Establishment of a STAT6 Reporter Assay for Screening Environmental Toxicants Affecting Allergic Airway Inflammation

Air pollution-associated allergic airway inflammation is an increasing public health concern. Interleukin‑4 (IL‑4) and interleukin‑13 (IL‑13), which activate the Signal Transducer and Activator of Transcription 6 (STAT6) pathway, a central mediator of allergic airway inflammation, may modulate the respiratory toxicities of pollutants. The present study describes the generation and validation of a stable STAT6 luciferase reporter assay in human airway epithelial cells for evaluating environmental toxicants that modulate STAT6 signaling. Human bronchial epithelial 16HBE14o^- cells were transduced with a STAT6-responsive luciferase reporter using a lentiviral vector, followed by optimization of puromycin selection and multiplicity of infection, and monoclonal isolation by limiting dilution. A stable clone with strong and reproducible induction across serial passages was selected. Reporter responsiveness was validated by IL-4/IL-13 stimulation, and STAT6 dependence was confirmed using selective STAT6, STAT5, and STAT3 inhibitors. Assay performance was quantified by Z'-factor analysis, which indicated reproducible signal separation. Furthermore, the assay was applied to individual air-pollution constituents, and benzo[b]fluoranthene and particulate matter significantly increased STAT6 reporter activity. This method provides a scalable approach for measuring STAT6 activity in airway epithelial cells and for prioritizing environmental toxicants that modulate allergic airway signaling.