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Pseudomonas aeruginosa is a prevalent opportunistic human pathogen that, like other Gram-negative pathogens, secretes outer membrane vesicles. Vesicles are complex entities composed of a subset of envelope lipid and protein components that have been observed to interact with and be internalized by host cells. This study characterized the inflammatory responses to naturally produced P. aeruginosa vesicles and determined the contribution of vesicle Toll-like receptor (TLR) ligands and vesicle proteins to that response. Analysis of macrophage responses to purified vesicles by real-time PCR and enzyme-linked immunosorbent assay identified proinflammatory cytokines upregulated by vesicles. Intact vesicles were shown to elicit a profoundly greater inflammatory response than the response to purified lipopolysaccharide (LPS). Both TLR ligands LPS and flagellin contributed to specific vesicle cytokine responses, whereas the CpG DNA content of vesicles did not. Neutralization of LPS sensing demonstrated that macrophage responses to the protein composition of vesicles required the adjuvantlike activity of LPS to elicit strain specific responses. Protease treatment to remove proteins from the vesicle surface resulted in decreased interleukin-6 and tumor necrosis factor alpha production, indicating that the production of these specific cytokines may be linked to macrophage recognition of vesicle proteins. Confocal microscopy of vesicle uptake by macrophages revealed that vesicle LPS allows for binding to macrophage surfaces, whereas vesicle protein content is required for internalization. These data demonstrate that macrophage sensing of both LPS and protein components of outer membrane vesicles combine to produce a bacterial strain-specific response that is distinct from those triggered by individual, purified vesicle components.

infection and immunity

Naturally Produced Outer Membrane Vesicles from<i>Pseudomonas aeruginosa</i>Elicit a Potent Innate Immune Response via Combined Sensing of Both Lipopolysaccharide and Protein Components

Naturally Produced Outer Membrane Vesicles fromPseudomonas aeruginosaElicit a Potent Innate Immune Response via Combined Sensing of Both Lipopolysaccharide and Protein Components