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

Abstract

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.

Figure 1. ExoU increases IL-8 mRNA in A549 cells.

In (A), representative agarose gels obtained from three different semi-quantitative RT-PCR assays carried out in duplicate. In (B) and (C), graph represents the means ± SEM of values obtained by three different Real Time qRT-PCR assays performed in triplicate. **p<0.01 or ***p<0.001 when the values obtained from cultures infected with the ExoU-producing PA103 strain were compared with those obtained from the other cultures.

Figure 2. ExoU PLA₂ activity stimulates IL-8 secretion by P. aeruginosa-infected A549 cultures.

In (A) and (B), cells were infected with the different P. aeruginosa strains for 21 hours and the concentrations of IL-8 in supernatants were assessed by ELISA. In (B), Brefeldin A was added or not to the gentamicin-containing culture medium. The graphs show the means ± SEM of three assays performed in quadruplicate. ***p<0.001 when the values obtained from untreated PA103-infected cultures were compared with those from the other cultures.

Figure 3. ExoU promotes p65/p50 nuclear translocation and NF-κB-dependent transcriptional activity in P. aeruginosa-infected A549 cells.

In (A), representative EMSAs showing NF-κB nuclear translocation after 2 and 12 hours of P. aeruginosa infection or treatment with culture medium. To evaluate NF-κB inhibition by Bay 11-7082, some cultures were treated with 10 µM Bay 11-7082, 1 hour prior PA103 infection. As a control for non-specific interactions, nuclear extracts from PA103-infected cells were also incubated with a probe mutated in a single nucleotide (Mut). In (B), extracts obtained from PA103-infected cells were supershifted with specific antibodies against NF-κB subunits, p50 and p65. In “Non-infected cells” and “PA103-infected cells” lanes, antibodies were not added. EMSA and supershift assays were performed in triplicate. In (C), the graph shows the luciferase activity (in arbitrary values) of whole-cell lysates obtained from A549 cultures transfected with p6κB-LUC and pRL-CMV plasmids and then infected with P. aeruginosa strains or treated with cultured medium for 24 hours. Data represent means ± SEM of three different assays carried out in quadruplicate. **p<0.01 and ***p<0.001 when the values obtained from PA103ΔexoU-infected cultures and non-infected cultures, respectively, were compared with those obtained from PA103-infected cultures.

Figure 4. ExoU induces NF-κB binding in IL-8 promoter of A549 cells.

In (A), representative agarose gel showing PCR products of DNA obtained after chromatin immunoprecipitation with anti-p65 antibody (IP:anti-p65) or non-immunoprecipitated DNA (input) amplified with primers specific for NF-κB site in IL-8 promoter. In (B), graph shows the means ± SEM of values obtained by Real Time PCR of three different ChIP assays. ***p<0.001 when the values obtained in non-infected cultures or cultures infected with the ExoU deficient strain were compared with those obtained in cultures infected with the ExoU-producing strain for 14 hours.

Figure 5. Inhibition of NF-κB reduces IL-8 expression and secretion induced by ExoU in P. aeruginosa-infected A549 cultures.

In (A), representative agarose gels of RT-PCR assays in which A549 cells were treated or not with 10 µM Bay 11-7082, 1 hour prior infection with PA103 or PA103ΔexoU strains or treatment with culture medium (non-infected cells) for 18 hours. In (B), graph shows the means ± SEM of values obtained by three Real Time qRT-PCR assays. In (C), IL-8 concentrations detected in supernatants of cultures pretreated or not with 10 µM Bay 11-7082 for 1 hour and then exposed for 21 hours to PA103, PA103ΔexoU or culture medium, as assessed by ELISA. In (D), IL-8 concentrations, as detected by ELISA, in supernatants of cultures pretreated or not with 10 µM Bay 11-7082 and/or 10 µM SP600125 for 1 hour and then exposed for 21 hours to bacterial strains or culture medium. In (C) and (D), data represent means ± SEM of three assays performed in quadruplicate. ***p<0.001 when the values obtained from untreated PA103-infected cultures were compared with those from the other cultures.

Figure 6. ExoU activates p65/p50 NF-κB and increases IL-8 expression and secretion in HMEC-1 capillary endothelial cells.

In (A), representative agarose gels of three different semi-quantitative RT-PCR assays carried out in duplicate. In (B), graph shows the means ± SEM of values obtained in three Real Time qRT-PCR assays. **p<0.01 and ***p<0.001 when the values obtained from PA103ΔexoU-infected cultures and non-infected cultures, respectively, were compared with those obtained from cultures infected with the ExoU-producing strain. In (C), representative EMSAs showing NF-κB nuclear translocation after 2 and 12 hours of P. aeruginosa infection or treatment with culture medium. In (D), extracts obtained from PA103-infected cells were incubated with specific antibodies for NF-κB subunits, p50, p52, p65 or cRel, but only supershifted with p50 and p65. In non-infected cells, antibodies were not added. EMSA and supershift assays were performed in triplicate. In (E), IL-8 concentrations, detected by ELISA, in supernatants of cultures pretreated or not with 5 µM Bay 11-7082, for 1 hour, and exposed to PA103, PA103ΔexoU or culture medium, for 21 hours. The graph represents the means ± SEM of two assays performed in quadruplicate and shows ***p<0.001 when the values obtained from untreated PA103-infected cultures were compared with those from the other untreated cultures or with those from Bay 11-7082-treated cultures infected with the ExoU-producing strain.

Figure 7. ExoU induces NF-κB-dependent KC secretion and neutrophil infiltration in mice airways at 24 hours post-infection.

In (A), concentrations of KC assessed by ELISA, in (B) total leukocytes and in (C) neutrophils (PMN), both assessed by light microscopy, in BALF of mice pretreated or not with Bay 11-7082 at 20 mg/Kg, for 1 hour, and inoculated intratracheally with PA103, PA103ΔexoU or saline. The graphs show the mean values ± SEM of three independent assays (n = 16 for each group). *p<0,05 or **p<0.01 when the values obtained in untreated mice infected with PA103 strain were compared with the values obtained in the other groups.