Yadav Brijesh, Yadav Jagjit S
Pulmonary Pathogenesis and Immunotoxicology Laboratory, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
Nanomaterials (Basel). 2024 Jul 29;14(15):1273. doi: 10.3390/nano14151273.
There is a need for the assessment of respiratory hazard potential and mode of action of carbon nanotubes (CNTs) before their approval for technological or medical applications. In CNT-exposed lungs, both alveolar macrophages (MФs), which phagocytose CNTs, and alveolar epithelial type II cells (AECII cells), which show tissue injury, are impacted but cell-cell interactions between them and the impacted mechanisms are unclear. To investigate this, we first optimized an air-liquid interface (ALI) transwell coculture of human AECII cell line A549 (upper chamber) and human monocyte cell line THP-1 derived macrophages (lower chamber) in a 12-well culture by exposing macrophages to CNTs at varying doses (5-60 ng/well) for 12-48 h and measuring the epithelial response markers for cell differentiation/maturation (proSP-C), proliferation (Ki-67), and inflammation (IL-1β). In optimal ALI epithelial-macrophage coculture (3:1 ratio), expression of Ki-67 in AECII cells showed dose dependence, peaking at 15 ng/well CNT dose; the Ki-67 and IL-1β responses were detectable within 12 h, peaking at 24-36 h in a time-course. Using the optimized ALI transwell coculture set up with and without macrophages, we demonstrated that direct interaction between CNTs and MФs, but not a physical cell-cell contact between MФ and AECII cells, was essential for inducing immunotoxicity (proliferative and inflammatory responses) in the AECII cells.
在碳纳米管（CNTs）被批准用于技术或医学应用之前，有必要对其呼吸危害潜力和作用模式进行评估。在暴露于碳纳米管的肺部，吞噬碳纳米管的肺泡巨噬细胞（MФs）和显示组织损伤的肺泡II型上皮细胞（AECII细胞）均受到影响，但它们之间的细胞间相互作用以及受影响的机制尚不清楚。为了对此进行研究，我们首先通过将巨噬细胞暴露于不同剂量（5 - 60 ng/孔）的碳纳米管12 - 48小时，并测量细胞分化/成熟（前表面活性蛋白-C，proSP-C）、增殖（Ki-67）和炎症（IL-1β）的上皮反应标志物，在12孔培养板中优化了人AECII细胞系A549（上室）和人单核细胞系THP-1衍生的巨噬细胞（下室）的气液界面（ALI）Transwell共培养。在最佳ALI上皮-巨噬细胞共培养（3:1比例）中，AECII细胞中Ki-67的表达呈剂量依赖性，在碳纳米管剂量为15 ng/孔时达到峰值；在时间进程中，Ki-67和IL-1β反应在12小时内即可检测到，在24 - 36小时达到峰值。使用优化的ALI Transwell共培养体系，在有和没有巨噬细胞的情况下，我们证明碳纳米管与MФs之间的直接相互作用，而非MФ与AECII细胞之间的物理细胞接触，对于诱导AECII细胞中的免疫毒性（增殖和炎症反应）至关重要。
