Lipids: Effectors in Innate Immunity

描述（由申请人提供）：先天免疫已发展成为通过在数小时内预制或可用的效应器抵御微生物增殖和侵入体表的第一道防线。人们对粘膜体表的上皮细胞及其上覆的液体给予了很大的关注，这些液体富含粘蛋白和抗菌（多肽），例如溶菌酶。然而，尽管所有体液都含有脂质，包括游离脂肪酸和磷脂，但只有在肺部，与蛋白质相关的表面活性剂因子作为防御分子已被充分记录。根据大量关于脂肪酸抗菌活性的报道、某些传染病中脂肪酸谱的改变、肠道表面活性剂样颗粒的记录以及我们表明溶菌酶与选定脂肪酸之间协同作用的初步数据，我们提出，包括上皮细胞分泌的游离脂肪酸在内的脂质是宿主防御不可或缺的武器，可以独立或与其他防御分子协同作用。为了验证这一假设，我们将采用以下具体目标的3种方法。我们将决定是否：

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.