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Neutrophils are essential for host defense against infections, but they also play a key role in acute and chronic inflammation. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes the lymphoid-specific tyrosine phosphatase (Lyp) and a genetic single-nucleotide polymorphism of PTPN22 rs2476601 (R620W) has been associated with several human autoimmune diseases, including rheumatoid arthritis (RA). Here, we investigated the role of Lyp in TNFα-induced priming of neutrophil ROS production and in the development of arthritis using new selective Lyp inhibitors. Results show that Lyp-selective inhibitors (IC-11 and compound 8b), inhibited TNFα-induced priming of neutrophil superoxide anion production. TNFα induced an increase of Lyp protein expression in human neutrophils isolated from healthy donors. Key pathways involved in neutrophil priming were investigated. Lyp-selective inhibitors, inhibited TNFα-induced p47phox phosphorylation on Ser345, ERK1/2 phosphorylation and Pin1 activation. Interestingly, Lyp expression was increased in neutrophils isolated from synovial fluid of RA patients and Lyp inhibitors, I-C11 and compound 8b prevented superoxide anion production by endogenously primed neutrophils isolated from synovial fluid of RA. Moreover, IC-11 significantly prevented collagen antibody-induced arthritis in mice. These results show that Lyp expression is increased in inflammatory neutrophils, Lyp is involved in TNFα-induced excessive ROS production by neutrophils and its inhibition protected mice against arthritis. Inhibition of Lyp could be a therapeutic strategy in inflammatory arthritis., Competing Interests: Declaration of competing interest The authors declare no competing conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The increased production of reactive oxygen species (ROS) by human neutrophils can lead to oxidative imbalances and several diseases, such as inflammatory bowel disease (IBD). Opuntia ficus-indica (O. ficus-indica) is rich in bioactive substances with anti-inflammatory properties. This study aimed to identify the bioactive compounds present in aqueous cladodes extract (ACE) of O. ficus-indica using high-performance liquid chromatography (HPLC) and to test its effects on human neutrophil inflammatory functions and on ulcerative colitis (UC) induced by acetic acid (Aa) in rats. ROS production and degranulation by neutrophils were assessed by luminol-amplified chemiluminescence, enzymatic techniques, and western blotting. In vivo, the experiment involved seven groups of rats: a negative control group (NaCl), the acetic acid group (Aa), and groups treated with oral sulfasalazine (150 mg/kg) or various doses of ACE for 7 days. Colonic lesions were induced by an intra-rectal Aa injection, and inflammation was assessed. HPLC analysis identified gallic acid, catechin, caffeic acid, and ferulic acid as major compounds in ACE. In vitro, ACE inhibited neutrophil ROS production, including superoxide anion produced by NADPH oxidase, and significantly reduced myeloperoxidase activity and neutrophil degranulation. In vivo, ACE protected rats from Aa-induced histopathological damage of the colonic mucosa, significantly increased catalase, superoxide dismutase and reduced glutathione levels, and significantly suppressed the increases of plasma cytokines (TNF-α and IL-1β) observed in the Aa group. In conclusion, O. ficus-indica ACE has significant anti-inflammatory properties by restoring oxidative balance, indicating that it could be a potential source of therapeutic agents for inflammatory diseases, particularly UC., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Eugenol (Eug) is a polyphenol extracted from the essential oil of Syzygium aromaticum (L.) Merr. and Perry (Myrtaceae). The health benefits of eugenol in human diseases were proved in several studies. This work aims to evaluate the effect of eugenol on lung inflammatory disorders. For this, using human neutrophils, the antioxidant activity of eugenol was investigated in vitro. Furthermore, a model of LPS-induced lung injury in mice was used to study the anti-inflammatory effect of eugenol in vivo. Results showed that eugenol inhibits luminol-amplified chemiluminescence of resting neutrophils and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLF) peptide or phorbol myristate acetate (PMA). This effect was dose dependent and was significant from a low concentration of 0.1 µg/mL. Furthermore, eugenol inhibited myeloperoxidase (MPO) activity without affecting its degranulation. Eugenol has no scavenging effect on hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 - ). Pretreatment of mice with eugenol prior to the administration of intra-tracheal LPS significantly reduced neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) and decreased total proteins concentration. Moreover, eugenol clearly inhibited the activity of matrix metalloproteinases MMP-2 (21%) and MMP-9 (28%), stimulated by LPS administration. These results suggest that the anti-inflammatory effect of eugenol against the LPS-induced lung inflammation could be exerted via inhibiting myeloperoxidase and metalloproteinases activity. Thus, eugenol could be a promising molecule for the treatment of lung inflammatory diseases.

Ulcerative colitis is an inflammatory bowel disease with a complex aetiology characterised by abnormal immune responses and oxidative stress-induced tissue injury. Inflammatory cells play an important role in the progression of this pathology through the overproduction of reactive oxygen species (ROS) from various sources including the NADPH oxidases (NOXs). The aim of this study was to investigate the preventive effect of apocynin, a natural antioxidant molecule and a selective inhibitor of NOXs, on acetic acid (AA)-induced ulcerative colitis in rats. Our results first confirmed that apocynin has a high free radical scavenging capacity as well as a potent iron chelating ability. Oral pretreatment of rats with apocynin (200 mg/kg and 400 mg/kg) for 7 days prior to AA-induced colitis suppressed the increase in pro-oxidant markers in colonic homogenates and preserved colonic cytoarchitecture from acetic acid-induced damage. Oral administration of apocynin (200 mg/kg and 400 mg/kg) also reduced several systemic inflammatory markers such as alkaline phosphatase, iron, pro-inflammatory cytokines, C-reactive protein and myeloperoxidase. This study shows that apocynin protects rats from acetic acid-induced colonic inflammation and suggests that apocynin may have a promising beneficial effect in the prevention of ulcerative colitis., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

TNFα-mediated signaling pathways play a pivotal role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) by promoting phagocyte inflammatory functions, notably cytokine release and reactive oxygen species (ROS) production by NOX2. In contrast, interleukin-10 (IL-10), a powerful anti-inflammatory cytokine, potently shuts down phagocyte activation, making IL-10 an attractive therapeutic candidate. However, IL-10 therapy has shown limited efficacy in patients with inflammatory diseases. Here, we report that TNFα blocks IL-10 anti-inflammatory pathways in human monocytes, thereby prolonging inflammation. TNFα decreased IL-10-induced phosphorylation of STAT3 and consequently IL-10-induced expression of the major anti-inflammatory factor, SOCS3. Decreased STAT3 phosphorylation was due to a SHP1/2 phosphatase, as NSC-87877, a SHP1/2 inhibitor, restored STAT3 phosphorylation and prevented the TNFα-induced inhibition of IL-10 signaling. TNFα activated only SHP1 in human monocytes and this activation was NOX2-dependent, as diphenyleneiodonium, a NOX2 inhibitor, suppressed SHP1 activation and STAT3 dephosphorylation triggered by TNFα. ROS-induced activation of SHP1 was mediated by the redox-sensitive kinase, Lyn, as its inhibition impeded TNFα-induced SHP1 activation and STAT3 dephosphorylation. Furthermore, H 2 O 2 recapitulated TNFα-inhibitory activity on IL-10 signaling. Finally, NSC-87877 dampened collagen antibody-induced arthritis (CAIA) in mice. These results reveal that TNFα disrupts IL-10 signaling by inducing STAT3 dephosphorylation through a NOX2-ROS-Lyn-SHP1 axis in human monocytes and that inhibition of SHP1/2 in vivo protects against CAIA. These new findings might explain the poor efficacy of IL-10 therapy in patients with inflammatory diseases and suggest that anti-TNFα agents and SHP1/2 inhibitors could improve the therapeutic use of IL-10., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Inflammation is the body's response to insults, for instance, lung inflammation is generally caused by pathogens or by exposure to pollutants, irritants and toxins. This process involves many inflammatory cells such as epithelial cells, monocytes, macrophages and neutrophils. These cells produce and release inflammatory mediators such as pro-inflammatory cytokines, lipids and reactive oxygen species (ROS). Lung epithelial cells and phagocytes (monocytes, macrophages and neutrophils) produce ROS mainly by the NADPH oxidase NOX1 and NOX2, respectively. The aim of this study was to investigate the effects of two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium (DPI), on lipopolysaccharide (LPS)-induced lung inflammation in rats. Our results showed that apocynin and DPI attenuated the LPS-induced morphological and histological alterations of the lung, reduced edema and decreased lung permeability. The evaluation of oxidative stress markers in lung homogenates showed that apocynin and DPI inhibited LPS-induced NADPH oxidase activity, and restored superoxide dismutase (SOD) and catalase activity in the lung resulting in the reduction in LPS-induced protein and lipid oxidation. Additionally, apocynin and DPI decreased LPS-induced MPO activity in bronchoalveolar liquid and lung homogenates, TNF-α and IL-1β in rat plasma. NADPH oxidase inhibition could be a new therapeutic strategy for the treatment of inflammatory lung diseases.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47 PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox-/- CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox-/- CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation., (© 2022 by The American Society of Hematology.)

Metformin (1,1-dimethylbiguanide hydrochloride) is the most commonly used drug to treat type II diabetic patients. It is believed that this drug has several other beneficial effects, such as anti-inflammatory and anticancer effects. Here, we wanted to evaluate the effect of metformin on the production of reactive oxygen species (ROS) by human macrophages. Macrophages are generated in vivo from circulating monocytes depending on the local tissue environment. In vitro proinflammatory macrophages (M1) and anti-inflammatory macrophages (M2) can be generated by culturing monocytes in the presence of different cytokines, such as GM-CSF or M-CSF, respectively. We show that metformin selectively inhibited human monocyte differentiation into proinflammatory macrophages (M1) without inhibiting their differentiation into anti-inflammatory macrophages (M2). Moreover, we demonstrate that, in response to LPS, M2 macrophages produced ROS, which could be very harmful for nearby tissues, and metformin inhibited this process. Interestingly, metformin with LPS induced activation of the adenosine-monophosphate-activated protein kinase (AMPK) and pharmacological activation of AMPK by AICAR, a known AMPK activator, decreased ROS production, whereas the deletion of AMPK in mice dramatically enhanced ROS production in different types of immune cells. These results suggest that metformin exhibits anti-inflammatory effects by inhibiting the differentiation of human monocytes into M1 macrophages and by limiting ROS production by macrophages via the activation of AMPK.

Background & Aims: NADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process., Methods: The NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed., Results: We identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid-induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage., Conclusions: The ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophils are key cells of the innate immune and inflammatory responses. They are the first blood cells to migrate to the infection site where they release high amounts of reactive oxygen species (ROS) and several peptides and enzymes required for microbial killing. However, excessive neutrophil activation can induce tissue injury participating in inflammation, thus the characterization of the enzymes involved in neutrophil activation could help to identify new pharmacological targets to treat inflammation. The prolyl-isomerase Pin1 is a ubiquitous enzyme involved in several functions, however, its role in neutrophil functions is less known. In this study, we show that the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP or fMLF), a G-protein coupled receptor (GPCR) agonist-induced Pin1 activation in human neutrophils. PiB and juglone, two Pin1 inhibitors inhibited Pin1 activity in neutrophils and consequently inhibited fMLP-induced chemotaxis and -degranulation of azurophil and specific granules as measured by myeloperoxidase and neutrophil gelatinase-associated lipocalin (NGAL) release respectively. We also showed that PiB inhibited TNFα + fMLP-induced superoxide production, confirming the effect of juglone. These data show that inhibitors of Pin1 impaired key pro-inflammatory neutrophil functions elicited by GPCR activation and suggest that Pin1 could control neutrophil inflammatory functions.

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function., Competing Interests: Competing interests: The authors declare that they have no competing interest.

Polymorphonuclear neutrophils (PMNs) are the first phagocyte recruited and infected by Leishmania . They synthetize superoxide anions (O 2 - ) under the control of the NADPH oxidase complex. In Morocco, Leishmania major and L. tropica are the main species responsible for cutaneous leishmaniasis (CL). The impact of these parasites on human PMN functions is still unclear. We evaluated the in vitro capacity of primary Moroccan strains of L. major and L. tropica to modulate PMN O 2 - production and p47 phox phosphorylation status of the NADPH oxidase complex. PMNs were isolated from healthy blood donors, and their infection rate was measured by microscopy. O 2 - production was measured by superoxide dismutase-inhibitable reduction of cytochrome C. P47 phox phosphorylation was analyzed by Western blot using specific antibodies against Ser328 and Ser345 sites. Whereas we did not observe any difference in PMN infectivity rate, our results indicated that only L. tropica promastigotes inhibited both fMLF- and PMA-mediated O 2 - production independently of p47 phox phosphorylation. Leishmania soluble antigens (SLAs) from both species significantly inhibited O 2 - induced by fMLF or PMA. However, they only decreased PMA-induced p47phox phosphorylation. L. major and L. tropica modulated differently O 2 - production by human PMNs independently of p47 phox phosphorylation. The inhibition of ROS production by L. tropica could be a mechanism of its survival within PMNs that might explain the reported chronic pathogenicity of L. tropica CL.

Early leaf spot (ELS) and late leaf spot (LLS) are major fungal diseases of peanut that can severely reduce yield and quality. Development of acceptable genetic resistance has been difficult due to a strong environmental component and many major and minor QTLs. Resistance genes (R-genes) are an important component of plant immune system and have been identified in peanut. Association of specific R-genes to leaf spot resistance will provide molecular targets for marker-assisted breeding strategies. In this study, advanced breeding lines from different pedigrees were evaluated for leaf spot resistance and 76 candidate R-genes expression study was applied to susceptible and resistant lines. Thirty-six R-genes were differentially expressed and significantly correlated with resistant lines, of which a majority are receptor like kinases (RLKs) and receptor like proteins (RLPs) that sense the presence of pathogen at the cell surface and initiate protection response. The largest group was receptor-like cytoplasmic kinases (RLCKs) VII that are involved in pattern-triggered kinase signaling resulting in the production reactive oxygen species (ROS). Four R-genes were homologous to TMV resistant protein N which has shown to confer resistance against tobacco mosaic virus (TMV). When mapped to peanut genomes, 36 R-genes were represented in most chromosomes except for A09 and B09. Low levels of gene-expression in resistant lines suggest expression is tightly controlled to balance the cost of R-gene expression to plant productively. Identification and association of R-genes involved in leaf spot resistance will facilitate genetic selection of leaf spot resistant lines with good agronomic traits.

Superoxide anion production by neutrophils is essential for host defense against microbes. Superoxide anion generates other reactive oxygen species (ROS) that are very toxic for microbes and host cells, therefore their excessive production could induce inflammatory reactions and tissue injury. Cyclic adenosine monophosphate (cAMP) elevating agents are considered to be physiological inhibitors of superoxide production by neutrophils but the mechanisms involved in this inhibitory effect are poorly understood. Superoxide is produced by the phagocyte NADPH oxidase, a complex enzyme composed of two membrane subunits, gp91 phox or NOX2 and p22 phox , and four cytosolic components p47 phox , p67 phox , p40 phox , and Rac2. Except Rac2, these proteins are known to be phosphorylated upon neutrophil stimulation. Here we show that forskolin, an activator of the adenylate cyclase-cAMP-PKA pathway, induced phosphorylation of gp91phox/NOX2 and inhibited fMLF-induced NADPH oxidase activation in human neutrophils. H89, a PKA inhibitor prevented the forskolin-induced phosphorylation of gp91phox and restored NADPH oxidase activation. Furthermore, PKA phosphorylated the recombinant gp91phox/NOX2-cytosolic C-terminal region in vitro only on a few specific peptides containing serine residues, as compared to PKC. Interestingly, phosphorylation of NOX2-Cter by PKA alone did not induce interaction with the cytosolic components p47 phox , p67 phox and Rac2, however it induced inhibition of PKC-induced interaction. Furthermore, PKA alone did not induce NOX2 electron transfer activity, however it inhibited PKC-induced activation. These results suggest that PKA phosphorylates NOX2 in human neutrophils, a process essential to limit ROS production and inflammation under physiological conditions. Our data identify the cAMP-PKA-NOX2-axis as a critical gatekeeper of neutrophil ROS production., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, neutrophil function may be severely altered in conditions of iron overload, as observed in chronically transfused patients. Therefore, a tight regulation of neutrophil iron homeostasis seems to be critical for avoiding iron toxicity. Hepcidin is the key iron regulator in organisms; however, no studies have investigated its role in maintaining neutrophil iron homeostasis or characterized neutrophil function in patients with hereditary hemochromatosis (HH), a common iron overload genetic disorder that results from a defect in hepcidin production. To explore these issues, we studied 2 mouse models of iron overload: an experimentally induced iron overload model (EIO), in which hepcidin is increased, and a genetic HH model of iron overload with a deletion of hepatic hepcidin. We found that iron-dependent increase of hepatic hepcidin results in neutrophil intracellular iron trapping and consecutive defects in oxidative burst activity. In contrast, in both HH mouse models and HH patients, the lack of hepcidin expression protects neutrophils from toxic iron accumulation. Moreover, systemic iron overload correlated with a surprising neutrophil priming and resulted in a more powerful oxidative burst. Indeed, important factors in neutrophil priming and activation, such as tumor necrosis factor α (TNF-α), VCAM-1, and ICAM-1 are increased in the plasma of HH patients and are associated with an increase in HH neutrophil phagocytosis capacity and a decrease in L-selectin surface expression. This is the first study to characterize neutrophil iron homeostasis and associated functions in patients with HH., (© 2020 by The American Society of Hematology.)

Significance: Despite their intrinsic cytotoxic properties, mounting evidence indicates that reactive oxygen species (ROS) physiologically produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) of epithelial cells (NOX1, dual oxidase [DUOX]2) and phagocytes (NOX2) are critical for innate immune response and homeostasis of the intestinal mucosa. However, dysregulated ROS production could be a driving factor in inflammatory bowel diseases (IBDs). Recent Advances: In addition to NOX2, recent studies have demonstrated that NOX1- and DUOX2-derived ROS can regulate intestinal innate immune defense and homeostasis by impacting many processes, including bacterial virulence, expression of bacteriostatic proteins, epithelial renewal and restitution, and microbiota composition. Moreover, the antibacterial role of DUOX2 is a function conserved in evolution as it has been described in invertebrates, and lower and higher vertebrates. In humans, variants of the NOX2 , NOX1 , and DUOX2 genes, which are associated with impaired ROS production, have been identified in very early onset IBD, but overexpression of NOX/DUOX, especially DUOX2, has also been described in IBD, suggesting that loss-of-function or excessive activity of the ROS-generating enzymes could contribute to disease progression. Critical Issues: Therapeutic perspectives aiming at targeting NOX/DUOX in IBD should take into account the two sides of NOX/DUOX-derived ROS in intestinal inflammation. Hence, NOX/DUOX inhibitors or ROS inducers should be considered as a function of the disease context. Future Directions: A thorough understanding of the physiological and pathological regulation of NOX/DUOX in the gastrointestinal tract is an absolute pre-requisite for the development of therapeutic strategies that can modulate ROS levels in space and time.

Phagocytes, especially neutrophils, can produce reactive oxygen species (ROS), through the activation of the NADPH oxidase (NOX2). Although this enzyme is crucial for host-pathogen defense, ROS production by neutrophils can be harmful in several pathologies such as cardiovascular diseases or chronic pulmonary diseases. The ROS production by NOX2 involves the assembly of the cytosolic subunits (p67 phox , p47 phox , and p40 phox ) and Rac with the membrane subunits (gp91 phox and p22 phox ). Many studies are devoted to the activation of NOX2. However, the mechanisms that cause NADPH oxidase deactivation and thus terminate ROS production are not well known. Here we investigated the ability of class I phosphoinositide 3-kinases (PI3Ks) to sustain NADPH oxidase activation. The NADPH oxidase activation was triggered by seeding neutrophil-like PLB-985 cells, or human neutrophils on immobilized fibrinogen. Adhesion of the neutrophils, mediated by β2 integrins, induced activation of the NADPH oxidase and translocation of the cytosolic subunits at the plasma membrane. Inhibition of class I PI3Ks, and especially PI3Kβ, terminated ROS production. This deactivation of NOX2 is due to the release of the cytosolic subunits, p67 phox and p47 phox from the plasma membrane. Overexpression of an active form of Rac 1 did not prevent the drop of ROS production upon inhibition of class I PI3Ks. Moreover, the phosphorylation of p47 phox at S328, a potential target of kinases activated by the PI3K pathway, was unchanged. Our results indicate that the experimental downregulation of class I PI3K products triggers the plasma membrane NADPH oxidase deactivation. Release of p47 phox from the plasma membrane may involve its PX domains that bind PI3K products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neutrophils are key cells in innate immunity and inflammation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to enhance many neutrophil functions such as reactive oxygen species (ROS) production, degranulation and cell survival via the activation of the ERK1/2 pathway. ERK1/2 pathway activation is redox sensitive and could be modulated by ROS. In order to investigate whether NADPH oxidase NOX2-derived ROS could contribute to GM-CSF-induced ERK1/2 phosphorylation, we tested the effect of two selective NOX2 inhibitors, diphenylene iodonium (DPI) and apocynin. Results showed that, while both DPI and apocynin strongly inhibited neutrophil ROS production, only apocynin, but not DPI, inhibited GM-CSF-induced ERK1/2 phosphorylation, suggesting that ROS are not involved in this process. Apocynin did not affect GM-CSF-induced p38MAPKinase phosphorylation, another redox sensitive kinase. Interestingly, apocynin inhibited GM-CSF-induced MEK1/2 and AKT phosphorylation without affecting fMLF-induced phosphorylation of these proteins. GM-CSF is known to inhibit neutrophils apoptosis and to promote cell survival via the AKT-ERK1/2 pathway. In this regard, we found that apocynin also inhibited GM-CSF-induced anti-apoptotic effect in neutrophils. These results suggest that NADPH oxidase NOX2-derived ROS are not involved in GM-CSF-induced ERK1/2 phosphorylation and that apocynin inhibits GM-CSF-induced ERK1/2 phosphorylation pathway independently of its inhibitory action on NADPH oxidase NOX2. Thus, apocynin can exert an anti-inflammatory effect not only by limiting neutrophil ROS production but also by decreasing neutrophil survival at inflammatory site., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Excessive reactive oxygen species (ROS) production can induce tissue injury involved in a variety of neurodegenerative disorders such as neurodegeneration observed in pilocarpine-induced temporal lobe epilepsy. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist has beneficial effects in pilocarpine-induced temporal lobe epilepsy, when administered within minutes of seizure to avoid the harmful neurological lesions induced by pilocarpine. However, the enzymes involved in ROS productions and the effect of ketamine on this process remain less documented. Here we show that during pilocarpine-induced epilepsy in mice, the expression of the phagocyte NADPH oxidase NOX2 subunits (NOX2/gp91 phox , p22 phox , and p47 phox ) and the expression of myeloperoxidase (MPO) were dramatically increased in mice brain treated with pilocarpine. Interestingly, treatment of mice with ketamine before or after pilocarpine administration decreased this process, mainly when injected before pilocarpine. Finally, our results showed that pilocarpine induced p47 phox phosphorylation and H 2 O 2 production in mice brain and ketamine was able to inhibit these processes. Our results show that pilocarpine induced NOX2 activation to produce ROS in mice brain and that administration of ketamine before or after the induction of temporal lobe epilepsy by pilocarpine inhibited this activation in mice brain. These results suggest a key role of the phagocyte NADPH oxidase NOX2 and MPO in epilepsy and identify a novel effect of ketamine.

Neutrophils play a pivotal role in innate immunity and in the inflammatory reactions. Upon activation, neutrophils release several toxic molecules directed against microbial pathogens into the phagosome. These molecules include reactive oxygen species (ROS), myeloperoxidase, glucosidases, proteases, and antibacterial peptides. In resting cells these proteins and the enzyme responsible for ROS production (NOX2) are stored inside or at the membranes of different granules called azurophil or primary, specific or secondary, gelatinase or tertiary, and the secretory vesicles. Each granule has a specific density, content, and markers. Myeloperoxidase (MPO) is the azurophil granule marker, and the neutrophil-gelatinase-associated lipocalin (NGAL) is the specific granule marker. After cell activation by different stimuli, granule contents are released into the phagosome or in the extracellular space through a process called degranulation. Also during this process, membrane granules fuse with the phagosome and plasma membrane allowing expression of new markers at the cell surface. The degranulation can be assessed by measuring either the release of different proteins by neutrophils or the expression of granule markers at the plasma membrane. In this chapter, we describe the techniques used to measure degranulation of azurophil and specific neutrophil granules using different approaches such as measurement of MPO enzymatic activity and detection of MPO and NGAL proteins by SDS-PAGE and Western blot.

Eugenol is a polyphenol extracted from Syzygium aromaticum essential oil. It is known to have anti-inflammatory and chemoprotective properties as well as a potent anti-oxidant activity due the presence of its phenolic group. In this study, we examined the effects of eugenol on neutrophil superoxide production, a key process involved in innate immunity and inflammation. Superoxide anion generationin human neutrophils was measured by cytochrome c reduction assay. Western blotting was used to analyze the phosphorylation of, p47phox, MAPKinases (p38 and ERK1/2), MEK1/2 and Raf, key proteins involved in the activation of NADPH oxidase. Pretreatment of neutrophils by increasing concentrations (2.5 µg/mL-20 µg/mL) of eugenol for 30 min, inhibited significantly (p < 0.001) superoxide anion generation induced by the chemotactic peptide formyl-Met-Leu-Phe (fMLF) with an IC50 of 5 µg/mL. Phorbolmyristate acetate (PMA)-stimulated O2 - production was affected only at the highest eugenol concentration (20 µg/mL). Results showed that eugenol decreased the phosphorylation of p47phox onSer-345 and Ser-328, the translocation of p47phox to the membranesand the phosphorylation of Raf, MEK1/2 and ERK1/2 proteins. Taken together, our results suggest that eugenol inhibits the generation of superoxide anion by neutrophils via the inhibition of Raf/MEK/ERK1/2/p47phox-phosphorylation pathway.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology ( PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy., (© 2019 Ohayon et al.)

Production of superoxide anion and other reactive oxygen species (ROS) by neutrophils has a vital role in host defense against microbes. However, over-production can induce cell injury participating to inflammation. Superoxide anion is produced by the phagocyte NADPH oxidase/NOX2, a multicomponent enzyme system consisting of six proteins: two trans-membrane proteins (gp91 phox and p22 phox ) and four soluble cytosolic proteins (p40 phox , p67 phox , p47 phox , and the small G-proteins, Rac1/2). Phosphorylation of p47 phox on several serines regulates NADPH oxidase activation. LPS released by gram negative bacteria can enhance or prime neutrophil superoxide production in combination with other agonists such as the bacterial peptide formyl-Met-Leu-Phe (fMLP). Since the pathways involved in LPS-induced priming are not completely understood, we investigated the role of the prolyl cis/trans isomerase Pin1 in this process. Two different Pin1 inhibitors, PiB, and Juglone are able to block LPS-induced priming of ROS production by human neutrophils in a concentration dependent manner. PiB and Juglone did not inhibit LPS-induced CD11b translocation neither CD62L shedding. LPS induced an increase of Pin1 activity in neutrophils similar to TNFα and fMLP. Since the phosphorylation of p47 phox on Ser345 is critical for NADPH oxidase up-regulation, we investigated the effect of LPS on this process. Results show that LPS induced the phosphorylation of p47 phox mainly on serine 345 and induced the activation of p38MAPKinase and ERK1/2. These results suggest that the prolyl cis/trans isomerase Pin1 may control LPS-induced priming of superoxide production in human neutrophils. Pharmacological targeting of Pin1 could be a valuable approach in sepsis., (Copyright © 2019 Liu, Bedouhene, Hurtado-Nedelec, Pintard, Dang, Yu and El-Benna.)

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Peanut (Arachis hypogaea L.) is an important food and oilseed crop worldwide. Yield and quality can be significantly reduced by foliar fungal diseases, such as early and late leaf spot diseases. Acceptable levels of leaf spot resistance in cultivated peanut have been elusive due to environmental interactions and the proper combination of QTLs in any particular peanut genotype. Resistance gene analogs, as potential resistance (R)-genes, have unique roles in the recognition and activation of disease resistance responses. Novel R-genes can be identified by searches for conserved domains such as nucleotide binding site, leucine rich repeat, receptor like kinase, and receptor like protein from expressed genes or through genomic sequences. Expressed R-genes represent necessary plant signals in a disease response. The goals of this research are to identify expressed R-genes from cultivated peanuts that are naturally infected by early and late spot pathogens, compare these to the closest diploid progenitors, and evaluate specific gene expression in cultivated peanuts. Putative peanut R-genes (381) were available from a public database (NCBI). Primers were designed and PCR products were sequenced. A total of 214 sequences were produced which matched to proteins with the corresponding R-gene motifs. These R-genes were mapped to the genome sequences of Arachis duranensis and Arachis ipaensis, which are the closest diploid progenitors for tetraploid cultivated peanut, A. hypogaea. Identification and association of specific gene-expression will elucidate potential disease resistance mechanism in peanut and may facilitate the selection of breeding lines with high levels of leaf spot resistance.

The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22 phox and gp91 phox (renamed NOX2), which form the cytochrome b 558 , and four cytosolic proteins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32 P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91 phox /NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22 PHOX , and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme., (© 2018 Stichting European Society for Clinical Investigation Journal Foundation.)

Pseudomonas aeruginosa ( P.a ) is a pathogen causing significant morbidity and mortality, in particular, in hospital patients undergoing ventilation and in patients with cystic fibrosis. Among the virulence factors secreted or injected into host cells, the physiopathological relevance of type II secretions system (T2SS) is less studied. Although there is extensive literature on the destructive role of LasB in vitro on secreted innate immune components and on some stromal cell receptors, studies on its direct action on myeloid cells are scant. Using a variety of methods, including the use of bacterial mutants, gene-targeted mice, and proteomics technology, we show here, using non-opsonic conditions (thus mimicking resting and naïve conditions in the alveolar space), that LasB, an important component of the P.a T2SS is highly virulent in vivo , and can subvert alveolar macrophage (AM) activity and bacterial killing, in vitro and in vivo by downregulating important secreted innate immune molecules (complement factors, cytokines, etc.) and receptors (IFNAR, Csf1r, etc.). In particular, we show that LasB downregulates the production of C3 and factor B complement molecules, as well as the activation of reactive oxygen species production by AM. In addition, we showed that purified LasB impaired significantly the ability of AM to clear an unrelated bacterium, namely Streptococcus pneumoniae . These data provide a new mechanism of action for LasB, potentially partly explaining the early onset of P.a , alone, or with other bacteria, within the alveolar lumen in susceptible individuals, such as ventilated, chronic obstructive pulmonary disease and cystic fibrosis patients.

Purpose: Oleuropein and hydroxytyrosol are polyphenols that are extracted from olives and are major biological active components of olives and olive oil. Oleuropein and hydroxytyrosol exhibit interesting pharmacological effects on cells, and have been shown to have many health benefits such as anti-inflammatory effects. These effects were mainly attributed to their ability to scavenge the reactive oxygen species (ROS) produced by phagocytes such as neutrophils. The aim of this study was to investigate the effect of oleuropein and hydroxytyrosol on other neutrophil functions., Methods: Human neutrophils were isolated from healthy donors. ROS production was measured by luminol-amplified chemiluminescence. Degranulation was assessed by measuring myeloperoxidase activity and Western blots. Chemotaxis was assessed by the under-agarose chemotaxis assay. Phosphorylated proteins were assessed by gel electrophoresis and Western blots., Results: We show that in addition to their ROS scavenging effect, oleuropein and hydroxytyrosol significantly inhibited the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF)-induced degranulation of azurophilic and specific granules as measured by myeloperoxidase and lactoferrin release, respectively. We also show that oleuropein and hydroxytyrosol reduced fMLF-induced neutrophil chemotaxis. Interestingly, both agents impaired the fMLF-induced AKT, p38MAPKinase, and ERK1/2 phosphorylation, signaling molecules that are involved in pathways regulating neutrophil functions., Conclusion: Our data suggest that the anti-inflammatory properties of oleuropein and hydroxytyrosol are not only restricted to their ROS scavenging effect, but also involve the inhibition of two other major pro-inflammatory neutrophil functions.

Background: Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease caused by protozoa of the genus Leishmania. In Morocco, CL is a public health problem mainly caused by Leishmania major and Leishmania tropica, which are responsible for zoonotic and anthroponotic CL, respectively. Macrophages are the primary cells infected by Leishmania parasites and their capacity to produce nitric oxide (NO) is of critical importance for parasite elimination. To our knowledge, the role of NO on autochthonous infections has never been investigated before. In this study, we evaluated in vitro the capacity of autochthonous primary dermotropic strains of L. major and L. tropica to modulate NO production by J774-macrophages and determine the sensitivity of both species to exogenous NO., Methods: The infectivity of the J774 cell line was analyzed by optical microscopy. NO production by macrophages was measured by the Griess method. The sensitivity to NO by the two strains was assessed by the MTT assay using NO donors., Results: Our results show that the percentage of infected macrophages and the average number of parasites per macrophage were similar for L. major and L. tropica strains. While L. tropica significantly inhibited NO production induced by LPS and IFN-γ stimulation in J774 macrophages, L. major did not affect it. However, soluble Leishmania antigens (SLAs) from both autochthonous primary strains significantly inhibited the production of NO by J774-macrophages in a dose-dependent manner. Finally, our results demonstrated that promastigotes and amastigotes from both strains are sensitive to SNAP NO donor in a dose-dependent manner, although L. tropica demonstrated an increased sensitivity., Conclusions: Our results suggest a differential ability of L. major and L. tropica strains to modulate the capacity of macrophages to produce NO. The increased ability of L. tropica to inhibit NO production by macrophages might come as a necessity due to its higher sensitivity to NO donor. Our results provide one explanation for the tendency of L. tropica to cause chronic lesions and may contribute to the different physiopathology of CL in Morocco.

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system, acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study, we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox, and not on gp91phox/NOX2, as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover, we revealed that NOX5 expression was strongly increased during Mo-DC differentiation, but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC, and at a lower level in plasmacytoid DC. Interestingly, NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation, and thus could be critical for immunity and inflammation., (© 2017 by The American Society of Hematology.)

Reactive oxygen species (ROS) are produced by numerous biological systems and by several phagocytes such as neutrophils and macrophages. ROS include mostly superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radical, which are involved in a variety of biological processes such as immunity, inflammation, apoptosis and cell signaling. Thus, there is a need for a sensitive and reliable method to measure ROS. The luminol-amplified chemiluminescence technique is widely used to measure ROS production by neutrophils; however, it is unclear which ROS species are detected by this technique. In this study, we show that Xanthine/Xanthine oxidase (XXO), a known superoxide-producing system, stimulated a luminol-amplified chemiluminescence in the absence of horseradish peroxidase (HRPO), while the presence of HRPO enhanced the response. Both reactions were inhibited by superoxide dismutase (SOD), but not by catalase, confirming that superoxide anion, and not hydrogen peroxide, is the species oxidizing luminol to produce chemiluminescence. Glucose/Glucose oxidase (GGO), a known hydrogen peroxide-producing system, did not induce luminol-amplified chemiluminescence in the absence of HRPO; however, addition of HRPO resulted in a chemiluminescence response, which was inhibited by catalase, but not by SOD. Myeloperoxidase (MPO), isolated from human neutrophils, was also able to enhance the superoxide- and hydrogen peroxide-dependent luminol-amplified chemiluminescence. The production of ROS by stimulated human neutrophils was detected by luminol-amplified chemiluminescence, which was only partially inhibited by SOD and catalase. Interestingly, adding HRPO to stimulated neutrophils increased the luminol-amplified chemiluminescence, which was strongly inhibited by SOD, but not by catalase. These results show that (a) luminol-amplified chemiluminescence is able to detect superoxide anion in the absence of peroxidases, but not hydrogen peroxide; (b) in the presence of peroxidases, luminol-amplified chemiluminescence is able to detect both superoxide anion and hydrogen peroxide; and, Competing Interests: None., ((c) luminol-amplified chemiluminescence detects mainly superoxide anion produced by neutrophils, especially in the presence of HRPO.)

Aflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B 1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47 phox , p67 phox , p40 phox , and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA 11,22,28 ]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47 phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47 phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called 'the respiratory burst', and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with 'priming' agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Peanut is one of the most important oil crops worldwide. We used insertion-deletion (InDel) markers to assess the genetic diversity and population structure in cultivated peanut. Fifty-four accessions from North China were genotyped using 48 InDel markers. The markers amplified 61 polymorphic loci with 1 to 8 alleles and an average of 2.6 alleles per marker. The polymorphism information content values ranged from 0.0364 to 0.9030, with an average of 0.5038. Population structure and neighbor-joining (NJ) tree analyses suggested that all accessions could be divided into four clusters (A1-A4), using the NJ method. Likewise, four subpopulations (G1-G4) were identified using STRUCTURE analysis. A principal component analysis was also used and results concordant with the other analysis methods were found. A multi-linear stepwise regression analysis revealed that 13 InDel markers correlated with five measured agronomical traits. Our results will provide important information for future peanut molecular breeding and genetic research.

Polymorphonuclear neutrophils are key players in host defense against pathogens through the robust production of superoxide anion by the NADPH oxidase and the release of antibacterial proteins from granules. However, inappropriate release of these agents in the extracellular environment induces severe tissue injury, thereby contributing to the physiopathology of acute and chronic inflammatory disorders. Many studies have been carried out to identify molecules capable of inhibiting phagocyte functions, in particular superoxide anion production, for therapeutic purposes. In the present study, we show that thymoquinone (TQ), the major component of the volatile oil from Nigella sativa (black cumin) seeds strongly inhibits fMLF-induced superoxide production and granules exocytosis in neutrophils. The inhibition of superoxide anion was not due to a scavenger effect, as TQ did not inhibit superoxide anion produced by the xanthine/xanthine oxidase system. Interestingly, TQ impaired the phosphorylation on Ser-304 and Ser-328 of p47(PHOX), a cytosolic subunit of the NADPH oxidase. TQ also attenuated specific and azurophilic granule exocytosis in fMLF-stimulated neutrophils as evidenced by decreased cell surface expression of gp91(PHOX) and CD11b, and release of myeloperoxidase. Furthermore, both the PKC and MAPK pathways, which are involved in p47(PHOX) phosphorylation and granules exocytosis, respectively, were inhibited by TQ in fMLF-stimulated neutrophils. Finally, in a model of pleurisy induced by λ-carrageenan in rats, TQ reduced neutrophil accumulation in the pleural space, showing that it not only inhibits PMN functions in vitro, but also exhibits anti-inflammatory properties in vivo. Thus, TQ possesses promising anti-inflammatory therapeutic potential., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, reactive oxygen species (ROS) are produced by different systems which are involved in endothelial cells injury, ultimately leading to severe organ dysfunctions. The aim of this work was to study the effect of ROS produced by hypoxanthine-xanthine oxidase (Hx-XO) on the adhesion of human umbilical vein endothelial cells (HUVEC) and on the signaling pathways involved. Results show that Hx-XO-derived ROS induced an increase in HUVEC adhesion in the early stages of the process (less than 30 min), followed by a decrease in adhesion in the later stages of the process. Interestingly, Hx-XO-derived ROS induced the same biphasic effect on the phosphorylation of the focal adhesion kinase (FAK), a nonreceptor tyrosine kinase critical for cell adhesion, but not on ERK1/2 phosphorylation. The biphasic effect was not seen with ERK1/2 where a decrease in phosphorylation only was observed. Wortmannin, a PI3-kinase inhibitor, inhibited ROS-induced cell adhesion and FAK phosphorylation. Orthovanadate, a protein tyrosine phosphatase inhibitor, and Resveratrol (Resv), an antioxidant agent, protected FAK and ERK1/2 from dephosphorylation and HUVEC from ROS-induced loss of adhesion. This study shows that ROS could have both stimulatory and inhibitory effects on HUVEC adhesion and FAK phosphorylation and suggests that PI3-kinase and tyrosine phosphatase control these effects.

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p ≤ 0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng · g(-1) of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng · g(-1). This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.