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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.

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.

Agricultural workers, especially those who work in swine confinement facilities, are at increased risk for developing pulmonary diseases including asthma, chronic obstructive pulmonary disease, and chronic bronchitis due to exposures to fumes, vapors, and organic dust. Repetitive exposure to agricultural dust leads to unresolved inflammation, a common underlying mechanism that worsens lung disease. Besides occupational exposure to dusts, diet also significantly contributes to inflammation and disease progression. Since DHA (docosahexaenoic acid), a polyunsaturated omega-3 fatty acid and its bioactive metabolites have key roles in inflammation resolution, we rationalized that individuals chronically exposed to organic dusts can benefit from dietary modifications. Here, we evaluated the role of DHA in modifying airway inflammation in a murine model of repetitive exposure to an aqueous extract of agricultural dust (three-week exposure to swine confinement dust extract, HDE) and after a one-week resolution/recovery period. We found that mice fed a high DHA diet had significantly increased bronchoalveolar lavage fluid (BALF) levels of DHA-derived resolvins and lower TNFα along with altered plasma levels of endocannabinoids and related lipid mediators. Following the one-week recovery we identified significantly reduced BALF cellularity and cytokine/chemokine release along with increased BALF amphiregulin and resolvins in DHA diet-fed versus control diet-fed mice challenged with HDE. We further report observations on the effects of repetitive HDE exposure on lung Ym1+ and Arg-1+ macrophages. Overall, our findings support a protective role for DHA and identify DHA-derived resolvins and endocannabinoids among the potential mediators of DHA in altering airway inflammation in chronic agricultural dust exposure.

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.

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 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.

Assembly of the C1d projection of the central microtubule pair apparatus in mammalian motile cilia requires the ciliary protein CFAP54. Loss of the C1d projection in mice lacking CFAP54 impairs ciliary motility and cilia-driven fluid flow and results in a primary ciliary dyskinesia phenotype., Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.

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 β-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.

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.

Respiratory viral infections cause devastating airway inflammation and remodeling in asthmatic children and adults. Type 2 innate lymphoid cells (ILC2) have been characterized individually in both asthma and viral infections. We hypothesize that ILC2 are activated by IL-33 in our model, and thereby enhance the asthmatic phenotype in the lung. Asthma induction was performed in eight week old, female BALB/c mice using ovalbumin (OVA) sensitization and airway challenge. After the last airway challenge, mice were infected with RSV (3,250 TCID50 U/mouse). Lungs were collected for ILC2 sorting, immune cell quantitation, and analysis of IL-4, IL-13, IL-33 and IL-25 on days 1–4 of infection. A subset of lungs were collected and fixed for histological analysis. Results show that mucus production (PAS+ cells) and lymphocytic recruitment were enhanced with RSV infection and asthma. The expression of MUC5AC, CLCA3, IL-25 and IL-33 mRNA levels were increased at least two-fold in the lungs of OVA+RSV mice compared to RSV-infected alone. We detected comparable numbers of ILC2 in BAL fluid from both OVA+ and OVA+RSV mice, yet an evaluation of total lung lymphocytes yielded more ILC2 in the OVA+RSV mice as compared to OVA alone. ILC2 from OVA+RSV mice were sorted and treated with IL-33 for 24 hours. These cells produced greater IL-4 mRNA than cells isolated from OVA only mice. Our results describe a potential role for ILC2 in the exacerbation of asthma with RSV infection. Future studies are planned to determine the exact contribution of ILC2 to type 2 immunity and pulmonary function in our asthma-viral infection model.