Abstract
Accurately interpreting short-lived signals is challenging within noisy cellular microenvironments. It is not known if cells can distinguish different signaling molecules under transient exposure. Here, we explored the temporal limits of signal detection by single macrophages. Microfluidic experiments monitoring NF-kB dynamics showed that macrophages strongly respond even to 1-second pulses of cytokine and pathogen ligands. Information theory showed that short-pulse response is highly specific to the stimulating ligand, comparable to that under long-term stimulation. Macrophages were mainly sensitive to the duration of cytokines, and the dose of pathogen ligands. Stimulus duration altered the ranking of response strengths among different pathogenic ligands. Mathematical modeling showed that receptor cooperativity is crucial for robust responses to transient signals, while receptor pathway variability leads to accurate signaling in fluctuating environments. These findings demonstrate that dynamic transcription factor specificity is preserved across varying signal durations and uncover network-level mechanisms that accurately distinguish transiently encountered threats.