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

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.The innate immune response to Gram-negative bacteria is dominated by recognition of lipopolysaccharide (LPS). LPS is sensed by the Toll-like receptor 4 (TLR4) complex, and numerous studies have focused on both how LPS sensitivity drives effective inflammatory responses, leading to the clearance of infection, as well as how uncontrolled TLR4 signaling can lead to LPS toxicity and play a role in septic shock (20, 39, 48). Much of this research has been performed using purified LPS in the experimental treatments, and yet it is unlikely that pure LPS is shed from bacteria in the context of an infection. Instead, LPS has been found to be shed from the bacteria in the form of outer membrane vesicles.Outer membrane vesicles are spherical, selective portions of outer membrane and periplasm that are naturally secreted by all Gram-negative bacteria (7, 31, 49). Vesicles are produced at all stages of bacterial growth and have been detected in infected human tissues (7, 23, 28). Vesicle production has been identified as an independent bacterial stress response pathway that is activated when bacteria are exposed to environmental stress, such as might be experienced during colonization of host tissues (35).Compared to preparations of pure LPS, natural outer membrane vesicles are heterogeneous proteoliposomes of a larger dimension (50 to 250 nm in diameter) than liposomes composed solely of LPS (4, 7). Natural outer membrane vesicles are heterogeneous complexes of pathogen-associated molecular patterns (PAMPs), such as LPS, flagellin, and CpG DNA, as well as other outer membrane proteins, virulence factors, and envelope lipids (7, 31). The molecular composition of vesicles varies with the bacterial strain of origin, while LPS structure remains relatively constant within a bacterial species. The combination of PAMPs, virulence factors, and other outer membrane components results in vesicles that are particularly laden with molecules that can be recognized by the immune system. In the present study we have focused on characterizing macrophage innate immune responses to the combined signals presented by the heterogeneous PAMP ligands presented in the native context of outer membrane vesicles.Studies of host immune responses to outer membrane vesicles have mainly addressed the generation of antibodies to vesicle components. Notably, a protective antibody response is elicited by outer membrane vesicles generated from Neisseria meningitidis and Vibrio cholerae (17, 18, 38, 40, 47). In contrast, there are relatively few studies characterizing how vesicles trigger the innate inflammatory response. Outer membrane vesicles from Helicobacter pylori and Pseudomonas aeruginosa have been shown to elicit interleukin-8 (IL-8) production by epithelial cells (3, 24), and Salmonella enterica serovar Typhimurium vesicles have been shown to activate dendritic cells to secrete IL-12 and tumor necrosis factor alpha (TNF-α) (1).This research has focused on outer membrane vesicles from the opportunistic pathogen P. aeruginosa. P. aeruginosa infects the respiratory tract of patients with nosocomial pneumonia, cystic fibrosis, or acute respiratory distress syndrome (14). Host responses to acute pulmonary infections with P. aeruginosa are characterized by an intense inflammatory response. Macrophage recognition of bacterial components initiates a cascade of proinflammatory cytokine secretion, resulting in large numbers of neutrophils infiltrating the lung to clear the bacteria (44).We hypothesize that naturally produced vesicles may play an important role in activating innate immune responses. Given their small size and high proportion of PAMPs, it is reasonable to expect that vesicles could easily infiltrate into infected tissues and stimulate widespread inflammation. In addition to TLR ligands, vesicles also contain a variety of protein virulence factors that may specialize vesicles for specific functions or types of host cell damage. P. aeruginosa vesicles have been shown to contain the adhesins OprF, OprG, and OprH, as well as virulence factors that include β-lactamase, hemolysin, phospholipase C, antimicrobial quinolones, and quorum-sensing molecules (3, 12, 25, 26, 31, 34). P. aeruginosa vesicle-associated proteins have also been shown to directly affect host cells. Vesicle-packaged CiF protein downregulates lung epithelial expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein (10, 33), while an aminopeptidase enriched in vesicles increases association of vesicles with cultured epithelial cells (2).The present study describes how vesicles, with their heterogeneous mixture of PAMPs, contribute to the innate immune response during P. aeruginosa infection. We have focused on vesicle-macrophage interactions, since macrophages are sentinel cells that initiate inflammatory defenses against colonizing bacteria. Our data demonstrate that macrophages are more sensitive to the potent stimulus of outer membrane vesicles from P. aeruginosa compared to equivalent levels of pure LPS. Neutralizing the LPS reactivity of the vesicles or altering the protein composition of the vesicles significantly impacted both the macrophage-vesicle interactions and the cytokine responses to the vesicles. Based on our findings, we conclude that a potent and distinct inflammatory response to vesicles is the cumulative effect of sensing vesicle-associated heterogeneous protein and LPS ligands by macrophages.