Resumo

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 synthesis and secretion. Semi-quantitative and Real Time RT-PCR showed that ExoU PLA2 activity increased IL-8 mRNA levels in P. aeruginosa-infected airway epithelial cells. ExoU PLA2 activity also led to higher IL-8 secretion in cell culture supernatants, as assessed by ELISA. Moreover, IL-8 was actively secreted by epithelial cells since the treatment with Brefeldin A, an inhibitor of intracellular protein transport and protein secretion, significantly reduced IL-8 concentrations in supernatants. Our assays also demonstrate that the ExoU-mediated IL-8 induction mainly depends on NF-kB pathway. First, ChIP assays clearly revealed that ExoU was able to promote p65 binding to NF-κB site in IL-8 promoter. Second, the treatment of cultures with the NF-kB inhibitor Bay 11-7082 led to a significant reduction in IL-8 mRNA levels and protein secretion induced by ExoU. Third, although the treatment with the JNK inhibitor SP600125 has reduced IL-8 secretion, the concentration of IL-8 detected in supernatants of cells simultaneously treated with SP600125 and Bay 11-7082 did not differ from the concentration detected in supernatants of cells only treated with Bay 11-7082. 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 the ExoU-producing strain. In conclusion, our data demonstrate that ExoU stimulates IL-8 expression and secretion by a NF-κB-dependent mechanism, contributing to the robust neutrophil infiltration observed during infection with the ExoU-producing P. aeruginosa strains.