Lysosomal acidity fine-tunes reactive species production in macrophages

Macrophages rely on lysosomes to balance microbial killing with self-protection, generating reactive oxygen and nitrogen species during phagocytosis. Using platinum nanoelectrodes, researchers at Wuhan University tracked these reactive molecules in real time, revealing that lysosomal pH acts as a chemical dial. Acidic conditions favor hydrogen peroxide, while mild alkalinization promotes nitric oxide, peroxynitrite, and nitrite. This precise pH-dependent control shapes immune responses, oxidative stress, and inflammatory signaling, offering insights for therapies targeting macrophage function.

Macrophages, the sentinels of the innate immune system, are tasked with a delicate balancing act: they must destroy invading pathogens while limiting damage to surrounding tissues. Central to this function is phagocytosis, the process by which macrophages engulf and neutralize microbes. During this process, macrophages generate reactive oxygen species (ROS) and reactive nitrogen species (RNS), highly reactive chemical molecules that serve both as microbicidal agents and as signaling mediators. While the importance of ROS and RNS in immune defense is well established, the precise mechanisms that regulate their production and timing within macrophages have remained poorly understood.

Recent research has highlighted lysosomes, membrane-bound organelles known as cellular waste disposers, as pivotal regulatory hubs in immune signaling. Lysosomes not only digest pathogens but also create microenvironments that influence the chemistry of ROS and RNS production. It is speculated that the acidity within lysosomes, typically maintained at low pH, might dictate which reactive species are generated and in what quantity. But how exactly does lysosomal pH control the chemical arsenal of macrophages during phagocytosis, and could pH manipulation alter ROS/RNS production?

To address this question, a research team led by Dr. Wei-Hua Huang from Wuhan University, China, and Dr. Christian Amatore from Xiamen University, China, developed a nanoelectrochemical sensor that enables real-time monitoring of ROS and RNS dynamics directly inside lysosomes. The study was published in Volume 8 of the journal Research on June 5, 2025.

The study showed that lysosomal acidity acts as a fine-tuning mechanism that directs the balance between different reactive species. When lysosomal pH dropped below 5.0, protonation of superoxide anions facilitated the conversion to hydrogen peroxide without changing the production rates of superoxide and nitric oxide precursors. This shift increased oxidative activity within the lysosome while keeping overall ROS generation controlled. Conversely, alkalinization of lysosomes to pH levels above 6.0 promoted higher initial nitric oxide production, which subsequently led to peroxynitrite and nitrite formation. Both acidic and alkaline conditions elevated oxidative stress and stimulated proinflammatory signaling, suggesting that deviations from the optimal lysosomal pH can have significant consequences for immune regulation.

This finding underscores that lysosomes are not passive containers but active chemical modulators, controlling the release and conversion of reactive molecules according to local acidity.

The interplay between ROS and RNS species is also an interesting part of the study. Acidic lysosomes favored hydrogen peroxide formation, optimal for killing certain bacteria, while mild alkalinization promoted peroxynitrite and nitrite accumulation, which may target other pathogens or signal to neighboring immune cells. This nuanced chemical regulation allows macrophages to tailor their microbicidal arsenal to specific microbial threats, an adaptive feature that had long been hypothesized but never directly visualized.

The use of nanoelectrochemical sensors is novel. Previous approaches relied on bulk cell measurements, which averaged ROS/RNS signals across the entire cell and obscured localized dynamics. The nanometer-scale electrodes penetrated the phagocytic cup without disrupting normal cell function, allowing repeated measurements over time. This precision enabled the researchers to map the kinetics of ROS and RNS in unprecedented detail, revealing that temporal regulation and chemical conversion within lysosomes are highly pH-dependent.

The study has therapeutic potential as well. Dysregulated lysosomal pH has been implicated in chronic inflammation, autoimmune disorders, and impaired microbial clearance. Modulating lysosomal acidity could therefore provide a targeted strategy to enhance or suppress macrophage activity. For example, stabilizing lysosomal pH in aged or immunocompromised individuals might boost pathogen clearance, while controlled alkalinization could reduce excessive oxidative stress in autoimmune diseases.

Overall, the study highlights lysosomal pH as a critical determinant of ROS and RNS homeostasis during phagocytosis. By providing real-time, nanoscale insights into reactive species dynamics, the research illuminates a previously hidden layer of immune regulation: how macrophages balance killing microbes while avoiding self-harm.