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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.

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.

Organic dust exposure in agricultural animal environments results in airway diseases. Dendritic cells (DCs) orchestrate inflammatory immune response in the airways, but little is known about how organic dust affects differentiation and maturation of monocyte-derived immature and mature DCs (iDCs, mDCs). Peripheral blood monocytes were differentiated in vitro into iDCs with granulocyte-macrophage colony stimulating factor + IL-4 (6 days) with and without swine facility organic dust extract (ODE, 0.1%). Unlike control iDCs, ODE-conditioned iDCs maintained key monocyte properties (increased mCD14, increased phagocytic ability) while expressing DC features [increased mCD83, HLA-DR, CD80, CD86, diminished cytokine (TNF-[alpha], IL-6) responsiveness]. At day 6, iDCs were cultured for an additional 48 h (days 7 and 8) with lipopolysaccharide (LPS) to induce mDCs. ODE-conditioned mDCs maintained high expression of [mCD14.sup.+] and elevated phagocytosis while their DC features weakened as evidenced by decreased CD11c, CD83, HLA-DR, CD86, and CCR7 expression and reduced lymphocyte-stimulating capacity. Similar results were observed when monocytes were exposed to ODE for only the first 48 h and with ODE depleted of endotoxin. Control iDCs exposed to ODE during the final 2 days of iDC maturation (days 7 and 8) did not differ from control (no ODE) iDCs in surface marker expression and phagocytic ability, but exhibited enhanced lymphocyte-stimulating capacity. Dust exposure alters monocyte differentiation to iDCs and prevents maturation of iDC to mDCs. The first 48 h of monocyte differentiation appears to be the susceptible period to exposure. Environmental exposures present during early monocyte differentiation may impact the critical balance of DCs in the lung. phagocytosis; lymphocyte proliferation; innate cell surface molecules; cytokines; swine doi: 10.1152/ajplung.00107.2009

Organic dust exposure in agricultural environments results in an inflammatory response that attenuates over time, but repetitive exposures can result in chronic respiratory disease. Animal models to study these mechanisms are limited. This study investigated the effects of single vs. repetitive dust-induced airway inflammation in mice by intranasal exposure method. Mice were exposed to swine facility dust extract (DE) or saline once and once daily for 1 and 2 wk. Dust exposure resulted in increased bronchoalveolar lavage fluid neutrophils and macrophages after single and repetitive exposures. Lavage fluid TNFot, IL-6, keratinocyte chemoattractant, and macrophage inflammatory protein-2 were significantly increased after single and repetitive dust exposures, but were dampened in 2-wk dust-exposed mice compared with single exposure. Dust exposure induced PKCot and -e activation in isolated tracheal epithelial cells but were dampened with repetitive exposures. Ex vivo stimulation of alveolar macrophages from 2-wk animals demonstrated reduced cytokine responsiveness and phagocytic ability. Significant lung pathology occurred with development of mixed mononuclear cellular aggregates (T and B lymphocytes, phagocytes) after repetitive dust exposure, a novel observation. Airway hyperresponsiveness to methacholine occurred after single dust exposure but resolved after 2 wk. Collectively, intranasal exposure to DE results in significant lung inflammatory and pathological responses marked by a modulated innate immune response to single and repetitive dust exposures that is associated with PKC activity. antigen presenting cell; phagocytosis; cytokines; aggregate; airway hyperresponsiveness

Agricultural work and other occupational exposures are responsible for ~15% of chronic obstructive pulmonary disease (COPD). COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms. However, the effect of agricultural dust exposure on signaling pathways that regulate airway injury and repair has not been well characterized. A key step in this process is migration of airway cells to restore epithelial integrity. We have previously shown that agents that activate the critical regulatory enzyme protein kinase C (PKC) slow cell migration during wound repair. Based on this observation and direct kinase measurements that demonstrate that dust extract from hog confinement barns (HDE) specifically activates the PKC isoforms PKC[alpha] and PKC[epsilon], we hypothesized that HDE would slow wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line BEAS-2B and transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKC isoforms to demonstrate that HDE slows wound closure in BEAS-2B and PKC[epsilon] DN cell lines. However, in PKC[alpha] DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKC[alpha] isoform is an important regulator of cell migration in response to agricultural dust exposure. protein kinase C; chronic obstructive pulmonary disease

Individuals exposed to dusts from concentrated animal feeding operations report increased numbers of respiratory tract symptoms, and bronchoalveolar lavage samples from such individuals demonstrate elevated lung inflammatory mediators, including interleukin (IL)-8 and IL-6. We previously found that exposure of bronchial epithelial cells to hog barn dusts resulted in a protein kinase C (PKC)-dependent increase in IL-6 and IL-8 release. We hypothesized that cattle feedlot dusts would also generate bronchial epithelial interleukin release in vitro. To test this, we used interleukin ELISAs and direct PKC isoform assays. We found that a dust extract from cattle feedlots [feedlot dust extract (FLDE)] augments PKC activity of human bronchial epithelial cells in vitro. A 5-10% dilution of FLDE stimulated a significant release of IL-6 and IL-8 at 6-24 h in a PKC-dependent manner vs. control medium-treated cells. An increase in PKC[alpha] activity was observed with 1 h of FLDE treatment, and PKC[epsilon] activity was elevated at 6 h of FLDE exposure. The PKC[alpha] inhibitor, Go-6976, did not inhibit FLDE-stimulated IL-8 and IL-6 release. However, the PKC[epsilon] inhibitor, Ro 31-8220, effectively inhibited FLDE-stimulated IL-8 and IL-6 release. Inhibition of FLDE-stimulated IL-6 and IL-8 was confirmed in a dominant-negative PKC[epsilon]-expressing BEAS-2B cell line but not observed in a PKC[alpha] dominant negative BEAS-2B cell line. These data support the hypothesis that FLDE exposure stimulates bronchial epithelial IL-8 and IL-6 release via a PKC[epsilon]-dependent pathway. feedlot dust extract

Byline: Fu-Qiang Wen (1), Tadashi Kohyama (1), C. Magnus Skold (2), Yun Kiu Zhu (3), Xiangde Liu (1), Debra J. Romberger (1), Julie Stoner (4), Stephen I. Rennard (1,5) Keywords: glucocorticoids; transforming growth factor-[beta] (TGF-[beta]); Smads; AP-1 complex; fibroblast Abstract: TGF-[beta] is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF-[beta] isoforms affect TGF-[beta] production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-[beta]1, -[beta]2, or -[beta]3. Post-culture media were collected for ELISA assays of TGF-[beta]1, -[beta]2, and -[beta]3 . TGF-[beta] mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-[beta]2 and -[beta]3 stimulated TGF-[beta]1 production significantly (p &lt 0.01 relative to control). TGF-[beta]1 stimulated TGF-[beta]2 production (p &lt 0.01 relative to control). TGF-[beta]3 was undetectable. Glucocorticoids significantly inhibited TGF-[beta]1 and TGF-[beta]2 production and reduced expression of the up-regulated TGF-[beta]1 and TGF-[beta]2 mRNA induced by exogenous TGF-[beta]1, -[beta]2, or -[beta]3 (p &lt 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-[beta]-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-[beta] isoforms may stimulate production of various TGF-[beta] isoforms in the lung. Glucocorticoids then may block TGF-[beta] production by modulating mRNA levels and c-Jun. Author Affiliation: (1) Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (2) Department of Medicine, Karolinska Institute, Stockholm, Sweden (3) Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China (4) Preventive and Societal Medicine Section, University of Nebraska Medical Center, Omaha, Nebraska (5) The Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, Nebraska, 68198-5125, USA Article History: Registration Date: 19/10/2004

Cigarette smoke exposure has been associated with a variety of diseases, including emphysema. The current study evaluated the interaction of cell density and cigarette smoke extract (CSE) on fibroblast contraction of collagen gels. Protein levels of transforming growth factor (TGF)-[beta]1, fibronectin, PG[E.sub.2], and TGF-[beta]1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 x [10.sup.5] cell/ml), only 5% CSE augmented contraction in higher-density cultures (3-5 x [10.sup.5] cells/ml). CSE also inhibited fibronectin and TGF-[beta]1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-[beta]1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-[beta]1 mRNA expression was inhibited in high-density cultures by 10% CSE, expression was increased in the presence of 5% CSE. These results suggest that CSE-induced inhibition of low-density fibroblast contraction is due to inhibition of fibronectin production, whereas CSE's stimulatory effect on high-density cells is the result of increased release of TGF-[beta]1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke. cigarette smoke extract; collagen gels; transforming growth factor-[beta]; lung

Byline: Fu-Qiang Wen (1), Tadashi Kohyama (1), C. Magnus Skold (2), Yun Kiu Zhu (3), Xiangde Liu (1), Debra J. Romberger (1), Julie Stoner (4), Stephen I. Rennard (1) Keywords: glucocorticoids; transforming growth factor-[beta] (TGF-[beta]); Smads; AP-1 complex; fibroblast Abstract: TGF-[beta] is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB-[beta] isoforms affect TGF-[beta] production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-[beta]1, -[beta]2, and -[beta]3. TGF-[beta] mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-[beta]2 and -[beta]3 stimulated TGF-[beta]1 production significantly (p &lt 0.01 relative to control). TGF-[beta]1 stimulated TGF-[beta]2 production (p &lt 0.01 relative to control). TGF-[beta]3 was undetectable. Glucocorticoids significantly inhibited TGF-[beta]1 and -[beta]2 production and reduced expression of the upregulated TGF-[beta]1 and -[beta]2 mRNA induced by exogenous TGF-[beta]1, -[beta]2 or -[beta]3 (p &lt 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-[beta]-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-[beta] isoforms may stimulate production of various TGF-[beta] isoforms in the lung. Glucocorticoids then may block TGF-[beta] production by modulating mRNA levels and c-Jun. Author Affiliation: (1) Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (2) Department of Medicine, Karolinska Institute, Stockholm, Sweden (3) Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China (4) Preventive and Societal Medicine Section, University of Nebraska Medical Center, Omaha, Nebraska Article History: Registration Date: 12/10/2004

Cigarette smoke, the major risk factor for the development of emphysema, contains over 4,700 chemical compounds, including free radicals and other oxidants ([10.sup.14]/puff). An imbalance between oxidants and antioxidants has been proposed in the pathogenesis of chronic obstructive pulmonary disease. Inhibition of repair processes has been suggested to be one pathway contributing to the development of emphysema. We hypothesized that cigarette smoke inhibition of repair might result from a shift of the oxidant/antioxidant balance in favor of oxidants. To evaluate this hypothesis, N-acetyl-L-cysteine (NAC), which serves as a substrate for glutathione (GSH) production, and buthionine sulfoximine (BSO), which inhibits GSH production, were incubated in the presence and absence of cigarette smoke extract (CSE) with fibroblasts in three-dimensional collagen gels. Neither agent alone altered gel contraction. CSE inhibition of gel contraction, however, was mitigated by NAC and potentiated by BSO. Parallel effects were observed on cigarette smoke inhibition of fibronectin production and mRNA expression as well as by changes in intracellular GSH content. Pretreatment of fibroblasts with NAC or BSO resulted in similar effects, suggesting that neither agent was acting directly on smoke but, rather, was altering cellular response to smoke. In conclusion, smoke inhibition of fibroblast repair, as reflected by collagen gel contraction and fibronectin production, may be modulated by intracellular GSH levels. chronic obstructive pulmonary disease; oxidants