Mizoguchi Izuru, Ohashi Mio, Chiba Yukino, Hasegawa Hideaki, Xu Mingli, Owaki Toshiyuki, Yoshimoto Takayuki
Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan.
Front Immunol. 2017 Aug 4;8:929. doi: 10.3389/fimmu.2017.00929. eCollection 2017.
The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several assays to predict the sensitizing potential of chemicals have been developed, these methods cannot distinguish chemical respiratory sensitizers and skin sensitizers. In the present study, we describe a novel assay that can discriminate respiratory sensitizers from chemical skin sensitizers by taking advantage of the fundamental difference between their modes of action, namely the development of the T helper 2 immune response, which is critically important for respiratory sensitization. First, we established a novel three-dimensional (3D) coculture system of human upper airway epithelium using a commercially available scaffold. It consists of human airway epithelial cell line BEAS-2B, immature dendritic cells (DCs) derived from human peripheral blood CD14 monocytes, and human lung fibroblast cell line MRC-5. Respective cells were first cultured in individual scaffolds and subsequently assembled into a 3D multi-cell tissue model to more closely mimic the situation. Then, three typical chemicals that are known respiratory sensitizers (ortho-phthaldialdehyde, hexamethylene diisocyanate, and trimellitic anhydride) and skin sensitizers (oxazolone, formaldehyde, and dinitrochlorobenzene) were added individually to the 3D coculture system. Immunohistochemical analysis revealed that DCs do not migrate into other scaffolds under the experimental conditions. Therefore, the 3D structure was disassembled and real-time reverse transcriptase-PCR analysis was performed in individual scaffolds to analyze the expression levels of molecules critical for Th2 differentiation such as OX40 ligand (OX40L), interleukin (IL)-4, IL-10, IL-33, and thymic stromal lymphopoietin. Both sensitizers showed similarly augmented expression of DC maturation markers (e.g., CD86), but among these molecules, OX40L expression in DCs was most consistently and significantly enhanced by respiratory sensitizers as compared to that by skin sensitizers. Thus, we have established a 3D coculture system mimicking the airway upper epithelium that may be successfully applied to discriminate chemical respiratory sensitizers from skin sensitizers by measuring the critical molecule for Th2 differentiation, OX40L, in DCs.
由于近期在研究中引入了动物实验的3R原则，化学安全测试中动物模型的使用将受到显著限制。尽管已经开发了几种预测化学物质致敏潜力的检测方法，但这些方法无法区分化学性呼吸道致敏剂和皮肤致敏剂。在本研究中，我们描述了一种新的检测方法，该方法可以利用化学性呼吸道致敏剂和皮肤致敏剂作用方式的根本差异，即辅助性T细胞2免疫反应的发展，来区分呼吸道致敏剂和皮肤致敏剂，而这种免疫反应对呼吸道致敏至关重要。首先，我们使用市售支架建立了一种新型的人上呼吸道上皮三维（3D）共培养系统。它由人气道上皮细胞系BEAS-2B、源自人外周血CD14单核细胞的未成熟树突状细胞（DCs）和人肺成纤维细胞系MRC-5组成。各细胞首先在单独的支架中培养，随后组装成3D多细胞组织模型，以更紧密地模拟实际情况。然后，将三种已知的典型呼吸道致敏剂（邻苯二甲醛、六亚甲基二异氰酸酯和偏苯三酸酐）和皮肤致敏剂（恶唑酮、甲醛和二硝基氯苯）分别添加到3D共培养系统中。免疫组织化学分析显示，在实验条件下，DCs不会迁移到其他支架中。因此，将3D结构拆解，并在各个支架中进行实时逆转录聚合酶链反应分析，以分析对Th2分化至关重要的分子如OX40配体（OX40L）、白细胞介素（IL）-4、IL-10、IL-33和胸腺基质淋巴细胞生成素的表达水平。两种致敏剂均显示DC成熟标志物（如CD86）的表达同样增强，但在这些分子中，与皮肤致敏剂相比，呼吸道致敏剂使DCs中OX40L的表达最持续且显著增强。因此，我们建立了一种模拟气道上皮的3D共培养系统，通过测量DCs中Th2分化的关键分子OX40L，该系统可能成功应用于区分化学性呼吸道致敏剂和皮肤致敏剂。
