Respiratory epithelial cells require Toll-like receptor 4 for induction of human b-defensin 2 by lipopolysaccharide

Background: The respiratory epithelium is a major portal of entry for pathogens and employs innate defense mechanisms to prevent colonization and infection. Induced expression of human beta-defensin 2 (HBD2) represents a direct response by the epithelium to potential infection. Here we provide evidence for the critical role of Toll-like receptor 4 (TLR4) in lipopolysaccharide (LPS)induced HBD2 expression by human A549 epithelial cells. Methods: Using RTPCR, fluorescence microscopy, ELISA and luciferase reporter gene assays we quantified interleukin-8, TLR4 and HBD2 expression in unstimulated or agonist-treated A549 and/ or HEK293 cells. We also assessed the effect of over expressing wild type and/ or mutant TLR4, MyD88 and/ or Mal transgenes on LPS-induced HBD2 expression in these cells. Results: We demonstrate that A549 cells express TLR4 on their surface and respond directly to Pseudomonas LPS with increased HBD2 gene and protein expression. These effects are blocked by a TLR4 neutralizing antibody or functionally inactive TLR4, MyD88 and/ or Mal transgenes. We further implicate TLR4 in LPS-induced HBD2 production by demonstrating HBD2 expression in LPS non-responsive HEK293 cells transfected with a TLR4 expression plasmid. Conclusion: This data defines an additional role for TLR4 in the host defense in the lung.