Capturing the dynamics of STAT6 macrophage polarization using bioluminescence temporal signatures

Signal transducer and activator of transcription 6 (STAT6) signaling is activated by interleukin 4 (IL-4) and IL-13 and drives alternative macrophage polarization, which is pivotal in wound healing, immunosuppression, and tumor progression. STAT6 functions by forming a phosphorylated homodimer and binding to STAT6-responsive promoter elements to regulate anti-inflammatory genes. Measuring STAT6 activity can serve as a proxy for assessing macrophage polarization. We developed a STAT6-responsive-element (RE) THP-1 reporter to assess STAT6 activation in response to inflammatory stimuli. We quantitatively measured macrophage polarization by using bioluminescence temporal spectrometry (BTS). Human THP-1 monocytes were transduced with lentivirus to express STAT6-RE-firefly luciferase (FLuc)-green fluorescent protein (GFP). The STAT6-RE accurately reported endogenous STAT6 activity, as confirmed by ELISA, western blotting, bioluminescence, and imaging techniques. We developed a systems model to connect emergent bioluminescence to the kinetics of relevant STAT6 cellular signaling events. These results indicate a probe for assessing macrophage alternative polarization, enhancing our understanding of the relationship between molecular mechanisms and macrophage polarization.