Interactions between superoxide and myeloperoxidase in neutrophil phagosomes

Neutrophils protect against microbial infection by ingesting bacteria into intracellular compartments called phagosomes, where microbes are killed through oxidative and non-oxidative mechanisms. During neutrophil activation, an NADPH oxidase complex assembles and reduces molecular oxygen to generate a massive burst of superoxide (O₂^.-) within the phagosome. Subsequently, the neutrophil enzyme myeloperoxidase (MPO) uses O₂^.- to generate microbiocidal hypochlorous acid (HOCl). Despite the importance of O₂^.- and HOCl in host defence, it remains undefined as to how these reactive oxygen species contribute to microbial killing in the phagosome. It has been established that O₂^.- preferentially reacts with MPO in the phagosome. However, it has not been defined how these interactions influence the ability of MPO to produce HOCl. To elucidate the mechanisms of bacterial killing in the neutrophil phagosome, I investigated the relationship between O₂^.- and MPO, and how these interactions influence HOCl production.

Oxidant production inside neutrophils that were ingesting bacteria or the yeast cell fragment zymosan was monitored through the fluorescent probes R19-S and hydroethidine (HE). Oxidation of R19-S to R19 demonstrated HOCl was produced in phagosomes, whilst the formation of fluorescent oxidised HE reported the production of a range of oxidants by neutrophils. Flow cytometry assays were used to assess oxidant production by neutrophils phagocytosing zymosan or S. aureus. The flow cytometry assays revealed two distinct populations of neutrophils with different oxidant producing qualities. Variation in oxidant production between phagosomes was observed via live cell fluorescent microscopy. Within the same neutrophil, some phagosomes were highly fluorescent whilst other phagosomes were non-fluorescent.

When neutrophils were treated with superoxide dismutase (SOD) to scavenge superoxide, oxidised HE fluorescence decreased in a dose dependent manner. This result confirmed that O₂^.- contributed to HE oxidation and SOD could access phagosomes. Pre-incubation of neutrophils with SOD significantly increased R19 fluorescence and HOCl production. Thus, O₂^.- is likely to limit the chlorination activity of MPO by binding to the enzyme and producing a redox form of MPO called Compound III. Oxidation of HE by zymosan stimulated neutrophils was analysed by liquid chromatography‑mass spectrometry (LC-MS) in a pilot study. The initial probe (HE), a dimer product (E⁺-E⁺), and O₂^.- (2‑OH‑E⁺) plus HOCl (2‑Cl‑HE) specific products were identified and monitored. Treatment of neutrophils with SOD limited HOCl production, demonstrated by a decrease in 2-Cl-HE formation. Superoxide may be required to promote MPO activity by reducing the enzyme’s redox form Compound II, which cannot oxidise chloride. The contrasting results observed with R19-S and HE suggests that the probes may interfere with MPO activity, or react with other oxidant species in the phagosome. However, these results demonstrate that O₂^.- reacts with MPO and influences HOCl production. Exposure of neutrophils to SOD enhanced E⁺-E⁺ dimer formation, which suggests that O₂^.- reduces HE^.+ radicals within neutrophils. O₂^.- could potentially react with other radicals in the phagosome, and the resultant products could be bactericidal. Further investigation of the oxidative chemistry within phagosomes may reveal new targets to enhance neutrophil-mediated killing of pathogens. This research will have important implications for host defence and human health.