Purification and characterization of a serine protease from organic dust and elucidation of its inductive effects on lung inflammatory mediators.

Organic dust inhalation is associated with the development of respiratory diseases. Serine protease activities in organic dusts were previously reported to contribute to the induction of lung inflammatory mediators however, the identities of the proteases and the mechanisms by which they induce inflammatory mediators are unknown. The goal of this study was to purify and characterize serine protease(s) from organic dust and elucidate mechanisms by which they induce lung inflammatory mediators. A serine protease was purified from poultry organic dust by benzamidine-agarose affinity chromatography. Mass spectrometry and amino-terminal sequence analysis identified the purified protease as chicken trypsin II-P29. Purified protease induced proinflammatory cytokine levels in Beas2B and NHBE epithelial and THP-1 macrophage cells. Treatment with the purified protease increased cellular and mitochondrial reactive oxygen species (ROS) generation. Induction of inflammatory mediators and ROS were suppressed by serine protease inhibitors and antioxidants. Purified protease activated protein kinase C (PKC), mitogen-activated protein kinase (MAPK)1/3 and MAPK14 signaling, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (Stat-3), and chemical inhibitors targeting these pathways suppressed induction of inflammatory mediators. Calcium mobilization studies showed that the purified protease activated protease-activated receptors (PAR) F2R, F2RL1, F2RL2, F2RL3, and F2R and F2RL1 knockdown suppressed the induction of inflammatory mediators. Intranasal instillation of purified protease increased lung chemokine (C-X-C motif) ligand (CXCL)1, interleukin (IL)-6, and tumor necrosis factor (TNF) levels in mice. Our studies have shown that chicken trypsin is a proinflammatory constituent of poultry organic dust, and induces lung inflammatory mediators via increased ROS and PAR activation in a cell signaling pathway involving PKC, MAPK1/3 and MAPK14, and NF-κB and Stat-3.NEW & NOTEWORTHY Inhalation of dust in industrial agricultural operations is linked to the development of lung diseases. Our studies have isolated for the first time a trypsin protease from poultry farm dust and have shown that it stimulates lung inflammation. The protease stimulates the production of oxidants and cell signaling pathways to increase inflammatory mediator production. Targeting trypsin protease in poultry farm environment may be a useful strategy to counter the harmful effects of dust.

IL-22 regulates inflammatory responses to agricultural dust-induced airway inflammation.

IL-22 is a unique cytokine that is upregulated in many chronic inflammatory diseases, including asthma, and modulates tissue responses during inflammation. However, the role of IL-22 in the resolution of inflammation and how this contributes to lung repair processes are largely unknown. Here, we tested the hypothesis that IL-22 signaling is critical in inflammation resolution after repetitive exposure to agricultural dust. Using an established mouse model of organic dust extract-induced lung inflammation, we found that IL-22 knockout mice have an enhanced response to agricultural dust as evidenced by an exacerbated increase in infiltrating immune cells and lung pathology as compared to wild-type controls. We further identified that, in response to dust, IL-22 is expressed in airway epithelium and in Ym1+ macrophages found within the parenchyma in response to dust. The increase in IL-22 expression was accompanied by increases in IL-22 receptor IL-22R1 within the lung epithelium. In addition, we found that alveolar macrophages in vivo as well as THP-1 cells in vitro express IL-22, and this expression is modulated by dust exposure. Furthermore, subcellular localization of IL-22 appears to be in the Golgi of resting THP1 human monocytes, and treatment with dust extracts is associated with IL-22 release into the cytosolic compartment from the Golgi reservoirs during dust extract exposure. Taken together, we have identified a significant role for macrophage-mediated IL-22 signaling that is activated in dust-induced lung inflammation in mice.

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.

Sex-Specific Differences in Resolution of Airway Inflammation in Fat-1 Transgenic Mice Following Repetitive Agricultural Dust Exposure.

In agriculture industries, workers are at increased risk for developing pulmonary diseases due to inhalation of agricultural dusts, particularly when working in enclosed confinement facilities. Agricultural dusts inhalation leads to unresolved airway inflammation that precedes the development and progression of lung disease. We have previously shown beneficial effects of the omega-3 polyunsaturated fatty acid (ω-3 PUFA) DHA in protecting against the negative inflammatory effects of repetitive dust exposure in the lung. Dietary manipulation of pulmonary disease risk is an attractive and timely approach given the contribution of an increased ω-6 to ω-3 PUFA ratio to low grade inflammation and chronic disease in the Western diet. To prevent any confounding factors that comes with dietary supplementation of ω-3 PUFA (different sources, purity, dose, and duration), we employed a Fat-1 transgenic mouse model that convert ω-6 PUFA to ω-3 PUFA, leading to a tissue ω-6 to ω-3 PUFA ratio of approximately 1:1. Building on our initial findings, we hypothesized that attaining elevated tissue levels of ω-3 PUFA would attenuate agricultural dust-induced lung inflammation and its resolution. To test this hypothesis, we compared wild-type (WT) and Fat-1 transgenic mice in their response to aqueous extracts of agricultural dust (DE). We also used a soluble epoxide hydrolase inhibitor (sEH) to potentiate the effects of ω-3 PUFA, since sEH inhibitors have been shown to stabilize the anti-inflammatory P450 metabolites derived from both ω-3 and ω-6 PUFA and promote generation of specialized pro-resolving lipid mediators from ω-3 PUFA. Over a three-week period, mice were exposed to a total of 15 intranasal instillations of DE obtained from swine confinement buildings in the Midwest. We observed genotype and sex-specific differences between the WT vs. Fat-1 transgenic mice in response to repetitive dust exposure, where three-way ANOVA revealed significant main effects of treatment, genotype, and sex. Also, Fat-1 transgenic mice displayed reduced lymphoid aggregates in the lung following DE exposure as compared to WT animals exposed to DE, suggesting improved resilience to the DE-induced inflammatory effects. Overall, our data implicate a protective role of ω-3 FA in the lung following repetitive dust exposure.

Phenomenology and geographical gradients of atmospheric deposition in southwestern Europe: Results from a multi-site monitoring network.

This article presents the results of atmospheric deposition from a 15-sites network which cover remote, agricultural, urban and industrial areas in the Iberian Peninsula and the Balearic Islands, with the aim of exploring geographical, climatic and natural vs anthropogenic gradients. Annual average fluxes of global deposition, discriminating insoluble (3,5-20,7 g m-2 year-1) and soluble-inorganic (7,1-45,5 g m-2 year-1) aerosols are discussed, seasonal patterns are regarded, and an attempt to estimate the impact of the main sources is presented. The wide range of atmospheric deposition fluxes (DF) regarding soluble (DFSOL) and insoluble (DFINS) has been investigated taking into consideration the contribution from nearby to long-distance sources, such as African dust, or regional-to-nearby ones, which include agricultural dust in the Ebro Valley, industrial emissions at different parts, urban dust at all cities, or saline dust resuspension from a dissicated lake bed. DFSOL is made up of marine aerosols, prevailing in coastal areas, with few exceptions in the Ebro Valley; nitrogen-species, homogeneously distributed across the network, with few exceptions due to agricultural sources; mineral dust, enhanced in the Ebro Valley owing to regional and agricultural emissions; and phospathe, displaying comparable values to other studies in general, but three hotspots at regional background environments have been identified. DFINS particles followed the aridity pattern, especially where anthropogenic emissions take place. Our estimates indicate that the regional dust to DFINS in the Ebro Valley represented 23-30%, overpassing 50% at intensive agricultural areas. Similarly, urban-metropolitan contributions accounted for 37-45% at the four cities, and 55% at the industrial one. African dust deposition was enhanced in the Central Pyrenees (75-80%) as a result of the magnification of atmospheric washout processes, and in south-eastern Iberia (69%) owing to the higher frequency of dust outbreaks.

Swine barn dust stimulates CCL9 expression in mouse monocytes through PKC-delta activation.

Exposure to organic barn dusts has been shown to cause numerous lung problems to chronically exposed animal barn workers. Bacterial components in these dusts trigger innate immunity in the lungs that we are still trying to fully characterize. CCL9/MIP-1γ is constitutively expressed in high quantities in the mouse circulation, but at much lower levels in the lungs where it is inducible under certain circumstances. We show here that extracts from hog barn dusts (HDE) are capable of inducing significant increases of CCL9 mRNA and protein in RAW267.4 monocytic cells as well as in mouse lungs. We further show that incubation of CCL9 with HDE results in cleavage of CCL9, which others have shown to increase chemotactic signaling potential. Endotoxin and proteoglycan were determined to be the likely causes of this increase. We additionally present evidence for a role of PKC-delta in this activation. Addition of purified CCL9 protein to HDE treated cell culture resulted in a small, but significant reduction in KC production, suggesting a possible regulatory role for the chemokine.