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Trace element landscape of resting and activated human neutrophils on the sub-micrometer level
Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
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2015 (English)In: Metallomics, ISSN 1756-591X, Vol. 7, no 6, 996-1010 p.Article in journal (Refereed) Published
Abstract [en]

Every infection is a battle for trace elements. Neutrophils migrate first to the infection site and accumulate quickly to high numbers. They fight pathogens by phagocytosis and intracellular toxication. Additionally, neutrophils form neutrophil extracellular traps (NETs) to inhibit extracellular microbes. Yet, neutrophil trace element characteristics are largely unexplored. We investigated unstimulated and phorbol myristate acetate-stimulated neutrophils using synchrotron radiation X-ray fluorescence (SR-XRF) on the sub-micron spatial resolution level. PMA activates pinocytosis, cytoskeletal rearrangements and the release of NETs, all mechanisms deployed by neutrophils to combat infection. By analyzing Zn, Fe, Cu, Mn, P, S, and Ca, not only the nucleus but also vesicular granules were identifiable in the elemental maps. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) revealed a neutrophil-specific composition of Zn, Fe, Cu, and Mn in comparison with J774 and HeLa cells, indicating a neutrophil-specific metallome complying with their designated functions. When investigating PMA-activated neutrophils, the SR-XRF analysis depicted typical subcellular morphological changes: the transformation of nucleus and granules and the emergence of void vacuoles. Mature NETs were evenly composed of Fe, P, S, and Ca with occasional hot spots containing Zn, Fe, and Ca. An ICP-MS-based quantification of NET supernatants revealed a NETosis-induced decrease of soluble Zn, whereas Fe, Cu, and Mn concentrations were only slightly affected. In summary, we present a combination of SR-XRF and ICP-MS as a powerful tool to analyze trace elements in human neutrophils. The approach will be applicable and valuable to numerous aspects of nutritional immunity.

Place, publisher, year, edition, pages
2015. Vol. 7, no 6, 996-1010 p.
National Category
Microbiology in the medical area
URN: urn:nbn:se:umu:diva-102830DOI: 10.1039/c4mt00346bISI: 000356058300009PubMedID: 25832493OAI: diva2:810417

This article is part of themed collection: Metals in infectious diseases and nutritional immunity.

Available from: 2015-05-07 Created: 2015-05-07 Last updated: 2016-04-06Bibliographically approved
In thesis
1. Neutrophils versus Pathogenic Fungi: through the magnifying glass of nutritional immunity
Open this publication in new window or tab >>Neutrophils versus Pathogenic Fungi: through the magnifying glass of nutritional immunity
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neutrophils are among the first white blood cells recruited to the site of infection once microbial pathogens enter the host organism. At site, they perform a well-orchestrated chain of processes that aims to kill the microbial invader. Most prominent, neutrophils engulf microbes to inactivate them intracellularly, a process called phagocytosis. Alternatively, neutrophils can release neutrophil extracellular traps (NETs). NETs consist of chromatin decorated with antimicrobial effector proteins – a structure that can entangle bacteria and fungi. Neutrophils are crucial during fungal infections. This is reflected in the increased risk of fungal infections resulting of neutropenia. The concept of nutritional immunity describes every infection as a battle for resources. Those are mostly metal trace elements.

For a long time, neutrophils were seen as powerful, but “mindless”, killers with a limited set of actions and no transcriptional capacity, but this view is in the flux.

In the presented thesis, it was my goal to gain new insights into the interplay of neutrophils and fungi – with special attention to metal-nutritional aspects.

We compared human neutrophils lacking the ability to undergo NETosis, due to a non-functional NADPH complex, and neutrophils from the same person that were “cured” by gene therapy. We investigated those NETs and found that their inhibitory activity towards the mold A. nidulans depends on calprotectin, a known zinc-chelator.

Considering the high influx of neutrophils, we wanted to unravel the neutrophils’ contribution to the metal milieu at the site of infection and trace element changes resulting from NETosis. By combining synchrotron radiation XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial element distribution in activated neutrophils and NETs. Most strikingly, we found neutrophils to be exceptionally high in Fe and the process of NETosis to be reducing available Zn in the surrounding and the early phagosome, possibly by the formation of Zn-rich vesicles.

Using RNA-sequencing, we analyzed the interplay of the C. albicans and neutrophils face-to-face. We dissected their transcriptional profile and revealed a manifold response in neutrophils that include cytokine induction and cellular rearrangement. We further were the firsts to explore the transcriptional response of C. albicans to NETs. Our data indicates a distinct response compared to intact neutrophils or other known stress triggers. Metal homeostasis was affected in Candida in both set-ups.

In summary, this thesis provides new insights into the interaction of fungal pathogens with neutrophils and emphasizes the impact of nutritional aspects on this interplay. A deeper understanding of the nutritional immunity during fungal infection might open up new strategies to tackle fungal infections – a growing threat worldwide.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2015. 63 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1716
neutrophils, Candida albicans, nutritional immunity, metallome, Zn, Fe
National Category
Microbiology in the medical area
urn:nbn:se:umu:diva-102837 (URN)978-91-7601-261-1 (ISBN)
Public defence
2015-06-05, E04, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 11:14 (English)
Available from: 2015-05-13 Created: 2015-05-07 Last updated: 2015-05-19Bibliographically approved

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Niemiec, Maria JoannaBjörn, ErikSandblad, LindaUrban, Constantin F.
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Department of Clinical MicrobiologyMolecular Infection Medicine Sweden (MIMS)Department of ChemistryDepartment of Molecular Biology (Faculty of Medicine)
Microbiology in the medical area

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