Activity-Guided Identification of Pro-inflammatory Neutrophils with Dual Enzyme- and Metabolite-Activatable Fluorescent Probes

Abstract Neutrophils are the most abundant circulating leukocytes in the innate immune system. Primary neutrophils comprise multiple distinct subtypes that regulate antimicrobial defense and inflammatory diseases. Current fluorescent probes for imaging neutrophils cannot distinguish between activation mechanisms. In this work, we present dual enzyme- and metabolite-activatable fluorogenic probes that are selective for activated neutrophils prone to inflammatory responses. Our probes integrate, within a single scaffold, two neutrophil-associated activation mechanisms, that is, cathepsin G–mediated cleavage of FRET substrates and fluorophore oxidation by hypochlorous acid. This chemical design allows the selective identification of activated neutrophils over other immune cells from peripheral blood of human donors. This strategy provides a unique approach for in situ analysis of neutrophil heterogeneity in multiple pathologies, including different inflammatory diseases.