Oxidative Burst In Human Neutrophils Research Articles

Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis.

Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration.

Protective Role of Flavonoids against Intestinal Pro-Inflammatory Effects of Silver Nanoparticles.

Silver nanoparticles (AgNP) have been increasingly incorporated into food-related and hygiene products for their unique antimicrobial and preservative properties. The consequent oral exposure may then result in unpredicted harmful effects in the gastrointestinal tract (GIT), which should be considered in the risk assessment and risk management of these materials. In the present study, the toxic effects of polyethyleneimine (PEI)-coated AgNP (4 and 19 nm) were evaluated in GIT-relevant cells (Caco-2 cell line as a model of human intestinal cells, and neutrophils as a model of the intestinal inflammatory response). This study also evaluated the putative protective action of dietary flavonoids against such harmful effects. The obtained results showed that AgNP of 4 and 19 nm effectively induced Caco-2 cell death by apoptosis with concomitant production of nitric oxide, irrespective of the size. It was also observed that AgNP induced human neutrophil oxidative burst. Interestingly, some flavonoids, namely quercetin and quercetagetin, prevented the deleterious effects of AgNP in both cell types. Overall, the data of the present study provide a first insight into the promising protective role of flavonoids against the potentially toxic effects of AgNP at the intestinal level.

Chalcones as Modulators of Neutrophil Oxidative Burst under Physiological and High Glucose Conditions.

Several epidemiological studies indicate that neutrophils, under hyperglycemic conditions, are involved in the perpetuation of the inflammatory status, a characteristic of diabetes mellitus, leading to the production of prodigious quantities of reactive species and the release of neutrophil extracellular traps (NETs). Accordingly, our aim was to study the ability of a panel of 25 structurally related chalcones to modulate human neutrophil oxidative burst and the production of NETs under physiological and high glucose conditions. In general, all chalcones presented similar effects under physiological and high glucose conditions. 2',4-Dihydroxy-3-methoxychalcone (3), here studied for the first time, was the most active (IC50 ≤ 5 μM) on the inhibition of neutrophil oxidative burst, showing the importance of the presence of hydroxy substituents at the C-2' and C-4 positions of the A and B rings, respectively, and a 3-methoxy substituent at B ring of the chalcone scaffold. In the present experimental conditions, NETs release only occurred under high glucose levels. The pentahydroxylated chalcone 1 was the only one that was able to modulate the NETs release. This study provided important considerations about the chalcones' scaffold and their modulatory effect on human neutrophil activities at physiological and high glucose conditions, evidencing their potential use as complementary antidiabetic agents.

Biological Properties of a Novel Multifunctional Host Defense Peptide from the Skin Secretion of the Chaco Tree Frog, Boana raniceps.

In recent years, the number of new antimicrobial drugs launched on the market has decreased considerably even though there has been an increase in the number of resistant microbial strains. Thus, antimicrobial resistance has become a serious public health problem. Amphibian skin secretions are a rich source of host defense peptides, which generally are cationic and hydrophobic molecules, with a broad-spectrum of activity. In this study, one novel multifunctional defense peptide was isolated from the skin secretion of the Chaco tree frog, Boana raniceps. Figainin 2 (1FLGAILKIGHALAKTVLPMVTNAFKPKQ28) is cationic and hydrophobic, adopts an α-helical structure in 50% (v/v) trifluoroethanol (TFE), and is thermally stable. This peptide exhibited activity against Gram-negative and Gram-positive pathogenic bacteria arboviruses, T. cruzi epimastigotes; however, it did not show activity against yeasts. Figainin 2 also showed antiproliferative activity on cancer cells, is moderately active on human erythrocytes, and activates the oxidative burst in human neutrophils.

Bothrops moojeni venom and BmooLAAO-I downmodulate CXCL8/IL-8 and CCL2/MCP-1 production and oxidative burst response, and upregulate CD11b expression in human neutrophils

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

Characteristics of Attenuating Effects of Rebamipide, an Anti-ulcer Agent, on Oxidative Burst of Human Neutrophils

The aim of this study was to characterize the effects of rebamipide on the oxidative burst of human neutrophils. The neutrophil oxidative burst was measured in the presence of rebamipide and cimetidine using lucigenin- or luminol-dependent chemiluminescence (LgCL or LmCL). Rebamipide inhibited the LmCL response stimulated with opsonized zymosan, 12-myristate 13-acetate phorbol, and calcium ionophore in a dose-dependent manner, but the LgCL response was inhibited when neutrophils were stimulated with opsonized zymosan. LmCL response was also dose-dependently attenuated by rebamipide even in the presence of cimetidine. Thus, addition of rebamipide to H2-receptor antagonists can be considered for the treatment of gastric mucosal injury associated with oxidative stress.

The Orai Ca2+ channel inhibitor CM4620 targets both parenchymal and immune cells to reduce inflammation in experimental acute pancreatitis.

This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.

Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.

Pharmacological intervention with oxidative burst in human neutrophils.

In this study we investigated the effect of five therapeutically used drugs and four natural polyphenolic compounds on the mechanism of oxidative burst of human neutrophils concerning their participation in the generation of reactive oxygen species (ROS). The compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin > quercetin > curcumin > arbutin > dithiaden > carvedilol. The generation of intracellular reactive oxygen species in isolated neutrophils decreased in the same rank order, while carvedilol was ineffective. Scavenging of extracellular oxygen radicals followed the rank order of potency: N-feruloylserotonin > curcumin > quercetin > dithiaden. Arbutin and carvedilol had no effect. All compounds tested increased the activity of caspase-3 in cell-free system indicating a positive effect on apoptosis of neutrophils. Activation of protein kinase C was significantly decreased by dithiaden, curcumin, quercetin and N-feruloylserotonin. Carvedilol, dithiaden, quercetin and arbutin reduced activated neutrophil myeloperoxidase release more significantly compared with their less pronounced effect on superoxide generation The presented results are indicative of pharmacological intervention with neutrophils in pathological processes. Of particular interest was the effect of natural compounds. Intracellular inhibition of oxidative burst in isolated neutrophils by the drugs tested and natural antioxidants has to be further analysed since ROS play an important role in immunological responses of neutrophils.

The effects of berberine on reactive oxygen species production in human neutrophils and in cell-free assays.

The health benefits of berberine have been recognized for years. Even so, its effects on human neutrophils, the first line of immune defense, have not been reported. The purpose of this study was to investigate the effects of berberine on the human neutrophil oxidative burst. Reactive oxygen species production was analyzed by luminol-enhanced chemiluminescence. The analysis was performed in spontaneous and stimulated (phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP)) whole blood and isolated neutrophils in the presence or absence of berberine. The effects of berberine on oxidant production in cell-free assays were evaluated using luminescence (H2O2-peroxidase-luminol) and fluorescence (Oxygen Radical Absorbance Capacity – ORAC) techniques. Berberine decreased the production of reactive oxygen species in human whole blood and isolated neutrophils stimulated with either PMA or OZP with a different efficiency (EC50 was 69 μM and 197 μM for PMA and OZP, respectively). The effect was more pronounced in isolated neutrophils. Cell-free assays showed the antioxidant activity of berberine against peroxyl radicals and hydrogen peroxide. Based on our results, we suggest that the effects of berberine on reactive oxygen species production in human neutrophils are due to its antioxidant activity.

Effect of Dark Chocolate Extracts on Phorbol 12-Myristate 13-Acetate-Induced Oxidative Burst in Leukocytes Isolated by Normo-Weight and Overweight/Obese Subjects.

Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD) in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE) on in vitro free radical production, stimulated by phorbol 12-myristate 13-acetate (PMA), in leukocytes extracted from blood of normo-weight and overweight/obese subjects. Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the “stress” condition of the subjects.

6-Hydroxy-5,7-dimethoxy-flavone suppresses the neutrophil respiratory burst via selective PDE4 inhibition to ameliorate acute lung injury

Over-activated neutrophils produce enormous oxidative stress and play a key role in the development of acute and chronic inflammatory diseases. 6-Hydroxy-5,7-dimethoxy-flavone (UFM24), a flavone isolated from the Annonaceae Uvaria flexuosa, showed inhibitory effects on human neutrophil activation and salutary effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. UFM24 potently inhibited superoxide anion (O2•−) generation, reactive oxidants, and CD11b expression, but not elastase release, in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils. However, UFM24 failed to scavenge O2•− and inhibit the activity of subcellular NADPH oxidase. fMLF-induced phosphorylation of protein kinase B (Akt) was inhibited by UFM24. Noticeably, UFM24 increased cyclic adenosine monophosphate (cAMP) concentration and protein kinase (PK) A activity in activated human neutrophils. PKA inhibitors significantly reversed the inhibitory effects of UFM24, suggesting that the effects of UFM24 were through cAMP/PKA-dependent inhibition of Akt activation. Additionally, activity of cAMP-related phosphodiesterase (PDE)4, but not PDE3 or PDE7, was significantly reduced by UFM24. Furthermore, UFM24 attenuated neutrophil infiltration, myeloperoxidase activity, and pulmonary edema in LPS-induced ALI in mice. In conclusion, our data demonstrated that UFM24 inhibits oxidative burst in human neutrophils through inhibition of PDE4 activity. UFM24 also exhibited significant protection against endotoxin-induced ALI in mice. UFM24 has potential as an anti-inflammatory agent for treating neutrophilic lung damage.

Anvillea garcinii extract inhibits the oxidative burst of primary human neutrophils.

Background Anvillea garcinii Coss. & Durieu (Anv) plant is used as a traditional North African medicine against several diseases associated with inflammation. At inflammatory sites, reactive oxygen species (ROS) produced in excess by activated phagocyte NADPH oxidase (NOX2) can accentuate inflammatory responses. Thus, we investigated if Anv-water soluble polysaccharides could modulate primary human neutrophil oxidative burst in vitro.MethodsHuman neutrophils were isolated from fresh whole blood and O2 .- generation was measured by cytochrome c reduction assays. Western blots were used to analyse the translocation of PKC, p47phox (a key component of NOX2 activity) to neutrophil plasma membrane. Also, myeloperoxidase (MPO) release in the extracellular medium was studied by western blots. Flow cytometric analysis was used to detect CD11b membrane expression.ResultsWater soluble polysaccharides from Anv dose-dependently inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and phorbol myristate acetate (PMA)-induced O2 .- generation by human neutrophils. Moreover, Anv-polysaccharides strongly inhibited PMA-induced PKCβ and p47phox translocation to membranes and p47phox phosphorylation on Ser328, a main PKC target. In contrast, polysaccharides extract from Zygophyllum gaetulum plant, which is also used as a traditional North African medicine against inflammatory diseases, was ineffective on this PKCβ-p47phox pathway. Further, Anv inhibited important neutrophil degranulation markers corresponding to myeloperoxidase (MPO) release and CD11b membrane expression.ConclusionThe process of down-regulating NADPH oxidase by polysaccharides extracts from Anv provides new insights into the mechanism of Anv’s anti-inflammatory actions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1411-7) contains supplementary material, which is available to authorized users.

Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils

The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.

Caffeic acid derivatives isolated from the aerial parts of Galinsoga parviflora and their effect on inhibiting oxidative burst in human neutrophils

Four new caffeoyl -glucaric and -altraric acid derivatives along with eleven known compounds were isolated from aerial parts of Galinsonga parviflora. Their structures were elucidated by high-resolution spectroscopic studies. The four new compounds were determined as being 2,3,4,5-tetracaffeoylglucaric acid (1), 2,4,5-tricaffeoylglucaric acid (2), 2,3,4- or 3,4,5-tricaffeoylaltraric acid (3) and 2,3(4,5)-dicaffeoylaltraric acid (4). A reliable criterion for the determination of the linkage position of caffeic acids moieties in glucaric acid derivatives has been proposed, on the basis of detailed analysis of the respective J-couplings, including substitution and solvent influence on the observed values. All hexaric acids derivatives appeared as inhibitors of reactive oxygen species production by stimulated neutrophils.

Low-level laser therapy stimulates the oxidative burst in human neutrophils and increases their fungicidal capacity.

Low-level laser therapy (LLLT) is known to enhance mitochondrial electron transfer and ATP production; thus, this study asked whether LLLT could stimulate the oxidative burst in human neutrophils (PMN) and improve their ability to kill microorganisms. Blood from healthy human subjects was collected and PMN were isolated from the samples. PMN were treated in vitro with 660nm or 780nm CW laser light at 40mW power and increasing energies up to 19.2J and were subsequently incubated with Candida albicans cells. Generation of hydroxyl radicals, hypochlorite anions and superoxide anions by PMN were checked using fluorescent probes and chemiluminescence assays; a microbicidal activity assay against C. albicans was also performed. LLLT excited PMN to a higher functional profile, which was translated as superior production of reactive oxygen species (ROS) and increased fungicidal capacity. The most efficacious energy was 19.2J and, interestingly, the 660nm light was even more efficacious than 780nm at increasing the respiratory burst of PMN and the fungicidal capacity. Human neutrophils (PMN) were stimulated in vitro with 660nm or 780nm CW laser light at 40mW of power and a total energy of 19.2J. Low-level laser therapy (LLLT) excited PMN to a higher functional profile, which was translated as a superior production of reactive oxygen species (ROS) such as hydroxyl radicals (HO• ) and hypochlorite anions (ClO- ) (Figure) and increased fungicidal capacity against Candida albicans cells.

On the pharmacology of oxidative burst of human neutrophils.

The effect of three therapeutically used drugs and five polyphenolic compounds on the mechanism of oxidative burst was compared in whole blood and isolated neutrophils at cellular and molecular level. In 10 microM concentration, the compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin (N-f-5HT) > curcumin (CUR) > quercetin (QUER) > arbutin (ARB) > resveratrol (RES) > dithiaden (DIT) > carvedilol (CARV) > brompheniramine (BPA). The ratio between the percentage inhibition of extracellular versus intracellular chemiluminescence (CL) followed the rank order QUER > N-f-5HT > RES > CUR > DIT and is indicative of the positive effect of the compounds tested against oxidative burst of neutrophils, demonstrating suppression of reactive oxygen species extracellularly with minimal alteration of intracellular reactive oxygen species (ROS). Activation of protein kinase C was significantly decreased by DIT, CUR, QUER and N-f-5HT. CARV, DIT, QUER and ARB reduced activated neutrophil myeloperoxidase release more significantly compared with the effect on superoxide anion generation. All compounds tested increased the activity of caspase-3 in cell-free system. It is suggested that other regulatory mechanisms than protein kinase C might participate in the inhibition of neutrophil activation with the compounds tested. Different mechanisms are concerned in controlling the assembly of NADPH oxidase and the regulatory role of calcium ions is suggested. Compounds decreasing the amount of extracellular ROS generation, yet affecting but minimally intracellular ROS generation, are promising for further investigation in vivo.

Uncovering novel 3-hydroxy-4-pyridinone metal ion complexes with potential anti-inflammatory properties

Ligands of the 3-hydroxy-4-pyridinone (3,4-HPO) type, with one (Hmpp) or two methyl groups (Hdmpp), have been reported to possess biomedical, chemical and analytical applications. In this first screening study aiming to uncover new promising agents to mitigate the oxidative damage highly present in several metabolic disorders, such as diabetes mellitus, we assessed the potential of twelve 3,4-HPO metal ion complexes to modulate oxidative burst in human neutrophils. Metal ion 3,4-HPO complexes of Ni, Fe, V, Co, Cu and Zn were synthesized and tested up to 15μM. Among all the compounds, [Cu(mpp)2] and [Cu(dmpp)2] exhibited the highest scavenging capacity against superoxide radical (O2−) (IC50=0.36±0.07 and 0.30±0.06μM, respectively) and against hypochlorous acid (HOCl) (IC50=0.6±0.3 and 0.4±0.1μM, respectively). In the particular case of O2−, [Fe(mpp)3] and [Fe(dmpp)3] (both at 15μM) presented 35% and 22% of inhibition, respectively, while all the other compounds were neither able to scavenge O2− nor stimulate its production. Regarding the scavenging capacity against hydrogen peroxide (H2O2), all the compounds showed low efficiency (from 6–39%). Finally, with exception of [VO(mpp)2] and [VO(dmpp)2], all compounds exhibited scavenging activity against HOCl (39–81%) and the most efficient compounds were Cu(II) and Zn(II) complexes. Thus, these preliminary results uncover promising new metal ion complexes, inhibitors of neutrophil's oxidative burst, with potential anti-inflammatory properties, which may be seen as an useful strategy for further studies in the treatment of a number of diseases where oxidative damage is a serious issue.

Redox-active biflavonoids from Garcinia brasiliensis as inhibitors of neutrophil oxidative burst and human erythrocyte membrane damage

Ethnopharmacological relevanceGarcinia brasiliensis, a plant native to the Brazilian Amazon Rainforest, is used in traditional medicine to treat inflammation of the urinary tract, peptic ulcers, arthritis and other conditions. Aim of the studyThe purposes of this study were to analyze the chemical constituents of G. brasiliensis branches and leaves and to evaluate the potential of isolated compounds to act as inhibitors of both the oxidative burst of stimulated neutrophils and oxidative damage in human erythrocyte membranes to verify the antioxidant and anti-inflammatory effects of this plant. Materials and methodsNeutrophils were isolated from the blood of healthy donors by Ficoll–Paque density gradient centrifugation. Superoxide anion and total reactive oxygen species (ROS) produced by stimulated neutrophils were measured by WST-1 reduction and luminol-enhanced chemiluminescence assays, respectively. Radical-induced lipoperoxidation and hemolysis were performed using erythrocytes from the blood of healthy donors. Compounds were isolated from G. brasiliensis branches and leaves by HPLC microfractionation, and structure elucidation of the isolated compounds was performed based on NMR and HR-MS analyses. ResultsThe biflavonoids procyanidin, fukugetin, amentoflavone and podocarpusflavone isolated from G. brasiliensis showed potent inhibitory effects on the oxidative burst of human neutrophils, inhibiting ROS production by 50% at 1μmolL−1. These biflavonoids also proved to be potent inhibitors of hemolysis (with 88±7% inhibition at 50µmolL−1 for procyanidin) and lipid peroxidation in human erythrocytes, with a malondialdehyde level (a biomarker of oxidative stress) of 8.5±0.3nmol/mg Hb at 50µmolL−1 for procyanidin. ConclusionsThese findings indicate that the biflavonoids extracted from G. brasiliensis branches and leaves modulate oxidative stress via inhibition of NADPH oxidase and ROS production by stimulated human neutrophils. Furthermore, the biflavonoids exhibited potent inhibition of oxidant hemolysis and lipid peroxidation induced by AAPH in human erythrocytes. Therefore, these studies suggest the use of G. brasiliensis extract as an antioxidant and anti-inflammatory agent.

Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils.

Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function.