Particulate matter 2.5 (PM2.5) deposition in the lung’s alveolar capillary region (ACR) is significantly associated with respiratory disease development, yet the molecular mechanisms are not completely understood. Adverse responses that promote respiratory disease development involve orchestrated, intercellular signaling between multiple cell types within the ACR. We investigated the molecular mechanisms elicited in response to PM2.5 deposition in the ACR, in an in vitro model that enables intercellular communication between multiple resident cell types of the ACR. An in vitro, tri-culture model of the ACR, incorporating alveolar-like epithelial cells (NCI-H441), pulmonary fibroblasts (IMR90), and pulmonary microvascular endothelial cells (HULEC) was developed to investigate cell type-specific molecular responses to a PM2.5 exposure in an in-vivo-like model. This tri-culture in vitro model was termed the alveolar capillary region exposure (ACRE) model. Alveolar epithelial cells in the ACRE model were exposed to a suspension of diesel exhaust particulates (DEP) (20 µg/cm2) with an average diameter of 2.5 µm. Alveolar epithelial barrier formation, and transcriptional and protein expression alterations in the directly exposed alveolar epithelial and the underlying endothelial cells were investigated over a 24 h DEP exposure. Alveolar epithelial barrier formation was not perturbed by the 24 h DEP exposure. Despite no alteration in barrier formation, we demonstrate that alveolar epithelial DEP exposure induces transcriptional and protein changes in both the alveolar epithelial cells and the underlying microvascular endothelial cells. Specifically, we show that the underlying microvascular endothelial cells develop redox dysfunction and increase proinflammatory cytokine secretion. Furthermore, we demonstrate that alveolar epithelial MAPK signaling modulates the activation of NRF2 and IL-8 secretion in the underlying microvascular endothelial cells. Endothelial redox dysfunction and increased proinflammatory cytokine secretion are two common events in respiratory disease development. These findings highlight new, cell-type specific roles of the alveolar epithelium and microvascular endothelium in the ACR in respiratory disease development following PM2.5 exposure. Ultimately, these data expand our current understanding of respiratory disease development following particle exposures and illustrate the utility of multicellular in vitro systems for investigating respiratory tract health.

中文翻译：

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在柴油机尾气颗粒 (DEP) 暴露后的肺泡-微血管界面三培养模型中，上皮 MAPK 信号传导指导内皮 NRF2 信号传导和 IL-8 分泌

肺泡毛细血管区 (ACR) 中的颗粒物 2.5 (PM2.5) 沉积与呼吸系统疾病的发展显着相关，但其分子机制尚不完全清楚。促进呼吸道疾病发展的不良反应涉及 ACR 内多种细胞类型之间精心策划的细胞间信号传导。我们在体外模型中研究了 ACR 中 PM2.5 沉积引起的分子机制，该模型能够实现 ACR 多种驻留细胞类型之间的细胞间通讯。开发了 ACR 的体外三培养模型，其中包含肺泡样上皮细胞 (NCI-H441)、肺成纤维细胞 (IMR90) 和肺微血管内皮细胞 (HULEC)，以研究细胞类型特异性分子反应体内模型中的 PM2.5 暴露。这种三培养体外模型被称为肺泡毛细血管区域暴露（ACRE）模型。ACRE 模型中的肺泡上皮细胞暴露于平均直径为 2.5 µm 的柴油机尾气颗粒 (DEP) (20 µg/cm2) 悬浮液中。在 24 小时 DEP 暴露下，研究了肺泡上皮屏障的形成以及直接暴露的肺泡上皮和底层内皮细胞​​的转录和蛋白质表达变化。24 小时 DEP 暴露不会干扰肺泡上皮屏障的形成。尽管屏障形成没有改变，我们证明肺泡上皮 DEP 暴露会诱导肺泡上皮细胞和下面的微血管内皮细胞的转录和蛋白质变化。具体来说，我们表明潜在的微血管内皮细胞会出现氧化还原功能障碍并增加促炎细胞因子的分泌。此外，我们证明肺泡上皮 MAPK 信号传导调节底层微血管内皮细胞中 NRF2 的激活和 IL-8 的分泌。内皮氧化还原功能障碍和促炎细胞因子分泌增加是呼吸系统疾病发展中的两个常见事件。这些发现强调了 ACR 中的肺泡上皮和微血管内皮在 PM2.5 暴露后呼吸道疾病发展中的新的细胞类型特异性作用。最终，这些数据扩展了我们目前对颗粒暴露后呼吸道疾病发展的理解，并说明了多细胞体外系统在研究呼吸道健康方面的效用。