Neutralization of IL-33 modifies the type 2 and type 3 inflammatory signature of viral induced asthma exacerbation.

BACKGROUND: Respiratory viral infections are one of the leading causes of need for emergency care and hospitalizations in asthmatic individuals, and airway-secreted cytokines are released within hours of viral infection to initiate these exacerbations. IL-33, specifically, contributes to these allergic exacerbations by amplifying type 2 inflammation. We hypothesized that blocking IL-33 in RSV-induced exacerbation would significantly reduce allergic inflammation. METHODS: Sensitized BALB/c mice were challenged with aerosolized ovalbumin (OVA) to establish allergic inflammation, followed by RSV-A2 infection to yield four treatment groups: saline only (Saline), RSV-infected alone (RSV), OVA alone (OVA), and OVA-treated with RSV infection (OVA-RSV). Lung outcomes included lung mRNA and protein markers of allergic inflammation, histology for mucus cell metaplasia and lung immune cell influx by cytospin and flow cytometry. RESULTS: While thymic stromal lymphopoietin (TSLP) and IL-33 were detected 6 h after RSV infection in the OVA-RSV mice, IL-23 protein was uniquely upregulated in RSV-infected mice alone. OVA-RSV animals varied from RSV- or OVA-treated mice as they had increased lung eosinophils, neutrophils, group 2 innate lymphoid cells (ILC2) and group 3 innate lymphoid cells (ILC3) detectable as early as 6 h after RSV infection. Neutralized IL-33 significantly reduced ILC2 and eosinophils, and the prototypical allergic proteins, IL-5, IL-13, CCL17 and CCL22 in OVA-RSV mice. Numbers of neutrophils and ILC3 were also reduced with anti-IL-33 treatment in both RSV and OVA-RSV treated animals as well. CONCLUSIONS: Taken together, our findings indicate a broad reduction in allergic-proinflammatory events mediated by IL-33 neutralization in RSV-induced asthma exacerbation.

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.

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.

Loss of cAMP-dependent stimulation of isolated cilia motility by alcohol exposure is oxidant-dependent.

Alcohol exposure is associated with decreased mucociliary clearance, a key innate defense essential to lung immunity. Previously, we identified that prolonged alcohol exposure results in dysfunction of airway cilia that persists at the organelle level. This dysfunction is characterized by a loss of 3',5'-cyclic adenosine monophosphate (cAMP)-mediated cilia stimulation. However, whether or not ciliary dysfunction develops intrinsically at the organelle level has not been explored. We hypothesized that prolonged alcohol exposure directly to isolated demembranated cilia (axonemes) causes ciliary dysfunction. To test this hypothesis, we exposed isolated axonemes to alcohol (100 mM) for 1-24 h and assessed ciliary beat frequency (CBF) in response to cAMP at 1, 3, 4, 6, and 24 h post-exposure. We found that after 1 h of alcohol exposure, cilia axonemes do not increase CBF in response to cAMP. Importantly, by 6 h after the initial exposure to alcohol, cAMP-mediated CBF was restored to control levels. Additionally, we found that thioredoxin reverses ciliary dysfunction in axonemes exposed to alcohol. Finally, we identified, using a combination of a xanthine oxidase oxidant-generating system, direct application of hydrogen peroxide, and electron paramagnetic resonance, that hydrogen peroxide versus superoxide, is likely the key oxidant species driving alcohol-induced ciliary dysfunction in isolated axonemes. These data highlight the role of alcohol to stimulate local production of oxidants in the axoneme to cause ciliary dysfunction. Additionally, these data specifically add hydrogen peroxide as a potential therapeutic target in the treatment or prevention of alcohol-associated ciliary dysfunction and subsequent pneumonia.

An association between MMP-9 and impaired T cell migration in ethanol-fed BALB/c mice infected with respiratory syncytial virus-2A.

Matrix metalloproteinases are important for proper airway matrix structure and wound healing. These enzymes are also implicated in many airway diseases. Previously, chronic ethanol consumption was shown to prolong inflammation and delay viral clearance in respiratory syncytial virus (RSV)-infected mice. We hypothesize that alcohol alters anti-viral immunity by disrupting immune cell chemotaxis in the lung. BALB/c mice were randomly selected to consume 18% alcohol ad libitum for 8 weeks prior to infection with RSV-2A. Bronchoalveolar lavage (BAL) cell populations were measured by flow cytometry, and chemokines were detected by Western blot or ELISA. MMP-9 levels were determined by polymerase chain reaction (PCR) in mouse lungs and in BAL fluid by ELISA. T cells were acquired from the spleens of water-fed, non-infected control mice (CTRL); alcohol-fed, non-infected (ETOH); water-fed, RSV-infected (RSV); or ethanol-fed, RSV-infected (ETOH-RSV) 4 days after RSV infection. T cells were placed in a transmigration system where chemokines had been treated with and without activated MMP-9. Lymphocyte recruitment was significantly reduced in the BAL 4 days after RSV infection in ETOH-RSV mice, whereas chemokine levels were the highest in this group at all experimental time points examined in comparison to RSV (p < 0.05). MMP-9 mRNA and protein were detected at high levels in ETOH-RSV mice compared to RSV. Using ex vivo transmigration to CCL2 and CXCL10, T cell migration was not impaired between any of the treatment groups, yet when CCL2 and CXCL10 were treated with activated MMP-9, significantly fewer T cells migrated across collagen-coated 5-µm membranes (p < 0.05). Immune cell recruitment is necessary for viral clearance. We show that immune cells are decreased in the lungs of ETOH-RSV mice. In contrast to decreased cell recruitment, key inflammatory chemokines were elevated in the lungs of ETOH-RSV mice. These proteins may be prematurely degraded by MMP-9 in the lung, leading to defective immunity and reduced viral clearance.

Organic barn dust inhibits surfactant protein D production through protein kinase-c alpha dependent increase of GPR116.

Prolonged exposure to organic barn dusts can lead to chronic inflammation and a broad range of lung problems over time, mediated by innate immune mechanisms. The immune surfactant or collectin surfactant protein D (SP-D) is a crucial multifunctional innate immune receptor. Little work to date has examined the effect of such collectins in response to organic dusts. We provide evidence here that agricultural organic dusts can inhibit mRNA and protein expression of SP-D in a human alveolar epithelial cell line, and an in vivo mouse model. This inhibition was not a result of lipopolysaccharide (LPS) or peptidoglycans, the two most commonly cited immune active components of these dusts. We further show that inhibition of the signaling molecule protein kinase C alpha (PKCα) can reverse this inhibition implicating it as a mechanism of SP-D inhibition. Examination of the SP-D regulatory receptor GPR116 showed that its mRNA expression was increased in response to dust and inhibited by blocking PKCα, implicating it as a means of inhibiting SP-D in the lungs in response to organic dusts. This reduction shows that organic barn dust can reduce lung SP-D, thus leaving workers potentially at risk for a host of pathogens.

S-nitrosation of protein phosphatase 1 mediates alcohol-induced ciliary dysfunction.

Alcohol use disorder (AUD) is a strong risk factor for development and mortality of pneumonia. Mucociliary clearance, a key innate defense against pneumonia, is perturbed by alcohol use. Specifically, ciliated airway cells lose the ability to increase ciliary beat frequency (CBF) to ß-agonist stimulation after prolonged alcohol exposure. We previously found that alcohol activates protein phosphatase 1 (PP1) through a redox mechanism to cause ciliary dysfunction. Therefore, we hypothesized that PP1 activity is enhanced by alcohol exposure through an S-nitrosothiol-dependent mechanism resulting in desensitization of CBF stimulation. Bronchoalveolar S-nitrosothiol (SNO) content and tracheal PP1 activity was increased in wild-type (WT) mice drinking alcohol for 6-weeks compared to control mice. In contrast, alcohol drinking did not increase SNO content or PP1 activity in nitric oxide synthase 3-deficient mice. S-nitrosoglutathione induced PP1-dependent CBF desensitization in mouse tracheal rings, cultured cells and isolated cilia. In vitro expression of mutant PP1 (cysteine 155 to alanine) in primary human airway epithelial cells prevented CBF desensitization after prolonged alcohol exposure compared to cells expressing WT PP1. Thus, redox modulation in the airways by alcohol is an important ciliary regulatory mechanism. Pharmacologic strategies to reduce S-nitrosation may enhance mucociliary clearance and reduce pneumonia prevalence, mortality and morbidity with AUD.

Dimethylarginine dimethylaminohydrolase (DDAH) overexpression enhances wound repair in airway epithelial cells exposed to agricultural organic dust.

OBJECTIVE: Workers exposed to dusts from concentrated animal feeding operations have a high prevalence of pulmonary diseases. These exposures lead to chronic inflammation and aberrant airway remodeling. Previous work shows that activating cAMP-dependent protein kinase (PKA) enhances airway epithelial wound repair while activating protein kinase C (PKC) inhibits wound repair. Hog barn dust extracts slow cell migration and wound repair via a PKC-dependent mechanism. Further, blocking nitric oxide (NO) production in bronchial epithelial cells prevents PKA activation. We hypothesized that blocking an endogenous NO inhibitor, asymmetric dimethylarginine, by overexpressing dimethylarginine dimethylaminohydrolase mitigates the effects of hog dust extract on airway epithelial would repair. MATERIALS/METHODS: We cultured primary tracheal epithelial cells in monolayers from both wild-type (WT) and dimethylarginine dimethylaminohydrolase overexpressing C57Bl/6 (DDAH1 transgenic) mice and measured wound repair using the electric cell impedance sensing system. RESULTS: Wound closure in epithelial cells from WT mice occurred within 24 h in vitro. In contrast, treatment of the WT cell monolayers with 5% hog dust extract prevented significant NO-stimulated wound closure. In cells from DDAH1 transgenic mice, control wounds were repaired up to 8 h earlier than seen in WT mice. A significant enhancement of wound repair was observed in DDAH cells compared to WT cells treated with hog dust extract for 24 h. Likewise, cells from DDAH1 transgenic mice demonstrated increased NO and PKA activity and decreased hog dust extract-stimulated PKC. DISCUSSION/CONCLUSION: Preserving the NO signal through endogenous inhibition of asymmetric dimethylarginine enhances wound repair even in the presence of dust exposure.

Malondialdehyde-Acetaldehyde (MAA) Protein Adducts Are Found Exclusively in the Lungs of Smokers with Alcohol Use Disorders and Are Associated with Systemic Anti-MAA Antibodies.

BACKGROUND: Malondialdehyde (MDA) and acetaldehyde (AA) exist following ethanol metabolism and tobacco pyrolysis. As such, lungs of individuals with alcohol use disorders (AUDs) are a target for the effects of combined alcohol and cigarette smoke metabolites. MDA and AA form a stable protein adduct, malondialdehyde-acetaldehyde (MAA) adduct, known to be immunogenic, profibrotic, and proinflammatory. MAA adduct is the dominant epitope in anti-MAA antibody formation. We hypothesized that MAA-adducted protein forms in lungs of those who both abuse alcohol and smoke cigarettes, and that this would be associated with systemically elevated anti-MAA antibodies. METHODS: Four groups were established: AUD subjects who smoked cigarettes (+AUD/+smoke), smokers without AUD (-AUD/+smoke), AUD without smoke (+AUD/-smoke), and non-AUD/nonsmokers (-AUD/-smoke). RESULTS: We observed a significant increase in MAA adducts in lung cells of +AUD/+smoke versus -AUD/-smoke. No significant increase in MAA adducts was observed in -AUD/+smoke or in +AUD/-smoke compared to -AUD/-smoke. Serum from +AUD/+smoke had significantly increased levels of circulating anti-MAA IgA antibodies. After 1 week of alcohol that MAA-adducted protein is formed in the lungs of those who smoke cigarettes and abuse alcohol, leading to a subsequent increase in serum IgA antibodies. CONCLUSIONS: MAA-adducted proteins could play a role in pneumonia and other diseases of the lung in the setting of AUD and smoking.

Effect of low-level CO2 on innate inflammatory protein response to organic dust from swine confinement barns.

BACKGROUND: Organic hog barn dust (HDE) exposure induces lung inflammation and long-term decreases in lung function in agricultural workers. While concentrations of common gasses in confined animal facilities are well characterized, few studies have been done addressing if exposure to elevated barn gasses impacts the lung immune response to organic dusts. Given the well documented effects of hypercapnia at much higher levels we hypothesized that CO2 at 8 h exposure limit levels (5000 ppm) could alter innate immune responses to HDE. METHODS: Using a mouse model, C57BL/6 mice were nasally instilled with defined barn dust extracts and then housed in an exposure box maintained at one of several CO2 levels for six hours. Bronchiolar lavage (BAL) was tested for several cytokines while lung tissue was saved for mRNA purification and immunohistochemistry. RESULTS: Exposure to elevated CO2 significantly increased the expression of pro-inflammatory markers, IL-6 and KC, in BAL fluid as compared to dust exposure alone. Expression of other pro-inflammatory markers, such as ICAM-1 and matrix metalloproteinase-9 (MMP-9), were also tested and showed similar increased expression upon HDE + CO2 exposure. A chemokine array analysis of BAL fluid revealed that MIP-1γ (CCL9) shows a similar increased response to HDE + CO2. Further testing showed CCL9 was significantly elevated by barn dust and further enhanced by CO2 co-exposure in a dose-dependent manner that was noticeable at the protein and mRNA levels. In all cases, except for ICAM-1, increases in tested markers in the presence of elevated CO2 were only significant in the presence of HDE as well. CONCLUSIONS: We show that even at mandated safe exposure limits, CO2 is capable of enhancing multiple markers of inflammation in response to HDE.

Malondialdehyde-acetaldehyde (MAA) adducted surfactant protein induced lung inflammation is mediated through scavenger receptor a (SR-A1).

BACKGROUND: Co-exposure to cigarette smoke and alcohol leads to the generation of high concentrations of acetaldehyde and malondialdehyde in the lung. These aldehydes being highly electrophilic in nature react with biologically relevant proteins such as surfactant protein D (SPD) through a Schiff base reaction to generate SPD adducted malondialdehyde-acetaldehyde adduct (SPD-MAA) in mouse lung. SPD-MAA results in an increase in lung pro-inflammatory chemokine, keratinocyte chemoattractant (KC), and the recruitment of lung lavage neutrophils. Previous in vitro studies in bronchial epithelial cells and macrophages show that scavenger receptor A (SR-A1/CD204) is a major receptor for SPD-MAA. No studies have yet examined the in vivo role of SR-A1 in MAA-mediated lung inflammation. Therefore, we hypothesize that in the absence of SR-A1, MAA-induced inflammation in the lung is reduced or diminished. METHODS: To test this hypothesis, C57BL/6 WT and SR-A1 KO mice were nasally instilled with 50 µg/mL of SPD-MAA for 3 weeks (wks). After 3 weeks, bronchoalveolar lavage (BAL) fluid was collected and assayed for a total cell count, a differential cell count and CXCL1 (KC) chemokine. Lung tissue sections were stained with hematoxylin and eosin (H&E) and antibodies to MAA adduct. RESULTS: Results showed that BAL cellularity and influx of neutrophils were decreased in SR-A1 KO mice as compared to WT following repetitive SPD-MAA exposure. MAA adduct staining in the lung epithelium was decreased in SR-A1 KO mice. In comparison to WT, no increase in CXCL1 was observed in BAL fluid from SR-A1 KO mice over time. CONCLUSIONS: Overall, the data demonstrate that SR-A1/CD204 plays an important role in SPD-MAA induced inflammation in lung.

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.

Alcohol Decreases Organic Dust-Stimulated Airway Epithelial TNF-Alpha Through a Nitric Oxide and Protein Kinase-Mediated Inhibition of TACE.

BACKGROUND: Farm workers in rural areas consume more alcohol than those who reside in urban areas. Occupational exposures such as agricultural work can pose hazards on the respiratory system. It is established that hog barn dust induces inflammation in the airway, including the release of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8. We have shown that alcohol alters airway epithelial innate defense through changes in both nitric oxide (NO) and cAMP-dependent protein kinase A (PKA). Simultaneous exposure to hog barn dust and alcohol decreases inflammatory mediators, TNF-α, IL-6, and IL-8, in mice. Previously, mice exposed to both alcohol and hog barn dust showed a depleted amount of lymphocytes compared to mice exposed only to hog barn dust. Weakening of the innate immune response could lead to enhanced susceptibility to disease. In addition, mice that were co-exposed to hog barn dust and alcohol also experienced increased mortality. METHODS: Because we recently demonstrated that PKA activation inhibits the TNF-α sheddase, TNF-α-converting enzyme (TACE), we hypothesized that an alcohol-mediated PKA pathway blocks TACE activity and prevents the normative inflammatory response to hog barn dust exposure. To delineate these effects, we used PKA pathway inhibitors (adenylyl cyclase [AC], cAMP, and PKA) to modulate the effects of alcohol on dust-stimulated TNF-α release in the bronchial epithelial cell line, BEAS-2B. Alcohol pretreatment blocked TACE activity and TNF-α release in hog barn dust-treated cells. RESULTS: Alcohol continued to block hog barn dust-mediated TNF-α release in the presence of the particulate AC inhibitor, SQ22,536. The soluble adenylyl cyclase inhibitor, KH7, however, significantly increased the inflammatory response to hog barn dust. phosphodiesterase 4 inhibitors significantly elevated cAMP and enhanced alcohol-mediated inhibition of dust-stimulated TNF-α release. In addition, the NO synthase inhibitor, l-NMMA, also reversed the alcohol-blocking effect on dust-stimulated TNF-α. CONCLUSIONS: These data suggest that alcohol requires a soluble cyclase-generated cAMP-PKA pathway that is dependent upon the action of NO to inhibit TACE and TNF-α release. These findings support our observations that alcohol functions through a dual NO and PKA pathway in bronchial epithelial cells.

cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells.

Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8.

Protective role of CYP2E1 inhibitor diallyl disulfide (DADS) on alcohol-induced malondialdehyde-deoxyguanosine (M1dG) adduct formation.

BACKGROUND: Alcohol use disorders are often associated with lung disease. Alcohol exposure leads to the production of reactive oxygen species, lipid peroxidation, and formation of malondialdehyde (MDA) as well as to induce the expression of cytochrome p450 2E1 (CYP2E1). Likewise, cigarette smoking can lead to lung lipid peroxidation and formation of MDA. MDA can bind to DNA forming MDA-deoxyguanosine (M1dG) adducts, which have been implicated in alcohol-related cancers and cardiovascular disease. Because CYP2E1 regulates MDA production, and our previous studies have shown that alcohol and cigarette smoke can lead to MDA formation, we hypothesized that CYP2E1 would modulate M1dG adduct formation and single-strand DNA damage in alcohol- and cigarette smoke-exposed lung cells and tissue. METHODS: Normal human bronchial epithelial cells (HBECs) were pretreated with 10 µM diallyl disulfide (DADS) for 1 hour and treated with 80 mM ethanol (EtOH) ± 5% cigarette smoke extract (CSE) for 3 hours for comet assay and 6 hours for CYP2E1, MDA, and M1dG adduct assays. C57BL/6 mice were administered 20% EtOH ad libitum in drinking water for 8 weeks and exposed to whole-body cigarette smoke for 5 weeks. Mice were also fed a CYP2E1 inhibitor, DADS, at 1 µM/g of feed in their daily diet for 7 weeks. Whole lung tissue homogenate was used for CYP2E1, MDA, and M1dG adduct assays. RESULTS: EtOH exposure significantly increased HBEC olive tail moment. DADS pretreatment of HBECs attenuated this EtOH effect. EtOH also induced MDA and M1dG adduct formation, which was also significantly reduced by DADS treatment. CSE ± EtOH did not enhance these effects. In lung tissue homogenate of 8-week alcohol-fed mice, MDA and M1dG adduct levels were significantly elevated in comparison with control mice and mice fed DADS while consuming alcohol. No increase in MDA and M1dG adduct formation was observed in 5-week cigarette smoke-exposed mice. CONCLUSIONS: These findings suggest that CYP2E1 plays a pivotal role in alcohol-induced M1dG adducts, and the use of DADS as dietary supplement can reverse the effects of alcohol on M1dG formation.

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.

Co-exposure to cigarette smoke and alcohol decreases airway epithelial cell cilia beating in a protein kinase Cε-dependent manner.

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.

Non-typeable Haemophilus influenzae decreases cilia beating via protein kinase Cε.

BACKGROUND: Haemophilus influenzae infection of the nasal epithelium has long been associated with observations of decreased nasal ciliary beat frequency (CBF) and injury to the ciliated epithelium. Previously, we have reported that several agents that slow CBF also have the effect of activating protein kinase C epsilon (PKCε) activity in bronchial epithelial cells. The subsequent auto-downregulation of PKCε or the direct inhibition of PKCε leads to the specific detachment of the ciliated cells. METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures. CBF and motile points were measured and PKCε activity assayed. RESULTS: NTHi supernatant exposure significantly and rapidly decreased CBF in a dose-dependent manner within 10 minutes of exposure. After 3 hours of exposure, the number of motile ciliated cells significantly decreased. Direct measurement of PKCε activity revealed a dose-dependent activation of PKCε in response to NTHi supernatant exposure. Both CBF and PKCε activity changes were only observed in fresh NTHi culture supernatant and not observed in exposures to heat-inactivated or frozen supernatants. CONCLUSIONS: Our results suggest that CBF slowing observed in response to NTHi is consistent with the stimulated activation of PKCε. Ciliated cell detachment is associated with PKCε autodownregulation.

Hybrid malondialdehyde and acetaldehyde protein adducts form in the lungs of mice exposed to alcohol and cigarette smoke.

BACKGROUND: Most alcohol abusers smoke cigarettes and approximately half of all cigarette smokers consume alcohol. However, no animal models of cigarette and alcohol co-exposure exist to examine reactive aldehydes in the lungs. Cigarette smoking results in elevated lung acetaldehyde (AA) and malondialdehyde (MDA) levels. Likewise, alcohol metabolism produces AA via the action of alcohol dehydrogenase and MDA via lipid peroxidation. A high concentration of AA and MDA form stable hybrid protein adducts known as malondialdehyde-acetaldehyde (MAA) adducts. We hypothesized that chronic cigarette smoke and alcohol exposure in an in vivo mouse model would result in the in vivo formation of MAA adducts. METHODS: We fed C57BL/6 mice ad libitum ethanol (20%) in drinking water and exposed them to whole-body cigarette smoke 2 h/d, 5 d/wk for 6 weeks. Bronchoalveolar lavage fluid and lung homogenates were assayed for AA, MDA, and MAA adduct concentrations. MAA-adducted proteins were identified by Western blot and ELISA. RESULTS: Smoke and alcohol exposure alone elevated both AA and MDA, but only the combination of smoke+alcohol generated protein-adducting concentrations of AA and MDA. MAA-adducted protein (~500 ng/ml) was significantly elevated in the smoke+alcohol-exposed mice. Of the 5 MAA-adducted proteins identified by Western blot, 1 protein band immunoprecipitated with antibodies to surfactant protein D. Similar to in vitro PKC stimulation by purified MAA-adducted protein, protein kinase C (PKC) epsilon was activated only in tracheal epithelial extracts from smoke- and alcohol-exposed mice. CONCLUSIONS: These data demonstrate that only the combination of cigarette smoke exposure and alcohol feeding in mice results in the generation of significant AA and MDA concentrations, the formation of MAA-adducted protein, and the activation of airway epithelial PKC epsilon in the lung.