B-defensin protection of human oral epithelial cells

描述：人β防御素(HBDS)是一种表达在粘膜上皮细胞中的小的、阳离子的、抗菌的、天然的反应分子，是宿主抵御微生物致病的第一道防线。我们发现口腔上皮和细胞不仅表达组成性hBD1和诱导性hBD2和-3，而且后两者在正常完整的口腔上皮中总是“开启”的。这与身体的其他粘膜形成了鲜明的对比，在那里炎症或感染诱导了它们的表达。我们的数据表明，体内平衡的HBD表达可能是由于口腔组织暴露于特定的HBD诱导的共生菌。核梭杆菌是口腔中普遍存在的一种革兰氏阴性杆菌，它能诱导正常人口腔上皮细胞表达HBD-2和-3，而牙龈卟啉单胞菌则不能诱导HBD-2和HBD-3的表达。牙龈卟啉单胞菌是一种主要的革兰氏阴性机会性细菌，参与牙周病的发生，并能有效地侵袭口腔组织和口腔上皮细胞。我们的工作使我们建立了一个模型，表明核假单胞菌攻击的人类口腔上皮细胞可以保护自己免受牙龈卟啉单胞菌的入侵。我们假设这种保护是通过诱导hBD而产生的。有趣的是，核假单胞菌对hBD1、-2和-3具有抗性，而牙龈假单胞菌在低微摩尔浓度下对这三种药物都敏感。由于hBDS在保护口腔黏膜上皮中的作用尚未得到系统的研究，也没有认真考虑这些抗菌肽和其他NHOEC衍生抗菌肽的差异表达或诱导HBD保护的机制，我们提出了以下目标：(I)确定抑制hBD2或hBD3的产生是否会使NHOECs在核假单胞菌激活后容易受到牙龈假单胞菌的侵袭，(Ii)使用消减保护组学方法来确定在受到核杆菌和牙龈假单胞菌挑战的NHOECs中蛋白质的差异，以及(HI)解决在细菌攻击后是否需要可溶性HBD来保护NHOECs。有了我们的i)细菌挑战NHOEC单层的工作模型，ii)通过小的抑制性RNA策略在口腔上皮细胞中阻断hBDS的能力，iii。)除了生产重组hBDS的能力，以及iv)进行基于蛋白质组学的研究的能力，我们将能够进一步研究这些肽在口腔防御和稳定条件下所起的作用。本文提出的研究可能有助于更好地了解hBDS的作用机制及其对维持局部粘膜健康的贡献。

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.