Novel Reporter System Monitoring IL-18 Specific Signaling Can Be Applied to High-Throughput Screening
Novel Reporter System Monitoring IL-18 Specific Signaling Can Be Applied to High-Throughput Screening
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DOI：
10.3390/md18010060
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发表时间：
2020-01-01
期刊：
影响因子：
5.4
通讯作者：
Yonezawa, Tomo
中科院分区：
文献类型：
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作者：
Kurata, Riho;Shimizu, Kenji;Yonezawa, Tomo
最近，针对癌症、自身免疫性疾病和感染的免疫疗法已经是可行的和有前途的。因此，我们已经研究了人类γ δ T细胞是否可以应用于新的免疫疗法的可能性。我们先前建立了稳定维持NF κ B驱动的人分泌型胚胎碱性磷酸酶（SEAP）表达的细胞。该细胞可用于测定具有高标准动态范围和准确度的NFkB转录活性。由于IL-18是一种通过NFkB活化增强人γ δ T细胞的细胞毒性和活性的细胞因子，因此我们将重点放在IL-18的活性和信号转导上。在本研究中，我们对以前的报告细胞，可以确定转录活性的NF κ B表达的两个亚基组成的人IL-18受体进行了修改。经修饰的细胞以浓度依赖性方式响应于用人重组IL-18处理而分泌SEAP。我们还观察到在用小鼠重组IL-18处理的细胞中NFkB活性的浓度依赖性增强，尽管与人重组IL-18相比亲和力较低。我们先前还建立了稳定表达和分泌人重组IL-18的细胞，然后使用该测定验证来自细胞的条件培养基是否激活NF κ B转录活性。本校从大村湾附近海域的18，000多种海洋细菌、真菌、中草药植物等收集了大量的提取物，并从日本化学家那里收集了大量的合成化合物作为药物库。最后，为了鉴定模拟IL-18生物活性或对IL-18诱导的NFkB具有抑制作用的药物，我们使用来自海洋细菌的许多提取物和合成化合物进行了药物筛选。
Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL -18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL -18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL -18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. Our university has kept collecting many extracts from over 18,000 marine bacteria in our local sea around Omura bay fungi, plants for Chinese herbal medicine, and so on and also have kept gathering synthetic compounds from many Japanese chemists as drug libraries. Finally, in order to identify drugs mimicking IL -18 biological activity or possessing inhibitory effects on IL-18 -induced NFkB, we demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.