Abstract
As part of the innate immune response, neutrophils destroy microbes in a process called phagocytosis – the uptake of microbes into individual phagosome compartments. Within each phagosome, hypochlorous acid is produced from superoxide by the enzyme myeloperoxidase. Hypochlorous acid is a strong oxidant and is required for effective microbial killing. Despite the importance of hypochlorous acid, a lot remains unknown about how it kills microbes ingested by neutrophils. It would help to have a sensitive and specific assay to measure hypochlorous acid production inside neutrophil phagosomes. I have developed a fluorescent R19-S based assay to monitor the production of hypochlorous acid when neutrophils ingest microbes. I used this assay, alongside other established oxidant assays, to determine what influences the production of hypochlorous acid in neutrophil phagosomes. A potent suicide inhibitor of myeloperoxidase, AZM-198, significantly reduced hypochlorous acid production within the phagosome. Conversely, tryptamine, a reversible inhibitor of myeloperoxidase, only had a minor effect on hypochlorous acid production within the phagosome. Low concentrations of iodide enhanced the oxidation of R19-S in neutrophil phagosomes, suggesting the involvement of chloramines in oxidation within phagosomes. High concentrations of thiocyanate had a minimal impact on the R19 signal, suggesting thiocyanate cannot readily access the phagosome. I also used the R19-S assay and other oxidant assays in a clinical setting to investigate neutrophils from an immunocompromised patient. The patient’s neutrophils could generate normal levels of superoxide but had significantly decreased hypochlorous acid production. The cause of the identified reduced hypochlorous acid generation requires further investigation but demonstrates that the R19-S assay has potential clinical applications to screen patients for abnormal neutrophil oxidant production.