Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions. All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 μg/cm2 for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA). In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive. In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures.

中文翻译：

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评估一组白介素8报告基因肺上皮细胞系，以监测在不同细胞培养条件下暴露于氧化锌纳米粒子后的促炎反应。

稳定转染的肺上皮报道细胞系是替代复杂的实验技术以监测纳米颗粒（NP）暴露后促炎反应的一种有利替代方法。以前，报告细胞系已在淹没培养条件下使用，但是，目前尚不清楚它们与气-液界面（ALI）接触相结合的潜在用途。因此，本研究的目的是比较一组源自A549肺上皮的白细胞介素8启动子（pIL8）报告基因细胞系（即绿色或红色荧光蛋白（GFP，RFP）和荧光素酶（Luc））。在淹没和ALI条件下NPs暴露后，II型细胞样细胞。在浸没和ALI条件下，所有细胞系均以0.6和6.2μg/ cm2的氧化锌（ZnO）NP暴露3和16小时。根据理化特性，确定每种暴露情况下ZnO-NPs的细胞毒特性。在启动子水平上分析了所有细胞类型中IL-8的表达，并与mRNA（qRT-PCR）和蛋白质水平（ELISA）进行了比较。总之，在每种测试条件下，ZnO暴露后，每个报告细胞系均检测到急性促炎作用。就报告信号强度和TNF-α处理后的发作速度而言，pIL8-Luc细胞系最敏感。尽管仅在16小时之后，pIL8-GFP和pIL8-RFP都显示出对TNF-α的显着信号诱导。就ZnO-NP诱导的细胞毒性而言，pIL8-RFP细胞受到的影响最大，而pIL8-Luc则反应最少。综上所述，