Abstract Rationale Exposure to organic dust generated in industrial agricultural operations causes lung inflammation and is associated with the development of respiratory diseases such as bronchitis, hypersensitivity pneumonitis, asthma, and chronic obstructive pulmonary disease (COPD). Different components of organic dust have been identified, but the mechanisms by which they induce lung inflammation are not well understood. We previously reported that bacterial extracellular vesicles isolated from poultry organic dust induce neutrophilic lung inflammation (Am. J Physiol. Lung Cell. Mol. Physiol. 319: L893-L907, 2020). In this study we sought to identify the stimulatory constituents of bacterial EVs that trigger inflammatory responses in bronchial epithelial cells and understand the underlying mechanisms. Methods Bacterial EVs from poultry organic dust were isolated by ultracentrifugation. Potential stimulatory constituents of bacterial EVs such as DNA, RNA, and protein were inactivated using DNase, RNase, and proteinase K, respectively. Bacterial lipoproteins were inactivated using hydrogen peroxide or lipoprotein lipase (LPL) and lipoteichoic acid (LTA) was inactivated using platelet activating factor acetyl hydrolase (PAF-AH), hydrogen peroxide or LPL. Effects of bacterial EVs on inflammatory mediator protein levels in Beas2B bronchial epithelial cells were analyzed by ELISA and western blotting. Effects on cell toxicity were determined by MTS assay. Cellular reactive oxygen species (ROS) levels were measured by 2’,7’-dichlorofluorescein diacetate (DCFDA) labeling. Activations of NFκB-p65 and Stat3 were analyzed by western blotting. Results RNase and DNase treatments of bacterial EVs did not significantly affect induction of inflammatory mediators in Beas2B cells. In contrast, treatment with proteinase K, LPL, or PAF-AH, significantly reduced bacterial EVs ability to induce inflammatory mediators. Likewise, treatment with hydrogen peroxide reduced bacterial EVs induction of inflammatory mediators. None of the treatments affected Beas2B cell viability. Proteinase K, PAF-AH, LPL, and hydrogen peroxide treatments reduced bacterial EVs stimulation of cellular ROS generation and NFκB-p65 activation, whereas treatment with hydrogen peroxide reduced both NFκB-p65 and Stat3 activation. Conclusions Our studies have indicated that lipoproteins, LTA, and possibly LPS in bacterial EVs stimulate induction of inflammatory mediators in bronchial epithelial cells via a signaling pathway involving increased cellular ROS generation and activation of NFκB-p65 and Stat3 transcription factors. This abstract is funded by: CDC/NIOSH Grant U54 OH007541
Kusampudi et al. (Fri,) studied this question.