Redox Dependent Activation of Lung Epithelial STAT3 Is Required for Inducible Protection Against Bacterial Pneumonia

The lung epithelium is dynamic, capable of considerable structural and functional plasticity in response to pathogen challenges. Our lab has demonstrated that an inhaled combination of a Toll-like receptor (TLR)2/6 agonist and a TLR9 agonist (Pam2ODN) results in robust, protection against otherwise lethal Pseudomonas aeruginosa pneumonias. We have previously shown that intact epithelial TLR signaling and generation of multisource epithelial reactive oxygen species (ROS) are both required for the inducible protection. Further investigating the mechanisms underlying this phenomenon of inducible resistance, reverse phase protein array analysis demonstrated robust Signal transducer and activator of transcription 3 (STAT3) phosphorylation following treatment of lung epithelial cells, and we show here that Pam2ODN-induced STAT3 phosphorylation is interleukin (IL)-6-independent. We further found that therapeutic epithelial STAT3 activation is required inducible protection against P. aeruginosa pneumonia. Additional studies showed that inhibiting epithelial dual oxidases (DUOX) or scavenging ROS significantly reduced the Pam2ODN induction of STAT3 phosphorylation, suggesting a proximal role for ROS in inducible STAT3 activation. Dissecting these potential mechanisms, we analyzed the contributions of redox sensitive kinases and found that Pam2-ODN activated EGFR in a ROS-dependent manner that is required for inducible STAT3 activation. Taken together, we demonstrate that epithelial STAT3 is imperative for Pam2ODN’s function and describe a novel, redox based mechanism for its activation. These key mechanistic insights may facilitate strategies to leverage inducible epithelial resistance to protect susceptible patients during periods of peak vulnerability.