Date Published: April 19, 2019
Publisher: Public Library of Science
Author(s): Kyle D. Buchan, Tomasz K. Prajsnar, Nikolay V. Ogryzko, Nienke W. M. de Jong, Michiel van Gent, Julia Kolata, Simon J. Foster, Jos A. G. van Strijp, Stephen A. Renshaw, Pierre Boudinot.
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.
The enzyme Myeloperoxidase (MPO) enhances the microbicidal potential of neutrophils by converting hydrogen peroxide (H2O2) into the highly toxic antimicrobial compound hypochlorous acid (HOCl) , and by forming radicals by oxidating substrates including phenols, nitrate and tyrosine residues . MPO is located in the primary granules of neutrophils, which deliver MPO and other bactericidal compounds to invading pathogens by fusing with phagocytic vesicles, accelerating pathogen destruction. MPO is the most abundant protein in the primary granules of human neutrophils , and consequently neutrophils are able to produce high levels of HOCl to deliver a potent antimicrobial response that is capable of killing a broad variety of major pathogens [4–6]. Importantly, due to the activity of upstream NADPH oxidase, the phagocytic vacuole is thought to be relatively alkaline (~pH 9), and under such conditions MPO activity may be less efficient  than other neutrophil enzymes . MPO activity appears to be context-dependent, particularly during phagocytosis of large structures such as fungal hyphae  or bacterial biofilms . In these cases, the phagocytic vacuole does not fully close , causing MPO to act at the acidic pH of sites of inflammation (~pH 6) , at which it can function normally. This observation is supported by the fact that MPO is thought to play a role in the generation of neutrophil extracellular traps (NETs) , which are often induced in response to large targets . While the precise site of action of MPO is uncertain, it is clear that it plays a role in antimicrobial defence, as pathogens produce specific virulence factors targeting it . Beyond its role in bolstering the antimicrobial defence, MPO is also an important regulator of inflammation. The arrival of neutrophils at the wound site marks the initial steps of the anti-inflammatory response, as MPO is delivered to the wound site to consume H2O2 and reduce inflammatory signalling [16,17]. There is also a link between aberrant MPO activity and inflammatory conditions: overactivity is associated with cardiovascular disease, multiple sclerosis and glomerulonephritis [18–20], while MPO deficiency has been implicated in pulmonary fibrosis and atherosclerosis [21,22], highlighting its critical role in immune homeostasis. MPO deficiency is a relatively common condition affecting 1 in every 2,000–4,000 people across Europe and North America , with no major health risks apart from a susceptibility to Candida albicans infections . This observation is in stark contrast to people with chronic granulomatous disease (CGD), who lack a working NADPH oxidase. Those with CGD are unable to generate an effective phagocytic environment capable of destroying microbes [25,26]. Unlike MPO deficiency, those with CGD experience frequent life-threatening infections from a wide range of pathogens , and consequently, the role of MPO is less clear when observed in the context of other oxidative enzymes and compounds. Further studies are required to understand the complex roles of MPO in the immune system.
In this study, we created a transgenic line expressing a fluorescently-tagged human myeloperoxidase in zebrafish neutrophils. Expression in neutrophils was determined by observing expression of lyz:MPO-mEmerald in the fluorescent red neutrophil line, Tg(lyz:nfsB-mCherry)sh260, which expresses mCherry in the cytoplasm of zebrafish neutrophils. Both transgenes were expressed within the same cells (Figs 1 and 2), and TSA staining showed that the majority of MPO-mEmerald cells produced active peroxidase that colocalises with MPO-mEmerald signal (Fig 3) confirming that the lyz:MPO-mEmerald transgene labels neutrophils. However, as MPO localises with the primary granules of neutrophils, it was essential that the fluorescent signal observed in the lyz:MPO-mEmerald line should differ from the cytoplasmic signal observed in the Tg(lyz:nfsB-mCherry)sh260 line. This was observed in several instances; in double transgenic neutrophils, distinct areas of the cell remain unlabelled with mEmerald (Figs 1 and 4) suggesting that MPO is translated and trafficked to a subcellular location that is distinct from the cytoplasm. This observation is also evident in Airyscan confocal imaging (Fig 4C), where a large unlabelled area of a double-transgenic neutrophil is visible in the mEmerald channel. This is likely to be a region of the cell that is inaccessible to the primary granules, for example the nucleus, and could be verified using a fluorescent nuclear probe.
We have generated a transgenic zebrafish line expressing fluorescently labelled human MPO within its neutrophils. The enzyme is non-functional and does not interfere with neutrophil recruitment to sites of infection or inflammation, suggesting that it may be used to study granule dynamics in vivo without disrupting neutrophil behaviour. Additionally, the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line may be used to investigate processing and targeting of MPO during development, which is currently uncharacterised in vivo. Lastly, we provide a protocol for genotyping endogenous myeloperoxidase-null Spotless (mpxNL144) fish, which will prove useful in future studies investigating myeloperoxidase in the zebrafish.