TLR Ectodomains for Microbial Detection and Therapeutics

描述（由申请人提供）：Toll样受体（TLR）和NOD样受体（NLR）是先天免疫系统的关键组成部分，在先天免疫系统中，它们作为哨兵受体用于检测微生物和内源性危险信号。迄今为止，10个人类和13个小鼠TLR已被表征并显示识别微生物产物，如脂肽、鞭毛蛋白、脂磷壁酸、GPI锚、各种核酸和脂多糖。在许多情况下，由一系列富含亮氨酸的重复序列（LRR）组成的TLR的胞外域直接结合微生物衍生的化合物。由各种微生物化合物以及许多内源性危险信号触发的NLR使用LRR作为推定的配体结合结构域。除了哺乳动物类TLR/NLR外，紫海胆（Strongylocentrotus purpuratus）具有大大扩展的模式识别受体库，代替适应性免疫系统，具有222个TLR和203个NLR。这些动物生活在微生物丰富的沿海沃茨，这种扩大的受体库创造了一个更多样化的微生物配体，他们可以检测。我们建议使用高通量克隆技术和我们在生产重组TLR胞外域方面的成熟经验，以产生具有重组TLR和NLR衍生胞外域的微生物产物结合蛋白的稳健文库（目的1）。我们将利用这些蛋白质来产生针对哺乳动物TLR/NLR的单克隆抗体（Aim 2）。此外，新型TLR胞外域的微生物识别能力将用PAMP阵列表征（目的3）。最后，这些TLR胞外域将被开发用于诊断，用于检测生物流体中的微生物、微生物产物和内源性危险信号（目标4），并可能用作抗微生物疗法。该项目将向科学界提供重组TLR和NLR胞外域，用于在各种系统中快速表达TLR/NLR胞外域的工具，哺乳动物TLR和NLR的单克隆抗体，微生物产物和微生物的阵列文库，并将进一步开发这些模式识别受体家族作为临床应用的工具。我们向同事分发这些试剂的计划（目标5）是基于我们在TLR领域共享试剂的广泛历史记录。 相关性：先天免疫反应在许多人类疾病的发病机制中起着关键作用。我们的目标是开发微生物传感先天免疫受体作为探针，用于在实验室中先天免疫的进一步研究，用于开发用于检测临床样品中微生物的新方法，并作为可能的治疗方法用于先天免疫应答过度刺激引起的疾病。

DESCRIPTION (provided by applicant): Toll-like Receptors (TLRs) and NOD-like receptors (NLRs) are critical components of the innate immune system, where they serve as sentinel receptors for the detection of microbes and endogenous danger signals. To date, 10 human and 13 mouse TLRs have been characterized and shown to recognize microbial products such as lipopeptides, flagellin, lipoteichoic acid, GPI anchors, various nucleic acids, and lipopolysaccharides. In many cases, the ectodomains of the TLRs, which consists of a series of Leucine-rich repeats (LRRs), directly bind microbe-derived compounds. The NLRs, which are triggered by various microbial compounds as well as numerous endogenous danger signals, use LRRs as putative ligand binding domains. In addition to mammalian TLRs/NLRs, the purple sea urchin (Strongylocentrotus purpuratus) has a greatly expanded repertoire of pattern recognition receptors, in lieu of an adaptive immune system, with 222 TLRs and 203 NLRs. These animals live in microbe-rich coastal waters, and this expanded receptor repertoire creates an even more diverse set of microbial ligands that they can detect. We propose to use high throughput cloning technology and our proven experience in producing recombinant TLR ectodomains to generate a robust library of microbial product binding proteins, with recombinant TLR and NLR-derived ectodomains (Aim 1). We will harness these proteins to produce MAbs to the mammalian TLR/NLRs (Aim 2). Moreover, the microbial recognition capabilities of novel TLR ectodomains will be characterized with PAMP arrays (Aim 3). Finally, these TLR ectodomains will be developed for use in diagnostics, for detection of microbes, microbial products, and endogenous danger signals in biological fluids (Aim 4) and potentially as antimicrobial therapies. This project will deliver to the scientific community recombinant TLR and NLR ectodomains, tools for rapidly expressing TLR/NLR ectodomains in a variety of systems, MAbs for mammalian TLRs and NLRs, arrayed libraries of microbial products and microbes, and will further develop these families of pattern recognition receptors as tools for clinical applications. Our plan for distributing these reagents to colleagues (Aim 5) is based upon our documented extensive history of sharing reagents within the TLR field. RELEVANCE: The innate immune response plays a critical role in the pathogenesis of many human diseases. We aim to develop microbe-sensing innate immune receptors as probes to be used for further studies of innate immunity in the laboratory, for development of novel methods for detecting microbes in clinical samples, and as possible therapies for diseases caused by hyper-stimulation of innate immune response.