Effects of culture media on gene expression in reconstructed human epidermis and THP-1 monocytes for skin sensitization evaluation in co-culture systems

IF 2.6 3区医学 Q3 TOXICOLOGY

Toxicology in Vitro Pub Date : 2025-02-28 DOI:10.1016/j.tiv.2025.106035

Y. Sugimoto-Sawada , M. Yamashiro , M. Kono , H. Ikeda , H. Itagaki , K. Iijima

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Abstract

Co-culture with reconstituted epidermis formed by normal human epidermal keratinocytes (RhE) increases the expression of the skin sensitization markers CD54 and CD86 on the human monocytic leukemia cell line THP-1 without chemicals. Therefore, we investigated the effects of culture media [RPMI1640 for RhE; keratinization induction (KI) medium for THP-1], co-culture, and the responses to the skin sensitizer 2,4-dinitrochlorobenzene (DNCB) on gene expression in mono- and cocultures of RhE and THP-1 cells. Microarray and pathway analyses revealed that in mono-RhE, RPMI medium induced epidermal differentiation-related genes, whereas in monoculture THP-1 cells, KI medium upregulated inflammation-related genes. Surprisingly, the medium composition had a more significant impact than co-culture in both cells. However, crosstalk between RhE and THP-1 cells was observed upon DNCB exposure by comparing the differentially expressed gene sets. DNCB-treated THP-1 cells showed increased expression of NR4A1, NR4A2, NR4A3, SIK1, and HMOX1 in co-culture than in monoculture, and these gene expression patterns were confirmed by real-time RT-PCR. It has been suggested that danger signals from RhE, in response to DNCB, enhance the expression of these genes in THP-1 cells. We clarified the effects of the medium and co-culture and proposed these five genes as potential markers for skin sensitization evaluation.

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与正常人表皮角质细胞（RhE）形成的重建表皮共培养可增加人单核细胞白血病细胞系 THP-1 上皮肤过敏标志物 CD54/CD86 的表达，而无需化学试剂。因此，我们研究了培养基（RhE 为 RPMI1640；THP-1 为角质化诱导（KI）培养基）、共培养以及对皮肤致敏剂 2,4-二硝基氯苯（DNCB）的反应对 RhE 和 THP-1 细胞单培养和共培养基因表达的影响。微阵列和通路分析表明，在单培养RhE细胞中，RPMI培养基诱导表皮分化相关基因，而在单培养THP-1细胞中，KI培养基上调炎症相关基因。令人惊讶的是，在这两种细胞中，培养基成分的影响比共培养更显著。然而，通过比较差异表达基因组，可以观察到 RhE 和 THP-1 细胞在接触 DNCB 后的串扰。经 DNCB 处理的 THP-1 细胞在共培养中比在单培养中显示出更高的 NR4A1、NR4A2、NR4A3、SIK1 和 HMOX1 表达，这些基因表达模式通过实时 RT-PCR 得到了证实。有研究表明，来自 RhE 的危险信号在 DNCB 的作用下会增强 THP-1 细胞中这些基因的表达。我们阐明了培养基和共培养的影响，并建议将这五个基因作为皮肤过敏评估的潜在标记。

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来源期刊

Toxicology in Vitro 医学-毒理学

CiteScore

6.50

自引率

3.10%

发文量

181

审稿时长

65 days

期刊介绍： Toxicology in Vitro publishes original research papers and reviews on the application and use of in vitro systems for assessing or predicting the toxic effects of chemicals and elucidating their mechanisms of action. These in vitro techniques include utilizing cell or tissue cultures, isolated cells, tissue slices, subcellular fractions, transgenic cell cultures, and cells from transgenic organisms, as well as in silico modelling. The Journal will focus on investigations that involve the development and validation of new in vitro methods, e.g. for prediction of toxic effects based on traditional and in silico modelling; on the use of methods in high-throughput toxicology and pharmacology; elucidation of mechanisms of toxic action; the application of genomics, transcriptomics and proteomics in toxicology, as well as on comparative studies that characterise the relationship between in vitro and in vivo findings. The Journal strongly encourages the submission of manuscripts that focus on the development of in vitro methods, their practical applications and regulatory use (e.g. in the areas of food components cosmetics, pharmaceuticals, pesticides, and industrial chemicals). Toxicology in Vitro discourages papers that record reporting on toxicological effects from materials, such as plant extracts or herbal medicines, that have not been chemically characterized.