| 1. |
- Björnsdottir, Halla, et al.
(författare)
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Phenol-soluble Modulin α Peptide Toxins from aggressive Staphylococcus aureus induce rapid Formation of neutrophil extracellular Traps through a reactive Oxygen species-independent Pathway
- 2017
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin alpha (PSM alpha) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMa-induced NETs contained the typical protein markers and were able to capture microbes. The PSMa-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMa-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4 degrees C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMa peptides, a process that may be of importance for CA-MRSA virulence.
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| 2. |
- Crawford, Aaron C., et al.
(författare)
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Biphasic zinc compartmentalisation in a human fungal pathogen
- 2018
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Ingår i: PLoS Pathogens. - : PUBLIC LIBRARY SCIENCE. - 1553-7366 .- 1553-7374. ; 14:5
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Tidskriftsartikel (refereegranskat)abstract
- Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2 Delta growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrtl/Zrt2-cellular import, followed by Zrcl-dependent intracellular compartmentalisation.
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| 3. |
- De Samber, Bjorn, et al.
(författare)
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Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- High pressure frozen (HPF), cryo-substituted microtome sections of 2 mu m thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1-2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis.
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| 4. |
- Fuchs, Tobias A., et al.
(författare)
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Neutrophil extracellular traps
- 2018
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Ingår i: Inflammation. - : World Scientific. - 9789813109445 - 9789813109438 ; , s. 205-275
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Bokkapitel (refereegranskat)
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| 5. |
- Gillenius, Erik, et al.
(författare)
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The adhesive protein invasin of Yersinia pseudotuberculosis induces neutrophil extracellular traps via β1 integrins
- 2015
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Ingår i: Microbes and infection. - : Elsevier BV. - 1286-4579 .- 1769-714X. ; 17:5, s. 327-336
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Tidskriftsartikel (refereegranskat)abstract
- Yersinia pseudotuberculosis adhesive protein invasin is crucial for the bacteria to cross the intestine epithelium by binding to β1 integrins on M-cells and gaining access to the underlying tissues. After the crossing invasin can bind to β1 integrins on other cell surfaces, however effector proteins delivered by the type III secretion system Y. pseudotuberculosis efficiently inhibit potential immune responses induced by this interaction. Here, we use mutant Y. pseudotuberculosis strains lacking the type III secretion system and additionally invasin-expressing Escherichia coli to analyze neutrophil responses towards invasin. Our data reveals that invasin induces production of reactive oxygen species and release of chromatin into the extracellular milieu, which we confirmed to be neutrophil extracellular traps by immunofluorescence microscopy. This was mediated through β1 integrins and was dependent on both the production of reactive oxygen species and signaling through phosphoinositide 3-kinase. We therefore have gained insight into a potential role of integrins in inflammation and infection clearance that has not previously been described, suggesting that targeting of β1 integrins could be utilized as an adjunctive therapy against yersiniosis.
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| 6. |
- Ginley, Brandon G, et al.
(författare)
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Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil extracellular traps (NETs) are extracellular defense mechanisms used by neutrophils, where chromatin is expelled together with histones and granular/cytoplasmic proteins. They have become an immunology hotspot, implicated in infections, but also in a diverse array of diseases such as systemic lupus erythematosus, diabetes, and cancer. However, the precise assessment of in vivo relevance in different disease settings has been hampered by limited tools to quantify occurrence of extracellular traps in experimental models and human samples. To expedite progress towards improved quantitative tools, we have developed computational pipelines to identify extracellular traps from an in vitro human samples visualized using the ImageStream® platform (Millipore Sigma, Darmstadt, Germany), and confocal images of an in vivo mouse disease model of aspergillus fumigatus pneumonia. Our two in vitro methods, tested on n = 363/n =145 images respectively, achieved holdout sensitivity/specificity 0.98/0.93 and 1/0.92. Our unsupervised method for thin lung tissue sections in murine fungal pneumonia achieved sensitivity/specificity 0.99/0.98 in n = 14 images. Our supervised method for thin lung tissue classified NETs with sensitivity/specificity 0.86/0.90. We expect that our approach will be of value for researchers, and have application in infectious and inflammatory diseases.
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| 7. |
- Hosseinzadeh, Ava, 1978-
(författare)
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Modulation of neutrophil extracellular trap formation in health and disease
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 8. |
- Hosseinzadeh, Ava, et al.
(författare)
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Nicotine induces neutrophil extracellular traps
- 2016
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Ingår i: Journal of Leukocyte Biology. - 0741-5400 .- 1938-3673. ; 100:5, s. 1105-1112
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Hosseinzadeh, Ava, et al.
(författare)
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Stable Redox-Cycling Nitroxide Tempol has Antifungal and Immune-modulatory Properties
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Invasive mycoses remain underdiagnosed and difficult to treat. Hospitalized individuals with compromised immunity increase in number and constitute the main risk group for severe fungal infections. Current antifungal therapy is hampered by slow and insensitive diagnostics and frequent toxic side effects of standard antifungal drugs. Identification of new antifungal compounds with high efficacy and low toxicity is therefore urgently required. We investigated the antifungal activity of tempol, a cell-permeable nitroxide. To narrow down possible mode of action we used RNA-seq technology and metabolomics to probe for pathways specifically disrupted in the human fungal pathogen Candida albicans due to tempol administration. We found genes upregulated which are involved in iron homeostasis, mitochondrial stress, steroid synthesis, and amino acid metabolism. In an ex vivo whole blood infection, tempol treatment reduced C. albicans colony forming units and at the same time increased the release of pro-inflammatory cytokines, such as interleukin 8 (IL-8, monocyte chemoattractant protein-1, and macrophage migration inhibitory factor). In a systemic mouse model, tempol was partially protective with a significant reduction of fungal burden in the kidneys of infected animals during infection onset. The results obtained propose tempol as a promising new antifungal compound and open new opportunities for the future development of novel therapies.
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| 10. |
- Lopes, José Pedro, 1986-
(författare)
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Candida albicans adaption to host microenvironments drives immune evasion
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. Here we describe how mast cells acted as tissue sentinels and modulated initial antifungal immune responses. Mast cells response was able to reduce fungal viability and signaled for neutrophil infiltration to the tissue. Upon chemokine sensing circulating neutrophils are rapid infiltrating to the mucosal to help fight infection. A high number of infiltrating cells coupled with the formation of multicellular structures such as biofilm comes with induction of hypoxic and anoxic micro niches. We found that a persistence anoxia hampered neutrophil responses by affecting fungi sensing and consequent antifungal due to cell wall masking. Adaption to low oxygen seems is important for a successful host infection. Hypoxic and anoxic environments do not allow neutrophils to efficiently produce ROS. Neutrophil oxidative burst is essential for antifungal activity and many fungal pathogens evolved antioxidative factors to mediate survival during infection. We reasoned that targeting of fungal redox balances could be a new therapy approach. We have tested tempol, a redox-cycling nitroxide Tempol as a new antifungal drug. Tempol proved an efficient compound in our testing. We found that Tempol affected fundamental pathways for fungal homeostases such as glycolysis and steroid biosynthesis. Additionally, Tempol helped curve fungal infectivity in a mouse model and leads for an enhanced immune system cytokine profile in human blood. The results obtained proposed tempol as a valid new antifungal compound and open new opportunities for the future development of therapies. Efficient antifungal therapies are still urgent since only 6 classes of antimycotics exist and all with few restricted fungal targets. Since primarily fungal infections affect patients with other immunosuppressive conditions, which are undergoing treatment, we reasoned that repurposing drugs could offer clinical benefits. We performed a screening of two US Food and Drug Administration (FDA)–approved compound libraries for compounds with anti-Candida activity. From 844 drugs, 26 agents showed activity against C. albicans. We identified 7 new off-target drugs all with potent anti- C. albicans activity. The use of these new drugs could be prophylactic or to treat both conditions simultaneously offering, therefore the intended benefit.Overall, in this thesis work, we have focused on the sensing clearing and management of fungal pathogens. These findings open new doors for understanding better fungal pathogenicity and purpose valid new antifungal compounds that pave the way for future development of therapies.
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