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Nanoparticle effect on neutrophil produced myeloperoxidase.

ABSTRACT: Nanoparticles affect the immune system as they may interact directly with immune cells and activate them. However, it is possible that nanoparticles also interact with released cytokines and immunologically active enzymes. To test this hypothesis, the activity of myeloperoxidase released from activated neutrophils was measured in the presence of nanoparticles with different chemistry and size. In high concentrations of nanoparticles, myeloperoxidase activity is decreased whereas in low concentrations of nanoparticles the activity is increased. The effect of the nanoparticles on myeloperoxidase is dependent on the total protein concentration as low concentrations of bovine serum albumin together with nanoparticles further increase the myeloperoxidase activity. The results herein show that nanoparticles affect the immune response not only at the cellular level but also on released immune effectors. In particular, they show that the nanoparticle effect on myeloperoxidase activity in the neutrophil degranulation environment is the result of an intricate interplay between the enzyme and protein concentrations in the environment and the available surface area on the nanoparticle.

SUBMITTER: Sanfins E

PROVIDER: S-EPMC5773199 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Nanoparticle effect on neutrophil produced myeloperoxidase.

Sanfins Elodie E Correia Alexandra A B Gunnarsson Stefan S Vilanova Manuel M Cedervall Tommy T

PloS one 20180118 1

Nanoparticles affect the immune system as they may interact directly with immune cells and activate them. However, it is possible that nanoparticles also interact with released cytokines and immunologically active enzymes. To test this hypothesis, the activity of myeloperoxidase released from activated neutrophils was measured in the presence of nanoparticles with different chemistry and size. In high concentrations of nanoparticles, myeloperoxidase activity is decreased whereas in low concentra ...[more]

PMID: 29346422

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Project description:Myeloperoxidase (MPO), an important diprotomeric glycoprotein in neutrophil-mediated immunity, produces microbicidal hypohalous acids, but the underpinning glycobiology remains elusive. Deep characterisation of neutrophil-derived MPO (nMPO) using advanced mass spectrometry demonstrated that under-processed oligomannosidic- and hyper-truncated paucimannosidic- and N-acetyl-β-D-glucosamine (GlcNAc) core-type asparagine-linked glycans decorate the protein. Occlusion of Asn355 and Asn391 and sterical hindrance of Asn323- and Asn483-glycans located in the MPO dimerisation zone were found to shape the local glycan processing thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed extreme molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a lower-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-activated neutrophils demonstrated that MPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and rapidly degranulated, but surprisingly carries uniform glycosylation across conditions. Complete proMPO-to-MPO maturation evidently occur during early/mid-stage granulopoiesis. The conserved Asn355- and Asn391-sequons displayed elevated GlcNAc signatures and higher oxidation and chlorination activity of the secretory vesicle/plasma membrane-resident MPO relative to MPO from other granules. Endoglycosidase H-treated nMPO displaying Asn355-/Asn391-GlcNAcylation recapitulated the activity gain and showed increased thermal stability and polypeptide accessibility relative to untreated nMPO as measured by activity assays, circular dichroism and molecular dynamics. Endoglycosidase H-treated nMPO also demonstrated elevated ceruloplasmin-mediated inhibition relative to nMPO. Modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO:ceruloplasmin interface are critical for uninterrupted inhibition. We report on novel bimodal roles of the peculiar MPO glycosylation providing new insight into neutrophil glycobiology.

Project description:Babesiosis caused by the virulent tick-borne hemoprotozoan, Babesia rossi, results in a marked systemic inflammatory host response in dogs. Neutrophils form part of the innate immune response and contains myeloperoxidase (MPO) as the predominant component of the neutrophil lysosomal protein in azurophilic granules. The neutrophil myeloperoxidase index (MPXI), determined on the ADVIA hematology analyzer, is a quantitative estimate of intracellular MPO content. Objectives of this study were to: (a) compare MPXI in dogs with babesiosis with healthy control dogs; (b) compare MPXI in dogs that died from babesiosis with dogs that survived and controls; and (c) correlate the MPXI with the previously determined segmented and band neutrophil count and cytokine concentrations in dogs with babesiosis. Data for 140 dogs naturally infected with B. rossi and 20 healthy control dogs were retrospectively evaluated. Neutrophil counts and MPXI were determined on an ADVIA 2120 analyzer. Cytokine concentrations [interleukin (IL)-2, IL-6, IL-8, IL-10, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), and monocyte chemo-attractant protein-1 (MCP-1)] were determined using a canine-specific multiplex immunoassay. The mortality rate of the Babesia-infected dogs was 11% (15/140). MPXI was significantly higher in Babesia-infected dogs (P = 0.033), and in Babesia-infected non-survivors (P = 0.011), compared with healthy control dogs. In Babesia-infected dogs a significant positive correlation was found between MPXI and IL-10 (r = 0.211, P = 0.039) and a significant negative correlation was found between MPXI and IL-8 (r = -0.350, P < 0.001). In Babesia-infected non-survivors, significant positive correlations were found between MPXI and IL-2 (r = 0.616, P = 0.033), IL-6 (r = 0.615, P = 0.033), IL-18 (r = 0.613, P = 0.034), GM-CSF (r = 0.630, P = 0.028), and MCP-1 (r = 0.713, P = 0.009). In Babesia-infected survivors, a significant negative correlation was found between MPXI and IL-8 (r = -0.363, P = 0.001). MPXI was correlated with pro-inflammatory cytokines in Babesia-infected dogs that died. The potential of MPXI as a novel marker of inflammation and prognosis in dogs infected with B. rossi, thus warrants further investigation.

Project description: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.

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Nanoparticle effect on neutrophil produced myeloperoxidase.

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Nanoparticle effect on neutrophil produced myeloperoxidase.

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