Lipids: Effectors in Innate Immunity

描述（由申请人提供）：先天免疫已经发展成为通过在数小时内预先形成或可用的效应器对抗微生物增殖和侵入体表的第一线防御。已经给予了很多关注的上皮细胞内衬粘膜体表面和覆盖的流体，这是丰富的粘蛋白和抗微生物（多）肽，例如溶菌酶。然而，尽管所有体液也含有脂质，包括游离脂肪酸和磷脂，但仅在肺中，与蛋白质相关的表面活性因子已被证明是防御分子。支持的脂肪酸的抗微生物活性，在某些感染性疾病中的脂肪酸谱的改变，肠表面活性剂样颗粒的文件和我们的初步数据表明溶菌酶和选定的脂肪酸之间的协同作用的许多报告，我们提出，脂质，包括上皮细胞分泌的游离脂肪酸是一个不可缺少的手臂的主机防御独立或协同作用与其他防御分子。为了验证这一假设，我们将采取3种方法，具体目标如下。我们将确定： (1)上皮脂质分泌的抑制导致微生物增殖增强，以及（2）从鼻液中选择性去除脂质将降低流体的固有抗微生物活性，这可以在用提取的脂质再补充后逆转。研究的粘液组织部位为鼻窦区域和生殖道。体外模型将采用原代上皮细胞和现有的永生化细胞。细胞将在存在放射性标记的脂质的空气-液体界面处培养，存在和不存在促炎刺激物或脂质分泌抑制剂。正常鼻液中的脂质将通过C18键合硅胶固相萃取和乙腈洗脱选择性去除。将使用显色TLC板的射线照片和质谱技术鉴别和定量脂质。通过使用通常不会引起健康成人感染的细菌和酵母菌以及常见STD病原体测定CFU和微生物代谢活性，评估经处理和未经处理样本的抗菌活性。 (3)目的1和2中鉴定的上皮脂质效应物与微生物靶标结合。将记录候选脂质效应物与微生物的结合，并通过采用液体闪烁和放射性标记脂质的放射性结合研究以及表面等离子体共振光谱法进行定量。先天免疫的这一新概念可能通过提供慢性传染病遗传易感性和宿主病原体相互作用的新方面，并启动药物开发和营养建议的新方法，对传染病具有重大意义。

DESCRIPTION (provided by applicant): Innate immunity has evolved as the first line defense against microbial proliferation and invasion on body surfaces through effectors that are preformed or available within hours. Much attention has been given to the epithelial cells lining mucosal body surfaces and to the overlaying fluid, which is rich in mucins and antimicrobial (poly) peptides, for example lysozyme. However, though all body fluids also contain lipids, including free fatty acids and phospholipids, only in the lung, surfactant factor associated with proteins has been welt documented as defense molecule. Supported by numerous reports of antimicrobial activity of fatty acids, alterations of fatty acid profiles in certain infectious diseases, documentation of an intestinal surfactant-like particle and our preliminary data indicating a synergism between lysozyme and selected fatty acids, we propose that lipids including free fatty acids secreted by epithelial cells are an indispensable arm of host defense acting independently or in synergy with other defense molecules. To test this hypothesis we will take 3 approaches with the following specific aims. We will determine whether: (1) Inhibition of epithelial lipid secretion leads to enhanced microbial proliferation, and (2) selective removal of lipids from nasal fluids will decrease the fluid's inherent antimicrobial activity, which can be reversed after resupplementation with the extracted lipids. Mucosal sites investigated will be the sino-nasal region and the genital tract. In vitro models will employ primary epithelial cells and existing immortalized. Cells will be cultured at an air-liquid interface in the presence of radioactively labeled lipids with and without pro-inflammatory stimuli or lipid secretion inhibitors. Lipids from normal nasal fluid will be selectively removed by solid phase extraction with C18-bonded silica and eluted with acetonitrile. Lipids will be identified and quantified using radiographs of developed TLC plates and mass spectrometry techniques. Antimicrobial activity of treated and untreated samples will be assessed by determination of CFUs and microbial metabolic activity using bacteria and yeasts that do not routinely cause infection in the healthy adult and a common STD pathogen. (3) Epithelial lipid effectors identified in aim 1 and 2 bind to the microbial target. Binding of candidate lipid effectors to microbes will be documented and quantified with radioactive binding studies employing liquid scintillation and radiolabeled lipids and with surface plasmon resonance spectrometry. This new concept in innate immunity may have major significance in infectious diseases through providing new aspects in hereditary predisposition to chronic infectious diseases and host pathogen interactions and initiating new approaches to drug development and nutritional recommendations.