B-defensin protection of human oral epithelial cells

描述：人β防御素（hBDs）是一种在粘膜上皮中表达的小的、阳离子的、抗菌的先天反应分子，是宿主抵御微生物发病的第一道防线。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的口腔上皮中始终处于“开启”状态。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诱导hBD的共生细菌。核梭杆菌是一种普遍存在于口腔的革兰氏阴性菌，可诱导正常人口腔上皮细胞（NHOECs）中hBD-2和-3的表达，而牙龈卟啉单胞菌是一种主要的革兰氏阴性机会菌，参与牙周病的发病，并能有效侵入NHOECs和口腔组织，却不能。我们的工作使我们建立了一个模型，显示具核梭菌攻击的人口腔上皮细胞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hBDs引起的。有趣的是，具核假单胞菌对hBD1、-2和-3有抗性，而牙龈假单胞菌对低微摩尔浓度的所有三种药物都敏感。由于hBD在保护口腔黏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衍生的抗菌肽的差异表达或引发hBD保护的机制，我们提出以下目的：(1)确定抑制hBD2或hBD3的产生是否会使NHOECs在F. nucleatum激活后对P. gingivalis侵袭敏感；(2)使用减法蛋白质组学方法确定受F. nucleatum攻击的NHOECs与P. gingivalis攻击的NHOECs之间的蛋白质差异；（HI）确定可溶性hBD是否对细菌攻击后NHOECs的保护是必要的。有了我们的i)细菌挑战NHOEC单层的工作模型，ii)通过小抑制RNA策略阻断口腔上皮细胞中hBDs的能力，iii)产生重组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状态条件下的作用。本文提出的研究可以更好地理解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.