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Neutrophils versus Pathogenic Fungi: through the magnifying glass of nutritional immunity
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Constantin Urban)
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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1716
Keyword [en]
neutrophils, Candida albicans, nutritional immunity, metallome, Zn, Fe
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-102837ISBN: 978-91-7601-261-1 (print)OAI: oai:DiVA.org:umu-102837DiVA: diva2:810466
Public defence
2015-06-05, E04, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 11:14 (English)
Opponent
Supervisors
Available from: 2015-05-13 Created: 2015-05-07 Last updated: 2015-05-19Bibliographically approved
List of papers
1. Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent
Open this publication in new window or tab >>Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent
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2011 (English)In: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 127, no 5, 1243-1252 e.7 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function.

OBJECTIVE: To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD.

METHODS: Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or alpha-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils.

RESULTS: We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice.

CONCLUSION: Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.

Keyword
chronic granulomatous disease, gene therapy, neutrophil extracellular trap, calprotectin, Aspergillus infection
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-43206 (URN)10.1016/j.jaci.2011.01.021 (DOI)000290018600020 ()
Available from: 2011-04-22 Created: 2011-04-22 Last updated: 2017-12-11Bibliographically approved
2. Trace element landscape of resting and activated human neutrophils on the sub-micrometer level
Open this publication in new window or tab >>Trace element landscape of resting and activated human neutrophils on the sub-micrometer level
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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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-102830 (URN)10.1039/c4mt00346b (DOI)000356058300009 ()25832493 (PubMedID)
Note

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
3. Dual transcriptome of the immediate neutrophil and Candida albicans interplay
Open this publication in new window or tab >>Dual transcriptome of the immediate neutrophil and Candida albicans interplay
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2017 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, 696Article in journal (Refereed) Published
Abstract [en]

Background: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. Methods: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. Results: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil-and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. Conclusions: Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.

Keyword
Candida, Neutrophils, Dual transcriptome, Extracellular traps, NOD-like receptor pathway, Nutritional immunity, Morphotype
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-102836 (URN)10.1186/s12864-017-4097-4 (DOI)000409208200002 ()
Note

Originally published in manuscript form with title [RNA-Seq transcription profile of the neutrophil: Candida albicans in vitro interaction]

Errata BMC Genomics (2017) 18:696 DOI: 10.1186/s12864-017-4097-4

Available from: 2015-05-07 Created: 2015-05-07 Last updated: 2017-12-04Bibliographically approved

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