Contribution of Particle-Induced Lysosomal Membrane Hyperpolarization to Lysosomal Membrane Permeabilization

Lysosomal membrane permeabilization (LMP) has been proposed to precede nanoparticle-induced macrophage injury and NLRP3 inflammasome activation; however, the underlying mechanism(s) of LMP is unknown. We propose that nanoparticle-induced lysosomal hyperpolarization triggers LMP. In this study, a rapid non-invasive method was used to measure changes in lysosomal membrane potential of murine alveolar macrophages (AM) in response to a series of nanoparticles (ZnO, TiO₂, and CeO₂). Crystalline SiO₂ (micron-sized) was used as a positive control. Changes in cytosolic potassium were measured using Asante potassium green 2. The results demonstrated that ZnO or SiO₂ hyperpolarized the lysosomal membrane and decreased cytosolic potassium, suggesting increased lysosome permeability to potassium. Time-course experiments revealed that lysosomal hyperpolarization was an early event leading to LMP, NLRP3 activation, and cell death. In contrast, TiO₂- or valinomycin-treated AM did not cause LMP unless high doses led to lysosomal hyperpolarization. Neither lysosomal hyperpolarization nor LMP was observed in CeO₂-treated AM. These results suggested that a threshold of lysosomal membrane potential must be exceeded to cause LMP. Furthermore, inhibition of lysosomal hyperpolarization with Bafilomycin A1 blocked LMP and NLRP3 activation, suggesting a causal relation between lysosomal hyperpolarization and LMP.