Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.

Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells.

BACKGROUND: Persistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial cells. METHODS: The effects of dust extracts on inflammatory gene expression were analyzed by quantitative polymerase chain reaction (qPCR), enzyme linked immunosorbent (ELISA) and western blot assays. Oxidant stress was probed by dihydroethidium (DHE) labeling, and immunostaining for 4-hydroxynonenal (4-HNE). Effects on interleukin-8 (IL-8) promoter regulation were determined by transient transfection assay. RESULTS: Dust extracts contained trypsin and elastase activities, and activated protease activated receptor (PAR)-1 and -2. Serine protease inhibitors and PAR-1 or PAR-2 knockdown suppressed inflammatory gene induction. Dust extract induction of IL-8 gene expression was associated with increased DHE-fluorescence and 4-HNE staining, and antioxidants suppressed inflammatory gene induction. Protease inhibitors and antioxidants suppressed protein kinase C and NF-κB activation and induction of IL-8 promoter activity in cells exposed to dust extract. CONCLUSIONS: Our studies demonstrate that proteases and intracellular oxidants control organic dust induction of inflammatory gene expression in lung epithelial cells. Targeting proteases and oxidant stress may serve as novel approaches for the treatment of organic dust induced lung diseases. This is the first report on the involvement of oxidant stress in the induction of inflammatory gene expression by organic dust.

Gene expression profiling of the effects of organic dust in lung epithelial and THP-1 cells reveals inductive effects on inflammatory and immune response genes.

The intensification and concentration of animal production operations expose workers to high levels of organic dusts in the work environment. Exposure to organic dusts is a risk factor for the development of acute and chronic respiratory symptoms and diseases. Lung epithelium plays important roles in the control of immune and inflammatory responses to environmental agents to maintain lung health. To better understand the effects of organic dust on lung inflammatory responses, we characterized the gene expression profiles of A549 alveolar and Beas2B bronchial epithelial and THP-1 monocytic cells influenced by exposure to poultry dust extract by DNA microarray analysis using Illumina Human HT-12 v4 Expression BeadChip. We found that A549 alveolar and Beas2B bronchial epithelial and THP-1 cells responded with unique changes in the gene expression profiles with regulation of genes encoding inflammatory cytokines, chemokines, and other inflammatory proteins being common to all the three cells. Significantly induced genes included IL-8, IL-6, IL-1ß, ICAM-1, CCL2, CCL5, TLR4, and PTGS2. Validation by real-time qRT-PCR, ELISA, Western immunoblotting, and immunohistochemical staining of lung sections from mice exposed to dust extract validated DNA microarray results. Pathway analysis indicated that dust extract induced changes in gene expression influenced functions related to cellular growth and proliferation, cell death and survival, and cellular development. These data show that a broad range of inflammatory mediators produced in response to poultry dust exposure can modulate lung immune and inflammatory responses. This is the first report on organic dust induced changes in expression profiles in lung epithelial and THP-1 monocytic cells.

Transcriptional mechanisms and protein kinase signaling mediate organic dust induction of IL-8 expression in lung epithelial and THP-1 cells.

Exposure to the agricultural work environment is a risk factor for the development of respiratory symptoms and chronic lung diseases. Inflammation is an important contributor to the pathogenesis of tissue injury and disease. Cellular and molecular mechanisms mediating lung inflammatory responses to agricultural dust are not yet fully understood. We studied the effects of poultry dust extract on molecular regulation of interleukin-8 (IL-8), a proinflammatory cytokine, in A549 and Beas2B lung epithelial and THP-1 monocytic cells. Our findings indicate that poultry dust extract potently induces IL-8 levels by increasing IL-8 gene transcription without altering IL-8 mRNA stability. Increase in IL-8 promoter activity was due to enhanced binding of activator protein 1 and NF-κB. IL-8 induction was associated with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation and inhibited by PKC and MAPK inhibitors. IL-8 increase was not inhibited by polymyxin B or l-nitroarginine methyl ester, indicating lack of involvement of lipopolysaccharide and nitric oxide in the induction. Lung epithelial and THP-1 cells share common mechanisms for induction of IL-8 levels. Our findings identify key roles for transcriptional mechanisms and protein kinase signaling pathways for IL-8 induction and provide insights into the mechanisms regulating lung inflammatory responses to organic dust exposure.

Early secreted antigenic target of 6 kDa (ESAT-6) protein of Mycobacterium tuberculosis induces interleukin-8 (IL-8) expression in lung epithelial cells via protein kinase signaling and reactive oxygen species.

Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is critical for the virulence and pathogenicity of M. tuberculosis. IL-8, a major chemotactic cytokine for neutrophils and T lymphocytes, plays important roles in the development of lung injury. To further understand the role of ESAT-6 in lung pathology associated with tuberculosis development, we studied the effects of ESAT-6 on the regulation of IL-8 expression in lung epithelial cells. ESAT-6 induced IL-8 expression by increasing IL-8 gene transcription and mRNA stability. ESAT-6 induction of IL-8 promoter activity was dependent on nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding and sensitive to pharmacological inhibition of PKC and ERK and p38 MAPK pathways. ESAT-6 activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. Dimethylthiourea but not mannitol inhibited IL-8 induction by ESAT-6, further supporting the involvement of ROS in the induction of IL-8 expression. Exposure of mice to ESAT-6 induced localized inflammatory cell aggregate formation with characteristics of early granuloma concomitant with increased keratinocyte chemoattractant CXCL1 staining in bronchiolar and alveolar type II epithelial cells and alveolar macrophages. Our studies have identified a signal transduction pathway involving ROS, PKC, ERK, and p38 MAPKs and NF-κB and AP-1 in the ESAT-6 induction of IL-8 expression in lung epithelial cells. This has important implications for the understanding of lung innate immune responses to tuberculosis and the pathogenesis of lung injury in tuberculosis.

Passive cigarette smoke exposure inhibits ultraviolet light B-induced skin tumors in SKH-1 hairless mice by blocking the nuclear factor kappa B signalling pathway.

Chronic exposure to sunlight [ultraviolet light B (UVB) irradiation] is the most common cause of non-melanoma skin tumors. In the present study, we investigated the effects of passive cigarette smoke superimposed over UVB irradiation, on tumor development, skin pathology and matrix changes in SKH-1 hairless mice. Groups of mice were exposed to 0.1 J/cm(2) of UVB five times per week for 20 weeks and/or exposure to passive cigarette smoke from 40 cigarettes a day over the same time period. UVB exposure resulted in an average of four large squamous cell carcinomas (SCC) and 15 smaller papillomas per mouse, whereas exposing the mice to both UVB + passive cigarette smoke completely prevented SCC formation and averaged less than one small papilloma per mouse. Oxidative DNA damage was investigated and there were no significant changes in the levels of urinary DNA adducts between control, smoke, UV and UV + smoke groups with the exception of 8-oxo guanine which was significantly reduced in the presence of passive cigarette smoke. Immunohistochemistry results revealed that tumor necrosis factor receptor 2 (TNF-R2), glycogen synthase kinase-3 beta, nuclear factor kappa B (NF-kappaB)/p65, KI-67 and cyclooxygenase 2 (COX-2) were markedly up-regulated in the epithelium by UVB exposure, whereas passive smoke exposure combined with the UVB irradiation completely blocked the expression of these proteins. Our results suggest that passive smoke exposure prevents UVB-induced SCC in mice and dramatically reduces the incidence of non-malignant papillomas by altering the NF-kappaB signalling pathway of tumorigenesis.