Publications by authors named "Art J Heires"

29 Publications

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A high docosahexaenoic acid diet alters lung inflammation and recovery following repetitive exposure to aqueous organic dust extracts.

J Nutr Biochem 2021 Jun 12;97:108797. Epub 2021 Jun 12.

Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA; Pulmonary, Critical Care, Sleep and Allergy Division, University of Nebraska Medical Center, Omaha, Nebraska, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jnutbio.2021.108797DOI Listing
June 2021

A High Docosahexaenoic Acid Diet Alters the Lung Inflammatory Response to Acute Dust Exposure.

Nutrients 2020 Aug 4;12(8). Epub 2020 Aug 4.

Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, CA 92521, USA.

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.
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http://dx.doi.org/10.3390/nu12082334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468878PMC
August 2020

Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust.

Am J Physiol Lung Cell Mol Physiol 2020 01 6;318(1):L180-L191. Epub 2019 Nov 6.

Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska.

Exposure to agricultural bioaerosols can lead to chronic inflammatory lung diseases. Amphiregulin (AREG) can promote the lung repair process but can also lead to fibrotic remodeling. The objective of this study was to determine the role of AREG in altering recovery from environmental dust exposure in a murine in vivo model and in vitro using cultured human and murine lung fibroblasts. C57BL/6 mice were intranasally exposed to swine confinement facility dust extract (DE) or saline daily for 1 wk or allowed to recover for 3-7 days while being treated with an AREG-neutralizing antibody or recombinant AREG. Treatment with the anti-AREG antibody prevented resolution of DE exposure-induced airway influx of total cells, neutrophils, and macrophages and increased levels of TNF-α, IL-6, and CXCL1. Neutrophils and activated macrophages (CD11cCD11b) persisted after recovery in lung tissues of anti-AREG-treated mice. In murine and human lung fibroblasts, DE induced the release of AREG and inflammatory cytokines. Fibroblast recellularization of primary human lung mesenchymal matrix scaffolds and wound closure was inhibited by DE and enhanced with recombinant AREG alone. AREG treatment rescued the DE-induced inhibitory fibroblast effects. AREG intranasal treatment for 3 days during recovery phase reduced repetitive DE-induced airway inflammatory cell influx and cytokine release. Collectively, these studies demonstrate that inhibition of AREG reduced, whereas AREG supplementation promoted, the airway inflammatory recovery response following environmental bioaerosol exposure, and AREG enhanced fibroblast function, suggesting that AREG could be targeted in agricultural workers repetitively exposed to organic dust environments to potentially prevent and/or reduce disease.
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http://dx.doi.org/10.1152/ajplung.00039.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985879PMC
January 2020

Insufficient zinc intake enhances lung inflammation in response to agricultural organic dust exposure.

J Nutr Biochem 2019 08 3;70:56-64. Epub 2019 May 3.

The University of Nebraska Medical Center College of Medicine, Omaha, NE 68198. Electronic address:

Organic dust exposure particularly within hog confinement facilities is a significant cause of airway inflammation and lung disease. In a cohort of Midwestern veterans with COPD and agricultural work exposure we observed reduced zinc intakes which were associated with decreased lung function. Because insufficient zinc intake is common within the U.S. and a potent modulator of innate immune function, we sought to determine whether deficits in zinc intake would impact the airway inflammatory response to hog confinement facility dust extract (HDE). Adult male C57BL/6 mice were randomized to zinc deficient or matched zinc sufficient diets for 3 weeks and subsequently treated with intranasal HDE inhalation or saline once or daily for 3 weeks while maintained on specific diets. Lavage fluid and lung tissue was collected. Conditions of zinc deficiency were also studied in macrophages exposed to HDE. Single and repetitive HDE inhalation exposure resulted in increased influx of total cells and neutrophils, increased mediator hyper-responsiveness (TNFα, IL-6, CXCL1, and amphiregulin), and enhanced tissue pathology that was more pronounced in zinc deficient mice compared to normal dietary counterparts. Airway inflammation was most pronounced in zinc deficient mice treated with repetitive HDE for 3 weeks. Similarly, macrophages maintained in a zinc deficient environment exhibited increased CXCL1 and IL-23 production as a result of increased NF-κB activation. Conclusion: Given the relatively high incidence of dietary deficiencies in agriculture workers, we anticipate that zinc intake, or a lack thereof, may play an important role in modulating the host response to organic dust exposure.
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http://dx.doi.org/10.1016/j.jnutbio.2019.04.007DOI Listing
August 2019

Biphasic changes in airway epithelial cell EGF receptor binding and phosphorylation induced by components of hogbarn dust.

Exp Lung Res 2018 12 12;44(10):443-454. Epub 2019 Mar 12.

a Department of Pharmacology and Experimental Neuroscience , University of Nebraska Medical Center , Omaha , NE , USA.

Purpose Of The Study: Workers in enclosed hogbarns experience an increased incidence of airway inflammation and obstructive lung disease, and an aqueous hogbarn dust extract (HDE) induces multiple inflammation-related responses in cultured airway epithelial cells. Epidermal growth factor receptor (EGFR) phosphorylation and activation has been identified as one important mediator of inflammatory cytokine release from these cells. The studies here investigated both early and late phase adaptive changes in EGFR binding properties and subcellular localization induced by exposure of cells to HDE.

Materials And Methods: Cell surface EGFRs were quantified as binding to intact cells on ice. EGFR phosphorylation, expression, and localization were assessed with anti-EGFR antibodies and either blotting or confocal microscopy.

Results: In BEAS-2B and primary human bronchial epithelial cells, HDE induced decreases in cell surface EGFR binding following both 15-min and 18-h exposures. In contrast, H292 cells exhibited only the 15-min decrease, with binding near the control level at 18 hr. Confocal microscopy showed that the 15-min decrease in binding is due to EGFR endocytosis. Although total EGFR immunoreactivity decreased markedly at 18 hr in confocal microscopy with BEAS-2B cells, immunoblots showed no loss of EGFR protein. HDE stimulated EGFR phosphorylation at both 15 min and 18 hr in BEAS-2B cells and primary cells, but only at 15 min in H292 cells, indicating that the different EGFR binding changes among these cell types is likely related to their different time-dependent changes in phosphorylation.

Conclusions: These studies extend the evidence for EGFRs as important cellular targets for components of HDE and they reveal novel patterns of EGFR phosphorylation and binding changes that vary among airway epithelial cell types. The results provide both impetus and convenient assays for identifying the EGFR-activating components and pathways that likely contribute to hogbarn dust-induced lung disease in agricultural workers.
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http://dx.doi.org/10.1080/01902148.2019.1575931DOI Listing
December 2018

Bovine milk-derived extracellular vesicles enhance inflammation and promote M1 polarization following agricultural dust exposure in mice.

J Nutr Biochem 2019 02 3;64:110-120. Epub 2018 Nov 3.

Pulmonary, Critical Care, Sleep and Allergy Division, University of Nebraska Medical Center, Omaha, NE, 68198; VA Nebraska-Western Iowa Health Care System, Omaha, NE, 68105. Electronic address:

Occupational agricultural dust exposure can cause severe lung injury, including COPD and asthma exacerbations. Cell-derived extracellular vesicles can mediate inflammatory responses and immune activation, but the contribution of diet-derived extracellular vesicles to these processes is poorly understood. We investigated whether bovine milk-derived extracellular vesicles modulate inflammatory responses to agricultural dust exposures in a murine model. C57BL/6 mice were fed either an extracellular vesicle-enriched modification of the AIN-93G diet with lyophilized bovine milk (EV) or a control diet wherein the milk was presonicated, disrupting the milk extracellular vesicles and thereby leading to RNA degradation (DEV). Mice were maintained on the diets for 5-7 weeks and challenged with a single (acute) intranasal instillation of a 12.5% organic dust extract (DE) or with 15 instillations over 3 weeks (repetitive exposure model). Through these investigations, we identified significant interactions between diet and DE when considering numerous inflammatory outcomes, including lavage inflammatory cytokine levels and cellular infiltration into the lung airways. DE-treated peritoneal macrophages also demonstrated altered polarization, with EV-fed mouse macrophages exhibiting an M1 shift compared to an M2 phenotype in DEV-fed mice (IL-6, TNF, IL-12/23 all significantly elevated, and IL-10 and arginase decreased in EV macrophages, ex vivo). In complementary in vitro studies, mouse macrophages treated with purified milk-derived EV were found to express similar polarization phenotypes upon DE stimulation. These results suggest a role for dietary extracellular vesicles in the modulation of lung inflammation in response to organic dust which may involve macrophage phenotype polarization.
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http://dx.doi.org/10.1016/j.jnutbio.2018.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363837PMC
February 2019

Alcohol and cannabis use alter pulmonary innate immunity.

Alcohol 2019 11 10;80:131-138. Epub 2018 Nov 10.

University of Colorado, Anschutz Medical Campus, Aurora, CO 80045, United States.

Purpose: Cannabis use is increasing due to recent legislative changes. In addition, cannabis is often used in conjunction with alcohol. The airway epithelium is the first line of defense against infectious microbes. Toll-like receptors (TLR) recognize airborne microbes and initiate the inflammatory cytokine response. The mechanism by which cannabis use in conjunction with alcohol affects pulmonary innate immunity mediated by TLRs is unknown.

Methods: Samples and data from an existing cohort of individuals with alcohol use disorders (AUDs), along with samples from additional participants with cannabis use alone and with AUD were utilized. Subjects were categorized into the following groups: no alcohol use disorder (AUD) or cannabis use (control) (n = 46), AUD only (n = 29), cannabis use-only (n = 39), and AUD and cannabis use (n = 29). The participants underwent bronchoscopy with bronchoalveolar lavage (BAL) and airway epithelial brushings. We measured IL-6, IL-8, TNF⍺, and IL-10 levels in BAL fluid, and performed real-time PCR for TLR1-9 on the airway epithelial brushings.

Results: We found significant increases in TLR2 with AUD alone, cannabis use alone, and cannabis use with AUD, compared to control. TLR5 was increased in cannabis users compared to control, TLR6 was increased in cannabis users and cannabis users with AUD compared to control, TLR7 was increased in cannabis users compared to control, and TLR9 was increased in cannabis users compared to control. In terms of cytokine production, IL-6 was increased in cannabis users compared to control. IL-8 and IL-10 were increased in AUD only.

Conclusions: AUD and cannabis use have complex effects on pulmonary innate immunity that promote airway inflammation.
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http://dx.doi.org/10.1016/j.alcohol.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509015PMC
November 2019

Aging leads to dysfunctional innate immune responses to TLR2 and TLR4 agonists.

Aging Clin Exp Res 2019 Sep 7;31(9):1185-1193. Epub 2018 Nov 7.

Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198-5910, USA.

Background: Sepsis is more common in the elderly. TNF⍺ is recognized as an important mediator in sepsis and Toll-like receptors (TLRs) play an important role in initiating signaling cascades to produce TNF⍺. Little is known about how innate immunity is altered in healthy human aging that predisposes to sepsis.

Aims And Methods: We tested the hypothesis that aging dysregulates the innate immune response to TLR 2 and 4 ligands. We performed whole blood assays on 554 healthy subjects aged 40-80 years. TNFα production was measured at baseline and after stimulation with the TLR2 agonists: peptidoglycan, lipoteichoic acid, Pam3CysK, Zymosan A and the TLR4 agonist lipopolysaccharide (LPS). In a subset of subjects (n = 250), we measured Toll-like receptor (TLR) 2, 4 and MyD88 expression using real-time PCR.

Results And Discussion: We measured a 2.5% increase per year in basal secretion of TNFα with aging (n = 554 p = 0.02). Likewise, TNFα secretion was increased with aging after stimulation with peptidoglycan (1.3% increase/year; p = 0.0005) and zymosan A (1.1% increase/year p = 0.03). We also examined the difference between baseline and stimulated TNFα for each individual. We found that the increase was driven by the elevated baseline levels. In fact, there was a diminished stimulated response to LPS (1.9% decrease/year; p = 0.05), lipoteichoic acid (2.1% decrease/year p = 0.03), and Pam3CysK (2.6% decrease/year p = 0.0007). There were no differences in TLR or MyD88 mRNA expression with aging, however, there was an inverse relationship between TLR expression and stimulated TNFα production.

Conclusions: With aging, circulating leukocytes produce high levels of TNFα at baseline and have inadequate responses to TLR2 and TLR4 agonists. These defects likely contribute to the increased susceptibility to sepsis in older adults.
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http://dx.doi.org/10.1007/s40520-018-1064-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504629PMC
September 2019

Relationship of systemic IL-10 levels with proinflammatory cytokine responsiveness and lung function in agriculture workers.

Respir Res 2018 Sep 3;19(1):166. Epub 2018 Sep 3.

Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA.

Background: Agriculture workers are exposed to microbial component- and particulate matter-enriched organic dust aerosols. Whereas it is clear that exposure to these aerosols can lead to lung inflammation, it is not known how inflammatory responses are resolved in some individuals while others develop chronic lung disease. Interleukin (IL)-10 is an immunomodulatory cytokine that is recognized as a potent anti-inflammatory and pro-resolving factor. The objective of this study was to determine whether there is a relationship of systemic IL-10 and proinflammatory responses and/or respiratory health effects in humans with prior agriculture exposure.

Methods: This is a cross sectional study of 625 veterans with > 2 years of farming experience. Whole blood was stimulated with or without organic dust and measured for IL-6, TNFα and IL-10. Participants underwent spirometry and respiratory symptoms were assessed by questionnaire.

Results: We found that baseline IL-10 concentration from the whole blood assay was inversely associated with ΔTNF-α (r = - 0.63) and ΔIL-6 (r = - 0.37) levels. Results remained highly significant in the linear regression model after adjusting for age, sex, BMI, race, education, smoking status, and white blood cell count (ΔTNF-α, p < 0.0001; ΔIL-6, p < 0.0001). We found no association between chronic cough (p = 0.18), chronic phlegm (p = 0.31) and chronic bronchitis (p = 0.06) and baseline IL-10 levels using univariate logistic regression models. However, we did find that higher FEV/FVC was significantly associated with increased baseline IL-10 concentration.

Conclusions: Collectively, these studies support a potential role for IL-10 in modulating an inflammatory response and lung function in agriculture-exposed persons.
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http://dx.doi.org/10.1186/s12931-018-0875-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122449PMC
September 2018

Short term dynamics of the sputum microbiome among COPD patients.

PLoS One 2018 8;13(3):e0191499. Epub 2018 Mar 8.

University of Nebraska, Department of Food Science & Technology, Lincoln, NE, United States of America.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by incompletely reversible airflow obstruction. The complexity of the lung microbial community in COPD patients has been highlighted in recent years. Evidence suggests that transplantation, medications, age, and disease severity influence microbial community membership. However, the dynamics of the lung microbiome in stable COPD patients remain poorly understood. In this study, we completed a longitudinal 16S ribosomal RNA survey of the lung microbiome on replicate sputum samples collected from 4 former smokers with COPD (Stage 2) within a 2-day time period. Samples from each individual over the two-day period were similar based on α-diversity, principle component analysis and taxonomy at the phyla and genera level. Sputum samples from COPD patients were also collected between 2-9 months of follow-up. Data suggest an increased variability of the sputum microbiota when comparing samples collected ≤ 3 months compared to those collected ≥ 4 months; however, no statistically significant shifts in the abundance (>2-fold) of taxa between the two time points was observed. Bacterial composition and the number of operational taxonomic units (OTUs) remained similar over time. Results from this study suggest that the sputum microbiome is relatively stable in clinically stable COPD patients (Stage 2). This study furthers our understanding of the dynamics of the lung microbiome in COPD patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191499PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843169PMC
June 2018

Post-injury and resolution response to repetitive inhalation exposure to agricultural organic dust in mice.

Safety (Basel) 2017 21;3(1). Epub 2017 Feb 21.

Pulmonary, Critical Care, Sleep & Allergy Division; Department of Internal Medicine, University of Nebraska Medical Center (UNMC), 985990 Nebraska Medical Center, Omaha, NE 68198-5990, USA.

Inhalation of organic dusts in agricultural environments causes airway inflammatory diseases. Despite advances in understanding the airway response to dust-induced inflammation, less is known about the transition from lung injury to repair and recovery. The objective of this study was to define the post-inflammation homeostasis events following organic dust-induced lung injury. Using an established protocol, mice were intranasally treated with swine confinement facility organic dust extract (ODE) daily for 3 weeks (repetitive exposure) or treated daily with ODE for 3 weeks followed by no treatment for 1-4 weeks (recovery period) whereupon lavage fluid, lung tissue, and sera were processed. During recovery period, a significant decrease was observed in ODE-induced neutrophil levels after 1 week, lymphocytes at 2 weeks, and macrophages at 4 weeks in the lavage fluid. ODE-induced lung cellular aggregates and bronchiolar compartment inflammation were diminished, but persisted for 4 weeks post-injury. Alveolar inflammation resolved at 3 weeks. ODE-induced lung neutrophils were cleared by 3 weeks, B-cells by 2 weeks, and CD3CD4 and CD3CD8 T cells by 4 week recovery period. Collectively, these results identify important processes during recovery period following agricultural dust-induced inflammation, and present possible strategies for improving lung repair and resolution.
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http://dx.doi.org/10.3390/safety3010010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788309PMC
February 2017

Effects of Agricultural Organic Dusts on Human Lung-Resident Mesenchymal Stem (Stromal) Cell Function.

Toxicol Sci 2018 04;162(2):635-644

Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198.

Agricultural organic dust exposures trigger harmful airway inflammation, and workers experiencing repetitive dust exposures are at increased risk for lung disease. Mesenchymal stem/stromal cells (MSCs) regulate wound repair processes in the lung, and may contribute to either proresolution or -fibrotic lung responses. It is unknown how organic dust exposures alter lung-resident MSC activation and proinflammatory versus prorepair programs in the lung. To address this gap in knowledge, we isolated human lung-resident MSC from lung tissue. Cells were stimulated with aqueous extracts of organic dusts (DE) derived from swine confinement facilities and were assessed for changes in proliferative and migratory capacities, and production of proinflammatory and prorepair mediators. Through these investigations, we found that DE induces significant release of proinflammatory mediators TNF-α, IL-6, IL-8, and matrix metalloproteases, while also inducing the production of prorepair mediators amphiregulin, FGF-10, and resolvin D1. In addition, DE significantly reduced the growth and migratory capacities of lung-resident MSC. Together, these investigations indicate lung-resident MSC activation and wound repair activities are altered by organic dust exposures. These findings warrant future investigations to assess how organic dusts affect lung-resident mesenchymal stem/stromal cell function and impact airway inflammation, injury, and repair during agricultural aerosol exposures.
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http://dx.doi.org/10.1093/toxsci/kfx286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889032PMC
April 2018

Docosahexaenoic acid enhances amphiregulin-mediated bronchial epithelial cell repair processes following organic dust exposure.

Am J Physiol Lung Cell Mol Physiol 2018 03 2;314(3):L421-L431. Epub 2017 Nov 2.

Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska.

Injurious dust exposures in the agricultural workplace involve the release of inflammatory mediators and activation of epidermal growth factor receptor (EGFR) in the respiratory epithelium. Amphiregulin (AREG), an EGFR ligand, mediates tissue repair and wound healing in the lung epithelium. Omega-3 fatty acids such as docosahexaenoic acid (DHA) are also known modulators of repair and resolution of inflammatory injury. This study investigated how AREG, DHA, and EGFR modulate lung repair processes following dust-induced injury. Primary human bronchial epithelial (BEC) and BEAS-2B cells were treated with an aqueous extract of swine confinement facility dust (DE) in the presence of DHA and AREG or EGFR inhibitors. Mice were exposed to DE intranasally with or without EGFR inhibition and DHA. Using a decellularized lung scaffolding tissue repair model, BEC recolonization of human lung scaffolds was analyzed in the context of DE, DHA, and AREG treatments. Through these investigations, we identified an important role for AREG in mediating BEC repair processes. DE-induced AREG release from BEC, and DHA treatment following DE exposure, enhanced this release. Both DHA and AREG also enhanced BEC repair capacities and rescued DE-induced recellularization deficits. In vivo, DHA treatment enhanced AREG production following DE exposure, whereas EGFR inhibitor-treated mice exhibited reduced AREG in their lung homogenates. These data indicate a role for AREG in the process of tissue repair after inflammatory lung injury caused by environmental dust exposure and implicate a role for DHA in regulating AREG-mediated repair signaling in BEC.
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http://dx.doi.org/10.1152/ajplung.00273.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900355PMC
March 2018

Alcohol Inhibits Organic Dust-induced ICAM-1 Expression on Bronchial Epithelial Cells.

Safety (Basel) 2017 Mar 7;3(1). Epub 2017 Jan 7.

Pulmonary, Critical Care, Sleep & Allergy Division of the Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198; USA.

Aims: Exposure to dusts/bioaerosols in concentrated animal feeding operations (CAFOs) results in inflammatory lung diseases in workers. Hog CAFOs dust extract (HDE) increases expression of intercellular adhesion molecule-1 (ICAM-1), neutrophil adhesion, and TNFα release in bronchial epithelial cells. Alcohol consumption is increasingly recognized to impair lung immunity. We hypothesized that alcohol impairs HDE-induced TNFα, ICAM-1 expression and neutrophil adhesion by directly inhibiting TNFα converting enzyme (TACE) activity.

Methods: Bronchial epithelial cells (BEAS-2B) and primary human bronchial epithelial cells were pretreated with ethanol (EtOH) or TACE inhibitor. ICAM-1 surface expression, TNFα release, and TACE activity were analyzed following HDE stimulation. The effect of alcohol and TACE inhibition on HDE-regulated epithelial cell/neutrophil adhesion interactions was investigated. Finally, utilizing an established animal model, C57BL/6 mice were fed ethanol (20%) in drinking water for 8 wk followed by daily intranasal inhalation of HDE or saline during the final two weeks. Mice were sacrificed and lung sections immunostained for ICAM-1.

Results: Pretreatment with alcohol or TACE inhibitor significantly decreased HDE-induced ICAM-1 expression and TNFα release. HDE augmented neutrophil adhesion to epithelial cells, which was decreased with alcohol (32% decrease) or TACE inhibitor (55% decrease) pretreatment. TACE activity increased following HDE exposure, but TACE activity was inhibited following alcohol pretreatment. Alcohol-fed mice demonstrated decreased HDE-induced airway epithelium ICAM-1 expression.

Conclusions: Alcohol diminishes HDE-induced ICAM-1 expression, TNFα release, and neutrophil adhesion via inhibition of TACE activity. These results suggest that alcohol may be an important modulator of lung innate immune responses following CAFO exposure.
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http://dx.doi.org/10.3390/safety3010005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658133PMC
March 2017

Bioactive Lipid Mediators Regulate Lung Epithelial and Mesenchymal Cell Reparative Processes In Vitro.

Ann Am Thorac Soc 2017 09;14(Suppl_3):S252-S253

1 Pulmonary, Critical Care, Sleep, and Allergy Division, University of Nebraska Medical Center, Omaha, Nebraska; and.

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http://dx.doi.org/10.1513/AnnalsATS.201606-457MGDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711337PMC
September 2017

Interaction of CD14 haplotypes and soluble CD14 on pulmonary function in agricultural workers.

Respir Res 2017 03 16;18(1):49. Epub 2017 Mar 16.

Department of Internal Medicine and Veterans Nebraska Western Iowa Healthcare System, Omaha, NE, USA.

Background: Agricultural environments are contaminated with organic dusts containing bacterial components. Chronic inhalation of organic dusts is implicated in respiratory diseases. CD14 is a critical receptor for gram-negative lipopolysaccharide; however, its association with respiratory disease among agricultural workers is unknown. The objective of this study was to determine if serum soluble CD14 (sCD14) levels are associated with lung function among agricultural workers and if this association is modified by genetic variants in CD14.

Methods: This cross-sectional study included 584 veterans with >2 years of farming experience and that were between the ages of 40 and 80 years. Participants underwent spirometry and were genotyped for four tagging CD14 polymorphisms (CD14/-2838, rs2569193; CD14/-1720, rs2915863; CD14/-651, rs5744455; and CD14/-260, rs2569190). Serum sCD14 was assayed by ELISA.

Results: Subjects were 98% white males with a mean age 64.5 years. High soluble CD14 levels (> median sCD14) were associated decreased lung function (FEV/FVC, p = 0.011; % predicted FEV, p = 0.03). When stratified by COPD (yes/no) and smoking status (ever/never), high sCD14 levels (> median sCD14) were associated with low lung function among ever smokers with COPD (% predicted FEV, p = 0.0008; FEV/FVC, p = 0.0002). A similar trend was observed for never smokers with COPD; however, results did not reach statistical significance due to small sample size. There was a significant sCD14 x COPD/smoking interaction with lung function (% predicted FEV, p = 0.0498; FEV/FVC, p = 0.011). Regression models were adjusted for age, body mass index, education, sex, race and years worked on a farm. No association was found between CD14 polymorphisms/haplotypes (CD14/-2838; CD14/-1720; CD14/-651; CD14/-260) and sCD14 levels. The final model included the variables sCD14 and haplotypes and a haplotype x sCD14 interaction term. Individuals with the GTTG haplotype (CD14/-2838 → CD14/-260) and high sCD14 levels (> median sCD14) had on average 6.94 lower % predicted FEV than individuals with the GCCA haplotype and low sCD14 levels (≤ median sCD14, p = 0.03).

Conclusion: CD14 haplotypes and sCD14 are important mediators of lung function among those with COPD in this occupationally-exposed population.
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http://dx.doi.org/10.1186/s12931-017-0532-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353891PMC
March 2017

Systemic IL-6 Effector Response in Mediating Systemic Bone Loss Following Inhalation of Organic Dust.

J Interferon Cytokine Res 2017 01 22;37(1):9-19. Epub 2016 Nov 22.

1 Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center , The Nebraska Medical Center, Omaha, Nebraska.

Airway and skeletal diseases are prominent among agriculture workers. Repetitive inhalant exposures to agriculture organic dust extract (ODE) induces bone deterioration in mice; yet the mechanisms responsible for connecting the lung-bone inflammatory axis remain unclear. We hypothesized that the interleukin (IL)-6 effector response regulates bone deterioration following inhalant ODE exposures. Using an established intranasal inhalation exposure model, wild-type (WT) and IL-6 knockout (KO) mice were treated daily with ODE or saline for 3 weeks. ODE-induced airway neutrophil influx, cytokine/chemokine release, and lung pathology were not reduced in IL-6 KO animals compared to WT mice. Utilizing micro-computed tomography, analysis of tibia showed that loss of bone mineral density, volume, and deterioration of bone micro-architecture, and mechanical strength induced by inhalant ODE exposures in WT mice were absent in IL-6 KO animals. Compared to saline treatments, bone-resorbing osteoclasts and bone marrow osteoclast precursor populations were also increased in ODE-treated WT but not IL-6 KO mice. These results show that the systemic IL-6 effector pathway mediates bone deterioration induced by repetitive inhalant ODE exposures through an effect on osteoclasts, but a positive role for IL-6 in the airway was not demonstrated. IL-6 might be an important link in explaining the lung-bone inflammatory axis.
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http://dx.doi.org/10.1089/jir.2016.0048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240004PMC
January 2017

β2-Adrenergic agonists attenuate organic dust-induced lung inflammation.

Am J Physiol Lung Cell Mol Physiol 2016 07 17;311(1):L101-10. Epub 2016 May 17.

Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska; Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska; Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska.

Agricultural dust exposure results in significant lung inflammation, and individuals working in concentrated animal feeding operations (CAFOs) are at risk for chronic airway inflammatory diseases. Exposure of bronchial epithelial cells to aqueous extracts of hog CAFO dusts (HDE) leads to inflammatory cytokine production that is driven by protein kinase C (PKC) activation. cAMP-dependent protein kinase (PKA)-activating agents can inhibit PKC activation in epithelial cells, leading to reduced inflammatory cytokine production following HDE exposure. β2-Adrenergic receptor agonists (β2-agonists) activate PKA, and we hypothesized that β2-agonists would beneficially impact HDE-induced adverse airway inflammatory consequences. Bronchial epithelial cells were cultured with the short-acting β2-agonist salbutamol or the long-acting β2-agonist salmeterol prior to stimulation with HDE. β2-Agonist treatment significantly increased PKA activation and significantly decreased HDE-stimulated IL-6 and IL-8 production in a concentration- and time-dependent manner. Salbutamol treatment significantly reduced HDE-induced intracellular adhesion molecule-1 expression and neutrophil adhesion to epithelial cells. Using an established intranasal inhalation exposure model, we found that salbutamol pretreatment reduced airway neutrophil influx and IL-6, TNF-α, CXCL1, and CXCL2 release in bronchoalveolar lavage fluid following a one-time exposure to HDE. Likewise, when mice were pretreated daily with salbutamol prior to HDE exposure for 3 wk, HDE-induced neutrophil influx and inflammatory mediator production were also reduced. The severity of HDE-induced lung pathology in mice repetitively exposed to HDE for 3 wk was also decreased with daily salbutamol pretreatment. Together, these results support the need for future clinical investigations to evaluate the utility of β2-agonist therapies in the treatment of airway inflammation associated with CAFO dust exposure.
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http://dx.doi.org/10.1152/ajplung.00125.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967192PMC
July 2016

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

Alcohol Clin Exp Res 2016 Feb;40(2):273-83

Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska.

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.
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http://dx.doi.org/10.1111/acer.12967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656047PMC
February 2016

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

Alcohol Clin Exp Res 2015 Sep 24;39(9):1691-7. Epub 2015 Jul 24.

University of Colorado School of Medicine, Denver, Colorado.

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.
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http://dx.doi.org/10.1111/acer.12803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843766PMC
September 2015

Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

Am J Physiol Lung Cell Mol Physiol 2015 Aug 19;309(4):L388-99. Epub 2015 Jun 19.

Pulmonary, Critical Care, Sleep and Allergy Division, University of Nebraska Medical Center, Omaha, Nebraska; Veterans Affairs Nebraska-Western Iowa Healthcare System, Omaha, Nebraska; Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska; and.

Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease.
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http://dx.doi.org/10.1152/ajplung.00025.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538230PMC
August 2015

Maresin-1 reduces airway inflammation associated with acute and repetitive exposures to organic dust.

Transl Res 2015 Jul 13;166(1):57-69. Epub 2015 Jan 13.

Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Neb; VA Nebraska-Western Iowa Health Care System, Research Service and Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Neb. Electronic address:

Agriculture industry workers are at a higher risk for chronic bronchitis and obstructive pulmonary diseases, and current therapeutics are not entirely effective. We previously found that the specialized proresolving lipid mediator maresin-1 (MaR1) reduced proinflammatory cytokine release and intracellular adhesion molecule-1 (ICAM-1) expression in bronchial epithelial cells exposed to extracts of organic dust (DE) derived from swine confinement facilities in vitro. The objective of this study was to determine whether MaR1 is effective at limiting lung inflammation associated with acute and repetitive exposures to DE in an established murine model of inhalant dust exposures. C57Bl/6 mice were treated with MaR1 or vehicle control and intranasally instilled with DE once or daily for 3 weeks. Bronchioalveolar lavage fluid was analyzed for total and differential cell counts and proinflammatory cytokine levels, and lung tissues were assessed for histopathology and ICAM-1 expression. In both single and repetitive DE exposure studies, MaR1 significantly decreased bronchoalveolar lavage neutrophil infiltration, interleukin 6, tumor necrosis factor α, and chemokine C-X-C motif ligand 1 levels without altering repetitive DE-induced bronchioalveolar inflammation or lymphoid aggregate formation. Lung tissue ICAM-1 expression was also reduced in both single and repetitive exposure studies. These data suggest that MaR1 might contribute to an effective strategy to reduce airway inflammatory diseases induced by agricultural-related organic dust environmental exposures.
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http://dx.doi.org/10.1016/j.trsl.2015.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458456PMC
July 2015

The omega-3 fatty acid docosahexaenoic acid attenuates organic dust-induced airway inflammation.

Nutrients 2014 Nov 27;6(12):5434-52. Epub 2014 Nov 27.

Pulmonary, Critical Care, Sleep and Allergy Division, University of Nebraska Medical Center, Omaha, NE 68198, USA.

Workers exposed to organic dusts from concentrated animal feeding operations (CAFOs) are at risk for developing airway inflammatory diseases. Available preventative and therapeutic measures for alleviating dust-induced lung disease are inadequate. Because omega-3 fatty acids can mitigate inflammatory processes, we aimed to determine whether nutritional supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) could reduce the airway inflammatory consequences of exposures to organic dust. Aqueous extracts of organic dusts from swine CAFOs (ODE) were utilized. In DHA-pretreated human bronchial epithelial cells, lung fibroblasts, monocyte cell cultures, and precision-cut murine lung slices, we found that DHA pretreatment dose-dependently decreased ODE-induced inflammatory cytokine production. To determine the in vivo significance of DHA, C57BL/6 mice were orally administered DHA for seven days prior to treatment with intranasal ODE or saline inhalations. Animals treated with 2 mg DHA demonstrated significant reductions in ODE-induced bronchial alveolar lavage neutrophil influx and pro-inflammatory cytokine/chemokine production compared to mice exposed to ODE alone. Collectively, these data demonstrate that DHA affects several lung cells to reduce the airway inflammatory response to organic dust exposures. Dietary supplementation with DHA may be an effective therapeutic strategy to reduce the airway inflammatory consequences in individuals exposed to agriculture dust environments.
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http://dx.doi.org/10.3390/nu6125434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276977PMC
November 2014

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

Am J Physiol Lung Cell Mol Physiol 2014 Oct 22;307(8):L643-51. Epub 2014 Aug 22.

VA Nebraska-Western Iowa Health Care System Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska; Department of Environmental, Agricultural, and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska;

Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8.
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http://dx.doi.org/10.1152/ajplung.00373.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200387PMC
October 2014

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

Am J Physiol Lung Cell Mol Physiol 2014 May 7;306(9):L829-39. Epub 2014 Mar 7.

988090 Nebraska Medical Center, Omaha, NE 68198.

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.
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http://dx.doi.org/10.1152/ajplung.00364.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116420PMC
May 2014

Maresin-1 reduces the pro-inflammatory response of bronchial epithelial cells to organic dust.

Respir Res 2013 May 10;14:51. Epub 2013 May 10.

Background: Exposure to organic dust causes detrimental airway inflammation. Current preventative and therapeutic measures do not adequately treat resulting disease, necessitating novel therapeutic interventions. Recently identified mediators derived from polyunsaturated fatty acids exhibit anti-inflammatory and pro-resolving actions. We tested the potential of one of these mediators, maresin-1 (MaR1), in reducing organic dust-associated airway inflammation.

Methods: As bronchial epithelial cells (BECs) are pivotal in initiating organic dust-induced inflammation, we investigated the in vitro effects of MaR1 on a human BEC cell line (BEAS-2B). Cells were pretreated for 1 hour with 0-200 nM MaR1, followed by 1-24 hour treatment with 5% hog confinement facility-derived organic dust extract (HDE). Alternatively, a mouse lung slice model was utilized in supportive cytokine studies. Supernatants were harvested and cytokine levels determined via enzyme-linked immunosorbent assays. Epithelial cell protein kinase C (PKC) isoforms α and ϵ, and PKA activities were assessed via radioactivity assays, and NFκB and MAPK-related signaling mechanisms were investigated using luciferase vector reporters.

Results: MaR1 dose-dependently reduced IL-6 and IL-8 production following HDE treatment of BECs. MaR1 also reduced HDE-stimulated cytokine release including TNF-α in a mouse lung slice model when given before or following HDE treatment. Previous studies have established that HDE sequentially activates epithelial PKCα and PKCϵ at 1 and 6 hours, respectively that regulated TNF-α, IL-6, and IL-8 release. MaR1 pretreatment abrogated these HDE-induced PKC activities. Furthermore, HDE treatment over a 24-hour period revealed temporal increases in NFκB, AP-1, SP-1, and SRE DNA binding activities, using luciferase reporter assays. MaR1 pretreatment did not alter the activation of NFκB, AP-1, or SP-1, but did reduce the activation of DNA binding at SRE.

Conclusions: These observations indicate a role for MaR1 in attenuating the pro-inflammatory responses of BECs to organic dust extract, through a mechanism that does not appear to rely on reduced NFκB, AP-1, or SP-1-related signaling, but may be mediated partly through SRE-related signaling. These data offer insights for a novel mechanistic action of MaR1 in bronchial epithelial cells, and support future in vivo studies to test MaR1's utility in reducing the deleterious inflammatory effects of environmental dust exposures.
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http://dx.doi.org/10.1186/1465-9921-14-51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668181PMC
May 2013

Airway epithelial epidermal growth factor receptor mediates hogbarn dust-induced cytokine release but not Ca2+ response.

Am J Respir Cell Mol Biol 2011 Oct 25;45(4):882-8. Epub 2011 Mar 25.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800, USA.

A subset of workers in swine confinement facilities develops chronic respiratory disease. An aqueous extract of dust from these facilities (hogbarn dust extract [HDE]) induces IL-6 and IL-8 release and several other responses in isolated airway epithelial cells. The cell membrane receptors by which HDE initiates these responses have not been identified. Because several other inhaled agents induce airway epithelial cell responses through epidermal growth factor receptor (EGFR) activation, we hypothesized that HDE would activate EGFRs and that EGFRs would be required for some of the responses to HDE. Exposure of Beas-2B cells to HDE caused EGFR phosphorylation and downstream ERK activation, and both responses were blocked by the EGFR-selective kinase inhibitor AG1478. AG1478 and EGFR-neutralizing antibody reduced HDE-stimulated IL-6 and IL-8 release by about half. Similar EGFR phosphorylation and requirement of EGFRs for maximal IL-6 and IL-8 release were found with primary isolates of human bronchial epithelial cells. Because HDE-stimulated IL-6 and IL-8 release involve the Ca(2+)-dependent protein kinase Cα, we hypothesized that HDE would induce intracellular Ca(2+) mobilization. HDE exposure induced intracellular Ca(2+) mobilization in Beas-2B cells and in primary cell isolates, but this response was neither mimicked by EGF nor inhibited by AG1478. Thus, HDE activates EGFRs and their downstream signaling, and EGFR activation is required for some but not all airway epithelial cell responses to HDE.
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http://dx.doi.org/10.1165/rcmb.2010-0419OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208609PMC
October 2011

Cigarette smoke extract increases C5a receptor expression in human bronchial epithelial cells.

J Pharmacol Exp Ther 2005 Jul 20;314(1):476-82. Epub 2005 Apr 20.

Pulmonary, Critical Care, and Sleep Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985815 Nebraska Medical Center, Omaha, NE 68198-5815, USA.

We have shown that exposing human bronchial epithelial cells (HBECs) to 5% cigarette smoke extract (CSE) up-regulates C5a anaphylatoxin receptor (C5aR) expression as determined by flow cytometric analysis and immunohistochemistry. In this study, we conducted whole-cell saturation studies to quantitate the receptor number. After exposing an HBEC line (BEAS-2B) to CSE, radiolabeled C5a bound saturably with Kd = 2.71 +/- 1.03 nM (n = 4) and Bmax = 15,044 +/- 5702 receptors/cells. Without 5% CSE, no C5a binding was detected. Competitive binding studies revealed two classes of sites with distinct affinities for C5a (Ki1 = 3.28 x 10(-16) M; Ki2 = 1.60 x 10(-9) M). BEAS-2Bs were transfected with wild-type (WT) or mutant dominant-negative (DN) protein kinase C-alpha (PKC-alpha) to investigate the relationship between PKC-alpha and C5aR availability and affinity. Western blot analysis revealed a 75-kDa lysate band from cells expressing WT and DN PKC-alpha, but DN cells exposed to 5% CSE had no functional PKC activity. Pretreatment with Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (PKC-alpha inhibitor) had no effect on DN but significantly decreased WT PKC activity. Competitive binding studies conducted on either WT or DN PKC-alpha-transfected cells also revealed two classes of binding sites for C5a having different affinities. There was a significant rightward shift of the binding curve when WT cells were pretreated with Gö6976. These data suggest that C5aR is detectable on bronchial epithelial cells exposed to CSE and that exposure to CSE increases the availability of C5a binding sites. The data also indicate that PKC-alpha may play an important role in modulating C5aR binding.
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http://dx.doi.org/10.1124/jpet.104.079822DOI Listing
July 2005

Smoke and C5a induce airway epithelial intercellular adhesion molecule-1 and cell adhesion.

Am J Respir Cell Mol Biol 2003 Oct 24;29(4):472-82. Epub 2003 Apr 24.

Division of Pulmonary and Critical Care Medicine, School of Allied Health Professions, University of Nebraska Medical Center/Nebraska Health Systems, Omaha, NE 68198-5300, USA.

The human bronchial epithelial cell is one of the first cell types to be exposed to the irritants and toxins present in inhaled cigarette smoke. The ability of the bronchial epithelium to modulate inflammatory and immune events in response to cigarette smoke is important in the pathogenesis of smoke-induced airway injury. We have shown that cigarette smoke extract and the complement anaphylatoxin C5a both independently induce increased expression of intercellular adhesion molecule (ICAM)-1 on airway epithelial monolayers compared with unstimulated cells in vitro. This enhanced ICAM-1 expression is associated with a greater capacity of the airway epithelial cells to bind mononuclear cells, a process that appears to require the proinflammatory cytokine tumor necrosis factor-alpha and protein kinase C intracellular signaling. Exposure of epithelial monolayers to the combination of cigarette smoke followed by C5a results in an additive response for ICAM-1 expression and mononuclear cell adhesion compared with smoke or C5a challenge alone. Inhibiting C5a receptor expression can attenuate these responses. These findings suggest that smoke exposure in some way enhances the functional responsiveness of the C5a receptor expressed on these airway epithelial cells for subsequent C5a-mediated increases in ICAM-1 expression and mononuclear cell adhesion. Our results may help explain the initiation and propagation of inflammatory events in vivo induced by chronic airway exposure to cigarette smoke.
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http://dx.doi.org/10.1165/rcmb.2002-0143OCDOI Listing
October 2003
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