Repetitive organic dust exposure in vitro impairs macrophage differentiation and function

Pulmonary, Critical Care, Sleep and Allergy Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Neb 68198-5300, USA.
The Journal of allergy and clinical immunology (Impact Factor: 11.48). 06/2008; 122(2):375-82, 382.e1-4. DOI: 10.1016/j.jaci.2008.05.023
Source: PubMed


Organic dust exposure in the agricultural industry results in significant airway disease and lung function decrease. Mononuclear phagocytes are key cells that mediate the inflammatory and innate immune response after dust exposure.
We sought to investigate the effect of organic dust extract (ODE) from modern swine operations on monocyte-derived macrophage (MDM) phenotype and function.
Peripheral blood monocytes were obtained by means of elutriation methodology (>99% CD14(+)) and differentiated into macrophages in the presence of GM-CSF (1 week) with and without ODE (0.1%). At 1 week, cells were analyzed by means of flow cytometry for cell-surface marker expression (HLA-DR, CD80, CD86, Toll-like receptor 2, Toll-like receptor 4, mCD14, and CD16), phagocytosis (IgG-opsonized zymosan particles), and intracellular killing of Streptococcus pneumoniae. At 1 week, MDMs were rechallenged with high-dose ODE (1%), LPS, and peptidoglycan (PGN), and cytokine levels (TNF-alpha, IL-6, IL-10, and CXCL8/IL-8) were measured. Comparisons were made to MDMs conditioned with heat-inactivated dust, endotoxin-depleted dust, LPS, and PGN to elucidate ODE-associated factors.
Expression of HLA-DR, CD80, and CD86; phagocytosis; and intracellular bacterial killing were significantly decreased with ODE-challenged versus control MDMs. Responses were retained after marked depletion of endotoxin. PGN, LPS, and PGN plus LPS significantly reduced MDM surface marker expression and, except for LPS alone, also reduced phagocytosis. ODE-challenged MDMs had significantly diminished cytokine responses (TNF-alpha, IL-6, and IL-10) after repeat challenge with high-dose ODE. Cross-tolerant cytokine responses were also observed.
Repetitive organic dust exposure significantly decreases markers of antigen presentation and host defense function in MDMs. Bacterial cell components appear to be driving these impaired responses.

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    • "Although not yet studied in the lung environment, MaR1 has shown utility in reducing neutrophil infiltration while increasing macrophage phagocytic capacities in a murine model of peritonitis [18]. Previously published data from our group suggest the recruitment and subsequent actions of macrophages in organic dust exposures are highly important in determining the outcomes of the pro-inflammatory insult, and pro-inflammatory cytokine production by BECs exposed to injurious stimuli such as organic dusts is key to the recruitment of these macrophages as well as neutrophils into the lung [19-23]. However, MaR1’s effects on BECs, along with other cells in the lung are currently unknown. "
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    ABSTRACT: 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.
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    • "Advances into understanding specific causative factors determined that PM size is important, with coarse PM inducing enhanced inflammation (Vogel et al., 2012). Endotoxin, signaling through TLR4 (Charavaryamath et al., 2008), remains an important causative agent, but recent studies show important roles for gram-positive bacteria (Nehme et al., 2009; Poole et al., 2008, 2010) and for targeting the TLR2 pathway. "
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    • "Human monocytes, neutrophils (PMNs) and red blood cells (RBCs) were isolated as previously described [32]. To prepare monocyte-derived macrophages (MDMs), isolated monocytes were cultured in DMEM/10% FCS supplemented with macrophage colony-stimulating factor (M-CSF) for 6 days [33]. Mature macrophages (3×105/well) were then plated in 24-well tissue culture plates and allowed to adhere for 2 h. "
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