mg/mL) were resolved by gel filtration using Sephadex G-200 and protein concentration of each fraction determined by Bradford assay. Fractions were immobilized onto nitrocellulose by slot blot and assayed by Western blot using antibodies to SPD and to MAA. Binding of SPD and SPD-MAA was determined fluorometrically using GFP-labeled Streptococcus pneumoniae (GFP-SP). Anti-bacterial aggregation of GFP-SP and macrophage bacterial phagocytosis were assayed by microscopy and permeability determined by bacterial phosphatase release. Viral injury was measured as LDH release in RSV-treated airway epithelial cells. Three sizes of SPD were resolved by gel chromatography as monomeric, trimeric, and multimeric forms. SPD multimer was the most prevalent, while the majority of SPD-MAA eluted as trimer and monomer. SPD dose-dependently bound to GFP-SP, but SPD-MAA binding to bacteria was significantly reduced. SPD enhanced, but MAA adduction of SPD prevented, both aggregation and macrophage phagocytosis of GFP-SP. Likewise, SPD increased bacterial permeability while SPD-MAA did not. In the presence of RSV, BEAS-2B cell viability was enhanced by SPD, but not protected by SPD-MAA. Our results demonstrate that MAA adduction changes the quaternary structure of SPD from multimer to trimer and monomer leading to a decrease in the native anti-microbial function of SPD. These findings suggest one mechanism for increased pneumonia observed in alcohol use disorders.

Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury.

Most individuals diagnosed with alcohol use disorders smoke cigarettes. Large concentrations of malondialdehyde and acetaldehyde are found in lungs co-exposed to cigarette smoke and alcohol. Aldehydes directly injure lungs and form aldehyde protein adducts, impacting epithelial functions. Recently, 2-(3-Amino-6-chloroquinolin-2-yl)propan-2-ol (ADX-102) was developed as an aldehyde-trapping drug. We hypothesized that aldehyde-trapping compounds are protective against lung injury derived from cigarette smoke and alcohol co-exposure. To test this hypothesis, we pretreated mouse ciliated tracheal epithelial cells with 0-100 µM of ADX-102 followed by co-exposure to 5% cigarette smoke extract and 50 mM of ethanol. Pretreatment with ADX-102 dose-dependently protected against smoke and alcohol induced cilia-slowing, decreases in bronchial epithelial cell wound repair, decreases in epithelial monolayer resistance, and the formation of MAA adducts. ADX-102 concentrations up to 100 µM showed no cellular toxicity. As protein kinase C (PKC) activation is a known mechanism for slowing cilia and wound repair, we examined the effects of ADX-102 on smoke and alcohol induced PKC epsilon activity. ADX-102 prevented early (3 h) activation and late (24 h) autodownregulation of PKC epsilon in response to smoke and alcohol. These data suggest that reactive aldehydes generated from cigarette smoke and alcohol metabolism may be potential targets for therapeutic intervention to reduce lung injury.

Perfluorocarbon Nanoemulsions Enhance Therapeutic siRNA Delivery in the Treatment of Pulmonary Fibrosis.

Local pulmonary administration of therapeutic siRNA represents a promising approach to the treatment of lung fibrosis, which is currently hampered by inefficient delivery. Development of perfluorooctylbromide (PFOB) nanoemulsions as a way of improving the efficiency of pulmonary polycation-based delivery of siRNA is reported. The results show that the polycation/siRNA/PFOB nanoemulsions are capable of efficiently silencing the expression of STAT3 and inhibiting chemokine receptor CXCR4-two validated targets in pulmonary fibrosis. Both in vitro and in vivo results demonstrate that the nanoemulsions improve mucus penetration and facilitate effective cellular delivery of siRNA. Pulmonary treatment of mice with bleomycin-induced pulmonary fibrosis shows strong inhibition of the progression of the disease and significant prolongation of animal survival. Overall, the study points to a promising local treatment strategy of pulmonary fibrosis.

Malondialdehyde-Acetaldehyde Adduct Formation Decreases Immunoglobulin A Transport across Airway Epithelium in Smokers Who Abuse Alcohol.

Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor-ß (TGF-ß) down-regulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGF-ß, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGF-ß in human bronchoalveolar lavage fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human bronchial epithelial cells (HBECs) and mouse tracheal epithelial cells were treated with SPD-MAA and sIgA and TGF-ß was measured. Decreased sIgA and increased TGF-ß were observed in bronchoalveolar lavage from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBECs. Conversely, SPD-MAA stimulated TGF-ß release in both HBECs and mouse tracheal epithelial cells, but not in CD204 knockout mice. SPD-MAA stimulated TGF-ß in alveolar macrophage cells. These data show that MAA-adducted surfactant protein stimulates lung epithelial cell TGF-ß, down-regulates pIgR, and decreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.