Lipid Effector Molecules of Innate Immunity: Antimicrobial Cholesteryl Esters

描述(申请人提供)：传染病，包括呼吸道感染，仍然是一个主要的健康问题。在许多情况下，当固有的粘膜防御失效时，就会发生感染。抗菌肽已被公认为是第一道防线的重要组成部分。我们最近的工作提供了证据表明，宿主衍生的脂类代表了一类新的抗菌效应分子，这些分子是粘膜液固有的抗菌活性的组成部分，单独发挥杀菌活性，并与抗菌肽协同作用。我们首次进一步鉴定了胆固醇酯(CE‘s)为抗菌效应分子。然而，关于抗菌脂的调节、其作用方式以及它们与抗菌肽的相互作用，人们的认识存在很大差距。这项研究将回答以下重要问题：在呼吸道感染和炎症的背景下，抗菌素CE是如何调节的，哪些CE发挥最强的抗菌活性，以及它们如何与抗菌肽结合。为了确定上皮CE产生的关键调节因子和最有效的抗菌CE，我们将使用一个感染模型，该模型包含极化的呼吸道上皮细胞和机会性呼吸道病原体，当粘膜屏障功能受损时，这些病原体会导致感染。我们将评估参与CE生物合成的关键酶的表达，并量化抗菌CE对模式识别受体(如细菌细胞壁产物)和作用于上皮细胞的促炎细胞因子(如IL1b、IL17A)的配体的响应。我们将采用定量RT-PCR、生化分析和抗菌检测。我们将用从鼻腔粘膜中建立的原代上皮细胞作为手术多余材料来验证我们的结果，并用siRNA方法学确认关键调控因子。我们将利用抗菌试验、细菌对上皮细胞的攻击实验以及利用siRNA技术选择性地抑制效应分子的产生来剖析抗菌脂和抗菌肽对粘膜固有防御的相对贡献。在完成拟议的工作后，我们将能够研究抗菌脂在体外的作用模式，并进入动物模型。我们的研究具有创新性和很高的意义。天然防御的脂质臂是一个新兴的概念，这项研究承诺在多个方面对公共健康产生重大影响。更好地了解如何操纵抗菌脂的自然产生和分泌，特别是CE，可以为脂类生成不足引起的传染病提供新的免疫疗法。抗菌CE‘s及其协同作用抗菌肽的鉴定将为新型脂类抗生素的开发提供指导。最后，这项研究可能会导致发现新的致病微生物毒力因子，这些致病微生物可以绕过呼吸道和其他粘膜表面抗微生物脂类介导的宿主防御。

DESCRIPTION (provided by applicant): Infectious diseases including respiratory infections continue to be a major health concern. In many cases infections establish when the innate mucosal defense fails. Antimicrobial peptides have been recognized as important contributors to the first line of defense. Our recent work has provided evidence that host-derived lipids represent a new class of antimicrobial effector molecules that are integral to the inherent antimicrobial activity of mucosal fluids exerting bactericidal activity alone and in synergism with antimicrobial peptides. We have further identified, for the first time, cholesteryl esters (CE's) as antimicrobial effector molecules. However, there is a major gap in knowledge with respect to the regulation of antimicrobial lipids, their mode of action, and their interaction with antimicrobial peptides. The research proposed here will answer the important questions how antimicrobial CE's are regulated in the context of infection and inflammation in the respiratory tract, which of the CE's exert most potent antibacterial activity, and how they interface with antimicrobial peptides. To identify key regulators of epithelial CE production and the most potent antibacterial CE's we will use an infection model with polarized airway epithelial cells and opportunistic respiratory tract pathogens that cause infection when the mucosal barrier function is impaired. We will assess the expression of key enzymes involved in CE biosynthesis and quantify antibacterial CE's in response to ligands for pattern recognition receptors such as bacterial cell wall products, and pro-inflammatory cytokines that act on epithelial cells, for example IL1b, IL17A. We will employ quantitative RT-PCR, biochemical analysis, and antibacterial assays. We will validate our results with primary epithelial cells established from sino-nasal mucosa obtained as surgical excess material, and confirm key regulators with siRNA methodology. We will dissect the relative contributions of antimicrobial lipids and antimicrobial peptides to the mucosal innate defense employing antibacterial assays, and bacterial challenge experiments with epithelial cells and selective inhibition of effector molecule production with siRNA technology. Upon completion of the proposed work we will be in the position to study the mode of action of antimicrobial lipids in vitro and moving into an animal model. Our study is innovative and highly significant. The lipid-arm of innate defense is an emerging concept and this research promises a high impact on public health in multiple ways. A better understanding of how to manipulate the natural production and secretion of antimicrobial lipids, in particular CE's, can lead to novel immunotherapies for infectious diseases caused by a deficiency in lipid production. The identification of antibacterial CE's and synergistically acting antimicrobial peptides may guide the development of novel lipid-antibiotics. Last, not least, this research may lead to the discovery of novel virulence factors of pathogenic microbes that circumvent host defenses mediated by antimicrobial lipids in the respiratory tract and on other mucosal surfaces.