Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Level 3, Immunos Building, 138648, Singapore.
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, 117543, Singapore.
Toxicol In Vitro. 2021 Aug;74:105154. doi: 10.1016/j.tiv.2021.105154. Epub 2021 Mar 25.
The human cell line activation test (h-CLAT) is an OECD approved (Test No. 442E) assay to identify novel skin sensitizers. h-CLAT simulates dendritic cell activation in the skin sensitization pathway and is based on the measurement of CD54 and CD86 overexpression on monocytic, leukemic THP-1 cells. However, the current h-CLAT markers show inconsistent results with moderate and weak sensitizers. Moreover, these markers have accessory roles in cell adhesion and signaling rather than a direct role in cellular inflammation. Therefore, we have explored other inflammation-related markers in this study. PBMCs comprises a mixture of cells that resemble the complex immunological milieu in adults and were primarily used to identify markers. PBMCs (n = 10) and THP-1 cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB, strong) and NiCl (Ni, moderate) sensitizers or DMSO (control) and incubated for 24 h. The samples were subjected to RNA sequencing to obtain logfold change in gene expression. DNCB and NiCl significantly upregulated 80 genes in both cell types. Of these, CD109, CD181, CD183, CLEC5A, CLEC8A & CD354 were experimentally validated. DNCB and Ni but not isopropyl alcohol (non-sensitizer) significantly induced the expression of all novel markers except CLEC8A. Moreover, the percentage induction of all novel markers except CLEC8A satisfied the OECD acceptance criteria. In summary, we identified five novel markers that may supplement the current repertoire of h-CLAT markers.
人细胞系激活测试（h-CLAT）是一种经 OECD 批准的（测试编号 442E）方法，用于识别新型皮肤致敏剂。h-CLAT 模拟了皮肤致敏途径中的树突状细胞激活，基于对单核细胞白血病 THP-1 细胞上 CD54 和 CD86 过表达的测量。然而，当前的 h-CLAT 标志物与中度和弱致敏剂的结果不一致。此外，这些标志物在细胞黏附和信号传递中具有辅助作用，而不是在细胞炎症中具有直接作用。因此，我们在本研究中探索了其他与炎症相关的标志物。
外周血单核细胞（PBMCs）包含类似于成人复杂免疫环境的细胞混合物，主要用于识别标志物。用 1-氯-2,4-二硝基苯（DNCB，强致敏剂）和 NiCl（Ni，中致敏剂）或 DMSO（对照）处理 PBMCs（n=10）和 THP-1 细胞，并孵育 24 小时。对样品进行 RNA 测序以获得基因表达的对数倍变化。DNCB 和 NiCl 在两种细胞类型中均显著上调了 80 个基因。其中，CD109、CD181、CD183、CLEC5A、CLEC8A 和 CD354 通过实验验证。DNCB 和 Ni（而非异丙醇，非致敏剂）显著诱导除 CLEC8A 之外的所有新型标志物的表达。此外，除 CLEC8A 之外的所有新型标志物的诱导百分比均满足 OECD 接受标准。
总之，我们确定了五个可能补充当前 h-CLAT 标志物组合的新型标志物。
