Abstract
The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae—which express a non-functional zebrafish myeloperoxidase—the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development.
Highlights
The enzyme Myeloperoxidase (MPO) enhances the microbicidal potential of neutrophils by converting hydrogen peroxide (H2O2) into the highly toxic antimicrobial compound hypochlorous acid (HOCl) [1], and by forming radicals by oxidating substrates including phenols, nitrate and tyrosine residues [2]
This observation is supported by the fact that MPO is thought to play a role in the generation of neutrophil extracellular traps (NETs) [13], which are often induced in response to large targets [14]
There is a link between aberrant MPO activity and inflammatory conditions: overactivity is associated with cardiovascular disease, multiple sclerosis and glomerulonephritis [18,19,20], while MPO deficiency has been implicated in pulmonary fibrosis and atherosclerosis [21,22], highlighting its critical role in immune homeostasis
Summary
The enzyme Myeloperoxidase (MPO) enhances the microbicidal potential of neutrophils by converting hydrogen peroxide (H2O2) into the highly toxic antimicrobial compound hypochlorous acid (HOCl) [1], and by forming radicals by oxidating substrates including phenols, nitrate and tyrosine residues [2]. MPO activity appears to be context-dependent, during phagocytosis of large structures such as fungal hyphae [9] or bacterial biofilms [10] In these cases, the phagocytic vacuole does not fully close [11], causing MPO to act at the acidic pH of sites of inflammation (~pH 6) [12], at which it can function normally. MPO deficiency is a relatively common condition affecting 1 in every 2,000–4,000 people across Europe and North America [23], with no major health risks apart from a susceptibility to Candida albicans infections [24] This observation is in stark contrast to people with chronic granulomatous disease (CGD), who lack a working NADPH oxidase. Further studies are required to understand the complex roles of MPO in the immune system
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