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Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA
Published on: May 12, 2023
A transgenic zebrafish line for in vivo visualisation of neutrophil myeloperoxidase
Kyle D Buchan1, Tomasz K Prajsnar1, Nikolay V Ogryzko1,2
1The Bateson Centre and Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Western Bank, Sheffield, United Kingdom.
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.
Insights
Researchers developed a new zebrafish model to visualize myeloperoxidase (MPO) enzyme activity in neutrophils. This tool aids in studying MPO
Area of Science:
- Immunology
- Cell Biology
- Genetics
Background:
- Neutrophil myeloperoxidase (MPO) generates antimicrobial compounds like hypochlorous acid (HOCl), crucial for pathogen destruction and inflammation regulation.
- MPO's precise role in infection prevention is unclear, though deficiency increases candidiasis risk. MPO dysregulation is linked to inflammatory diseases like atherosclerosis.
- Understanding MPO localization within neutrophils is vital for elucidating its dual role in disease prevention and exacerbation.
Purpose of the Study:
- To develop a novel in vivo tool for visualizing myeloperoxidase (MPO) activity and neutrophil granule dynamics.
- To create a transgenic zebrafish line expressing human MPO fused to a fluorescent tag for tracking its subcellular localization.
- To validate the utility of this new model for studying MPO function without disrupting neutrophil behavior.
Main Methods:
- Generated a transgenic zebrafish line (Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496) using Tol2 transgenesis, expressing human MPO-mEmerald under the neutrophil-specific lyz promoter.
- Confirmed transgene expression and observed MPO-mEmerald localization within neutrophil-like subcellular granules.
- Utilized Spotless (mpxNL144) mutant zebrafish larvae to assess the impact of the MPO-mEmerald transgene on neutrophil migration to inflammatory sites.
Main Results:
- Successfully established a transgenic zebrafish line expressing human MPO fused to a fluorescent marker (mEmerald) in neutrophils.
- Observed MPO-mEmerald localized to subcellular granules within zebrafish neutrophils, mimicking human MPO localization.
- Demonstrated that the MPO-mEmerald transgene does not impede neutrophil migration in zebrafish larvae with non-functional endogenous MPO.
Conclusions:
- A new transgenic zebrafish line expressing functional human MPO allows for in vivo visualization of neutrophil granule dynamics.
- This model provides a valuable tool for studying MPO processing, maturation, and function in neutrophil-related diseases without altering neutrophil behavior.
- Further research using this model can enhance understanding of MPO's role in innate immunity and inflammatory conditions.
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