B28-21 Regulation of Organic Dust-induced Lung Inflammation by Toll-like Receptors 1/2 and 4

Abstract Rationale Industrial animal farming operations produce high levels of aerosolized organic dust. Organic dust is a complex mixture containing animal dander and waste, feed particles, microbes and microbial products. Organic dust exposure is a risk factor for the development of respiratory diseases such as occupational asthma, bronchitis, organic dust toxic syndrome, hypersensitivity pneumonitis, and chronic obstructive pulmonary disease (COPD). Toll like receptors (TLRs) are transmembrane pattern recognition receptors (PRRs) that bind microbial and endogenous molecules to modulate immune and inflammatory responses. As organic dust contains microbial bioactive molecules, we hypothesized that TLRs regulate organic dust induced lung inflammation. The role of TLRs in the regulation of lung inflammation induced by organic dust is not fully understood. Methods Beas2B bronchial epithelial cell cultures were treated with aqueous extracts of poultry farm dust (dust extract, DE) or bacterial extracellular vesicles (bacterial EVs) isolated from poultry farm dust. Expression of TLRs and inflammatory mediators (pro-IL-1β, ICAM-1, IL-6, IL-8/CXCL1, TNF-α) was analyzed by real-time qRT-PCR, western blotting and ELISA. The role of TLRs was investigated by studying the effects of chemical inhibitors, neutralizing antibodies, and siRNA mediated knock down on the induction of inflammatory mediators. Cellular ROS generation was assessed by DCFDA staining and NF-κB and Stat3 phosphorylation were analyzed by Western blotting. TLR2 knockout mice (B6.129-Tlr2tm1Kir/J, Jackson Laboratory) were exposed to bacterial EVs via intranasal instillation to study the effects on lung inflammation in vivo. Results Dust extract and bacterial EVs increased TLR2, TLR4, and inflammatory mediators’ expression in Beas2B cells. The TLR1/2 inhibitor CuCPT22 and TLR4 inhibitor resatorvid (TAK-242), TLR2-neutralizing antibodies, and TLR2 knockdown reduced induction of inflammatory mediators’ expression. Moreover, MyD88 knockdown also reduced induction of inflammatory mediators. Inhibition of inflammatory mediators by CuCPT22 and resatorvid was associated with decreased cellular ROS levels, ΝFκΒ and Stat3 phosphorylation, along with reduced aryl hydrocarbon receptor (AhR) expression. Bacterial EVs induction of lung CXCL1 and IL-6 levels were reduced in TLR2 knockout mice. Conclusions Our study demonstrated that organic dust extract and bacterial EVs isolated from organic dust activate TLR2 and TLR4 signaling pathways to modulate lung inflammatory responses. Activation of TLRs leads to increased expression of inflammatory mediators via enhanced cellular ROS production and activation of downstream NFκB, Stat3, and AhR signaling pathways. Studies in TLR2 knockout mice indicated that in addition to TLR2, other PRR signaling pathways may also be involved in the regulation of organic dust induced inflammatory responses. This abstract is funded by: CDC/NIOSH Grant U54 OH007541