Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan.
Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, Setagaya-ku, Tokyo, 158-8501, Japan.
Sci Rep. 2017 Oct 20;7(1):13661. doi: 10.1038/s41598-017-13890-3.
We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.
我们使用表达人 Toll 样受体 9（hTLR9）的 HEK-Blue hTLR9 细胞，该细胞在响应 CpG DNA 时会释放分泌型碱性磷酸酶（SEAP），以评估核酸候选药物的免疫学特性。我们的初步研究表明，磷酸二酯 CpG DNA 几乎不会诱导 HEK-Blue hTLR9 细胞中任何 SEAP 的分泌。因此，在本研究中，我们开发了转染有人巨噬细胞清道夫受体-1（hMSR1）的 HEK-Blue hTLR9 细胞，该细胞是一种细胞表面 DNA 受体，并确定 HEK-Blue hTLR9/hMSR1 细胞是否对磷酸硫代（PS）CpG DNA 和磷酸二酯（PO）CpG DNA 作出反应。我们选择了 PS CpG2006 作为单链 PO CpG DNA（ssCpG），以及一种包含 ssCpG 的四足样结构 DNA（tetraCpG）作为 TLR9 配体的模型。用 Alexa Fluor 488 标记的配体进行流式细胞术分析。与 mock 转染的 HEK-Blue hTLR9 细胞不同，HEK-Blue hTLR9/hMSR1 细胞有效地摄取了所有三种 CpG DNA。SEAP 的释放几乎与摄取量成正比。用抗 hMSR1 抗体处理 HEK-Blue hTLR9/hMSR1 细胞可显著减少 ssCpG 和 tetraCpG 的摄取。总之，重建 TLR9 介导的 CpG DNA 反应可以用于评估具有不同物理化学性质的核酸候选药物的毒性。
