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Hyperglycemia Induces Neutrophil Extracellular Traps Formation Through an NADPH Oxidase-Dependent Pathway in Diabetic Retinopathy.

ABSTRACT: Neutrophil extracellular traps (NETs), the product of NETosis, is found to localize pathogens and crystals in immune response. Recent studies have found that excessive NETs lead to disease conditions such as diabetes and its complications like diabetic retinopathy (DR). However, the correlation between NETs and high glucose or DR remains unclear. Here, we found NETs level was significantly increased in the serum of diabetic patients, especially in proliferation diabetic retinopathy (PDR) patients. High glucose dramatically increased NETs production in diabetic individuals with time prolonging. The activation of NADPH oxidase was involved in the NETs process which is triggered by high glucose. Moreover, we verified the infiltration of neutrophils in the eyes and adhesion to vascular endothelial cells in diabetic rat models. NETs formation was observed in the vitreous bodies and retinas of diabetic individuals, which indicates NETs may play a role in the pathogenesis of diabetic retinopathy. Furthermore, anti-VEGF therapy downregulates NETs production indicating that NADPH oxidase-derived ROS may be another signaling pathway involved in anti-VEGF therapy.

SUBMITTER: Wang L

PROVIDER: S-EPMC6331470 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:The inflammatory microenvironment is commonly characterized by extracellular acidosis (pH < 7.35). Sensitivity to pH, CO2 or bicarbonate concentrations allows neutrophils to react to changes in their environment and to detect inflamed areas in the tissue. One important antimicrobial effector mechanism is the production of neutrophil extracellular traps (NETs), which are released during a programmed reactive oxygen species (ROS)-dependent cell death, the so-called NETosis. Although several functions of neutrophils have been analyzed under acidic conditions, the effect of extracellular acidosis on NETosis remains mainly unexplored and the available experimental results are contradictory. We performed a comprehensive study with the aim to elucidate the effect of extracellular acidosis on ROS-dependent NETosis of primary human neutrophils and to identify the underlying mechanisms. The study was performed in parallel in a CO2-bicabonate-buffered culture medium, which mimics in vivo conditions, and under HEPES-buffered conditions to verify the effect of pH independent of CO2 or bicarbonate. We could clearly show that extracellular acidosis (pH 6.5, 6.0, and 5.5) and intracellular acidification inhibit the release of ROS-dependent NETs upon stimulation of neutrophils with phorbol myristate acetate and immobilized immune complexes. Moreover, our findings suggest that the diminished NET release is a consequence of reduced ROS production and diminished glycolysis of neutrophils under acidic conditions. It was suggested previously that neutrophils can sense the border of inflamed tissue by the pH gradient and that a drop in pH serves as an indicator for the progress of inflammation. Following this hypothesis, our data indicate that an acidic inflammatory environment results in inhibition of extracellular operating effector mechanisms of neutrophils such as release of ROS and NETs. This way the release of toxic components and tissue damage can be avoided. However, we observed that major antimicrobial effector mechanisms such as phagocytosis and the killing of pathogens by neutrophils remain functional under acidic conditions.

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Hyperglycemia Induces Neutrophil Extracellular Traps Formation Through an NADPH Oxidase-Dependent Pathway in Diabetic Retinopathy.

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