DNA vector augments inflammation in epithelial cells via EGFR-dependent regulation of TLR4 and TLR2

Gene delivery applications to treat lung diseases are, in some instances, suboptimal due to deleterious host inflammatory reactions. Current DNA plasmids (pDNA) exert toxicity in part via unmethylated CpG motifs that stimulate Toll-like receptor (TLR)9-expressing leukocytes; however, the airway epithelial response has not been well defined. 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 lipopolysaccharide (LIPS) and lipoteichoic acid (LTA), recognized by TLR4 and TLR2, respectively. Cells primed with pDNA synergistically expressed IL-8 mRNA and protein in response to LIPS and LTA (3- to 5-fold). A similar induction was also observed for IL-1 beta, IL-6, colony-stimulating factor (CSF)-1, and granulocyte macrophage-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, signaling occurs via CpG/TLR9-dependent and -independent modules. As epidermal growth factor receptor (EGFR) signaling 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 extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase inhibitor also blocked IL-8 release, implicating Erk as a key mediator of EGFR signaling. Our findings identify a novel EGFR-dependent mechanism for regulating TLR, and show that targeted disruption of EGFR signaling ameliorates the airway epithelial inflammatory response to pDNA. Targeting the EGFR system may improve the efficiency, tolerability, and safety of gene therapy strategies.