Agricultural dust derived bacterial extracellular vesicle mediated inflammation is attenuated by DHA.

Dietary long-chain omega-3 polyunsaturated fatty acids (n-3 PUFA) and their pro-resolving metabolites are protective against atherosclerotic disease, and ameliorate systemic inflammatory conditions including lupus erythematosus, psoriasis, and bronchial asthma. Organic bioaerosol inhalation is a common and injurious hazard associated with agricultural occupations such as work in swine concentrated animal feeding operations (CAFOs) and is known to increase the risk for developing respiratory conditions such as asthma and COPD. Nearly all cells secrete membrane-bound vesicles (extracellular vesicles, EVs) that have the capacity to transmit protein, nucleic acid, and lipid signaling mediators between cells. Using a polymer-based isolation technique (ExoQuick, PEG) followed by ultracentrifugation, EVs were isolated from CAFO dust extracts, and were quantified and partially characterized. Here, we investigated the role of the n-3 PUFA docosahexaenoic acid (DHA) as a component of n-6 to n-3 PUFA mixtures used to recapitulate physiologically relevant dietary ratios in the resolution of inflammatory injury caused by exposure to EVs carried by agricultural organic dust in vitro. Primary human bronchial epithelial cells, fibroblasts and monocyte-derived macrophages were exposed to EVs isolated from swine CAFO dust. Cells were treated with mixtures of n-6 and n-3 PUFA during recovery from the EV-induced injury. CAFO dust extract (DE) was found to contain EVs that contributed significantly to the overall consequences of exposure to complete DE. DHA-rich PUFA ratios inhibited DE-derived EV-induced proinflammatory cytokine release dose-dependently. DHA-rich PUFA ratios also reversed the damaging effects of EVs on recellularization of lung matrix scaffolds, accelerated wound healing, and stimulated the release of pro-resolution mediators. These results underscore the importance of n-3 PUFA as anti-inflammatory compounds during recovery from EV-laden environmental dust exposure in the context of cellular responses in vitro, warranting future translational studies.

Relationships of serum CC16 levels with smoking status and lung function in COPD.

BACKGROUND: The club cell secretory protein (CC16) has anti-inflammatory and antioxidant effects, and low CC16 serum levels have been associated with both risk and progression of COPD, yet the interaction between smoking and CC16 on lung function outcomes remains unknown. METHODS: Utilizing cross-sectional data on United States veterans, CC16 serum concentrations were measured by ELISA and log transformed for analyses. Spirometry was conducted and COPD status was defined by post-bronchodilator FEV1/FVC ratio < 0.7. Smoking measures were self-reported on questionnaire. Multivariable logistic and linear regression were employed to examine associations between CC16 levels and COPD, and lung function with adjustment for covariates. Unadjusted Pearson correlations described relationships between CC16 level and lung function measures, pack-years smoked, and years since smoking cessation. RESULTS: The study population (N = 351) was mostly male, white, with an average age over 60 years. An interaction between CC16 and smoking status on FEV1/FVC ratio was demonstrated among subjects with COPD (N = 245, p = 0.01). There was a positive correlation among former smokers and negative correlation among current or never smokers with COPD. Among former smokers with COPD, CC16 levels were also positively correlated with years since smoking cessation, and inversely related with pack-years smoked. Increasing CC16 levels were associated with lower odds of COPD (ORadj = 0.36, 95% CI 0.22-0.57, Padj < 0.0001). CONCLUSIONS: Smoking status is an important effect modifier of CC16 relationships with lung function. Increasing serum CC16 corresponded to increases in FEV1/FVC ratio in former smokers with COPD versus opposite relationships in current or never smokers. Additional longitudinal studies may be warranted to assess relationship of CC16 with smoking cessation on lung function among subjects with COPD.

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

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.

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

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.

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

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 (CD11c+CD11bhi) 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.

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

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.

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

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.

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

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.

Alcohol and cannabis use alter pulmonary innate immunity.

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.

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

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 FEV1/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.

Short term dynamics of the sputum microbiome among COPD patients.

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.

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

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.

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

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.

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

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.

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

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 ad libitum 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.

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

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 (FEV1/FVC, p = 0.011; % predicted FEV1, 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 FEV1, padj = 0.0008; FEV1/FVC, padj = 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 FEV1, pinter = 0.0498; FEV1/FVC, pinter = 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 FEV1 than individuals with the GCCA haplotype and low sCD14 levels (≤ median sCD14, padj = 0.03). CONCLUSION: CD14 haplotypes and sCD14 are important mediators of lung function among those with COPD in this occupationally-exposed population.

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

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 CD3+CD4+ and CD3+CD8+ 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.

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

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.

ß2-Adrenergic agonists attenuate organic dust-induced lung inflammation.

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. ß2-Adrenergic receptor agonists (ß2-agonists) activate PKA, and we hypothesized that ß2-agonists would beneficially impact HDE-induced adverse airway inflammatory consequences. Bronchial epithelial cells were cultured with the short-acting ß2-agonist salbutamol or the long-acting ß2-agonist salmeterol prior to stimulation with HDE. ß2-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 ß2-agonist therapies in the treatment of airway inflammation associated with CAFO dust exposure.