Tombácz Kata, Mwangi Duncan, Werling Dirk, Gibson Amanda J
1 Department of Pathobiology and Population Sciences, The Royal Veterinary College, University of London, North Mymms, Hatfield, UK.
2 Zoetis InC, VMRD, Kalamazoo, MI, USA.
Innate Immun. 2017 May;23(4):329-335. doi: 10.1177/1753425917695445. Epub 2017 Jan 1.
PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.
模式识别受体（PRRs）是固有免疫系统的哨兵，其中Toll样受体（TLRs）最为重要。针对TLR配体相互作用的检测已被用于深入了解其功能和信号通路。由于不同物种在配体识别方面存在显著差异，因此有必要对这些工具进行调整以适用于其他物种的TLRs。在本研究中，我们描述了一种适用于分析单个PRR的物种特异性细胞检测方法。将人胚胎肾293T细胞与牛TLR2一起稳定转染了NF-κB诱导型报告基因分泌型胚胎碱性磷酸酶（SEAP）。我们将SEAP反应与现有的荧光素酶NF-κB报告基因检测进行比较，以与白细胞介素-8（IL-8）的产生进行相关性分析。两种方法在刺激后均检测到剂量依赖性反应，且与IL-8分泌具有良好的相关性。SEAP检测法检测到的刺激剂浓度低于IL-8分泌水平。荧光素酶检测法在所有配体浓度下均产生高非特异性背景。在所有测试的检测方法中，我们发现牛特异性SEAP报告基因检测法最为便捷，且能在最短时间内得出结果。所建立的报告细胞系将非常适合用于牛的快速、高通量TLR配体筛选。
