Bacterial DNA induces pulmonary damage via TLR-9 through cross-talk with neutrophils.

Bacterial DNA (bDNA) contains hypo-methylated “CpG” repeats that can be recognized by toll-like receptor (TLR)-9 as a pathogen-associated molecular pattern (PAMP). The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMN) are thought to play a key role in the genesis of septic acute lung injury (ALI). We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of ALI. Using a bio-capacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMN. These permeability changes are inhibited by chloroquine, suggesting TLR9-dependency. When PMN were pre-incubated with bDNA and applied to EC or when bDNA was applied to EC without PMN, no permeability changes were detected. To study the underlying mechanisms we evaluated the effects of bDNA on PMN-EC adherence. bDNA significantly increased PMN adherence to EC in association with up-regulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. bDNA stimulation of TLR9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk which is required for injury to occur.