Agricultural workers are at risk for the development of acute and chronic lung diseases due to their exposure to organic agricultural dusts. A diet intervention using the omega-3 fatty acid docosahexaenoic acid (DHA) has been shown to be an effective therapeutic approach for alleviating a dust-induced inflammatory response. We thus hypothesized a high-DHA diet would alter the dust-induced inflammatory response through the increased production of specialized pro-resolving mediators (SPMs). Mice were pre-treated with a DHA-rich diet 4 weeks before being intranasally challenged with a single dose of an extract made from dust collected from a concentrated swine feeding operation (HDE). This omega-3-fatty-acid-rich diet led to reduced arachidonic acid levels in the blood, enhanced macrophage recruitment, and increased the production of the DHA-derived SPM Resolvin D1 (RvD1) in the lung following HDE exposure. An assessment of transcript-level changes in the immune response demonstrated significant differences in immune pathway activation and alterations of numerous macrophage-associated genes among HDE-challenged mice fed a high DHA diet. Our data indicate that consuming a DHA-rich diet leads to the enhanced production of SPMs during an acute inflammatory challenge to dust, supporting a role for dietary DHA supplementation as a potential therapeutic strategy for reducing dust-induced lung inflammation.

Motile cilia on airway cells are necessary for clearance of mucus-trapped particles out of the lung. Ciliated airway epithelial cells are uniquely exposed to oxidants through trapping of particles, debris and pathogens in mucus and the direct exposure to inhaled oxidant gases. Dynein ATPases, the motors driving ciliary motility, are sensitive to the local redox environment within each cilium. Several redox-sensitive cilia-localized proteins modulate dynein activity and include Protein Kinase A, Protein Kinase C, and Protein Phosphatase 1. Moreover, cilia are rich in known redox regulatory proteins and thioredoxin domain-containing proteins that are critical in maintaining a balanced redox environment. Importantly, a nonsense mutation in TXNDC3, which contains a thioredoxin motif, has recently been identified as disease-causing in Primary Ciliary Dyskinesia, a hereditary motile cilia disease resulting in impaired mucociliary clearance. Here we review current understanding of the role(s) oxidant species play in modifying airway ciliary function. We focus on oxidants generated in the airways, cilia redox targets that modulate ciliary beating and imbalances in redox state that impact health and disease. Finally, we review disease models such as smoking, asthma, alcohol drinking, and infections as well as the direct application of oxidants that implicate redox balance as a modulator of cilia motility.

ALCOHOL DRIVES OXIDATION-DEPENDENT DESENSITIZATION OF AIRWAY CILIA MOTILITY RESPONSIVENESS Michael E. Price Ph.D. University of Nebraska Medical Center 2018 Supervisor: Joseph H. Sisson, M. D. Alcohol abuse, which can impair clearance of pathogens and debris from the airway, is associated with an increase in complications and a higher mortality rate during the progression of pneumonia. With prolonged alcohol exposure, mucociliary clearance (MCC), which depends on the coordinated beating of cilia, is dysfunctional. Effective MCC relies on cAMP-dependent protein kinase (PKA) activation of ciliary beat frequency (CBF). PKA activation and stimulation of CBF are blunted with chronic exposure to alcohol. This phenomenon is known as alcohol-induced ciliary dysfunction (AICD). Previous studies have shown that concomitant feeding of mice with alcohol and antioxidants prevents AICD, suggesting a key role for oxidant signaling in AICD. We hypothesized that increased cilia-associated oxidant driven phosphatase activity plays a key role in the link between alcohol-driven oxidant production and cilia dysfunction. To test this hypothesis we assessed phosphatase activity and key checkpoints of ciliary function, such as CBF-and PKA responsiveness in isolated cilia, intact cell, tissue and animal models of AICD. Alcohol increased S-nitrosation in bronchoalveolar fluid and protein phosphatase 1 (PP1) activity in tracheal rings from mice drinking alcohol for 6 weeks. Furthermore, alcohol-stimulation of PP1 activity and S-nitrosation persisted at the tissue, cell and isolated cilia organelle level. Inhibition of PP1 reversed AICD, demonstrating a key role for PP1 in AICD. Structure based studies indicate that PP1 contains an oxidant sensitive active site. We identified PP1 cysteine 155 (PP1) as a key S-nitrosated residue in AICD and generated and expressed PP1 mutants in

Alcohol intake has been inconsistently associated with lung function levels in cross-sectional studies. The goal of our study was to determine whether longitudinally assessed light-to-moderate alcohol intake is associated with levels and decline of lung function. We examined data from 1333 adult participants in the population-based Tucson Epidemiological Study of Airway Obstructive Disease. Alcohol intake was assessed with four surveys between 1972 and 1992. Subjects who completed at least two surveys were classified into longitudinal drinking categories ("never", "inconsistent", or "persistent drinker"). Spirometric lung function was measured in up to 11 surveys between 1972 and 1992. Random coefficient models were used to test for differences in lung function by drinking categories. After adjustment for sex, age, height, education, BMI categories, smoking status, and pack-years, as compared to never-drinkers, persistent drinkers had higher FVC (coefficient: 157 mL, p < 0.001), but lower FEV1/FVC ratio (-2.3%, p < 0.001). Differences were due to a slower decline of FVC among persistent than among never-drinkers (p = 0.003), and these trends were present independent of smoking status. Inconsistent drinking showed similar, but weaker associations. After adjustment for potential confounders, light-to-moderate alcohol consumption was associated with a significantly decreased rate of FVC decline over adult life. (C) 2017 Elsevier Inc. All rights reserved.

Schmidt, Kyle; Price, Michael; Gard, Andrew P MD; Surdell, Daniel L MD; Sisson, Joseph MD; Thorell, William E MD Author Information

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.

While many genetic defects have been determined to compromise ciliary function, the detrimental effects of alcohol and other environmental factors can also cause ciliary dysfunction—a condition we call an "acquired ciliopathy." In this chapter, we focus on the impact of alcohol exposure on ciliary function. Alcohol-induced ciliary dysfunction (AICD) occurs with prolonged alcohol consumption and results in compromised lung function, in part, due to loss of airway cilia responsiveness resulting in impaired mucociliary clearance. In particular, we detail the impact of alcohol (ethanol) on the mammalian lung and airway cilia, discussing the effects of both brief and prolonged alcohol exposure. We summarize recent work demonstrating AICD in the model protist, Chlamydomonas reinhardtii, and the discovery of specific ciliary targets of alcohol. This chapter concludes with a section discussing new questions, future directions, and potential treatments for AICD.

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 β-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.

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.

Excessive alcohol consumption impairs mucociliary clearance, in part, by compromising ciliary movement. Our previous study found alcohol reduces ciliary beat frequency in Chlamydomonas through a mechanism that involves the β and γ heavy chains of the outer dynein arm (ODA). Moreover, we identified DC1, a subunit of the ODA-docking complex (ODA-DC), as the first ciliary target for alcohol. DC1 phosphorylation is alcohol sensitive and correlates with alcohol-induced ciliary dysfunction (AICD). Furthermore, DC1 phosphorylation is disrupted in the absence of the central pair and ODA. These results implicate a role for DC1 phosphorylation in regulating the ODA activity and mediating AICD. In our current study, we identified four alcohol-sensitive phosphosites in DC1: S33, T73, T351, and S628. Mutations of these sites rescue the assembly of the ODA-DC and ODA, resulting in wild-type swimming velocities. When cells were challenged with alcohol, we determined that three sites, S33, T351, and S628, are critical for mediating the ciliary slowing effects of alcohol. This result is consistent with our pharmacological studies, which reveal that both PP1 and PKA activities are required for AICD.

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. (C) 2018 Elsevier Inc. All rights reserved.

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.

Sex differences in asthma phenotypes and prevalence have been well described. In experimental animal models of asthma, female mice have increased airway hyperresponsiveness, eosinophil influx, and increased type 2 cytokine production (ie, interleukin [IL] 4, IL-5, and IL-13) in the lungs after allergen challenge when compared with males. Although CD4+ TH2 cells are known to produce type 2 cytokines, the type 2 innate lymphoid cells (ILC2s) are better described for producing much larger quantities of IL-5 and IL-13 compared with TH2 cells.

The prevalence of asthma in humans is increased in females compared to males after puberty, but the reasons for this remain unclear. The rare type 2 innate lymphoid cells (ILC2) have been recently implicated as drivers of allergic asthma. The objective of this study was to investigate sex differences in an experimental murine model with a focus on sex-specific ILC2 effects. Male and female BALB/c mice were sensitized and subsequently challenged with aerosolized ovalbumin (OVA) on days 17-21 for serum, bronchoalveolar lavage fluid (BALF), and lung tissue collection, and challenged for an additional 5 days for airway hyper-responsiveness (AHR). As compared to saline, OVA challenged mice demonstrated increased serum OVA-IgE, AHR, levels of type 2 cytokines (IL-4, IL-5, IL-13), and cellular influx of eosinophils, B cells, dendritic cells, T cells, and ILC2. Sex differences were demonstrated such that female mice had increased serum OVA-specific IgE, lung nitric oxide levels, and AHR as compared to males. As compared to male mice, dendritic cells were decreased in BALF and B cells were increased in the BALF and lung tissues of OVA challenged females. Female OVA challenged mice also demonstrated lower frequency of ILC2 in BALF, yet increased frequency in whole lung tissue. There was a significant increase in the levels of IL-5 and IL-13 produced ex vivo by IL-33 stimulated, isolated lung ILC2 from female saline and OVA challenged mice as compared to males. Taken together, the study highlights a potential sex-specific difference in ILC2 localization and activation in experimental allergic asthma.

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.