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2612218 
Journal Article 
Abstract 
House dust mite directly activates STAT3 in human airway epithelial cells and mediates pro-inflammatory cytokine generation 
Murray, L; Han, E; Kravitz, J; Fields, E; Simon, AR 
2010 
Yes 
American Journal of Respiratory and Critical Care Medicine
ISSN: 1073-449X
EISSN: 1535-4970 
181 
A1443 
English 
WOS:000208771000444 
is part of a larger document 3452678 Proceedings of the American Thoracic Society 2010 International Conference, May 14-19, 2010, New Orleans
Rationale: The airway epithelium is a key regulator of allergic inflammation. We have previously determined that house dust mite (HDM) administration in mice activates STAT3 in the airway epithelium. Furthermore, mice with STAT3 deficiency in the airway epithelium were protected from HDM-induced allergic inflammation and airway hyperresponsiveness. In vitro, HDM has been shown to directly activate the MAPK pathway in airway epithelial cells, and we hypothesize that HDM also directly activates STAT3 in these cells and that this activation regulates inflammatory cytokine generation.

Methods: Quiescent normal human bronchial epithelial cells (NHBE) or BEAS2B cells were grown in submerged cultures and treated with HDM extract (10 μg or 50 μg). STAT3 knockdown was performed by infecting cells with a non-targeting control or shSTAT3 lentivirus. Total lysates were obtained and processed for Western blot using phosphospecific STAT3, EGFR and STAT3 antibodies. Total RNA was processed for quantitative real-time PCR using primers for CCL17, IL-6 and IL-8. Cytokine levels in the conditioned media was determined by a multiplex ELISA assay.

Results: HDM treatment resulted in a rapid (5 min) and prolonged (2 hr) activation of STAT3 in both BEAS2B and NHBE cells. STAT3 activation at both early and late timepoints was significantly decreased when cells were pre-treated with the EGF Receptor inhibitor AG1478. In addition, pre-treatment of cells with a Src kinase inhibitor (SU6656), but not the Jak kinase inhibitor (Tyrphostin A1), signficantly blocked HDM-induced STAT3 activation. Pre-treating cells with the antioxidant DPI also significantly attenuated HDM-induced STAT3 activation at timepoints starting at 30 minutes. HDM-induced EGFR activation occurred with kinetics similar to that seen for STAT3. To evaluate downstream targets of HDM-induced STAT3 activation, STAT3 expression was knocked down via shRNA. HDM treatment from 4 to 24 hours significantly increased the generation of CCL17 (TARC), IL-6 and IL-8 in BEAS2B cells compared to control, as determined by Q-RT-PCR. The HDM-induced increase in CCL17 and IL-8 was significantly decreased in cells with STAT3 knockdown. Conditioned media obtained from these cells demonstrated an increase in HDM-induced IL-8 and IL-6 generation, but not CCL17. Both HDM-induced IL-8 and IL-6 levels were significantly lower in cells with STAT3 deficiency.

Conclusion: Our studies define a novel pathway in human airway epithelial cells downstream of HDM that signals via the EGFR-ROS-Src pathway to STAT3, and which induces the generation of cytokines important for allergic inflammation. Thus targeting STAT3 in the airway may have therapeutic potential in asthma. 
American Thoracic Society 2010 International Conference 
New Orleans, LA 
May 14-19, 2010