The Impact of Various Reactive Oxygen Species on the Formation of Neutrophil Extracellular Traps

The formation of neutrophil extracellular traps (NETs) depends on the generation of reactive oxygen species (ROS). Previous studies revealed that both NADPH oxidase and myeloperoxidase (MPO) are required for NET release. However, the contribution of various ROS as well as the role of mitochondria-derived ROS has not been addressed so far. In the present study we aimed to investigate in a systematic and comprehensive manner the contribution of various ROS and ROS-generating pathways to the PMA-induced NET release. By using specific inhibitors, the role of both NADPH oxidase- and mitochondria-derived ROS as well as the contribution of superoxide dismutase (SOD) and MPO on the NET release was assessed. We could demonstrate that NADPH oxidase function is crucial for the formation of NETs. In addition, we could clearly show the involvement of MPO-derived ROS in NET release. Our results, however, did not provide evidence for the role of SOD- or mitochondria-derived ROS in NET formation

A természetben megtalálható deutérium biológiai jelentősége: A deutériumdepletio daganatellenes hatása

The concentration of deuterium is about 150 ppm (over 16 mmol/L) in surface water and more than 10 mmol/L in living organisms. Experiments with deuterium depleted water (30+/-5 ppm) revealed that due to D-depletion various tumorous cell lines (PC-3, human prostate, MDA, human breast, HT-29, human colon, M14, human melanoma) required longer time to multiply in vitro. DDW caused tumor regression in xenotransplanted mice (MDA and MCF-7, human breast, PC-3) and induced apoptosis in vitro and in vivo. Deuterium depleted water (25+/-5 ppm) induced complete or partial tumor regression in dogs and cats with spontaneous malignancies, it was registered as anticancer for veterinary use in 1999 (Vetera-DDW-25 A.U.V., 13/99 FVM). The hypodermic preparation of the registered veterinary drug was successfully tested in clinical investigations. Under the permission of the Hungarian Institute of Pharmacology (No. 5621/40/95) a randomized, double blind controlled, human Phase II clinical trial with prostate cancer was performed, in compliance with GCP principles, which exhibited a significant difference between the control and treated groups with respect to the examined parameters, median survival time and the extension of life-span. We suggest that cells are able to regulate the D/H ratio and the changes in the D/H ratio can trigger certain molecular mechanisms having a key role in cell cycle regulation. We suppose that not the shift in the intracellular pH, but the concomitant increase in the D/H ratio is the real trigger for the cells to enter into S phase. The decrease of D concentration can intervene in the signal transduction pathways thus leading to tumor regression. Deuterium depletion may open new perspectives in cancer treatment and prevention helping to increase the effectiveness of current oncotherapies

β-lactam antibiotic-induced release of lipoteichoic acid from Staphylococcus aureus leads to activation of neutrophil granulocytes

BACKGROUND: Polymorphonuclear neutrophil granulocytes (PMN) are phagocytes of the first line of antimicrobial defense. Previously we demonstrated that lipoteichoic acid (LTA) from Staphylococcus aureus (S. aureus) directly activates neutrophil granulocytes. Others have reported that exposure of S. aureus to β-lactam antibiotics leads to LTA release. In the present study we addressed the question whether exposure of S. aureus to β-lactam antibiotics or antibiotics of other groups results in the generation of PMN-stimulating activity and whether this activity can be attributed to LTA. METHODS: S. aureus were exposed to flucloxacillin, a β-lactam antibiotic or to the protein synthesis-inhibitors erythromycin and gentamicin, or to ciprofloxacin, a gyrase inhibitor. Supernatants of the antibiotic-treated bacteria were assayed for their LTA content and for their effect on PMN functions. RESULTS: We observed that exposure of S. aureus to flucloxacillin and, to a lesser degree to ciprofloxacin, but not to erythromycin or gentamicin led to LTA release. Co-incubation of neutrophil granulocytes with LTA-containing supernatants led to PMN activation as assed by morphological changes, release of IL-8, delay of spontaneous apoptosis and enhanced phagocytic activity. Depletion of LTA from the supernatants markedly reduced their PMN-activating capacity. CONCLUSION: The findings suggest that, via the activation of PMN, antibiotic-induced LTA release from S. aureus leads to enhanced antimicrobial activity of the innate immune defense mechanisms

Proinflammatory Stimuli Enhance Phagocytosis of Apoptotic Cells by Neutrophil Granulocytes

Recently, we have reported that, in addition to macrophages, also neutrophil granulocytes can phagocytose apoptotic neutrophils. Based on this finding, we hypothesized that “cannibalistic” neutrophils at sites of acute infection/inflammation play a major role in the clearance of apoptotic neutrophils. Since at sites of infection/inflammation neutrophils are exposed to microbial constituents and proinflammatory cytokines, in the present study we analyzed the effect of TLR-ligands and cytokines on the ability of neutrophils to phagocytose apoptotic cells in vitro. We observed that exposure to ligands of TLR2 (Malp2, Pam3CSK4), TLR4 (LPS), TLR7/TLR8 (R848), and TLR9 (ODN 2006) led to increased phagocytosis of apoptotic cells by neutrophils. In addition, proinflammatory cytokines such as TNF and GM-CSF strongly enhanced the uptake of apoptotic cells by neutrophils. These results support the hypothesis that neutrophils acquire the ability to phagocytose apoptotic cells at sites of acute infection/inflammation and thereby can contribute to the resolution of inflammation

Dimethylfumarate Impairs Neutrophil Functions

Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates—in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases

A Mitochondrial Polymorphism Alters Immune Cell Metabolism and Protects Mice from Skin Inflammation

Several genetic variants in the mitochondrial genome (mtDNA), including ancient polymorphisms, are associated with chronic inflammatory conditions, but investigating the functional consequences of such mtDNA polymorphisms in humans is challenging due to the influence of many other polymorphisms in both mtDNA and the nuclear genome (nDNA). Here, using the conplastic mouse strain B6-mtFVB, we show that in mice, a maternally inherited natural mutation (m.7778G > T) in the mitochondrially encoded gene ATP synthase 8 (mt-Atp8) of complex V impacts on the cellular metabolic profile and effector functions of CD4+ T cells and induces mild changes in oxidative phosphorylation (OXPHOS) complex activities. These changes culminated in significantly lower disease susceptibility in two models of inflammatory skin disease. Our findings provide experimental evidence that a natural variation in mtDNA influences chronic inflammatory conditions through alterations in cellular metabolism and the systemic metabolic profile without causing major dysfunction in the OXPHOS system

Early Production of the Neutrophil-Derived Lipid Mediators LTB4 and LXA4 Is Modulated by Intracellular Infection with Leishmania major

Recruitment of neutrophil granulocytes to sites of infectious tissue damage is an early event in innate immune responses. Following chemotactic signals neutrophils establish a first line of defense in a swarm-like manner. Intracellular pathogens such as Leishmania major can, however, evade neutrophil-mediated killing and survive inside neutrophils. To achieve this the parasites evolved potent evasion mechanisms. Since neutrophils are a major source of inflammation regulating lipid mediators, we hypothesized that intracellular infection modifies the release of pro- and anti-inflammatory lipid mediators like leukotriene B4 (LTB4) and lipoxin A4 (LXA4), respectively. In the present study, we demonstrated in vitro that L. major-infected primary human neutrophils release an increased amount of LTB4, whereas LXA4 liberation is reduced during the first hours of infection. To investigate whether lipid mediator modulation is a common feature in intracellular infections, we tested the impact of an infection with Anaplasma phagocytophilum. Similarly to L. major, neutrophil infection with A. phagocytophilum led to an enhanced release of LTB4 and decreased LXA4 production. Together, our findings indicate that intracellular infections modulate the lipid mediator profile of neutrophils. This effect is likely to contribute to the survival of the pathogens in neutrophils and to the outcome of the infections