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: 7093136
• 负责人: AARON WEINBERG
• 金额: $ 35.22万
• 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/7093136
• 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：人类β防御素 (hBD) 是粘膜上皮细胞中表达的小型、阳离子、抗菌、先天反应分子，是宿主对抗微生物发病机制的第一道防线。我们已经证明，口腔上皮和细胞不仅表达组成型 hBD1 和诱导型 hBD2 和 -3，而且后两者在正常完整口腔上皮中始终“开启”。这与炎症或感染诱导其表达的身体其他粘膜形成鲜明对比。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 诱导共生细菌所致。具核梭杆菌是口腔中普遍存在的革兰氏阴性细菌，可在正常人口腔上皮细胞 (NHOEC) 中诱导 hBD-2 和 -3 表达，而牙龈卟啉单胞菌是一种主要的革兰氏阴性机会菌，参与牙周病的发生，并且有效地侵入 NHOEC 和口腔组织，但没有。我们的工作使我们建立了一个模型，表明具核梭杆菌攻击的人类口腔上皮细胞受到保护，免受牙龈卟啉单胞菌的侵袭。我们假设这种保护是通过诱导 hBD 引起的。有趣的是，具核梭杆菌对 hBD1、-2 和 -3 具有抗性，而牙龈卟啉单胞菌对低微摩尔浓度的所有三种药剂均敏感。由于 hBD 在保护口腔粘膜上皮中的作用尚未得到系统研究，也没有认真考虑这些和其他 NHOEC 衍生抗菌肽的差异表达或引起 hBD 保护的机制，我们提出以下目标： (I) 确定抑制 hBD2 或 hBD3 的产生是否会使 NHOEC 在具核梭杆菌激活后容易受到牙龈卟啉单胞菌的侵袭，(II) 使用消减法蛋白质组学方法以确定具核梭杆菌与牙龈卟啉单胞菌攻击的 NHOEC 中的蛋白质差异，并 (HI) 确定可溶性 hBD 是否是细菌攻击后保护 NHOEC 所必需的。凭借我们的 i) 受细菌挑战的 NHOEC 单层工作模型，ii) 通过小抑制性 RNA 策略阻断口腔上皮细胞中 hBD 的能力，iii.) 生产重组 hBD 的能力，以及 iv) 进行基于蛋白质组学研究的能力，我们将能够进一步研究这些肽在口腔防御和稳态条件中发挥的作用。本文提出的研究可以帮助人们更好地了解 hBD 的功能机制及其对维持局部粘膜健康的贡献。