Dual-Locked FluorescenceProbe for Monitoring theDynamic Transition of Pulmonary Macrophages

Pulmonary macrophages undergo dynamic changes in population, proportion, and polarization during respiratory diseases. Monitoring these changes is critical for understanding their roles in pathology, improving the diagnosis, and guiding drug development. However, current analytic methods based on tissue biopsy are invasive and static, limiting their ability to provide such dynamic information. Herein, we report a dual-locked macrophage-specific renal-clearable probe (DMRP_NOCas) for the dynamic monitoring of pulmonary macrophages during influenza A virus (IAV) infection. DMRP_NOCas activates fluorescence in the presence of two biomarkers (caspase-1 and NO) only coexpressed by M1 macrophages. To optimize the NO reactivity, the scaffold of DMRP_NOCas is screened from the hemicyanine derivatives with an <i>o</i>-phenylenediamine group positioned differently on the indole ring. Notably, the para-substituted o-phenylenediamine demonstrates a higher NO-activated fluorescence compared to its meta-substituted counterpart. This enhancement, as revealed by quantum chemical calculations, is attributed to differential inhibition of twisted intramolecular charge transfer induced by the NO reaction. DMRP_NOCas specifically distinguishes M1 macrophages from other leukocytes including T cells, neutrophils, and M2 macrophages, a capability unmatched by single-locked control probes and other reported probes. Consequently, DMRP_NOCas enables in vivo dynamic monitoring of pulmonary macrophages, uncovering extensive recruitment and M1 polarization of monocyte-derived macrophages within 48 h of IAV infection. This process is accompanied by a significant reduction in alveolar macrophages. These findings provide new insights into macrophage-mediated pulmonary inflammation and underscore the potential of dual-locked probes for precise diagnosis and monitoring of pathological processes.