An Environment-Sensitive Turn-On Fluorescent Probe Enables Detection of PAD4 Activation in Response to Distinct NETosis Stimuli

Protein arginine deiminase 4 (PAD4) is a crucial regulator of human neutrophils, playing a central role in the innate immune response as well as the initiation and progression of several immune diseases, including rheumatoid arthritis, cancer, and neurodegeneration. Current approaches for detecting PAD4 activity employ methods that cannot be used in living cells, such as immunodetection or chemical probes that need fixed material or harsh experimental conditions. As a result, they provide limited information on the real-time dynamics of PAD4 activation and localization during neutrophil activation. Here, we present a turn-on activity-based probe that fluoresces exclusively upon binding to active PAD4. This probe allows for the real-time imaging of intracellular PAD4 activity in live human neutrophils during NETosis, a specialized form of cell death, under wash-free conditions. Our results indicate that PAD4 is active in NETosis triggered by both NADPH oxidase-dependent and NADPH oxidase-independent mechanisms; however, the activation dynamics and localization vary depending on the NETosis stimulus. Our probes are valuable tools to further provide insight into the mechanistic differences in NETosis induction and progression, as well as the role of PAD4 in the innate immune system.