Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Oxidative stress associated with aging activates protein kinase Cε, leading to cilia slowing.

Older people are four times more likely to develop pneumonia than younger people. As we age, many components of pulmonary innate immunity are impaired, including slowing of mucociliary clearance. Ciliary beat frequency (CBF) is a major determinant of mucociliary clearance, and it slows as we age. We hypothesized that CBF is slowed in aging because of increased oxidative stress, which activates PKCε signaling. We pharmacologically inhibited PKCε in ex vivo mouse models of aging. We measured a slowing of CBF with aging that was reversed with inhibition using the novel PKC inhibitor, Ro-31-8220, as well as the PKCε inhibitor, PKCe141. Inhibition of PKCε using siRNA in mouse trachea also returned CBF to normal. In addition, antioxidants decrease PKCε activity and speed cilia. We also aged wild-type and PKCε KO mice and measured CBF. The PKCε KO mice were spared from the CBF slowing of aging. Using human airway epithelial cells from younger and older donors at air-liquid interface (ALI), we inhibited PKCε with siRNA. We measured a slowing of CBF with aging that was reversed with siRNA inhibition of PKCε. In addition, we measured bead clearance speeds in human ALI, which demonstrated a decrease in bead velocity with aging and a return to baseline after inhibition of PKCε. In summary, in human and mouse models, aging is associated with increased oxidant stress, which activates PKCε and slows CBF.

Alcohol drives S-nitrosylation and redox activation of protein phosphatase 1, causing bovine airway cilia dysfunction.

Individuals with alcohol (ethanol)-use disorders are at increased risk for lung infections, in part, due to defective mucociliary clearance driven by motile cilia in the airways. We recently reported that isolated, demembranated bovine cilia (axonemes) are capable of producing nitric oxide (∙NO) when exposed to biologically relevant concentrations of alcohol. This increased presence of ∙NO can lead to protein S-nitrosylation, a posttranslational modification signaling mechanism involving reversible adduction of nitrosonium cations or ∙NO to thiolate or thiyl radicals, respectively, of proteins forming S-nitrosothiols (SNOs). We quantified and compared SNO content between isolated, demembranated axonemes extracted from bovine tracheae, with or without in situ alcohol exposure (100 mM × 24 h). We demonstrate that relevant concentrations of alcohol exposure shift the S-nitrosylation status of key cilia regulatory proteins, including 20-fold increases in S-nitrosylation of proteins that include protein phosphatase 1 (PP1). With the use of an ATP-reactivated axoneme motility system, we demonstrate that alcohol-driven S-nitrosylation of PP1 is associated with PP1 activation and dysfunction of axoneme motility. These new data demonstrate that alcohol can shift the S-nitrothiol balance at the level of the cilia organelle and highlight S-nitrosylation as a novel signaling mechanism to regulate PP1 and cilia motility.

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.

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.

Inhibition of protein phosphatase 1 reverses alcohol-induced ciliary dysfunction.

Airway mucociliary clearance is a first-line defense of the lung against inhaled particles and debris. Among individuals with alcohol use disorders, there is an increase in lung diseases. We previously identified that prolonged alcohol exposure impairs mucociliary clearance, known as alcohol-induced ciliary dysfunction (AICD). Cilia-localized enzymes, known as the ciliary metabolon, are key to the pathogenesis of AICD. In AICD, cyclic nucleotide-dependent ciliary kinases, which modulate phosphorylation to regulate cilia beat, are desensitized. We hypothesized that alcohol activates cilia-associated protein phosphatase 1 (PP1) activity, driving phosphorylation changes of cilia motility regulatory proteins. To test this hypothesis we identified the effects of prolonged alcohol exposure on phosphatase activity, cilia beat, and kinase responsiveness and cilia-associated phosphorylation targets when stimulated by ß-agonist or cAMP. Prolonged alcohol activated PP1 and blocked cAMP-dependent cilia beat and protein kinase A (PKA) responsiveness and phosphorylation of a 29-kDa substrate of PKA. Importantly, prolonged alcohol-induced phosphatase activation was inhibited by the PP1 specific inhibitor, inhibitor-2 (I-2), restoring cAMP-stimulated cilia beat and PKA responsiveness and phosphorylation of the 29-kDa substrate. The I-2 inhibitory effect persisted in tissue, cell, and isolated cilia-organelle models, highlighting the association of ciliary metabolon-localized enzymes to AICD. Prolonged alcohol exposure drives ciliary metabolon-localized PP1 activation. PP1 activation modifies phosphorylation of a 29-kDa protein related to PKA activity. These data reinforce our previous findings that alcohol is acting at the level of the ciliary metabolon to cause ciliary dysfunction and identifies PP1 as a therapeutic target to prevent or reverse AICD.

Alcohol-induced ciliary dysfunction targets the outer dynein arm.

Alcohol abuse results in an increased incidence of pulmonary infection, in part attributable to impaired mucociliary clearance. Analysis of motility in mammalian airway cilia has revealed that alcohol impacts the ciliary dynein motors by a mechanism involving altered axonemal protein phosphorylation. Given the highly conserved nature of cilia, it is likely that the mechanisms for alcohol-induced ciliary dysfunction (AICD) are conserved. Thus we utilized the experimental advantages offered by the model organism, Chlamydomonas, to determine the precise effects of alcohol on ciliary dynein activity and identify axonemal phosphoproteins that are altered by alcohol exposure. Analysis of live cells or reactivated cell models showed that alcohol significantly inhibits ciliary motility in Chlamydomonas via a mechanism that is part of the axonemal structure. Taking advantage of informative mutant cells, we found that alcohol impacts the activity of the outer dynein arm. Consistent with this finding, alcohol exposure results in a significant reduction in ciliary beat frequency, a parameter of ciliary movement that requires normal outer dynein arm function. Using mutants that lack specific heavy-chain motor domains, we have determined that alcohol impacts the ß- and γ-heavy chains of the outer dynein arm. Furthermore, using a phospho-threonine-specific antibody, we determined that the phosphorylation state of DCC1 of the outer dynein arm-docking complex is altered in the presence of alcohol, and its phosphorylation correlates with AICD. These results demonstrate that alcohol targets specific outer dynein arm components and suggest that DCC1 is part of an alcohol-sensitive mechanism that controls outer dynein arm activity.

MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment.

Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement.

TLR2 and TLR4 Expression and Inflammatory Cytokines are Altered in the Airway Epithelium of Those with Alcohol Use Disorders.

BACKGROUND: The lung has a highly regulated system of innate immunity to protect itself from inhaled microbes and toxins. The first line of defense is mucociliary clearance, but if invaders overcome this, inflammatory pathways are activated. Toll-like receptors (TLRs) are expressed on the airway epithelium. Their signaling initiates the inflammatory cascade and leads to production of inflammatory cytokines such as interleukin (IL)-6 and IL-8. We hypothesized that airway epithelial insults, including heavy alcohol intake or smoking, would alter the expression of TLRs on the airway epithelium. METHODS: Bronchoscopy with bronchoalveolar lavage and brushings of the airway epithelium was performed in otherwise healthy subjects who had normal chest radiographs and spirometry. A history of alcohol use disorders (AUDs) was ascertained using the Alcohol Use Disorders Identification Test (AUDIT), and a history of cigarette smoking was also obtained. Age, gender, and nutritional status in all groups were similar. We used real-time polymerase chain reaction (PCR) to quantitate TLR1 to 9 and enzyme-linked immune assay to measure tumor necrosis factor-α, IL-6, and IL-8. RESULTS: Airway brushings were obtained from 26 nonsmoking/non-AUD subjects, 28 smoking/non-AUD subjects, 36 smoking/AUD subjects, and 17 nonsmoking/AUD subjects. We found that TLR2 is up-regulated in AUD subjects, compared to nonsmoking/non-AUD subjects, and correlated with their AUDIT scores. We also measured a decrease in TLR4 expression in AUD subjects that correlated with AUDIT score. IL-6 and IL-8 were also increased in bronchial washings from AUD subjects. CONCLUSIONS: We have previously demonstrated in normal human bronchial epithelial cells that in vitro alcohol exposure up-regulates TLR2 through a NO/cGMP/PKG-dependent pathway, resulting in up-regulation of inflammatory cytokine production after Gram-positive bacterial product stimulation. Our current translational study confirms that TLR2 is also up-regulated in humans with AUDs.

Use of a novel cell adhesion method and digital measurement to show stimulus-dependent variation in somatic and oral ciliary beat frequency in Paramecium.

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.

Motile cilia harbor serum response factor as a mechanism of environment sensing and injury response in the airway.

Nonmotile primary cilia are recognized as important sensory organelles during development and normal biological functioning. For example, recent work demonstrates that transcriptional regulators of the sonic hedgehog signaling pathway localize to primary cilia and participate in sensing and transducing signals regarding the cellular environment. In contrast, motile cilia are traditionally viewed as mechanical machinery, vital for the movement of solutes and clearance of bacteria and debris, but not participants in cellular sensing and signaling mechanisms. Recently, motile cilia were found to harbor receptors responsible for sensing and responding to environmental stimuli. However, no transcription factors are known to be regulated by cilia localization as a sensing mechanism in vertebrates. Using a mouse model of organic dust-induced airway inflammation, we found that the transcription factor serum response factor (SRF) localizes to motile cilia of airway epithelial cells and alters its localization in response to inflammatory stimuli. Furthermore, inhibition of SRF signaling using the small molecule CCG-1423 reduces organic dust-induced IL-8 release from bronchial epithelial cells and stimulates cilia beat frequency in ciliated mouse tracheal epithelial cells. Immunohistochemical analyses reveal that SRF localizes to the cilia of mouse brain ependymal and ovarian epithelial cells as well. These data reveal a novel mechanism by which a transcription factor localizes to motile cilia and modulates cell activities including cilia motility and inflammation response. These data challenge current dogma regarding motile cilia functioning and may lead to significant contributions in understanding motile ciliary signaling dynamics, as well as mechanisms involving SRF-mediated responses to inflammation and injury.

Deletion of airway cilia results in noninflammatory bronchiectasis and hyperreactive airways.

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.

Smoke extract impairs adenosine wound healing: implications of smoke-generated reactive oxygen species.

Adenosine concentrations are elevated in the lungs of patients with asthma and chronic obstructive pulmonary disease, where it balances between tissue repair and excessive airway remodeling. We previously demonstrated that the activation of the adenosine A2A receptor promotes epithelial wound closure. However, the mechanism by which adenosine-mediated wound healing occurs after cigarette smoke exposure has not been investigated. The present study investigates whether cigarette smoke exposure alters adenosine-mediated reparative properties via its ability to induce a shift in the oxidant/antioxidant balance. Using an in vitro wounding model, bronchial epithelial cells were exposed to 5% cigarette smoke extract, were wounded, and were then stimulated with either 10 µM adenosine or the specific A2A receptor agonist, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA; 10 µM), and assessed for wound closure. In a subset of experiments, bronchial epithelial cells were infected with adenovirus vectors encoding human superoxide dismutase and/or catalase or control vector. In the presence of 5% smoke extract, significant delay was evident in both adenosine-mediated and CPCA-mediated wound closure. However, cells pretreated with N-acetylcysteine (NAC), a nonspecific antioxidant, reversed smoke extract-mediated inhibition. We found that cells overexpressing mitochondrial catalase repealed the smoke extract inhibition of CPCA-stimulated wound closure, whereas superoxide dismutase overexpression exerted no effect. Kinase experiments revealed that smoke extract significantly reduced the A2A-mediated activation of cyclic adenosine monophosphate-dependent protein kinase. However, pretreatment with NAC reversed this effect. In conclusion, our data suggest that cigarette smoke exposure impairs A2A-stimulated wound repair via a reactive oxygen species-dependent mechanism, thereby providing a better understanding of adenosine signaling that may direct the development of pharmacological tools for the treatment of chronic inflammatory lung disorders.

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.

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 reduces airway hyperresponsiveness (AHR) and allergic airway inflammation in mice.

There is very limited knowledge about the effects of alcohol on airway hyperresponsiveness and inflammation in asthma. Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that alcohol exposure will alter airway hyperresponsiveness (AHR) and pulmonary inflammation in a mouse model of allergic asthma. To test this hypothesis, BALB/c mice were fed either 18% alcohol or water and then sensitized and challenged with ovalbumin (OVA). AHR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause, respectively. Airway inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluid (BALF), cytokine levels in BALF, lung histology, and serum immunoglobulin E (IgE) levels. Alcohol feeding significantly blocked methacholine-induced increases in AHR compared with water-fed controls. Alcohol feeding significantly reduced total cell numbers (64%) as well as the number of eosinophils (84%) recruited to the lungs of these mice. Modest changes in lung pathology were also observed. Alcohol exposure led to a reduction of IgE in the serum of the EtOH OVA mice. These data demonstrate that alcohol exposure blunts AHR and dampens allergic airway inflammation indices in allergic mice and suggest that there may be an important role for alcohol in the modulation of asthma. These data provide an in vivo basis for previous clinical observations in humans substantiating the bronchodilator properties of alcohol and for the first time demonstrates an alcohol-induced reduction of allergic inflammatory cells in a mouse model of allergic asthma.

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.

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.

Protein kinase C epsilon is important in modulating organic-dust-induced airway inflammation.

Organic dust samples from swine confinement facilities elicit pro-inflammatory cytokine/chemokine release from bronchial epithelial cells and monocytes, dependent, in part, upon dust-induced activation of the protein kinase C (PKC) isoform, PKCε. PKCε is also rapidly activated in murine tracheal epithelial cells following in vivo organic dust challenges, yet the functional role of PKCε in modulating dust-induced airway inflammatory outcomes is not defined. Utilizing an established intranasal inhalation animal model, experiments investigated the biologic and physiologic responses following organic dust extract (ODE) treatments in wild-type (WT) and PKCε knock-out (KO) mice. We found that neutrophil influx increased more than twofold in PKCε KO mice following both a one-time challenge and 3 weeks of daily challenges with ODE as compared with WT mice. Lung pathology revealed increased bronchiolar and alveolar inflammation, lymphoid aggregates, and T cell influx in ODE-treated PKCε KO mice. Airway hyperresponsiveness to methacholine increased in PKCε KO + ODE to a greater magnitude than WT + ODE animals. There were no significant differences in cytokine/chemokine release elicited by ODE treatment between groups. However, ODE-induced nitric oxide (NO) production differed in that ODE exposure increased nitrate levels in WT mice but not in PKCε KO mice. Moreover, ODE failed to upregulate NO from ex vivo stimulated PKCε KO lung macrophages. Collectively, these studies demonstrate that PKCε-deficient mice were hypersensitive to organic dust exposure and suggest that PKCε is important in the normative lung inflammatory response to ODE. Dampening of ODE-induced NO may contribute to these enhanced inflammatory findings.