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

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

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

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 (\u3e2-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

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

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

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

β2 Adrenergic Receptor Agonists And Steroids Inhibit Cafo Dust-Mediated Bronchial Epithelial Inflammation In Vitro

Rationale: Agricultural workers employed in concentrated animal feeding operations (CAFOs) are repeatedly exposed to aerosolized dust, and are susceptible to inflammatory lung diseases including COPD. Aqueous extracts of agricultural dust collected from swine CAFOs (HDE) have been shown to be potent inflammatory stimulators in human cells and in vivo mouse models. HDE exposure augments proinflammatory cytokine release, ICAM-1 expression, PKC activation, and lymphocyte and neutrophil adhesion to airway epithelial cells. Because 2 adrenergic receptor agonists (β-agonists) alone, and in combination with corticosteroids, have been used to treat COPD, we examined the ability of these agents to diminish the inflammatory effects of HDE in human epithelial cells and mouse lungs in vitro. Methods: Immortalized human bronchial epithelial cells (Beas-2B) were grown in serum-free medium lacking the growth supplements hydrocortisone and epinephrine (LHC-7HE). Mouse lung slice cultures were prepared by making precise 150-μm sections through agarose-infused intact mouse lungs, and cultured for up to two weeks in serum-free medium. Cultured cells or lung slice cultures were pretreated with one of several -agonists [salbutamol,10 M, (SB); salmeterol, 0.01–10 M, (SM)) alone or in combination with the steroid fluticasone propionate (1 M, (FP)] for one hour prior to challenge with a 5% solution of HDE for an additional 24 hours. Supernatant medium was assayed for human IL-6 and IL-8, or for murine IL-6, KC, MIP-2, and TNF- by ELISA. Cells were harvested for ICAM-1 expression by flow cytometry, and cell lysates were analyzed for protein kinase A (PKA) activity. Results: Pretreatment of epithelial cells with the long-acting -agonist SM significantly inhibited the HDE-induced release of inflammatory cytokines both time- and dose-dependently (63%, IL-8; and 72%, IL-6, at 10 M SM; from 12 to 24 hours). Pretreatment with the steroid FP in combination with SM further decreased the proinflammatory effect. Likewise, mouse lung slice cultures treated with SM or the short-acting SB, alone and in combination with FP, exhibited a blunted response to subsequent 24-h HDE challenge for all four cytokines measured (a 48-71% decrement, depending on the cytokine measured). In addition, SB pretreatment markedly decreased the HDE-enhanced ICAM-1 surface expression on Beas-2B cells, whereas intracellular PKA activity was significantly increased. Conclusions: Long-acting β-agonists alone, or in combination with steroids, are a potentially useful therapy for the treatment or prevention of chronic inflammatory lung disease in workers exposed to dust from CAFO buildings

Cytokine Responses In Whole Blood Assay In Veterans With Agriculture Work Exposures

RATIONALE: Agriculture exposures are associated with increased risk of chronic obstructive pulmonary disease (COPD). Endotoxin exposure in agricultural environments is associated with lung disease. Our objective was to evaluate responsiveness to endotoxin stimulation in an in vitro whole blood assay and its association with COPD in an agriculturally exposed cohort. METHODS: Veterans with ≥ two years agricultural work exposure participated in this case-control study. Cases are veterans with COPD defined as FEV1/FVC ratio \u3c 70% on spirometry post-bronchodilator. Controls did not have COPD as assessed by spirometry nor other significant lung conditions. Whole blood assay (WBA) was done with 2 ml heparinized blood mixed with 2 ml of media and stimulated with increasing doses of LPS (0 – 1 ng/ml). Samples were incubated at 37°C for 24 hours, centrifuged, supernatants harvested and frozen until assayed for cytokines by ELISA. Cytokine values were log transformed. Repeated measures ANOVA was used to examine the association between LPS dose and group (case or control) and the interaction of dose with group. All p values were adjusted for age. RESULTS: There were 272 participants, 110 cases with COPD and 158 controls. Baseline demographics (gender, age, race) were similar between cases and controls. LPS-stimulated TNF-α production showed a significant dose effect (padjadj=0.0119) with controls having a greater capacity for TNF-α production than cases. There was no evidence of a significant interaction between dose and group (padj=0.0803). Tukey post hoc comparisons revealed a significant difference between cases and controls at the 0 ng/mL (padj=0.0146), 0.01 ng/mL (padj=0.0233) and 0.1 ng/mL (padj=0.0369) LPS doses. For IL-6, there was a significant dose effect (padjadj=0.0108) and 0.001 ng/mL LPS (padj=0.0164) doses with COPD subjects having a greater capacity for IL-6 production. CONCLUSIONS: In veterans with agriculture exposures, persons with COPD demonstrated increased IL-6 at baseline and in response to endotoxin stimulation in a whole blood assay compared to those without COPD; whereas the opposite was observed with TNF-α levels

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 (\u3e2-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