Exposure effects beyond the epithelial barrier: trans-epithelial induction of oxidative stress by diesel exhaust particulates in lung fibroblasts in an organotypic human airway model

In vitro bronchial epithelial monoculture models have been pivotal in defining the adverse effects of inhaled toxicant exposures; however, they are only representative of one cellular compartment and may not accurately reflect the effects of exposures on other cell types. Lung fibroblasts exist immediately beneath the bronchial epithelial barrier and play a central role in lung structure and function, as well as disease development and progression. We tested the hypothesis that in vitro exposure of a human bronchial epithelial cell barrier to the model oxidant diesel exhaust particulates caused trans-epithelial oxidative stress in the underlying lung fibroblasts using a human bronchial epithelial cell and lung fibroblast co-culture model. We observed that diesel exhaust particulates caused trans-epithelial oxidative stress in underlying lung fibroblasts as indicated by intracellular accumulation of the reactive oxygen species hydrogen peroxide, oxidation of the cellular antioxidant glutathione, activation of NRF2, and induction of oxidative stress responsive genes. Further, targeted antioxidant treatment of lung fibroblasts partially mitigated the oxidative stress response gene expression in adjacent human bronchial epithelial cells during diesel exhaust particulate exposure. This indicates that exposure induced oxidative stress in the airway extends beyond the bronchial epithelial barrier and that lung fibroblasts are both a target and a mediator of the adverse effects of inhaled chemical exposures despite a lack of direct exposure to the inhaled material. These findings illustrate the value of co-culture models and suggest that trans-epithelial exposure effects should be considered in inhalation toxicology research and testing.

Methods
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体外支气管上皮单层培养模型在明确吸入性毒物暴露的不良效应方面具有关键作用；然而，此类模型仅能代表单一细胞区室，无法准确反映暴露对其他细胞类型的影响。肺成纤维细胞紧邻支气管上皮屏障下方分布，在肺组织结构与功能、疾病发生发展过程中均发挥核心作用。本研究采用人支气管上皮细胞与肺成纤维细胞共培养模型，验证了如下假说：将人支气管上皮细胞屏障暴露于模式氧化性污染物——柴油机尾气颗粒物（diesel exhaust particulates）时，可引发下方肺成纤维细胞发生跨上皮氧化应激。研究结果显示，柴油机尾气颗粒物可诱导下方肺成纤维细胞产生跨上皮氧化应激，具体表现为活性氧物种过氧化氢在细胞内蓄积、细胞抗氧化剂谷胱甘肽发生氧化、核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2，NRF2）被激活，以及氧化应激响应基因的表达上调。此外，在柴油机尾气颗粒物暴露过程中，对肺成纤维细胞实施靶向抗氧化治疗，可部分缓解邻近人支气管上皮细胞中氧化应激响应基因的表达水平。上述结果表明，气道内暴露诱导产生的氧化应激可突破支气管上皮屏障；且尽管肺成纤维细胞未直接接触吸入性物质，但其既是吸入性化学暴露不良效应的作用靶点，同时也是此类效应的介导因子。本研究结果证实了共培养模型的科研价值，并提示在吸入毒理学研究与检测工作中，应纳入跨上皮暴露效应的考量。

实验方法
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