Xu Feng, Xu Zhihao, Zhang Rong, Wu Zuqun, Lim Jae-Hyang, Koga Tomoaki, Li Jian-Dong, Shen Huahao
Department of Respiratory Medicine, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China.
Respir Res. 2008 Jan 31;9(1):16. doi: 10.1186/1465-9921-9-16.
Nontypeable Haemophilus influenzae (NTHi) is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) via activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B in pulmonary alveolar epithelial cells.
Human alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA), and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The roles of Toll-like receptor (TLR) 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 in vitro and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice in vivo. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors.
NTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell in vitro and in the lungs of mice in vivo. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B. Moreover, the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-kappa B. However, NTHi-induced DNA binding activity of NF-kappa B was not affected by the inhibition of p38 MAPK.
NTHi induces COX-2 and PGE2 expression in a p38 MAPK and NF-kappa B-dependent manner through TLR2 in lung epithelial cells in vitro and lung tissues in vivo. The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.
不可分型流感嗜血杆菌（NTHi）是一种重要的呼吸道病原体，被认为是慢性阻塞性肺疾病的感染触发因素，但其与人类肺上皮细胞的分子相互作用仍不清楚。在此，我们验证了以下假设：NTHi通过激活肺泡上皮细胞中的p38丝裂原活化蛋白激酶（MAPK）和核因子（NF）-κB来诱导环氧化酶（COX）-2和前列腺素E2（PGE2）的表达。
用不同浓度的NTHi感染人肺泡上皮A549细胞。通过蛋白质免疫印迹分析检测p38 MAPK的磷酸化，用电泳迁移率变动分析（EMSA）评估NF-κB的DNA结合活性，分别用逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检测COX-1和2 mRNA以及PGE2蛋白的表达。分别在体外过表达TLR2和TLR4的HEK293细胞以及在体内使用TLR2和TLR4基因敲除小鼠的NTHi诱导的肺炎小鼠模型中，研究Toll样受体（TLR）2和TLR4（分别是肺上皮细胞中众所周知的NTHi识别受体和革兰氏阴性菌受体）对NTHi诱导的COX-2表达的作用。此外，通过使用p38 MAPK和NF-κB的特异性化学抑制剂，研究p38 MAPK和NF-κB对NTHi诱导的COX-2和PGE2表达的作用。
NTHi以剂量依赖性方式诱导A549细胞中COX-2 mRNA表达，但不诱导COX-1 mRNA表达。用COX-2特异性抑制剂预处理可显著降低NTHi感染导致的PGE2表达增强，但COX-1抑制剂则无此作用。在体外上皮细胞和体内小鼠肺中，NTHi诱导的COX-2表达由TLR2介导。NTHi诱导p38 MAPK磷酸化并上调NF-κB的DNA结合活性。此外，p38 MAPK和NF-κB的特异性抑制剂可显著抑制COX-2和PGE2的表达。然而，p38 MAPK的抑制并不影响NTHi诱导的NF-κB的DNA结合活性。
在体外肺上皮细胞和体内肺组织中，NTHi通过TLR2以p38 MAPK和NF-κB依赖性方式诱导COX-2和PGE2表达。全面了解内源性抗炎PGE2的作用及其调节将为解决肺部细菌感染中的炎症带来新的见解。
