Essential Role of Transcription Factor Nuclear Factor-κB in Regulation of Interleukin-8 Gene Expression by Nitrite Reductase from Pseudomonas aeruginosa in Respiratory Epithelial Cells

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases. Recently, we have reported that nitrite reductase from P. aeruginosa induces the production of IL-8 in respiratory cells, including bronchial epithelial cells. To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5′-flanking region of the IL-8 gene and then exposed to nitrite reductase. Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity. This increased IL-8 gene expression was inhibited by mutating the nuclear factor-κB (NF-κB) binding element. Nitrite reductase enhanced nuclear localization of the NF-κB binding complex. Furthermore, nitrite reductase induced the degradation of IκBα, the major cytoplasmic inhibitor of NF-κB, and the expression of IκBα mRNA. These data support the critical role of the activation of NF-κB in nitrite reductase-induced IL-8 gene expression in airway epithelium.

铜绿假单胞菌（Pseudomonas aeruginosa）持续感染可升高白细胞介素8 (IL-8) 水平，并使慢性气道疾病患者的气道内出现致密的中性粒细胞浸润。近期本团队已有研究报道，铜绿假单胞菌来源的亚硝酸还原酶（nitrite reductase）可诱导包括支气管上皮细胞（bronchial epithelial cells）在内的呼吸道细胞产生IL-8。为阐明亚硝酸还原酶诱导呼吸道细胞IL-8表达的分子机制，本研究将携带有IL-8基因5'侧翼区（5′-flanking region）系列缺失片段的质粒转染至A549上皮细胞（A549 epithelial cells），随后用亚硝酸还原酶处理细胞。实验结果显示，亚硝酸还原酶可显著增强IL-8基因启动子驱动的报告基因活性（reporter activity）。该IL-8基因表达上调现象可通过突变核因子κB (NF-κB) 结合元件（binding element）得到抑制。亚硝酸还原酶可增强NF-κB结合复合物的核定位（nuclear localization）。此外，亚硝酸还原酶可诱导NF-κB的主要胞质抑制剂κB抑制蛋白α (IκBα) 发生降解，同时上调IκBα mRNA的表达。上述实验数据证实，NF-κB的激活在亚硝酸还原酶诱导气道上皮细胞IL-8基因表达的过程中发挥关键作用。