Hirschfeld Josefine, Dommisch Henrik, Skora Philipp, Horvath Gabor, Latz Eicke, Hoerauf Achim, Waller Tobias, Kawai Toshihisa, Jepsen Søren, Deschner James, Bekeredjian-Ding Isabelle
Center for Dental and Oral Medicine, Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany.
Institute for Dental and Oral Medicine, Department of Periodontology and Synoptic Dentistry, Charité Berlin, Aßmannhauser Straße 4-6, D-14197 Berlin, Germany; Department of Oral Health Sciences, University of Washington, Seattle, WA 98195, USA.
Int J Med Microbiol. 2015 Jun-Aug;305(4-5):453-63. doi: 10.1016/j.ijmm.2015.04.002. Epub 2015 Apr 20.
Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins.
Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation.
Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation.
Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter-patient variability suggests NET formation as a potential factor influencing the individual course of disease.
口腔生物膜是牙周炎和龋齿等高度常见口腔疾病的病原体。此外，宿主免疫反应被认为在疾病发生中起关键作用。已知中性粒细胞是宿主对宿主表面细菌攻击的关键反应因子。中性粒细胞胞外陷阱（NETs）作为一种新型抗菌防御策略，在过去几年中受到越来越多的关注。在此，我们研究了中性粒细胞向牙菌斑的流入以及口腔细菌触发生物膜内NETs和细胞内蛋白质释放的能力。
从参与实验性牙龈炎研究的全身健康受试者中采集龈上生物膜和全唾液。通过免疫荧光分析生物膜，随后进行共聚焦和荧光显微镜检查。此外，通过酶联免疫吸附测定法评估生物膜悬液和唾液中细胞因子和免疫相关蛋白的浓度。用从培养的生物膜中分离出的12种细菌或脂多糖刺激从血液中获得的中性粒细胞，以监测NET的形成。
在菌斑样本和唾液中检测到中性粒细胞、NETs、中性粒细胞相关蛋白（髓过氧化物酶、弹性蛋白酶-2、组织蛋白酶G、杀菌肽LL-37）、白细胞介素-8、白细胞介素-1β和肿瘤坏死因子。所有测试的细菌种类以及从每个供体的菌斑中分离出的多微生物样本均诱导NETs和白细胞介素-8的释放。NET形成的程度在不同受试者之间有所不同，并且与菌斑评分或局部炎症的临床体征无关。
我们的研究结果表明，中性粒细胞被吸引到牙生物膜中，在其中它们被纳入并被微生物刺激释放NETs和免疫刺激蛋白。因此，中性粒细胞和NETs可能参与宿主生物膜控制，尽管它们的具体作用需要进一步阐明。此外，患者间的变异性表明NET形成是影响个体疾病进程的潜在因素。
