Nanoelectrochemical Monitoring of pH-Regulated Reactive Oxygen and Nitrogen Species Homeostasis in Macrophages Lysosomes during Phagocytosis

Macrophages participate in the immune system by recognizing and engulfing foreign bodies inside phagosomes, which fuse with lysosomes in their cytoplasm to form mature phagolysosomes. Lysosomes have an acidic interior and generate and release reactive oxygen and nitrogen species (ROS/RNS) to destroy the endocytosed entities. It has been previously reported that intra-lysosomal pH plays an essential role in the regulation of ROS/RNS. However, the exact regulatory mechanism remains to be elucidated. Taking advantage of the large number of active lysosomes distributed along the phagocytic lumen during frustrated phagocytosis of glass fibers by macrophages, the intensity of 4 primary ROS/RNS released fluxes (ONOO^-, H₂O₂, NO, and NO₂ ^-) was monitored with platinum nanoelectrochemical sensors, thereby revealing the important role of intra-lysosomal pH on ROS/RNS fluxes after pharmacological modulations. Acidification (pH <5.0) does not alter the rate of production of ROS/RNS precursors (superoxide ions, O₂ ^•-, and parent NO) but promotes O₂ ^•- protonation, leading to an increase of H₂O₂ release. In contrast, the initial production of NO, which subsequently increased the release of ONOO^- and NO₂ ^-, was enhanced by alkalinization (pH >6.0). The resulting increased oxidative stress was associated with massive proinflammatory cytokine release. Taken together, these results provide important information about the impact of lysosomal pH on ROS/RNS regulation.