Rationale: Gene delivery applications to treat lung diseases are, in some instances, sub-optimal due to deleterious host inflammatory reactions. Current DNA plasmids (pDNA) exert toxicity in part via unmethylated CpG motifs that stimulate TLR9 expressing leukocytes; however the airway epithelial response has not been well defined. Methods: Bronchial epithelial cells (BEAS-2B) were exposed to pDNA complexes and inflammatory mediators were measured. As patients with inflammatory lung disease are susceptible to infectious exacerbations, we also evaluated the reciprocal inflammatory response to pDNA and bacterial components LPS and LTA, recognised by TLR4 and TLR2 respectively. Results: Cells primed with pDNA synergistically expressed IL-8 mRNA and protein in response to LPS and LTA (3-5 fold). A similar induction was also observed for IL-1, IL-6, CSF-1 and GM-CSF. Their synergistic elevation was associated with an increase in TLR4 and TLR2 levels. Methylation of pDNA only partially reduced (25-30%) IL-8 release; hence signalling occurs via CpG/TLR9 dependent and independent modules. As EGFR signalling has been implicated in bronchial IL-8 expression, we assessed whether pDNA priming events were coordinated via EGFR. AG1478 (EGFR inhibitor) restored normal TLR4/2 levels and also suppressed synergistic release of IL-8. The Erk MAPK inhibitor also blocked IL-8 release, implicating Erk as a key mediator of EGFR signalling. Conclusions: Our findings identify a novel EGFR-dependent mechanism for regulating TLR, and show that targeted disruption of EGFR signalling ameliorates the airway epithelial inflammatory response to pDNA. Targeting the EGFR system may improve the efficiency, tolerability and safety of gene therapy strategies.