B-defensin protection of human oral epithelial cells

产品说明：人β防御素（hBD）是在粘膜上皮中表达的小的阳离子抗微生物先天性应答分子，作为宿主防御微生物致病的第一道防线发挥作用。我们已经表明，口腔上皮和细胞不仅表达组成型hBD 1和诱导型hBD 2和-3，但后两者总是“上”在正常完整的口腔上皮。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的表达可能是由于暴露的口腔组织特定hBD诱导口腔细菌。具核梭杆菌（Fusobacterium nucleatum）是口腔中普遍存在的革兰氏阴性细菌，其诱导正常人口腔上皮细胞（NHOEC）中hBD-2和hBD-3的表达，而牙龈卟啉单胞菌（Porphyromonas gingivalis）是参与牙周病起始的主要革兰氏阴性机会主义者，其有效地侵入NHOEC和口腔组织，但不诱导hBD-2和hBD-3的表达。我们的工作使我们建立了一个模型，表明F。保护受核质攻击的人口腔上皮细胞免受牙龈卟啉单胞菌侵袭。我们认为这种保护是通过诱导hBD引起的。有趣的是，F。具核单胞菌对hBD 1、hBD-2和hBD-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试剂均敏感。由于hBD在保护口腔粘膜上皮中的作用还没有被系统地研究，也没有认真考虑这些和其他NHOEC衍生的抗微生物肽的差异表达或引起hBD保护的机制，我们提出以下目的：（I）确定抑制hBD 2或hBD 3的产生是否使NHOEC对牙龈卟啉单胞菌在F.（II）使用消减蛋白质组学方法来确定用F.（III）解决可溶性hBD是否是细菌攻击后保护NHOEC所必需的。利用我们的i）适当的细菌攻击的NHOEC单层的工作模型，ii）通过小抑制性RNA策略阻断口腔上皮细胞中的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的研究的能力，我们将能够进一步研究这些肽在口腔的防御和稳态条件中发挥的作用。本文提出的研究可以更好地理解hBD发挥作用的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.