B-defensin protection of human oral epithelial cells

B-防御素对人口腔上皮细胞的保护作用

• 批准号: 6985500
• 负责人: AARON WEINBERG
• 金额: $ 36.07万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6985500
• 关键词: Bacteroides gingivalis; Fusobacterium nucleatum; bacteria infection mechanism; bactericidal immunity; biological products; clinical research; cytoprotection; defensins; flow cytometry; host organism interaction; human subject; immunocytochemistry; mass spectrometry; oral bacteria; oral mucosa; pathologic process; proteomics; receptor expression; small interfering RNA; surface enhanced laser desorption ionization; tissue /cell culture; two dimensional gel electrophoresis; western blottings

DESCRIPTION: Human beta defensins (hBDs), small, cationic, antimicrobial, innate response molecules expressed in mucosal epithelia, function as a first line of host defense against microbial pathogenesis. We have shown that oral epithelium and cells not only express the constitutive hBD1 and the inducible hBD2 and -3, but that the latter two are always "on" in normal intact oral epithelium. This is in stark contrast to other mucosae of the body where inflammation or infection induces their expression. Our data suggest that homeostatic hBD expression may be due to the exposure of the oral tissue to specific hBD-inducing commensal bacteria. Fusobacterium nucleatum, a ubiquitous Gram negative bacterium of the oral cavity, induces hBD-2 and -3 expression in normal human oral epithelial cells (NHOECs), while Porphyromonas gingivalis, a major Gram negative opportunist involved in the initiation of periodontal disease, and which efficiently invades NHOECs and oral tissues, does not. Our work has led us to establish a model showing that F. nucleatum challenged human oral epithelial cells are protected from Porphyromonas gingivalis invasion. We posit that this protection is elicited through induction of hBDs. Interestingly, F. nucleatum is resistant to hBD1, -2 and -3, while P. gingivalis is sensitive to all three agents in low micromolar concentrations. Since the role of hBDs in protecting the oral mucosal epithelium has not been systematically studied, nor has serious consideration been given to differential expression of these and other NHOEC derived antimicrobial peptides or the mechanisms by which hBD protection is elicited, we propose the following Aims: (I) Determine if inhibiting the production of hBD2 or hBD3 renders NHOECs susceptible to P. gingivalis invasion following F. nucleatum activation, (II) Use a subtractive proteomics approach to determine the protein differences in NHOECs challenged with F. nucleatum versus P. gingivalis and (HI) Resolve whether soluble hBD is necessary for protection of NHOECs following bacterial challenge. With our i) working model of bacterially challenged NHOEC monolayers in place, ii) ability to block hBDs in oral epithelial cells through small inhibitory RNA strategies, iii.) ability to produce recombinant hBDs, and iv) capabilities in conducting proteomics based studies, we will be able to investigate further the role these peptides play in defense and steady state conditions of the oral cavity. Studies proposed herein could lead to a better understanding of the mechanisms through which hBDs function and their contribution to the maintenance of localized mucosal health.

描述：人β防御素（HBD），小型，阳离子，抗菌，先天反应分子，在粘膜上皮中表达，是针对微生物发病机理的第一线宿主防御。我们已经表明，口服上皮和细胞不仅表达构成性HBD1和可诱导的HBD2和-3，而且后两个在正常完整的口服上皮中总是“ on”。这与身体的其他粘膜呈鲜明对比，在炎症或感染中会诱导其表达。我们的数据表明，稳态HBD表达可能是由于口腔组织暴露于特定的HBD诱导共生细菌。核杆菌是口腔中普遍存在的革兰氏阴性细菌，在正常的人口腔上皮细胞（NHOEC）中诱导HBD -2和-3表达，而卟啉单胞菌，而卟啉单胞菌则是一种涉及周期性疾病的主要人物的主要革兰氏负面的负面机会，并且有效地涉及NHOEC，或者不得不受益于NHOEC，或者或无与伦比。我们的工作使我们建立了一个模型，表明F. nucleatum挑战了人口腔上皮细胞受到保护免受牙龈毒性牙龈入侵的保护。我们认为，这种保护是通过诱导HBD引起的。有趣的是，F. nutleatum对HBD1，-2和-3具有抗性，而牙龈疟原虫在低微摩尔浓度下对所有三种药物敏感。由于HBD在保护口腔粘膜上皮中的作用尚未系统地研究，因此也没有认真考虑这些和其他NHOEC衍生的抗菌肽的差异表达，或引起HBD保护的机制或引起HBD保护的机制，我们提出了以下目的：（i）确定抑制HBD2或HBD2的生产。 （ii）（ii）使用减法蛋白质组学方法来确定F. noecs的蛋白质差异与牙龈斑no相比，（HI）解决可溶性HBD是否对于在细菌挑战后保护NHOEC是必需的。借助我们的i）ii）通过小型抑制性RNA策略阻止口腔上皮细胞中的HBD的NHOEC单层的工作模型，III。本文提出的研究可能会更好地理解HBD功能及其对维持局部粘膜健康的贡献的机制。