Ultraviolet irradiation increases green fluorescence of dihydrorhodamine (DHR) 123: false-positive results for reactive oxygen species generation

Djiadeu, Pascal; Azzouz, Dhia; Khan, Meraj A.; Kotra, Lakshmi P.; Sweezey, Neil; Palaniyar, Nades

doi:10.1002/prp2.303

Cited by 18 publications

(16 citation statements)

References 19 publications

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“…To determine the source of ROS production during UV-induced NETosis, we studied both types of ROS. hypochlorous acid (HOCl) produced during NOX-dependent NETosis is known to oxidise non-fluorescent DHR123 molecules to green fluorescent R123 molecules 34 . DHR123 plate reader assays showed that PMA, but not UV exposure, induced NOX-dependent ROS production (Fig.…”

Section: Resultsmentioning

confidence: 99%

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis

Azzouz

Khan

Sweezey

et al. 2018

Cell Death Discovery

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NETosis is a unique form of neutrophil death that differs from apoptosis and necrosis. However, whether NETosis and apoptosis can occur simultaneously in the same neutrophil is unknown. In this paper, we show that increasing doses of ultraviolet (UV) irradiation increases NETosis, which is confirmed by myeloperoxidase colocalisation to neutrophil extracellular DNA. Increasing UV irradiation increases caspase 3 activation, mitochondrial reactive oxygen species (ROS) generation and p38, but not ERK, phosphorylation. Inhibition of mitochondrial ROS production and p38 activation, but not NADPH oxidase (NOX) activity, suppresses UV-induced NETosis, indicating that UV induces NOX-independent NETosis. Like classical NOX-dependent and -independent NETosis, UV-induced NETosis requires transcriptional firing for chromatin decondensation. Cell death-specific inhibitor studies indicate that UV-mediated NETosis is not apoptosis, necrosis or necroptosis. Collectively, these studies indicate that increasing doses of UV irradiation induce both apoptosis and NETosis simultaneously, but the ultimate outcome is the induction of a novel form of NOX-independent NETosis, or “ApoNETosis”.

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Section: Resultsmentioning

confidence: 99%

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis

Azzouz

Khan

Sweezey

et al. 2018

Cell Death Discovery

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“…The e − generated by Nox reacts with molecular O 2 to generate superoxide among other ROS, which are important for NETosis ( 15 , 16 ). Therefore, we measured intracellular ROS by DHR123 as previously described ( 5 , 7 , 14 ). Neutrophils were preloaded with DHR123 and activated with or without PMA at six different pH levels.…”

Section: Resultsmentioning

confidence: 99%

“…DHR123 dye (Scientific Inc., MA, USA) was used to measure the intracellular ROS production. The neutrophils were preloaded with DHR123 (20 μM) for 10 min as per manufacturer’s instructions with brief modification as we reported earlier ( 5 , 7 , 14 ). After washing the extracellular DHR123 dye, cells were resuspended in fresh RPMI (10 mM HEPES) media and 50 μL of 50,000 cells, seeded into 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

et al. 2018

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Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

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“…The effect of salt on ROS production by neutrophils was assessed by DHR123 dye (Life Technologies). ROS oxidize DHR123 into R123, which emits a green fluorescence signal ( 36 ); thus, the amount of the green fluorescence signal indicates the amount of ROS production. Cells were preincubated with 25 µM of DHR123 dye for 15 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

et al. 2018

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Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

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Ultraviolet irradiation increases green fluorescence of dihydrorhodamine (DHR) 123: false-positive results for reactive oxygen species generation

Cited by 18 publications

References 19 publications

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

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