TLR Ectodomains for Microbial Detection and Therapeutics

描述（由申请人提供）：toll样受体（TLRs）和nod样受体（NLRs）是先天免疫系统的关键组成部分，它们作为哨兵受体检测微生物和内源性危险信号。迄今为止，已有10个人类tlr和13个小鼠tlr被鉴定并显示可识别微生物产物，如脂肽、鞭毛蛋白、脂质胆酸、GPI锚点、各种核酸和脂多糖。在许多情况下，由一系列富亮氨酸重复序列（lrr）组成的tlr的外结构域直接结合微生物衍生的化合物。NLRs是由多种微生物化合物和许多内源性危险信号触发的，将lrs作为假定的配体结合域。除了哺乳动物的tlr / nlr外，紫海胆（Strongylocentrotus purpuratus）也有大量的模式识别受体，而不是适应性免疫系统，有222个tlr和203个nlr。这些动物生活在微生物丰富的沿海水域，这种扩大的受体库创造了一套更多样化的微生物配体，它们可以检测到。我们建议使用高通量克隆技术和我们在生产重组TLR外结构域方面的成熟经验来生成一个强大的微生物产物结合蛋白库，其中包括重组TLR和nlr衍生的外结构域（目的1）。我们将利用这些蛋白生产针对哺乳动物TLR/ nlr的单克隆抗体（目标2）。此外，新型TLR外结构域的微生物识别能力将通过PAMP阵列进行表征（Aim 3）。最后，这些TLR外结构域将被开发用于诊断、检测微生物、微生物产物和生物流体中的内源性危险信号（Aim 4），并可能作为抗菌疗法。该项目将为科学界提供重组TLR和NLR外域、在各种系统中快速表达TLR/NLR外域的工具、用于哺乳动物TLR和NLR的单克隆抗体、微生物产品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。我们将这些试剂分发给同事的计划（目标5）是基于我们在TLR领域广泛记录的共享试剂历史。

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.