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

<p>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.</p

ExoU increases IL-8 mRNA in A549 cells.

<p>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.</p

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

<p>In (A), representative EMSAs showing NF-κB nuclear translocation after 2 and 12 hours of <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i> 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 PA103Δ<i>exoU</i>-infected cultures and non-infected cultures, respectively, were compared with those obtained from PA103-infected cultures.</p

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

<p>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, PA103Δ<i>exoU</i> 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.</p

Inhibition of NF-κB reduces IL-8 expression and secretion induced by ExoU in <i>P. aeruginosa</i>-infected A549 cultures.

<p>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 PA103Δ<i>exoU</i> 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, PA103Δ<i>exoU</i> 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.</p

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

<p>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 PA103Δ<i>exoU</i>-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 <i>P. aeruginosa</i> 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, PA103Δ<i>exoU</i> 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.</p

ExoU PLA₂ activity stimulates IL-8 secretion by <i>P. aeruginosa</i>-infected A549 cultures.

<p>In (A) and (B), cells were infected with the different <i>P. aeruginosa</i> 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.</p

LPC triggers IL-8 production through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected and stimulated as described on Figure 1. After 20 hours of incubation, IL-8 production was measured by the ELISA assay. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits LPS-induced ERK activation.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of 1 µg/mL LPS or in the presence or absence of the indicated concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere (<b>A</b>, <b>D</b>, <b>E</b>). In parallel HEK 293A cells with TLR constructs as indicated (<b>B</b>, <b>C</b>). Each group received expression constructs for TLR4 (<b>B</b>) or both TLR2 and TLR1 (<b>C</b>), as well as MD-2, CD14 and CD36 plasmids. The cells were then incubated in the absence or presence of 100 ng/mL LPS or 1 nM Pam3CSK4 (P3C) and 10 or 100 µM of LPC, for 40 min at 37 °C in a 5% CO₂ atmosphere. After incubation either macrophages or HEK cells were homogenized, the protein levels was determined and samples evaluated by Western blot with the use of antibodies against p-ERK (<b>A</b>, <b>B</b>, <b>C</b>), p-JNK (<b>D</b>) and p-P38 (<b>E</b>). Loading controls were run with the use of antibodies raised towards actin. Experiments were performed at least two times with different animals and samples.</p

LPC triggers NF-қB activation through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected in three different groups. Groups A and B received expression constructs for TLR4 (<b>A</b>) or TLR2 and TLR1 (<b>B</b>). Both also received MD-2, CD14, and CD36 constructs and the ELAM-1-firefly luciferase and β-actin-<i>Renilla</i> luciferase reporter plasmids. The third group (<b>C</b>) received only the empty vector pDisplay and the luciferase reporter plasmids. Groups A and B were separately stimulated with 0.1, 1, 10, 100 and 200 µM of different types of LPC (Sigma; C14:0, C16:0, C18:0, and C18:1), 100 ng/mL of LPS and 1 nM of Pam3CSK4 (P3C). Group C was stimulated with LPS, Pam3Cys or 0.1, 1, 10 and 100 µM of LPC (C16:0). The agonists were diluted in DMEM medium with 10% bovine fetal serum. After 4 h of incubation, luciferase activity was measured and expressed as the ratio of NF-қB-dependent firefly luciferase activity to the control <i>Renilla</i> luciferase activity. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p