An in vitro alveolar model allows for the rapid assessment of chemical respiratory sensitization with modifiable biomarker endpoints

Abstract

Diisocyanates are commonly used in polyurethanes where use includes industrial, commercial, and residential applications and can exist as respirable contaminants. These respirable contaminants exist in the air we breathe. Yet, there is no rapid assay available to test for potential respiratory sensitizers. To assess these hazards, as well as to decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. We describe an easy, reliable, verified cell culture model that can be adopted by any lab capable of performing molecular toxicology. The architecturally relevant alveolar model consists of epithelial cells, macrophage cells, and dendritic cells in a simply maintained submerged system ideal for high-throughput testing. Exposures to contaminants that verify biomarker identification include a known pulmonary sensitizer (isophorone diisocyanate) and a positive control for cellular activation (phorbol 12-myristate 13-acetate/ionomycin). The mitochondrial reactive oxygen species generation and cytostructural changes were assessed with confocal laser scanning microscopy; cell morphology was assessed with scanning electron microscopy; biochemical reactions were assessed via protein arrays; genetic alterations were assessed via gene arrays; and cell surface activation markers were assessed via flow cytometry. Results showed that compared to untreated cultures, isophorone diisocyanate increased markers for dendritic cell activation, trafficking, and antigen presentation; number and length of dendritic protrusions; oxidative stress; and genetic and cytokine expression of neutrophil chemoattractants. The chemokines and cytokines CCL7, CXCL5, IL-6, and IL-8 were identified as biomarkers indicative of respiratory sensitization. By including multiple methods to assess endpoints, the in vitro model described can serve as a high-throughput assay to identify substances which may lead to respiratory sensitization.

Keywords: Cellular activation; Immunotoxicology; In vitro: Dendritic cells; Pulmonary exposure; Sensitization.