Central role of PAFR signalling in ExoU-induced NF-κB activation.

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU-driven NF-κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF-κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR-dependent NF-κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR-/- or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU-induced NF-κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF-κB inhibitor Bay 11-7082, or transfection with IκBα negative-dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11-7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF-κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.

Contribution of the platelet activating factor signaling pathway to cerebral microcirculatory dysfunction during experimental sepsis by ExoU producing Pseudomonas aeruginosa.

Intravital microscopy was used to assess the involvement of ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, in dysfunction of cerebral microcirculation during experimental pneumosepsis. Cortical vessels from mice intratracheally infected with low density of the ExoU-producing PA103 P. aeruginosa strain exhibited increased leukocyte rolling and adhesion to venule endothelium, decreased capillar density and impaired arteriolar response to vasoactive acetylcholine. These phenomena were mediated by the platelet activating factor receptor (PAFR) pathway because they were reversed in mice treated with a PAFR antagonist prior to infection. Brains from PA103-infected animals exhibited a perivascular inflammatory infiltration that was not detected in animals infected with an exoU deficient mutant or in mice treated with the PAFR antagonist and infected with the wild type bacteria. No effect on brain capillary density was detected in mice infected with the PAO1 P. aeruginosa strain, which do not produce ExoU. Finally, after PA103 infection, mice with a targeted deletion of the PAFR gene exhibited higher brain capillary density and lower leukocyte adhesion to venule endothelium, as well as lower increase of systemic inflammatory cytokines, when compared to wild-type mice. Altogether, our results establish a role for PAFR in mediating ExoU-induced cerebral microvascular failure in a murine model of sepsis.

ExoU activates NF-κB and increases IL-8/KC secretion during Pseudomonas aeruginosa infection.

ExoU, a Pseudomonas aeruginosa cytotoxin injected into host cytosol by type III secretion system, exhibits a potent proinflammatory activity that leads to a marked recruitment of neutrophils to infected tissues. To evaluate the mechanisms that account for neutrophil infiltration, we investigated the effect of ExoU on IL-8 secretion and NF-κB activation. We demonstrate that ExoU increases IL-8 mRNA and protein levels in P. aeruginosa-infected epithelial and endothelial cell lines. Also, ExoU induces the nuclear translocation of p65/p50 NF-κB transactivator heterodimer as well as NF-κB-dependent transcriptional activity. ChIP assays clearly revealed that ExoU promotes p65 binding to NF-κB site in IL-8 promoter and the treatment of cultures with the NF-κB inhibitor Bay 11-7082 led to a significant reduction in IL-8 mRNA levels and protein secretion induced by ExoU. These results were corroborated in a murine model of pneumonia that revealed a significant reduction in KC secretion and neutrophil infiltration in bronchoalveolar lavage when mice were treated with Bay 11-7082 before infection with an ExoU-producing strain. In conclusion, our data demonstrate that ExoU activates NF-κB, stimulating IL-8 expression and secretion during P. aeruginosa infection, and unveils a new mechanism triggered by this important virulence factor to interfere in host signaling pathways.