Requirement Of Reactive Oxygen Species Research Articles

49 - Inhibition of Amputation-Induced Reactive Oxygen Species Blocks Salamander Tail Regeneration

Very little is known about signaling mechanisms that initiate and sustain tissue regeneration responses after injury. Here, we investigated the requirement of reactive oxygen species (ROS) and NOX activity for tail regeneration using a Mexican axolotl (Ambystoma mexicanum) embryo model. In vivo imaging using dihydroethidium (DHE) revealed a marked increase of ROS and superoxide anions O2●– only minutes after tail amputation. Surprisingly, ROS production was observed at 48 hours post amputation (hpa), long after wound healing. To establish that ROS production is dependent upon early NAPDH oxidase activity, we treated embryos with NOX inhibitor VAS2870 and observed a significant reduction in ROS at 6 hpa. We also observed that VAS2870 strongly inhibited tail regeneration at 72 hpa and 168 hpa. Our findings show that axolotl tail amputation induces ROS production at the amputation site and NOX activity is required for successful tail regeneration. Given the requirement of ROS production for Xenopus tadpole tail and zebrafish fin regeneration, our results suggest that ROS is a conserved mechanism of appendage regeneration.

Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways

Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish.

Contrasting Requirement for Reactive Oxygen Species for the Suppressive Function of Naturally Occurring Regulatory T Cells but Not Induced Regulatory T Cells.

AbstractAbstract 2146 Background:Naturally-occurring, thymus-derived regulatory T cells (nTregs) have clinically important roles in suppression of autoimmunity and graft versus host disease but are maladaptive in the tumor microenvironment where their activity results in the suppression of anti-tumor immunity. Induced regulatory T cells (iTregs) arise in the periphery under the influence of TGF-beta and have related phenotypic features and function. However, the mechanisms by which nTregs and iTregs impart suppression remain poorly understood. We have previously shown that NADPH oxidase derived reactive oxygen species (ROS) are important for nTreg suppressive function. We extend these observations and find distinct differences in the effect of antioxidants and NADPH oxidase inhibitors on blocking nTreg function but not iTreg function thus implying a role for ROS in the function of the former but not the latter. The contrasting requirement for ROS is further supported by distinct differences in expression of oxidant related genes in nTregs and iTregs. Methods:Murine nTregs were isolated by flow cytometric sorting for CD4+CD25++ T cells. Murine iTregs were induced from spleen sorted naïve T cells (CD4+CD44-) by incubating with TGF-beta and IL-2 and the resulting cells were sorted for the CD25++ subset. For suppression assays, nTregs and iTregs were incubated for three days with CFSE-labeled conventional T cells (Tc; CD4+CD25−) with antioxidants or NADPH oxidase inhibitors, as indicated. The proliferation of Tc co-incubated with Tregs was compared to their proliferation in identical conditions lacking Tregs in classical suppression assays. In some cases, nTregs and iTregs were co-incubated with CFSE-labeled Tc for two days in conditions identical to suppression assays to activate their suppressive function then resorted and subjected to RNA extraction and analysis of mRNA expression of a series of oxidant related genes. Results and Discussion:The antioxidants n-acetylcysteine (NAC; 1mM) and beta-mercaptoethanol (B-ME; 50 uM) had only small effects on suppression imparted by iTregs (p<0.01) while they completely blocked suppression by nTregs (see figure; p<0.0001). NADPH oxidase inhibitors VAS2870 (100nM) and diphenyleneiodonium (DPI; 10nM) demonstrated only mild effects on suppression by iTregs (p<0.01) while markedly reducing suppression by nTregs (p<0.001; see figure). iTregs derived from mice lacking a functional NADPH oxidase complex due to a mutation in the NADPH oxidase subunit NCF1 (NCF1−/−) demonstrated suppressive activity that was similar to wild type (NCF+/+) iTregs, thus genetically confirming no role for NADPH oxidase derived ROS for iTreg-mediated suppression. To evaluate dynamic functional differences between nTregs and iTregs, the expression of a series of ROS related genes was analyzed in purified Tregs with activated suppressive function. This demonstrated overexpression of two genes involved in oxidant detoxification in nTregs: sulfiredoxin1 (SRXN1; >16 fold overexpression in nTregs vs iTregs; mean of 3 experiments) and glutathione peroxidase 3 (GPX3; approximately 3-fold overexpression in nTregs vs iTregs; mean of 3 experiments). Overexpression of SRXN1 and GPX3 in nTregs was confirmed with additional unrelated assays. SRXN1 is a transcriptional target for the cytoprotective transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Therefore, we separately evaluated NRF2 expression and found approximately 2.5-fold mean overexpression in nTregs vs iTregs. Thus, because ROS is an important mediator of nTreg suppressive function we hypothesize that activation of NRF2, and downstream antioxidants such as SRXN1, are critical for the oxidant protection of nTregs themselves during suppression. Conclusions:Our findings illustrate fundamental differences in suppressive mechanisms employed by murine nTregs and iTregs and highlight a role for ROS in the former but not the latter. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation

Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron-enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level. We found that neurons exposed to a five minute period of glucose deprivation recovered oxygen utilization and lactate production using novel microphysiometry techniques. Using the non-toxic and energetically favorable five minute exposure, we developed a preconditioning paradigm where neurons are exposed to this brief OGD for three consecutive days. These cells experienced a 45% greater survival following an otherwise lethal event and exhibited a longer lasting window of protection in comparison to our previous in vitro preconditioning model using a single stress. As in other models, preconditioned cells exhibited mild caspase activation, an increase in oxidized proteins and a requirement for reactive oxygen species for neuroprotection. Heat shock protein 70 was upregulated during preconditioning, yet the majority of this protein was released extracellularly. We believe coupling this neuron-enriched multi-day model with microphysiometry will allow us to assess neuronal specific real-time metabolic adaptations necessary for preconditioning.

Reactive oxygen species promote raft formation in T lymphocytes

Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-γ in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCθ, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.

Flavonoids inhibit myelin phagocytosis by macrophages; a structure–activity relationship study

Demyelination is a characteristic hallmark of the neuro-inflammatory disease multiple sclerosis. During demyelination, macrophages phagocytose myelin and secrete inflammatory mediators that worsen the disease. Here, we investigated whether flavonoids, naturally occurring immunomodulating compounds, are able to influence myelin phagocytosis by macrophages in vitro. The flavonoids luteolin, quercetin and fisetin most significantly decreased the amount of myelin phagocytosed by a macrophage cell line without affecting its viability. ic 50 values for these compounds ranged from 20 to 80 μM. The flavonoid structure appeared to be essential for observed effects as flavonoids containing hydroxyl groups at the B-3 and B-4 positions in combination with a C-2,3 double bond were most effective. The capacity of the various flavonoids to inhibit phagocytosis correlated well with their potency as antioxidant, which is in line with the requirement of reactive oxygen species for the phagocytosis of myelin by macrophages. Our results implicate that flavonoids may be able to limit the demyelination process during multiple sclerosis.

Reactive oxygen species as second messengers? Induction of the expression of yeast catalase T gene by heat and hyperosmotic stress does not require oxygen.

It is shown that oxygen is not absolutely needed for stress-induced synthesis of catalase T in the yeast Saccharomyces cerevisiae. Yeast cells develop heat resistance after exposure to elevated temperatures in anoxia. The levels of catalase activity and thermotolerance are comparable to those in aerobically stressed cells. While these results obviously do not exclude a stress signaling role of reactive oxygen species in some systems, as postulated by other authors, they suggest that the question of the obligatory requirement for reactive oxygen species in other stress signaling systems should be rigorously re-investigated.

Requirement for Reactive Oxygen Species in Serum-induced and Platelet-derived Growth Factor-induced Growth of Airway Smooth Muscle

Reactive oxygen species have been recently identified as important mediators of mitogenic signaling in a number of cell types. We therefore explored their role in mediating mitogenesis of airway smooth muscle. The antioxidants catalase, N-acetylcysteine, and probucol significantly reduced proliferation in primary cultures of rat tracheal smooth muscle stimulated with fetal bovine serum or platelet-derived growth factor, without affecting cell viability or inducing apoptosis. N-Acetylcysteine also significantly reduced serum-stimulated elevation of c-Fos but did not prevent the normal mitogen-induced increase in c-fos mRNA. Fractionation of ribosomes by sucrose density centrifugation and subsequent dot-blot Northern analysis revealed that antioxidants reduced incorporation of c-fos mRNA into the heaviest polyribosomes, suggesting redox regulation of c-fos mRNA translation. Serum treatment of monolayers produced a small but reproducibly significant rise in superoxide dismutase-inhibitable reduction of ferricytochrome c by myocyte monolayers. Serum-induced ferricytochrome c reduction, cellular proliferation, and c-Fos elevation were decreased by the flavoprotein-dependent enzyme inhibitor dipheyleneiodonium. Growth responses to fetal bovine serum and superoxide dismutase-inhibitable reduction of ferricytochrome c were not different between cultured tracheal myocytes from wild-type versus gp91 phagocyte oxidase null mice. These results suggest that mitogen stimulation of airway smooth muscle induces signal transduction of cell proliferation that is in part dependent on generation of partially reduced oxygen species, generated by an NADH or NADPH oxidoreductase that is different from the oxidase in phagocytic cells.

Requirement of reactive oxygen species for cell cycle progression in vascular smooth muscle cells: [formula omitted] Bernard LassËgue, Andrew B. Chung, Sharon L. Grant, Kathy K. Griendling, Emory University, Atlanta, GA

Requirement of reactive oxygen species for cell cycle progression in vascular smooth muscle cells: [formula omitted] Bernard LassËgue, Andrew B. Chung, Sharon L. Grant, Kathy K. Griendling, Emory University, Atlanta, GA