Neutrophils mediate the early innate immune response through extracellular traps comprising intracellular protein and DNA. These traps play a pivotal role in both immunity against invading pathogens and the development of immunopathological reactions through the production of reactive oxygen species (ROS). Proteins serve as the main target for ROS, resulting in the formation of protein adducts. Herein, we report that the superoxide anion radical (O2˙-) plays a vital role in neutrophil function through sequential events involving 5-lipoxygenase (5-LOX) and NADPH oxidase (NOX). More specifically, differences in NOX homologs expression were observed post-stimulation with PMA and LPS. Differentiation conditions and O2˙- generation were confirmed using flow cytometry. Immunoblotting analysis confirmed the time-dependent expression of NOX underlying its requirement and 5-LOX-mediated lipid peroxidation events in neutrophil function. Protein-malondialdehyde (MDA) adducts formed were detected using immunoblotting, and quercetin was evaluated for its ability to scavenge free radicals through electron paramagnetic resonance (EPR) spin-trapping spectroscopy and results were confirmed with blotting analysis. Free radical-mediated protein oxidation events influence neutrophil function and protein adducts formed serve as markers of neutrophil activation upon infection and inflammation. The study warrants further corroboration and the study of specific proteins involved in neutrophil activation and their role in inflammation.

• Publication type
• Journal Article MeSH

Extracellular vesicles (EVs) are a type of cytoplasmic vesicles secreted by a variety of cells. EVs originating from cells have been known to participate in cell communication, antigen presentation, immune cell activation, tolerance induction, etc. These EVs can also carry the active form of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Hydrogen (NADPH) oxidase, which is very essential for the production of reactive oxygen species (ROS) and that can then modulate processes such as cell regeneration. The aim of this study is to characterize the EVs isolated from U-937 and THP-1 cells, identify the NADPH oxidase (NOX) isoforms, and to determine whether EVs can modulate NOX4 and NOX2 in monocytes and macrophages. In our study, isolated EVs of U-937 were characterized using dynamic light scattering (DLS) spectroscopy and immunoblotting. The results showed that the exogenous addition of differentiation agents (either phorbol 12-myristate 13-acetate (PMA) or ascorbic acid) or the supplementation of EVs used in the study did not cause any stress leading to alterations in cell proliferation and viability. In cells co-cultured with EVs for 72 h, strong suppression of NOX4 and NOX2 is evident when monocytes transform into macrophagic cells. We also observed lower levels of oxidative stress measured using immunoblotting and electron paramagnetic resonance spectroscopy under the EVs co-cultured condition, which also indicates that EVs might contribute significantly by acting as an antioxidant source, which agrees with previous studies that hypothesized the role of EVs in therapeutics. Therefore, our results provide evidence for NOX regulation by EVs in addition to its role as an antioxidant cargo.

• Keywords
• NADPH oxidase, NOX2, NOX4, extracellular vesicles, free radicals, macrophages, monocytes, reactive oxygen species,
• Publication type
• Journal Article MeSH

The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in various aspects of innate immune function, which involves respiratory bursts and inflammasome activation. These reactive species widely distributed within the cellular environment are short-lived intermediates that play a vital role in cellular signaling and proliferation and are likely to depend on their subcellular site of formation. NADPH oxidase complex of phagocytes is known to generate superoxide anion radical (O2 •-) that functions as a precursor for antimicrobial hydrogen peroxide (H2O2) production, and H2O2 is utilized by myeloperoxidase (MPO) to generate hypochlorous acid (HOCl) that mediates pathogen killing. H2O2 modulates the expression of redox-responsive transcriptional factors, namely NF-kB, NRF2, and HIF-1, thereby mediating redox-based epigenetic modification. Survival and function of immune cells are under redox control and depend on intracellular and extracellular levels of ROS/RNS. The current review focuses on redox factors involved in the activation of immune response and the role of ROS in oxidative modification of proteins in macrophage polarization and neutrophil function.

• Keywords
• inflammation, innate immune response, macrophage, neutrophils, oxidative stress, protein oxidation, reactive oxygen species,
• MeSH
• Hypochlorous Acid MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress MeSH
• Hydrogen Peroxide * MeSH
• Immunity, Innate MeSH
• Superoxides * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Hypochlorous Acid MeSH
• Hydrogen Peroxide * MeSH
• Superoxides * MeSH

In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.

• Keywords
• Antioxidants, Human cells, Pro-oxidant, Reactive oxygen species, Vitamin C,
• Publication type
• Journal Article MeSH

Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.

• Keywords
• antioxidants, bioactive compounds, macrophage, malondialdehyde, monocyte, nutraceuticals, phorbol 12-myristate 13-acetate, protein modification, reactive oxygen species, redox reactions,
• MeSH
• Humans MeSH
• Malondialdehyde MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress * MeSH
• Reactive Oxygen Species pharmacology   MeSH
• Free Radicals metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Malondialdehyde MeSH
• Reactive Oxygen Species MeSH
• Free Radicals MeSH