Resumo

The virulence factor ExoU is a potent Pseudomonas aeruginosa phospholipase A2 injected into host cytosol by type III secretion system. ExoU is expressed by only 30% of clinical isolates and plays an important role in pneumonia and invasive acute infections. In previous studies, we have shown that ExoU activates NF-κB, stimulating IL-8 secretion and neutrophil recruitment. In the present study, we used human airway epithelial cells and mice infected by wild type or ExoU-deficient mutant strains of P. aeruginosa to evaluate the mechanisms of ExoU-driven NF-κB activation. Luciferase reporter assays showed that NF-κB activation results from the action of soluble factors released by infected cells and that platelet-activating factor (PAF) has a crucial role. Western blot assays showed that ExoU PLA2 activity reduced IκBα levels on cytosol and increased nuclear p65. These events, as well as IL8 expression, were inhibited by anti-PAFR treatment, indicating that ExoU activates the canonical NF-κB pathway via PAFR. Since PAFR expression could be regulated by NF-kB, we next performed ChIP assays that showed increased p65 occupation of PAFR promoter when cells were infected with the ExoU-producing strain. Luciferase assays performed in cultures transfected with different plasmid constructs revealed that ExoU promotes p65 binding to the three κB sites in PAFR promoter. Moreover, treatment of cultures with the NF-kB inhibitor Bay 11-7082 or transfection with IκBα negative-dominant plasmid led to a significant reduction in PAFR mRNA levels, as assessed by qRT-PCR. Together these results demonstrate that NF-κB also regulates PAFR expression. The in vitro data were corroborated in a murine model of pneumonia that revealed that mice treated with the PAF antagonist WEB 2086 prior to infection with the ExoU-producing strain exhibited a significant reduction in p65 nuclear translocation in airway epithelial cells, as assessed by ELISA and confocal microscopy. Also, qRT-PCR assays showed that NF-kB inhibition by treatment with Bay 11-7082 before infection decreased the ExoU-induced PAFR expression. In conclusion, our data demonstrate that ExoU activates NF-κB by PAF-PAFR-dependent mechanism, which enhances PAFR expression, during infection with P. aeruginosa.