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Modulation of neutrophil extracellular trap formation in health and disease
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Constantin F. Urban)ORCID iD: 0000-0003-3069-0295
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The critical prompt innate immune response is highly built upon the influx of neutrophils from the blood stream to the site of infection. In the battlefield, neutrophils sense pathogen-associated molecular patterns (PAMPs) through their pattern-recognition receptors (PRRs) to launch a number of responses with the goal to defeat the invading pathogen. Neutrophils’ wide spectrum of responses ranges from reactive oxygen species production (ROS), phagocytosis, cytokine and chemokine secretion, and neutrophil extracellular trap (NET) formation. The NET scaffold is composed of nuclear chromatin which is armed with antimicrobial proteins. DNA traps are able to ensnare and kill microbes in the extracellular space and NET release concurs with cell death of the neutrophil. An increasing body of literature describes that NETs impose deleterious effects on the host itself in addition to their antimicrobial activity. These hazardous effects mainly stem from pro-inflammatory and tissue-destructive activity of NETs. These two diverse outcomes of NETs result in a series of effects on both host and pathogen. Therefore, it seems rational that NET formation is tightly regulated and not happening spontaneously. The opportunistic fungal pathogen Candida albicans captured and killed by NETs. This fungus has the remarkable ability to grow as budding yeast or as filamentous hyphae, and reversibly alternate between these morphotypes. Hyphae are the tissue-destructive, invasive and pro-inflammatory form of C. albicans, whereas yeast is the proliferative, non-invasive form. Hence, it is important to find out how neutrophils discriminate between distinct growth forms of C. albicans and how NET release is regulated in this regard.

To assess neutrophils responses towards each growth form of C. albicans, the mere ratio of each fungal morphotypes is an insufficient measure to describe comparable amounts used in infection experiments; we therefore used dry mass of fungal cells to serve as a common denominator for amounts of fungal cells with different morphotypes. As assessment of dry mass is laborious, we developed a quick correlative method, which quantified fungal metabolic activity corresponding to the actual dry mass. We applied this method in consecutive studies investigating the neutrophil responses specific to different morphotypes of C. albicans.

Positive and negative regulators of NET formation were investigated for this thesis in a mechanistic fashion. To identify how NET release is negatively regulated during C. albicans infection we focused on anti-inflammatory receptors on neutrophils. We observed that adenosine signals via adenosine receptor reduces the amount of NETs exclusively in response to C. albicans hyphae, the invasive, pro-inflammatory form. We identified adenosine receptor A3 as the responsible receptor suggesting that targeting of adenosine A3 would be a promising approach to control invasive fungal infection, since particularly during immune reconstitution invasive mycoses are frequently accompanied by hyperinflammation which additionally worsens the patient’s state.

As unbalanced inflammation is harmful to the host, a situation reflected in autoimmune diseases, such as systemic lupus erythematosus, we aimed to find molecules, which are able to inhibit NET formation. Thus, we introduced the non-toxic agent tempol’’. During ROS-depended stimulation of NET formation via C. albicans and phorbol esters, the stable redox-cycling nitroxide tempol efficiently blocked NET induction. We therefore proposed tempol as a potential treatment during inflammatory disorders where NET formation is out of balance. In quest for positive regulators of NET formation we found the major addictive component of tobacco and electronic cigarettes, nicotine, as compelling direct inducer of NET release. Interestingly, nicotine is associated with exacerbated inflammatory diseases exerting its pro-inflammatory activity via acetylcholine receptor by targeting protein kinase B (known as Akt) activation with no effect on NADPH oxidase complex in a ROS independent fashion. In consideration of neutrophils role in smoking-related diseases we propose targeting Akt could lower the undesirable effect of NET. 

In conclusion, this thesis identified new modulators of NET formation in response to fungal infection and more broadly to other NET-inducing stimuli, which might have implications in forthcoming therapies.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2015. , 87 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1768
Keyword [en]
neutrophils, Candida albicans, Adenosine, Tempol, Nicotine
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-112867ISBN: 978-91-7601-381-6 (print)OAI: oai:DiVA.org:umu-112867DiVA: diva2:883140
Public defence
2016-01-15, Major Groove, Byggnad 6L, Molecular Biology Department, Umeå University, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2015-12-18 Created: 2015-12-16 Last updated: 2015-12-17Bibliographically approved
List of papers
1. Novel Insight into Neutrophil Immune Responses by Dry Mass Determination of Candida albicans Morphotypes
Open this publication in new window or tab >>Novel Insight into Neutrophil Immune Responses by Dry Mass Determination of Candida albicans Morphotypes
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, e77993Article in journal (Refereed) Published
Abstract [en]

The common fungal pathogen Candida albicans has the ability to grow as a yeast or as a hypha and can alternate between these morphotypes. The overall biomass of both morphotypes increases with growth. However, only yeasts, but not hyphae, exist as discrete cellular entities. Multiplicity of infection (MOI) is a useful parameter to determine the initial inoculum of yeasts for in vitro infection assays. Since the amount of hyphae is difficult to quantify, comparable starting conditions in such assays cannot be determined accurately for yeasts and hyphae using MOI. To circumvent this problem, we have established a set of correlation coefficients to convert fungal metabolic activity and optical density to dry mass. Using these correlations, we were able to accurately compare ROS production and IL-8 release by polymorphonuclear neutrophils upon infection with equal dry mass amounts of yeast and hyphal morphotypes. Neutrophil responses depended on the initial form of infection, irrespective of C. albicans wild-type yeasts transforming to hyphal growth during the assay. Infection with a high mass of live C. albicans yeasts resulted in lower neutrophil ROS and this decrease stems from efficient ROS detoxification by C. albicans without directly affecting the phagocyte ROS machinery. Moreover, we show that dead C. albicans induces significantly less ROS and IL-8 release than live fungi, but thimerosal-killed C. albicans were still able to detoxify neutrophil ROS. Thus, the dry mass approach presented in this study reveals neutrophil responses to different amounts and morphotypes of C. albicans and serves as a template for studies that aim to identify morphotype-specific responses in a variety of immune cells.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-83630 (URN)10.1371/journal.pone.0077993 (DOI)000326334500087 ()
Funder
Swedish Research Council, K2012-99X-21961-01-3
Available from: 2013-12-05 Created: 2013-12-03 Last updated: 2017-12-06Bibliographically approved
2. Adenosine is a drugable negative regulator of neutrophil activity during Candida albicans infection
Open this publication in new window or tab >>Adenosine is a drugable negative regulator of neutrophil activity during Candida albicans infection
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-113401 (URN)
Available from: 2015-12-17 Created: 2015-12-17 Last updated: 2015-12-18Bibliographically approved
3. Stable Redox-Cycling Nitroxide Tempol Inhibits NET Formation
Open this publication in new window or tab >>Stable Redox-Cycling Nitroxide Tempol Inhibits NET Formation
2012 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, 391Article in journal (Refereed) Published
Abstract [en]

To prevent the spread of pathogens neutrophils as the first line of defense are able to release Neutrophil Extracellular Traps (NETs), a recently discovered form of immune response. Reactive oxygen species (ROS) have been shown to be essential for many different induction routes of NET formation. Therefore, pharmacological inhibition of ROS generation has implications for research and medicine related to NETs. The application of diphenylene iodonium (DPI), an inhibitor of NADPH oxidase activity, is limited due to its toxicity to host cells as well as microbes. Therefore, we investigated the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) a membrane-permeable radical scavenger on NET formation triggered by phorbol esters and Candida albicans. We quantified the amount of NETs with two complementary methods, using a microscopic analysis and an online fluorescence-based assay. In line with removal of ROS, Tempol reduced the amount of NET formation by neutrophils challenged with those stimuli significantly. Since Tempol efficiently blocks NET formation in vitro, it might be promising to test the effect of Tempol in experimental models of disorders in which NETs probably have hazardous effects.

Place, publisher, year, edition, pages
Frontiers Media S.A, 2012
Keyword
neutrophils, neutrophil extracellular traps, reactive oxygen species, nitroxide, Tempol, NET inhibition
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-64711 (URN)10.3389/fimmu.2012.00391 (DOI)000209501300384 ()23269921 (PubMedID)
Available from: 2013-02-01 Created: 2013-02-01 Last updated: 2017-12-06Bibliographically approved
4. Nicotine induces neutrophil extracellular traps
Open this publication in new window or tab >>Nicotine induces neutrophil extracellular traps
2016 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 100, no 5, 1105-1112 p.Article in journal (Refereed) Published
Abstract [en]

NETs serve to ensnare and kill microbial pathogens. However, NETs can at the same time contribute to tissue damage and excessive inflammation. Nicotine is a major toxic agent and has been associated with exacerbated inflammatory diseases. The current study aimed at investigating the role of nicotine, the addictive component of tobacco and electronic cigarettes, on triggering NET formation. We report that nicotine induces neutrophils to release NETs in a dose-dependent manner. Nicotine-induced NET formation is mediated via nicotine acetylcholine receptors, depends on Akt and PAD4 activation, but is Nox2-independent, as demonstrated by pharmacological inhibition of Nox2 and by use of Nox2-deficient mouse neutrophils. These findings demonstrate that nicotine induces NETs, which may in turn contribute to smoking-related diseases.

Keyword
tobacco, protein kinase B, ROS, nicotinic acetylcholine receptor
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-113405 (URN)10.1189/jlb.3AB0815-379RR (DOI)000386007500028 ()27312847 (PubMedID)
Available from: 2015-12-17 Created: 2015-12-17 Last updated: 2017-12-01Bibliographically approved
5. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases
Open this publication in new window or tab >>Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases
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2013 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 9, e1003614Article in journal (Refereed) Published
Abstract [en]

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-80699 (URN)10.1371/journal.ppat.1003614 (DOI)000324922300032 ()24039581 (PubMedID)
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved
6. Role of YopK in Yersinia pseudotuberculosis Resistance Against Polymorphonuclear Leukocyte Defense
Open this publication in new window or tab >>Role of YopK in Yersinia pseudotuberculosis Resistance Against Polymorphonuclear Leukocyte Defense
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2013 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 1, 11-22 p.Article in journal (Refereed) Published
Abstract [en]

The enteropathogen Y. pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type three secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte (PMN, or neutrophil) defense. A yopK mutant, which is attenuated in the mouse infection model where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and ROS production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction PMN death, whereas the mutant caused a necrotic-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism where YopK functions to prevent unintended Yop delivery and thereby PMN disruption resulting in necrotic like cell death, which would enhance the inflammatory response favoring the host.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-61273 (URN)10.1128/IAI.00650-12 (DOI)000316298000002 ()23090955 (PubMedID)
Available from: 2012-11-07 Created: 2012-11-07 Last updated: 2017-12-07Bibliographically approved

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