Co-culture of human alveolar epithelial (A549) and macrophage (THP-1) cells to study the potential toxicity of ambient PM2.5: a comparison of growth under ALI and submerged conditions

Abstract

Fine particulate matter (PM_2.5) in the ambient atmosphere is strongly associated with detrimental health effects. However, these particles from various sources and regions are unlikely equally toxic. While animal studies are impractical for high-throughput toxicity testing, appropriate <i>in vitro</i> models are urgently needed. Co-culture of A549 and THP-1 macrophages grown at air-liquid interface (ALI) or under submerged conditions was exposed to same concentrations of ambient PM_2.5 to provide accurate comparisons between culture methods. Following 24-h incubation with PM_2.5 collected in Harbin in China, biological endpoints being investigated include cytotoxicity, reactive oxygen species (ROS) levels and pro-inflammatory mediators. The co-culture grown under submerged condition demonstrated a significant increase in ROS levels and all tested pro-inflammatory indicators [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] in mRNA expression and released protein levels. Similar but a declining response trend was observed using the same PM_2.5 incubation after grown at ALI. We further observed a significant increase of PM_2.5-induced phosphorylation of p38 MAPK and activation of NF-κB p65 in a dose-dependent trend for co-cultures grown under submerged condition. These results provide important implications that culture conditions (ALI versus submerged) can induce different extents of biological responses to ambient PM_2.5; the co-culture grown at ALI is less likely to produce false-positive results than submerged culture. Hence, culture conditions should be discussed when comparing <i>in vitro</i> methods used for high-throughput PM_2.5 toxicity assessment in future.