Haarmann Helge, Steiner Tamara, Schreiber Frauke, Heinrich Annina, Zweigner Janine, N'Guessan Philippe Dje, Slevogt Hortense
Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany; Department of Internal Medicine/Infectious Diseases, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Department of Internal Medicine/Infectious Diseases, Charité - Universitätsmedizin Berlin, Berlin, Germany; Neurological Rehabilitation Center for Children and Adolescents, Helios Klinikum Hohenstücken, Brandenburg, Germany.
Biochem Biophys Res Commun. 2015 Nov 6;467(1):46-52. doi: 10.1016/j.bbrc.2015.09.126. Epub 2015 Sep 28.
Bacterial colonisation with Moraxella catarrhalis may partly sustain chronic inflammation in the lower airways of patients with chronic obstructive pulmonary disease (COPD). In addition, this bacterium causes infectious exacerbations of COPD, which often necessitate treatment with antibiotics. Antimicrobial peptides are the body's own antibiotic substances with bactericidal and bacteriostatic, as well as immunomodulatory function. In particular, human beta-defensin 3 (hBD-3) exerts an antimicrobial effect against an extraordinarily broad spectrum of pathogens. We therefore investigated the role of hBD-3 in infections of pulmonary epithelial cells with M. catarrhalis.
The antimicrobial activity of hBD-3 vs. M. catarrhalis was evaluated in an antimicrobial susceptibility assay. We analyzed hBD-3 secretion of M. catarrhalis-infected pulmonary epithelial cells using ELISA. The role of M. catarrhalis-specific virulence factors, toll-like receptors (TLR) 2 and 4, MAPK pathways, and transcription factors AP-1 and NF-κB in the induction and regulation of hBD-3 expression were explored with specific inhibitors, small interference RNA, Western Blot, and chromatin immunoprecipitation (ChIP) assays.
HBD-3 exhibited a strong bactericidal effect against M. catarrhalis. M. catarrhalis induced hBD-3 expression in pulmonary epithelial cells, which was dependent on M. catarrhalis membranous lipoolygosaccharide (LOS), while the surface proteins UspA1 and UspA2 were not involved. Gene silencing of TLR2, but not TLR4, led to a reduced hBD-3 secretion after stimulation with M. catarrhalis or M. catarrhalis LOS. Inhibition of MAPKs ERK1/2 and JNK, but not p38, reduced hBD-3 secretion. HBD-3 expression was mediated through the recruitment of AP-1 to the hBD-3 gene promoter and was independent of NF-κB.
The immune response of pulmonary epithelial cells towards M. catarrhalis involves secretion of hBD-3, which has a bactericidal effect against this pathogen. Binding of M. catarrhalis virulence factor LOS to TLR2 causes an ERK1/2- and JNK-dependent induction of AP-1-related transcription of the hBD-3 gene, resulting in the production and secretion of hBD-3.
卡他莫拉菌的细菌定植可能在一定程度上维持慢性阻塞性肺疾病（COPD）患者下呼吸道的慢性炎症。此外，这种细菌会引发COPD的感染性加重，这通常需要使用抗生素进行治疗。抗菌肽是人体自身具有杀菌、抑菌以及免疫调节功能的抗生素物质。特别是，人β-防御素3（hBD-3）对极其广泛的病原体具有抗菌作用。因此，我们研究了hBD-3在卡他莫拉菌感染肺上皮细胞中的作用。
在抗菌药敏试验中评估hBD-3对卡他莫拉菌的抗菌活性。我们使用酶联免疫吸附测定（ELISA）分析卡他莫拉菌感染的肺上皮细胞中hBD-3的分泌情况。使用特异性抑制剂、小干扰RNA、蛋白质免疫印迹法（Western Blot）和染色质免疫沉淀（ChIP）试验，探讨卡他莫拉菌特异性毒力因子、Toll样受体（TLR）2和4、丝裂原活化蛋白激酶（MAPK）途径以及转录因子活化蛋白-1（AP-1）和核因子κB（NF-κB）在hBD-3表达的诱导和调节中的作用。
HBD-3对卡他莫拉菌表现出强大的杀菌作用。卡他莫拉菌诱导肺上皮细胞中hBD-3的表达，这依赖于卡他莫拉菌的膜脂寡糖（LOS），而表面蛋白UspA1和UspA2不参与其中。TLR2基因沉默而非TLR4基因沉默，导致在用卡他莫拉菌或卡他莫拉菌LOS刺激后hBD-3分泌减少。抑制MAPKs的细胞外信号调节激酶1/2（ERK1/2）和应激活化蛋白激酶（JNK），而非p38，可减少hBD-3分泌。HBD-3的表达是通过AP-1募集到hBD-3基因启动子介导的，且独立于NF-κB。
肺上皮细胞对卡他莫拉菌的免疫反应涉及hBD-3的分泌，hBD-3对该病原体具有杀菌作用。卡他莫拉菌毒力因子LOS与TLR2结合导致ERK1/2和JNK依赖性诱导hBD-3基因的AP-1相关转录，从而导致hBD-3的产生和分泌。
