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 ( mpx NL144 ) 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 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.
Staphylococcus aureus infects approximately 30% of the human population and causes a spectrum of pathologies ranging from mild skin infections to life-threatening invasive diseases. The strict host specificity of its virulence factors has severely limited the accuracy of in vivo models for the development of vaccines and therapeutics. To resolve this, we generated a humanised zebrafish model and determined that neutrophil-specific expression of the human C5a receptor conferred susceptibility to the S. aureus toxins PVL and HlgCB, leading to reduced neutrophil numbers at the site of infection and increased infection-associated mortality. These results show that humanised zebrafish provide a valuable platform to study the contribution of human-specific S. aureus virulence factors to infection in vivo that could facilitate the development of novel therapeutic approaches and essential vaccines.
26Staphylococcus aureus (S. aureus) possesses high host-specificity, including many human-27 specific virulence factors. Current efforts towards vaccine development have largely failed, 28 citing inappropriate infection models and insufficient understanding of staphylococcal 29 virulence. We sought to create a humanised zebrafish infection model susceptible to human-30 specific virulence factors, focusing on the human C5a receptor (C5AR1) which is targeted by 31 three human-specific staphylococcal virulence factors, CHIPS, PVL, and HlgCB. We 32 demonstrated that the zebrafish C5a receptor (C5aR) responds to serum-derived zebrafish 33C5a, mediates phagocyte recruitment, and is not targeted by any adapted staphylococcal 34 virulence factors. In vivo expression of C5AR1 in zebrafish neutrophils conferred susceptibility 35 to PVL and HlgCB and enhanced S. aureus infection. Lastly, we designed a humanised 36 zebrafish C5aR with only three amino acid changes that maintains endogenous signalling 37 capability yet gained sensitivity to CHIPS-mediated inhibition. We show that a partially 38 humanised zebrafish is a valuable model for investigating host-specific virulence factors. 39 126 C5aR-signalling assay 127 U937 cells were incubated with 2 mM Fluo-3AM (Thermo Fisher) in RPMI with 0.05% human 128 serum albumin (HSA) at room temperature under constant agitation for 10 min, then washed 129 and suspended to 3 million cells per mL in RPMI/0.05% HSA followed by data acquisition on 130 a FACSVERSE (BD Biosciences). Basal fluorescence level for each sample was determined 131 during the first ten seconds, followed by addition of the stimulus while continuing the 132 7 acquisition to measure signalling-induced cytosolic Ca 2+ release by increased Fluo-3AM 133 fluorescence. 134 135
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