Pseudomonas aeruginosa Induces Interleukin-8 (IL-8) Gene Expression in Human Conjunctiva through the Recruitment of Both RelA and CCAAT/Enhancer-binding Protein β to the IL-8 Promoter

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-B 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 ␤ (C/EBP␤) via p38MAPK and activating NF-B. The simultaneous involvement of RelA and C/EBP␤ and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. ReChIP experiments showed that RelA and C/EBP␤ 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/EBP␤ and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing a wide range of acute and chronic infections prevalently in susceptible hosts. Immunocompromised or mechanically ventilated people, transplant recipients, or patients bearing malignancies, cystic fibrosis, or human immunodeficiency virus infection all are at increased risk of being affected by P. aeruginosa (1-6). Typically the pulmonary and urinary tracts, burns, wounds, eye, and blood are the main targets. The bacterium can colonize the ocular epithelium by means of fimbrial attachment to sialic acid receptors. If host defenses are compromised in any way, as in hospitalized preterm and low birth weight infants, the bacterium can proliferate rapidly and induce devastating endophthalmitis (7, 8) or extensive tissue damage with permanent scarring and irreversible loss of vision (9). In some cases, this destructive eye disease is associated with or followed by systemic complications (10 -14). These features and the development of antibiotic resistance (12, 15, 16) continue to be problematic from a treatment perspective. The role of various proinflammatory cytokines in the regulation of host defense by epithelia during infections caused by P. aeruginosa has been extensively studied. It has been shown that several P. aeruginosa virulence factors, both cellassociated and extracellular products, are potent inducers of proinflammatory mediators, such as IL-1, 2 IL-6, IL-8, and IL-10 (17-26). In particular, IL-8 overexpression has been shown to induce ulcer formation in the cornea th...

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