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.

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.

Combined Collagen-Induced Arthritis and Organic Dust-Induced Airway Inflammation to Model Inflammatory Lung Disease in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by extra-articular involvement including lung disease, yet the mechanisms linking the two conditions are poorly understood. The collagen-induced arthritis (CIA) model was combined with the organic dust extract (ODE) airway inflammatory model to assess bone/joint-lung inflammatory outcomes. DBA/1J mice were intranasally treated with saline or ODE daily for 5 weeks. CIA was induced on days 1 and 21. Treatment groups included sham (saline injection/saline inhalation), CIA (CIA/saline), ODE (saline/ODE), and CIA + ODE (CIA/ODE). Arthritis inflammatory scores, bones, bronchoalveolar lavage fluid, lung tissues, and serum were assessed. In DBA/1J male mice, arthritis was increased in CIA + ODE > CIA > ODE versus sham. Micro-computed tomography (µCT) demonstrated that loss of BMD and volume and deterioration of bone microarchitecture was greatest in CIA + ODE. However, ODE-induced airway neutrophil influx and inflammatory cytokine/chemokine levels in lavage fluids were increased in ODE > CIA + ODE versus sham. Activated lung CD11c+ CD11b+ macrophages were increased in ODE > CIA + ODE > CIA pattern, whereas lung hyaluronan, fibronectin, and amphiregulin levels were greatest in CIA + ODE. Serum autoantibody and inflammatory marker concentrations varied among experimental groups. Compared with male mice, female mice showed less articular and pulmonary disease. The interaction of inhalation-induced airway inflammation and arthritis induction resulted in compartmentalized responses with the greatest degree of arthritis and bone loss in male mice with combined exposures. Data also support suppression of the lung inflammatory response, but increases in extracellular matrix protein deposition/interstitial disease in the setting of arthritis. This coexposure model could be exploited to better understand and treat RA-lung disease. © 2019 American Society for Bone and Mineral Research.

Sex differences impact the lung-bone inflammatory response to repetitive inhalant lipopolysaccharide exposures in mice.

Skeletal health consequences associated with inflammatory diseases of the airways significantly contribute to morbidity. Sex differences have been described independently for lung and bone diseases. Repetitive inhalant exposure to lipopolysaccharide (LPS) induces bone loss and deterioration in male mice, but comparison effects in females are unknown. Using an intranasal inhalation exposure model, 8-week-old C57BL/6 male and female mice were treated daily with LPS (100 ng) or saline for 3 weeks. Bronchoalveolar lavage fluids, lung tissues, tibias, bone marrow cells, and blood were collected. LPS-induced airway neutrophil influx, interleukin (IL)-6 and neutrophil chemoattractant levels, and bronchiolar inflammation were exaggerated in male animals as compared to female mice. Trabecular bone micro-CT imaging and analysis of the proximal tibia were conducted. Inhalant LPS exposures lead to deterioration of bone quality only in male mice (not females) marked by decreased bone mineral density, bone volume/tissue volume ratio, trabecular thickness and number, and increased bone surface-to-bone volume ratio. Serum pentraxin-2 levels were modulated by sex differences and LPS exposure. In proof-of-concept studies, ovarectomized female mice demonstrated LPS-induced bone deterioration, and estradiol supplementation of ovarectomized female mice and control male mice protected against LPS-induced bone deterioration findings. Collectively, sex-specific differences exist in LPS-induced airway inflammatory consequences with significant differences found in bone quantity and quality parameters. Male mice demonstrated susceptibility to bone loss and female animals were protected, which was modulated by estrogen. Therefore, sex differences influence the biologic response in the lung-bone inflammatory axis in response to inhalant LPS exposures.

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.

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.

Vitamin D supplementation protects against bone loss following inhalant organic dust and lipopolysaccharide exposures in mice.

Systemic bone loss is associated with airway inflammatory diseases; yet, strategies to halt disease progression from inhalant exposures are not clear. Vitamin D might be a potentially protective approach against noxious respirable environmental exposures. We sought to determine whether vitamin D supplementation represents a viable lung- and bone-protective strategy following repetitive inhalant treatments with organic dust extract (ODE) or lipopolysaccharide (LPS) in mice. C57BL/5 mice were maintained on diets with low (1 IU/D/g) or high (10 IU/D/g) vitamin D for 5 weeks and treated with ODE from swine confinement facilities, LPS, or saline daily for 3 weeks per established intranasal inhalation protocol. Lungs, hind limbs, and sera were harvested for experimental outcomes. Serum 25-hydroxyvitamin D levels were tenfold different between low and high vitamin D treatment groups with no differences between inhalant agents and saline treatments. Serum calcium levels were not affected. There was no difference in the magnitude of ODE- or LPS-induced inflammatory cell influx or lung histopathology between high and low vitamin D treatment groups. However, high vitamin D treatment reversed the loss of bone mineral density, bone volume, and bone micro-architecture deterioration induced by ODE or LPS as determined by micro-CT analysis. Bone-resorbing osteoclasts were also reduced by high vitamin D treatment. In the low vitamin D treatment groups, ODE induced the greatest degree of airway inflammatory consequences, and LPS induced the greatest degree of bone loss. Collectively, high-concentration vitamin D was protective against systemic bone loss, but not airway inflammation, resulting from ODE- or LPS-induced airway injury.

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.