| 1. |
- Björnsdottir, Halla, et al.
(författare)
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Inhibition of phospholipase A(2) abrogates intracellular processing of NADPH-oxidase derived reactive oxygen species in human neutrophils.
- 2013
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Ingår i: Experimental cell research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 319:5, s. 761-774
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Tidskriftsartikel (refereegranskat)abstract
- Upon activation of human neutrophils, superoxide can be produced at two cellular sites; either in the plasma membrane, giving extracellular release of oxidants, or in intracellular organelles, resulting in oxidants being retained in the cell. The involvement of phospholipase A(2) (PLA(2)) in phorbol myristate acetate (PMA)-induced activation of the two pools of NADPH-oxidase was investigated using a variety of PLA(2) inhibitors and the oxidase activity was measured by luminol/isoluminol-amplified chemiluminescence (CL). Two of the seven inhibitors were without effect, two inhibitors inhibited both intra- and extracellular ROS production equally, and three inhibitors inhibited intracellular but not extracellular CL. Using another technique to measure ROS, PHPA oxidation, we found that intracellular ROS production was unaltered with the three last inhibitors, indicating that PLA(2) is not involved in the NADPH-oxidase activity per se, but in the intracellular processing of the radicals necessary for the CL reaction to take place. The PLA(2) inhibitors did not abolish the activity of myeloperoxidase (MPO), an enzyme necessary for intracellular CL to occur. Instead, we suggest that these PLA(2) inhibitors block heterotypic granule fusion and prohibit the colocalization of ROS and MPO needed for intracellular CL activity.
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| 2. |
- Björnsdottir, Halla
(författare)
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Intracellular radicals in neutrophils - processing and functional implications
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neutrophils are the most abundant leukocyte in human blood and essential components of our defense against microbial pathogens. These cells can neutralize microbial pathogens by phagocytosis, which involves engulfment and degradation of microbes intracellularly, as well as by the formation of neutrophil extracellular traps (NETs), which are structures released from neutrophils made up of DNA and proteins that capture microbes extracellularly. One characteristic of neutrophils is that they can produce massive amounts of reactive oxygen species (ROS) upon activation of a specialized enzyme system, the NADPH oxidase. The ROS can be produced at different cellular sites, inside the phagosome, intracellularly inside granules, and at the plasma membrane leading to the release of ROS extracellularly. Whereas ROS produced inside phagosomes are crucial for microbial killing, much less is known about intracellular ROS produced inside granules, which is therefore in focus in this thesis. Neutrophils contain multiple types of granules that are storage organelles for soluble proteins, receptors, and effector molecules. Part of the NADPH oxidase is found in granule membranes and upon activation, ROS can be produced inside granules where they may be processed by myeloperoxidase (MPO) to yield other types of ROS. In paper I, MPO- processing of intracellular ROS was shown to be dependent on phospholipase A2 (PLA2) activity. However, PLA2 was not directly involved in the processing but rather indirectly by mediating the fusion of different granule types, which enables the ROS and MPO to meet inside the cell. It has previously been suggested that the autoinflammatory disorder SAPHO syndrome, characterized by neutrophil dermatosis and typically sterile inflammation of the bone, is associated with neutrophils lacking the production of intracellular ROS. In paper IV, four patients with SAPHO syndrome were investigated with respect to ROS production and other neutrophil functions. All patients, however, produced normal amounts of intracellular ROS demonstrating that decreased intracellular ROS production is not a general feature of SAPHO syndrome. In paper II and III, the role of intragranular ROS for the formation of NETs was studied. Paper II demonstrates that intragranular ROS are essential to drive active NET formation and that intracellular processing of these ROS by MPO is a critical step. Paper III shows that NETs are not only the result of an active process but can also be induced by alternative means, e.g., by cytotoxic peptides released from bacteria. Unlike the process described in the literature and in paper II, this type of NET formation was not dependent on ROS or MPO. In conclusion, the processing of intracellularly produced ROS in neutrophils has been characterized and both production and processing were found to be essential for active NET formation. Further, an alternative mechanism of NET formation was described that is independent of ROS production.
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| 3. |
- Björnsdottir, Halla, et al.
(författare)
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Neutrophil NET formation is regulated from the inside by myeloperoxidase-processed reactive oxygen species.
- 2015
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Ingår i: Free radical biology & medicine. - : Elsevier BV. - 1873-4596 .- 0891-5849. ; 89, s. 1024-1035
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil extracellular traps (NETs) are mesh-like DNA fibers clad with intracellular proteins that are cast out from neutrophils in response to certain stimuli. The process is thought to depend on reactive oxygen species (ROS) generated by the phagocyte NADPH-oxidase and the ROS-modulating granule enzyme myeloperoxidase (MPO), but when, how, and where these factors contribute is so far uncertain. The neutrophil NADPH-oxidase can be activated at different cellular sites and ROS may be produced and processed by MPO within intracellular granules, even in situations where a phagosome is not formed, e.g., upon stimulation with phorbol myristate acetate (PMA).
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| 4. |
- 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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| 5. |
- Björnsdottir, Halla, et al.
(författare)
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Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry.
- 2016
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Ingår i: Data in brief. - : Elsevier BV. - 2352-3409. ; 6, s. 386-93
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Tidskriftsartikel (refereegranskat)abstract
- Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015)[1]. Described herein are data that show the quantification of such heterotypic granule-granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils.
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| 6. |
- Bylund, Johan, 1975, et al.
(författare)
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Measurement of respiratory burst products, released or retained, during activation of professional phagocytes.
- 2014
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Ingår i: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 1124, s. 321-38
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Forskningsöversikt (refereegranskat)abstract
- Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2 (-)) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst is of great importance, and a large number of methods have been used for this purpose. A selection of methods, including chemiluminescence amplified by luminol or isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of PHPA, are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically oriented research on innate immune mechanisms and inflammation.
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| 7. |
- Dahlgren, Claes, 1949, et al.
(författare)
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Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes.
- 2020
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Ingår i: Methods in molecular biology (Clifton, N.J.) Vol 2087.. - New York, NY : Springer. - 1940-6029. - 9781071601532 ; , s. 301-324
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Bokkapitel (refereegranskat)abstract
- Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increased cellular consumption of molecular oxygen (O2). The O2 molecules consumed are reduced by electrons delivered by a membrane localized NADPH-oxidase that initially generate one- and two electron reduced superoxide anions (O2-) and hydrogen peroxide (H2O2), respectively. These oxidants can then be processed into other highly reactive oxygen species (ROS) that can kill microbes, but that may also cause tissue destruction and drive other immune cells into apoptosis. The development of basic techniques to measure and quantify ROS generation by phagocytes is of great importance, and a large number of methods have been used for this purpose. A selection of methods (including chemiluminescence amplified by luminol or isoluminol, absorbance change following reduction of cytochrome c, and fluorescence increase upon oxidation of PHPA) are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to diagnosis of diseases linked to the NADPH-oxidase and more clinically oriented research on innate immune mechanisms and inflammation.
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| 8. |
- Khamzeh, Arsham, et al.
(författare)
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High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2
- 2024
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Ingår i: Journal of Leukocyte Biology. - : Oxford University Press. - 1938-3673 .- 0741-5400. ; 115:3, s. 536-546
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Tidskriftsartikel (refereegranskat)abstract
- Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.
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| 9. |
- Kwiecinski, Jakub, 1985, et al.
(författare)
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Staphylokinase controls Staphylococcus aureus biofilm formation and detachment through host plasminogen activation.
- 2016
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Ingår i: The Journal of infectious diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 213:1, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In this study, we investigated the contribution of fibrin and staphylokinase to biofilm formation. Both in clinical S. aureus isolates and in laboratory strains, high staphylokinase-producing strains formed less biofilm than strains that lacked staphylokinase, suggesting that staphylokinase prevents biofilm formation. Additionally, staphylokinase induced detachment of mature biofilms. This effect depended on plasminogen activation by staphylokinase. Host-derived fibrin, the main substrate cleaved by staphylokinase-activated plasminogen, was a major component of biofilm matrix and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Staphylokinase also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for staphylokinase-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.
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| 10. |
- Sanchez Klose, Felix Peter, 1989, et al.
(författare)
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A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function.
- 2021
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 16:12
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Tidskriftsartikel (refereegranskat)abstract
- Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.
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