[Show abstract][Hide abstract] ABSTRACT: 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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-015-0272-9) contains supplementary material, which is available to authorized users.
Full-text · Article · Sep 2015 · Respiratory research
[Show abstract][Hide abstract] ABSTRACT: Objective:
To evaluate the relationship between alcohol consumption and the risk of acute exacerbation of COPD (AECOPD).
Methods and measurements:
We conducted a secondary analysis of data previously collected in a large, multicenter trial of daily azithromycin in COPD. To analyze the relationship between amount of baseline self-reported alcohol consumption in the past 12 months and subsequent AECOPD, we categorized the subjects as minimal (<1 drink/month), light-to-moderate (1-60 drinks/month), or heavy alcohol users (>60 drinks/month). The primary outcome was time to first AECOPD and the secondary outcome was AECOPD rate during the 1-year study period.
Of the 1,142 enrolled participants, 1,082 completed baseline alcohol questionnaires and were included in this analysis. Six hundred and forty-five participants reported minimal alcohol intake, 363 reported light-to-moderate intake, and 74 reported heavy intake. There were no statistically significant differences in median time to first AECOPD among minimal (195 days), light-to-moderate (241 days), and heavy drinkers (288 days) (P=0.11). The mean crude rate of AECOPD did not significantly differ between minimal (1.62 events per year) and light-to-moderate (1.44 events per year) (P=0.095), or heavy drinkers (1.68 events per year) (P=0.796). There were no significant differences in hazard ratios for AECOPD after adjustment for multiple covariates.
Among persons with COPD at high risk of exacerbation, we found no significant relationship between self-reported baseline alcohol intake and subsequent exacerbations. The number of patients reporting heavy alcohol intake was small and further study is needed to determine the effect of heavy alcohol intake on AECOPD risk.
Preview · Article · Aug 2015 · International Journal of COPD
[Show abstract][Hide abstract] ABSTRACT: 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-, IL-6, and IL-8. ResultsAirway 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 invitro 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.
No preview · Article · Jul 2015 · Alcoholism Clinical and Experimental Research
[Show abstract][Hide abstract] ABSTRACT: 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 3–7 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.
[Show abstract][Hide abstract] ABSTRACT: When Paramecium encounter 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 of 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.This article is protected by copyright. All rights reserved.
No preview · Article · Jul 2014 · Journal of Eukaryotic Microbiology
[Show abstract][Hide abstract] ABSTRACT: Non-motile 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 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.
Full-text · Article · Mar 2014 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and the third most common reason for hospitalization in the elderly. One reason that the elderly may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however little is known about the mechanism(s). We compared the CBF in BALB/c and C57BL/6 mice aged 2 months, 12 months and 24 months and found that CBF diminishes with age. Both the 12 and 24-month cilia retained their ability to be stimulated by the β2 agonist, procaterol. To help determine the mechanism of ciliary slowing, we measured PKCα and PKCε activity. There were no activity differences in PKCα; however, we demonstrate a significantly higher PKCε activity in the 12- and 24-month mice than the 2-month mice. The increase in activity is likely due to a nearly 3-fold increase in PKCε protein in the lung during aging. To strengthen the connection between PKCε activation and ciliary slowing, we treated 2-month tracheas with the PKCε agonist, 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with β2 agonists. Conclusions: In this mouse model of aging, we show that the decreases in CBF are related to an increase in PKCε activity.
No preview · Article · Jan 2014 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: The debilitating and persistent effects of ICU-acquired delirium and weakness warrant testing of prevention strategies. The purpose of this study was to evaluate the effectiveness and safety of implementing the Awakening and Breathing Coordination, Delirium monitoring/management, and Early exercise/mobility bundle into everyday practice.
Eighteen-month, prospective, cohort, before-after study conducted between November 2010 and May 2012.
Five adult ICUs, one step-down unit, and one oncology/hematology special care unit located in a 624-bed tertiary medical center.
Two hundred ninety-six patients (146 prebundle and 150 postbundle implementation), who are 19 years old or older, managed by the institutions' medical or surgical critical care service.
Awakening and Breathing Coordination, Delirium monitoring/management, and Early exercise/mobility bundle.
For mechanically ventilated patients (n = 187), we examined the association between bundle implementation and ventilator-free days. For all patients, we used regression models to quantify the relationship between Awakening and Breathing Coordination, Delirium monitoring/management, and Early exercise/mobility bundle implementation and the prevalence/duration of delirium and coma, early mobilization, mortality, time to discharge, and change in residence. Safety outcomes and bundle adherence were monitored. Patients in the postimplementation period spent three more days breathing without mechanical assistance than did those in the preimplementation period (median [interquartile range], 24 [7-26] vs 21 [0-25]; p = 0.04). After adjusting for age, sex, severity of illness, comorbidity, and mechanical ventilation status, patients managed with the Awakening and Breathing Coordination, Delirium monitoring/management, and Early exercise/mobility bundle experienced a near halving of the odds of delirium (odds ratio, 0.55; 95% CI, 0.33-0.93; p = 0.03) and increased odds of mobilizing out of bed at least once during an ICU stay (odds ratio, 2.11; 95% CI, 1.29-3.45; p = 0.003). No significant differences were noted in self-extubation or reintubation rates.
Critically ill patients managed with the Awakening and Breathing Coordination, Delirium monitoring/management, and Early exercise/mobility bundle spent three more days breathing without assistance, experienced less delirium, and were more likely to be mobilized during their ICU stay than patients treated with usual care.
Full-text · Article · Jan 2014 · Critical care medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Background. Vest chest physiotherapy (VCPT) enhances airway clearance in cystic fibrosis (CF) by an unknown mechanism. Because cilia are sensitive to nitric oxide (NO), we hypothesized that VCPT enhances clearance by changing NO metabolism. Methods. Both normal subjects and stable CF subjects had pre- and post-VCPT airway clearance assessed using nasal saccharin transit time (NSTT) followed by a collection of exhaled breath condensate (EBC) analyzed for NO metabolites (NO x ). Results. VCPT shorted NSTT by 35% in normal and stable CF subjects with no difference observed between the groups. EBC NO x concentrations decreased 68% in control subjects after VCPT (before = 115 ± 32 μ M versus after = 37 ± 17 μ M; P < 0.002). CF subjects had a trend toward lower EBC NO x . Conclusion. We found an association between VCPT-stimulated clearance and exhaled NO x levels in human subjects. We speculate that VCPT stimulates clearance via increased NO metabolism.
Full-text · Article · Nov 2013 · Pulmonary Medicine
[Show abstract][Hide abstract] ABSTRACT: 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 Clara cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of Clara cells, since these cells are involved in airway repair. Clara cells may be prevented from differentiating into respiratory epithelial cells due to a lack of IFT88 protein that is necessary to form a single non-motile 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.
Full-text · Article · Nov 2013 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: The airway epithelium is exposed to alcohol during drinking through direct exhalation of volatized ethanol from the bronchial circulation. Alcohol exposure leads to a rapid increase in the cilia beat frequency (CBF) of bronchial epithelial cells followed by a chronic desensitization of cilia stimulatory responses. This effect is governed in part by the nitric oxide regulation of cyclic guanosine and adenosine monophosphate-dependent protein kinases (PKG and PKA) and is not fully understood. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is implicated in the pathogenesis of several pulmonary disorders. We hypothesized that the inhibition of nitric oxide synthase by ADMA blocks alcohol-stimulated increases in CBF. To test this hypothesis, ciliated primary bovine bronchial epithelial cells (BBEC) were preincubated with ADMA (100 µ M) and stimulated with 100 mM ethanol. CBF was measured and PKA assayed. By 1 hr, ethanol activated PKA, resulting in elevated CBF. Both alcohol-induced PKA activation and CBF were inhibited in the presence of ADMA. ADMA alone had no effect on PKA activity or CBF. Using a mouse model overexpressing the ADMA-degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), we examined PKA and CBF in precision-cut mouse lung slices. Alcohol-stimulated increases in lung slice PKA and CBF were temporally enhanced in the DDAH mice versus control mice.
Full-text · Article · Nov 2013 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Previously we have shown that chronic alcohol intake causes alcohol-induced ciliary dysfunction (AICD), leading to non-responsive airway cilia. AICD likely occurs through the downregulation of nitric oxide (NO) and cyclic nucleotide-dependent kinases, protein kinase G (PKG) and protein kinase A (PKA). Studies by others have shown that dietary supplementation with the antioxidants N-acetylcysteine (NAC) and procysteine prevent other alcohol-induced lung complications. This led us to hypothesize that dietary supplementation with NAC or procysteine prevents AICD. To test this hypothesis, C57BL/6 mice drank an alcohol/water solution (20% w/v) ad libitum for 6 weeks and were concurrently fed dietary supplements of either NAC or procysteine. Ciliary beat frequency (CBF) was measured in mice tracheas, and PKG/PKA responsiveness to β-agonists and NOx levels were measured from bronchoalveolar lavage (BAL) fluid. Long-term alcohol drinking reduced CBF, PKG and PKA responsiveness to β-agonists, and lung NOx levels in BAL fluid. In contrast, alcohol-drinking mice fed NAC or procysteine sustained ciliary function and PKG and PKA responsiveness to β-agonists. However, BAL NO levels remained low despite antioxidant supplementation. We also determined that removal of alcohol from the drinking water for as little as 1 week restored ciliary function, but not PKG and PKA responsiveness to β-agonists. We conclude that dietary supplementation with NAC or procysteine protects against AICD. In addition, alcohol removal for 1 week restores cilia function independent of PKG and PKA activity. Our findings provide a rationale for the use of antioxidants to prevent damage to airway mucociliary functions in chronic alcohol-drinking individuals.
No preview · Article · Oct 2013 · Alcohol (Fayetteville, N.Y.)