Bioluminescence temporal signatures of monocyte differentiation reveals changes in phenotype due to GM-CSF, M-CSF,...

Human THP-1 monocyte-like cells are a commonly used model for monocyte-derived macrophages (MoDM) in vitro, and significant literature has demonstrated how MoDM phenotype depends on the type, concentration, and duration of differentiation stimuli. However, far less attention has been given to how the choice of differentiation protocols actively reprograms transcriptional signaling networks during the differentiation process. Monocytes begin transcriptional and signaling rewiring immediately upon exposure to differentiation cues, suggesting that these early events condition how macrophages later respond to inflammatory and polarizing stimuli. As a result, differentiation protocols should be considered not as equally neutral preparatory steps, but as biologically active variables that modulate downstream signaling behavior. We compared three differentiation stimuli: PMA, GM-CSF, and M-CSF, to investigate how each agent influenced the temporal activity of key transcription factors. We generated THP-1 cells expressing bioluminescent reporters for CMV, NF-κB and STAT6, enabling longitudinal, quantitative assessment of transcription factor activity throughout differentiation. We found that PMA and GM-CSF enhanced NF-κB activation, whereas M-CSF led to higher STAT6 activity. We also observed that PMA induced early spikes in NF-κB activity and GM-CSF delayed NF-κB activation at later differentiation stages, revealing distinct temporal signaling programs. Differentiation stimuli also induced early-stage downregulation of CMV promoter activity, underscoring global transcriptional remodeling during macrophage maturation. Cytokine assays following secondary polarization demonstrated that CSF-differentiated macrophages exhibited greater responsiveness and dynamic range compared with PMA-differentiated cells. Together, these findings demonstrate that THP-1 differentiation protocols encode stimulus- and time-dependent transcriptional states that directly impact macrophage function. This study establishes temporal transcription factor profiling as a powerful framework for selecting differentiation strategies and experimental timepoints, improving biological relevance and reproducibility in in vitro macrophage models.