Abstract
The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-kappaB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein beta (C/EBPbeta) via p38MAPK and activating NF-kappaB. The simultaneous involvement of RelA and C/EBPbeta and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPbeta act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPbeta and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.
Highlights
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing a wide range of acute and chronic infections prevalently in susceptible hosts
Our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBP and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription
P. aeruginosa Induces IL-8 Production—To determine whether whole P. aeruginosa induces the synthesis of IL-8 protein in conjunctival cells, primary and Chang cultures were treated with different concentrations of either heat-killed or live bacteria, and IL-8 levels were measured in the supernatants
Summary
Cells and Culture Conditions—Experiments were performed in adherence to the Helsinki Declaration. Primary conjunctival epithelial cells were cultured as described previously [29]. Primary cultured conjunctival epithelial cells of passages 1–3 were used for the experiments. Reverse Transcription-PCR—RNA was extracted with TRIzol (Invitrogen) from cultured cells (3 ϫ 106 cells/well) stimulated with heat-killed or live bacterium. Chromatin Immunoprecipitation (ChIP) Assay—The ChIP enzymatic assay (Active Motif) was carried out, and the sheared chromatin samples were used for immunoprecipitation with 2 g of anti-c-Jun, anti-RelA, anti-C/EBP, or anti-NFKB1 antibodies (Santa Cruz Biotechnology) overnight at 4 °C.
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