Activation Of Human Neutrophils Research Articles

Montelukast, Leukotriene Inhibitor, Reduces LPS-Induced Acute Lung Inflammation and Human Neutrophil Activation

ObjectivesSome pro-inflammatory lipids derived from 1 lipooxygenase enzyme are potent neutrophil chemoattractant, a cell centrally involved in acute respiratory distress syndrome (ARDS); a syndrome lacking effective treatment. Considering the beneficial effects of the leukotriene receptor inhibitor, montelukast, on other lung diseases, whether montelukast attenuates inflammation in a mouse model of ARDS, and whether it reduces LPS stimulated activation of human neutrophils was investigated. MethodsThirty-five C57Bl/6 mice were distributed into control (PBS)+24h, LPS+24h (10μg/mouse), control+48h, LPS+48h, and LPS 48h+Montelukast (10mg/kg). In addition, human neutrophils were incubated with LPS (1μg/mL) and treated with montelukast (10μM). ResultsOral-tracheal administration of montelukast significantly attenuated total cells (P<.05), macrophages (P<.05), neutrophils (P<.01), lymphocytes (P<.001) and total protein levels in BAL (P<.05), as well as IL-6 (P<.05), CXCL1/KC (P<.05), IL-17 (P<.05) and TNF-α (P<.05). Furthermore, montelukast reduced neutrophils (P<.001), lymphocytes (P<.01) and macrophages (P<.01) in the lung parenchyma. In addition, montelukast restored BAL VEGF levels (P<.05). LTB4 receptor expression (P<.001) as well as NF-κB (P<.001), a downstream target of LPS, were also reduced in lung parenchymal leukocytes. Furthermore, montelukast reduced IL-8 (P<.001) production by LPS-treated human neutrophils. ConclusionIn conclusion, montelukast efficiently attenuated both LPS-induced lung inflammation in a mouse model of ARDS and in LPS challenged human neutrophils.

Nutraceutical Extract from Dulse (Palmaria palmata L.) Inhibits Primary Human Neutrophil Activation.

Palmaria palmata L. (Palmariaceae), commonly known as “dulse”, is a red alga that grows on the northern coasts of the Atlantic and Pacific oceans, and is widely used as source of fiber and protein. Dulse is reported to contain anti-inflammatory and antioxidant compounds, albeit no study has investigated these effects in primary human neutrophils. Implication strategies to diminish neutrophil activation have the potential to prevent pathological states. We evaluated the ability of a phenolic dulse extract (DULEXT) to modulate the lipopolysaccharide (LPS)-mediated activation of primary human neutrophils. Intracellular reactive oxygen species (ROS) were measured by fluorescence analysis and nitric oxide (NO) production using the Griess reaction. Inflammatory enzymes and cytokines were detected by ELISA and RT-qPCR. The results show that DULEXT diminished the neutrophil activation related to the down-regulation of TLR4 mRNA expression, deceased gene expression and the LPS-induced release of the chemoattractant mediator IL-8 and the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. ROS, NO, and myeloperoxidase (MPO) were also depressed. The data indicated that DULEXT has the potential to disrupt the activation of human primary neutrophils and the derived inflammatory and prooxidant conditions, and suggest a new role for Palmaria palmata L. in the regulation of the pathogenesis of health disorders in which neutrophils play a key role, including atherosclerosis.

Inula helenium and Grindelia squarrosa as a source of compounds with anti-inflammatory activity in human neutrophils and cultured human respiratory epithelium.

During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1β and TNF-α) release and downregulation of adhesive molecules. During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. The effect on IL-8, IL-1β and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of β2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1β release comparable with budesonide, used as a positive control. The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I.helenium and G.squarrosa for a treatment of inflammation-based diseases in respiratory tract.

DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils.

Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation. Neutrophils harbor two distinct pools of the membrane-localized oxidase components, one expressed in the plasma membrane and one in the membranes of intracellular granules. Assembly of active oxidase at either type of membrane leads to release of extracellular ROS or to the production of ROS inside intracellular compartments, respectively. The cytoplasmic NADPH oxidase subunit p40phox seems selectively critical for the ability to generate intracellular ROS, and the recent characterization of patients with p40phox deficiency implies that selective loss of intracellular neutrophil ROS leads to disease with pronounced hyperinflammatory features, suggesting that these ROS are critical for regulation of inflammation. This study aimed at characterizing two pharmacological NADPH oxidase inhibitors, the newly described GSK2795039 and the widely used diphenyleneiodonium (DPI), focusing on their abilities to inhibit human neutrophil ROS production extra- and intracellularly. Whereas GSK2795039 blocked extra- and intracellular NADPH oxidase activity equally, DPI was found to selectively interfere with intracellular ROS production. Selectivity for the intracellular NADPH oxidase was evident as a lower IC50 value, faster onset, and irreversibility of inhibition. We found no evidence of direct interactions between DPI and p40phox, but the selectivity of DPI confirms that regulation of NADPH oxidase activity in neutrophils differs depending on the subcellular localization of the enzyme. This information may be used to pharmacologically mimic p40phox deficiency and to further our understanding of how intracellular ROS contribute to health and disease.

Poly(ε-caprolactone) Scaffolds Doped with c-Jun N-terminal Kinase Inhibitors Modulate Phagocyte Activation.

The modulation of phagocyte responses is essential for successful performance of biomaterials in order to prevent negative outcomes associated with inflammation. Herein, we developed electrospun poly(ε-caprolactone) (PCL) scaffolds doped with the novel potent c-Jun N-terminal kinase (JNK) inhibitors 11H-indeno[1,2-b]quinoxalin-11-one oxime (IQ-1) and 11H-indeno[1,2-b]quinoxalin-11-one O-(O-ethylcarboxymethyl) oxime(IQ-1E) as a promising approach for modulating phagocyte activation. Optimized electrospinning parameters allowed us to produce microfiber composite materials with suitable mechanical properties. We found that embedded compounds were bound to the polymer matrix via hydrophobic interactions and released in two steps, with release mostly controlled by Fickian diffusion. The fabricated scaffolds doped with active compounds IQ-1 and IQ-1E effectively inhibited phagocyte inflammatory responses. For example, they suppressed human neutrophil activation by the biomaterials, as indicated by decreased neutrophil reactive oxygen species (ROS) production and Ca2+ mobilization. In addition, they inhibited lipopolysaccharide (LPS)-induced NF-κB/AP-1 reporter activity in THP-1Blue cells and interleukin (IL)-6 production in MonoMac-6 cells without affecting cell viability. These effects were attributed to the released compounds rather than cell-surface interactions. Therefore, our study demonstrates that doping tissue engineering scaffolds with novel JNK inhibitors represents a powerful tool for preventing adverse immune responses to biomaterials as well as serves as a platform for drug delivery.

Kan-Lu-Hsiao-Tu-Tan, a traditional Chinese medicine formula, inhibits human neutrophil activation and ameliorates imiquimod-induced psoriasis-like skin inflammation.

Kan-Lu-Hsiao-Tu-Tan (KLHTT) is a popular traditional Chinese medicine for treating various inflammatory diseases. The aim of the present study was to investigate the anti-inflammatory effects of KLHTT on human neutrophils and its therapeutic potential in treating imiquimod (IMQ)-induced psoriasis-like skin inflammation. Spectrophotometry, flow cytometry, and microscopy with immunohistochemical staining were used to evaluate superoxide anion generation, elastase release, CD11b expression, adhesion, and neutrophil extracellular trap (NET) formation in activated human neutrophils. Reactive oxygen species (ROS) and reactive nitrogen species in cell-free systems were measured using a multi-well fluorometer or a spectrophotometer. A psoriasis-like skin inflammation was induced in mice using the IMQ cream. KLHTT suppressed superoxide anion generation, ROS production, CD11b expression, and adhesion in activated human neutrophils. In contrast, KLHTT failed to alter elastase release in activated human neutrophils. Additionally, KLHTT had an ROS-scavenging effect in the AAPH assay, but it did not scavenge superoxide anions directly in the xanthine/xanthine oxidase assay. Protein kinase C (PKC)-induced NET formation most commonly occurs through ROS-dependent mechanisms. KLHTT significantly inhibited phorbol 12-myristate 13-acetate, a PKC activator, inducing NET formation. Furthermore, topical KLHTT treatment reduced the area affected by psoriasis area and severity index (PASI) score and ameliorated neutrophil infiltration in IMQ-induced psoriasis-like skin inflammation in mice. Our data show that KLHTT has anti-neutrophilic inflammatory effects in inhibiting ROS generation and cell adhesion. KLHTT also mitigated NET formation, mainly via an ROS-dependent pathway. In addition, KLHTT reduced neutrophil infiltration and improved the severity of IMQ-induced psoriasis-like skin inflammation in mice. Therefore, KLHTT may prove to be a safe and effective psoriasis therapy in the future.

NMDA receptor modulation of glutamate release in activated neutrophils.

BackgroundNeutrophil depletion improves neurologic outcomes in experimental sepsis/brain injury. We hypothesized that neutrophils may exacerbate neuronal injury through the release of neurotoxic quantities of the neurotransmitter glutamate.MethodsReal-time glutamate release by primary human neutrophils was determined using enzymatic biosensors. Bacterial and direct protein-kinase C (Phorbol 12-myristate 13-acetate; PMA) activation of neutrophils in human whole blood, isolated neutrophils or human cell lines were compared in the presence/absence of N-Methyl-d-aspartic acid receptor (NMDAR) antagonists. Bacterial and direct activation of neutrophils from wild-type and transgenic murine neutrophils deficient in NMDAR-scaffolding proteins were compared using flow cytometry (phagocytosis, reactive oxygen species (ROS) generation) and real-time respirometry (oxygen consumption).FindingsBoth glutamate and the NMDAR co-agonist d-serine are rapidly released by neutrophils in response to bacterial and PMA-induced activation. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, d-serine and the respiratory burst by activated primary human neutrophils. A highly specific small-molecule inhibitor ZL006 that limits NMDAR-mediated neuronal injury also reduced ROS by activated neutrophils in a murine model of peritonitis, via uncoupling of the NMDAR GluN2B subunit from its' scaffolding protein, postsynaptic density protein-95 (PSD-95). Genetic ablation of PSD-95 reduced ROS production by activated murine neutrophils. Pharmacological blockade of the NMDAR GluN2B subunit reduced primary human neutrophil activation induced by Pseudomonas fluorescens, a glutamate-secreting Gram-negative bacillus closely related to pathogens that cause hospital-acquired infections.InterpretationThese data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells.FundGLA: Academy Medical Sciences/Health Foundation Clinician Scientist. AVG is a Wellcome Trust Senior Research Fellow.

Montelukast, Leukotriene Inhibitor, Reduces LPS-Induced Acute Lung Inflammation and Human Neutrophil Activation

ObjectivesSome pro-inflammatory lipids derived from 1 lipooxygenase enzyme are potent neutrophil chemoattractant, a cell centrally involved in acute respiratory distress syndrome (ARDS); a syndrome lacking effective treatment. Considering the beneficial effects of the leukotriene receptor inhibitor, montelukast, on other lung diseases, whether montelukast attenuates inflammation in a mouse model of ARDS, and whether it reduces LPS stimulated activation of human neutrophils was investigated. MethodsThirty-five C57Bl/6 mice were distributed into control (PBS)+24h, LPS+24h (10μg/mouse), control+48h, LPS+48h, and LPS 48h+Montelukast (10mg/kg). In addition, human neutrophils were incubated with LPS (1μg/mL) and treated with montelukast (10μM). ResultsOral-tracheal administration of montelukast significantly attenuated total cells (P<.05), macrophages (P<.05), neutrophils (P<.01), lymphocytes (P<.001) and total protein levels in BAL (P<.05), as well as IL-6 (P<.05), CXCL1/KC (P<.05), IL-17 (P<.05) and TNF-α (P<.05). Furthermore, montelukast reduced neutrophils (P<.001), lymphocytes (P<.01) and macrophages (P<.01) in the lung parenchyma. In addition, montelukast restored BAL VEGF levels (P<.05). LTB4 receptor expression (P<.001) as well as NF-κB (P<.001), a downstream target of LPS, were also reduced in lung parenchymal leukocytes. Furthermore, montelukast reduced IL-8 (P<.001) production by LPS-treated human neutrophils. ConclusionIn conclusion, montelukast efficiently attenuated both LPS-induced lung inflammation in a mouse model of ARDS and in LPS challenged human neutrophils.

Neisseria meningitidis-Induced Caspase-1 Activation in Human Innate Immune Cells Is LOS-Dependent.

Meningococcal disease such as sepsis and meningitidis is hallmarked by an excessive inflammatory response. The causative agent, Neisseria meningitidis, expresses the endotoxin lipooligosaccharide (LOS) that is responsible for activation of immune cells and the release of proinflammatory cytokines. One of the most potent proinflammatory cytokines, interleukin-1β (IL-1β), is activated following caspase-1 activity in the intracellular multiprotein complex called inflammasome. Inflammasomes are activated by a number of microbial factors as well as danger molecules by a two-step mechanism—priming and licensing of inflammasome activation—but there are no data available regarding a role for inflammasome activation in meningococcal disease. The aim of this study was to investigate if N. meningitidis activates the inflammasome and, if so, the role of bacterial LOS in this activation. Cells were subjected to N. meningitidis, both wild-type (FAM20) and its LOS-deficient mutant (lpxA), and priming as well as licensing of inflammasome activation was investigated. The wild-type LOS-expressing parental FAM20 serogroup C N. meningitidis (FAM20) strain significantly enhanced the caspase-1 activity in human neutrophils and monocytes, whereas lpxA was unable to induce caspase-1 activity as well as to induce IL-1β release. While the lpxA mutant induced a priming response, measured as increased expression of NLRP3 and IL1B, the LOS-expressing FAM20 further increased this priming. We conclude that although non-LOS components of N. meningitidis contribute to the priming of the inflammasome activity, LOS per se is to be considered as the central component of N. meningitidis virulence, responsible for both priming and licensing of inflammasome activation.

Identification of Residues Critical for FPR2 Activation by the Cryptic Peptide Mitocryptide-2 Originating from the Mitochondrial DNA-Encoded Cytochrome b.

Similar to bacteria, synthesis of mitochondrial DNA-encoded proteins requires an N-formylated methionine to initiate translation. Thus, the N-formylated methionine peptides originating from mitochondria should be recognized as danger signals. To date, only one such peptide, denoted as mitocryptide-2 (MCT-2), originating from the N-terminal of the mitochondrial cytochrome b, has been isolated from mammalian tissues. Human neutrophils express FPR1 and FPR2 that detect formyl peptides, and the precise structural determinants for receptor recognition remain to be elucidated. MCT-2 is known to activate neutrophils through FPR2 but not FPR1. The aim of this study was to elucidate the structural determinants of importance for receptor preference and human neutrophil activation in MCT-2 by generating a series of MCT-2 variants. We show that there is an absolute requirement for the N-formyl group and the side chain of Met1 at position 1 of MCT-2 but also the C terminus is of importance for MCT-2 activity. We also uncovered individual side chains that positively contribute to MCT-2 activity as well as those suppressed in the response. The MCT-2 peptide and its two polymorphic variants ([Thr7]MCT-2 and [Ser8]MCT-2) all activated neutrophils, but MCT-2 containing Ile7 and Asn8 was the most potent. We also show that some peptide variants displayed a biased FPR2-signaling property related to NADPH oxidase activation and β-arrestin recruitment, respectively. In conclusion, we disclose several critical elements in MCT-2 that are required for neutrophil activation and disclose structural insights into how FPR2 recognition of this mitochondrial DNA-derived peptide may increase our understanding of the role of FPR2 in aseptic inflammation.

Neutrophils are a main source of circulating suPAR predicting outcome in critical illness

BackgroundCirculating levels of soluble urokinase plasminogen activation receptor (suPAR) have been proposed as a prognostic biomarker in patients with critical illness and sepsis. However, the origin of suPAR in sepsis has remained obscure. We investigated the potential cellular sources of suPAR by analyzing membrane-bound urokinase plasminogen activator receptor (uPAR, CD87) and evaluated its clinical relevance in critically ill patients.MethodsWe studied 87 critically ill patients (44 with sepsis, 43 without sepsis) from the medical intensive care unit (ICU) in comparison to 48 standard care patients with infections and 27 healthy controls in a prospective single-center non-interventional cohort study. Cellular uPAR expression of different immune cell subsets (by flow cytometry from peripheral blood) and corresponding serum suPAR concentrations were determined upon ICU admission and at day 3. Furthermore, we analyzed the effects of serum from sepsis patients on the activation and uPAR cleavage of primary human neutrophils and macrophages in vitro.ResultsIn healthy controls, uPAR (CD87) expression was detected on nearly all blood neutrophils and monocytes, but only scarcely on lymphocytes. While uPAR expression on monocytes was maintained in ICU patients, only 58% of neutrophils from critically ill patients expressed uPAR, which was significantly lower than in healthy controls or standard care patients. Concomitantly, serum suPAR levels were significantly increased in ICU patients. We noted a clear inverse correlation between low neutrophilic uPAR and high serum suPAR in standard care and ICU patients, indicating that shedding of uPAR from activated neutrophils represents a main source of suPAR in systemic inflammation. Both low uPAR and high suPAR were closely associated with mortality in critically ill patients. Furthermore, serum from sepsis patients induced uPAR protein expression and subsequent receptor shedding on isolated primary neutrophils, but not on macrophages, in vitro.ConclusionsThe inverse correlation between low uPAR surface expression on neutrophils and high serum suPAR in critically ill patients supports that neutrophils are a main source of shed suPAR proteins in systemic inflammation. Furthermore, high suPAR levels and low neutrophilic uPAR expression predict mortality in ICU patients.

β-1,3-Glucan/CR3/SYK pathway-dependent LC3B-II accumulation enhanced the fungicidal activity in human neutrophils.

Since molecular genotyping has been established for the Candida species, studies have found that a single Candida strain (endemic strain) can persist over a long period of time and results in the spread of nosocomial invasive candidiasis without general characteristics of horizontal transmissions. Our previous study also found the existence of endemic strains in a cancer center in Tianjin, China. In the current study, we performed further investigation on endemic and non-endemic Candida albicans strains, with the aim of explaining the higher morbidity of endemic strains. In an in vivo experiment, mice infected with endemic strains showed significantly shorter survival time and higher kidney fungal burdens compared to mice infected with non-endemic strains. In an in vitro experiment, the killing percentage of neutrophils to endemic strains was significantly lower than that to non-endemic strains, which is positively linked to the ratio of LC3B-II/I in neutrophils. An immunofluorescence assay showed more β-1,3-glucan exposure on the cell walls of non-endemic strains compared to endemic strains. After blocking the β-glucan receptor (CR3) or inhibiting downstream kinase (SYK) in neutrophils, the killing percent to C. albicans (regardless of endemic and non-endemic strains) and the ratio of LC3B-II/I of neutrophils were significantly decreased. These data suggested that the killing capability of neutrophils to C. albicans was monitored by β-1,3-glucan via CR3/SYK pathway-dependent LC3B-II accumulation and provided an explanation for the variable killing capability of neutrophils to different strains of C. albicans, which would be beneficial in improving infection control and therapeutic strategies for invasive candidiasis.

Lupus-Associated Immune Complexes Activate Human Neutrophils in an FcγRIIA-Dependent but TLR-Independent Response.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nucleic acids and nucleoproteins. Anti-dsDNA Abs are considered a hallmark of SLE, and previous studies have indicated that nucleic acid-containing immune complexes (ICs) induce B cell and dendritic cell activation in a TLR-dependent process. How ICs containing nucleic acids affect neutrophil function has not been well investigated. In this study, we report that nucleic acid-containing ICs derived from the sera of SLE patients induce human and mouse neutrophil activation through TLR-independent mechanisms. Soluble ICs containing Sm/RNP, an RNA Ag, activate human neutrophils to produce reactive oxygen species (ROS) and IL-8. In contrast, ICs containing DNA have to be immobilized to efficiently activate neutrophils. We found that deleting TLR7 or TLR9, the receptors for RNA and DNA, had no effect on mouse neutrophil activation induced by RNA-containing and immobilized DNA-containing ICs. Binding of ICs are mediated through FcγRIIA and FcγRIIIB. However, neutrophil activation induced by RNA- and DNA-containing ICs requires FcγRIIA, as blocking FcγRIIA inhibited ROS release from neutrophils. RNA-containing ICs induce calcium flux, whereas TLR7/8 ligand R848 do not. Surprisingly, chloroquine inhibits calcium flux induced by RNA-containing ICs, suggesting that this lesser known function of chloroquine is involved in the neutrophil activation induced by ICs. These data indicate the SLE-derived ICs activate neutrophils to release ROS and chemokines in an FcγRIIA-dependent and TLR7- and TLR9-independent manner that likely contributes to local tissue inflammation and damage.

Targeting allosteric site of AKT by 5,7-dimethoxy-1,4-phenanthrenequinone suppresses neutrophilic inflammation.

BackgroundAcute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown.MethodsHuman neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI.FindingsCLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309–313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice.InterpretationOur results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

Influence of the Donor of Hydrogen Sulfide GYY4137 on the Activation of Human Neutrophils by E. coli Lipopolysaccharides

Influence of the Donor of Hydrogen Sulfide GYY4137 on the Activation of Human Neutrophils by E. coli Lipopolysaccharides

Influence of the Donor of Hydrogen Sulfide GYY4137 on the Activation of Human Neutrophils by E. coli Lipopolysaccharides

Lipopolysaccharides (LPS), components of the cell wall of gram-negative bacteria, activate neutrophils that trigger pathological processes, including gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellularglutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by LPS and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Cationic Liposomes Cause ROS Generation and Release of Neutrophil Extracellular Traps

Cationic liposomes are used as nanocarriers of drugs in chemotherapy, antibacterial therapy, and gene therapy. Positively charged stearylamine is commonly used in cationic liposome production. It has been shown that cationic liposomes, when applied intravenously, impact the functions and viability of blood cells, including neutrophils (innate immunity cells). In this work we studied the influence of phosphatidylcholine cationic liposomes containing stearylamine (SA liposomes) on the activity of human neutrophils–their ability of the formation of extracellular traps (NETs) and of the production of reactive oxygen species (ROS). The ratio of phosphatidylcholine to stearylamine in the liposomes was 9 : 1. Phosphatidylcholine liposomes (PC liposomes) were used as control. Liposomes were 140 ± 49 nm in diameter and differed in zeta-potential: PC liposomes had –1.74 ± 0.31 mV, while SA-liposomes had 11.40 ± 0.44 mV. NETs were visualized by light and fluorescent microscopy. It was found that the cells remained intact after a 90-min incubation with PC liposomes (control experiment), while incubation with SA liposomes caused an extrusion of thin DNA fibers into the extracellular space, i.e., the formation of NETs. ROS generation by neutrophils incubated with the liposomes and stimulated either with 0.5 mg/mL of zymozan, or with 40 nM of phorbol-12-miristate-13 acetate, or without stimulation was assessed by luminol-dependent chemiluminescence. It was shown that PC liposomes did not exert any significant effect on chemiluminescence of unstimulated and zymozan-stimulated neutrophils. PMA in the presence of liposomes did not exert any effect on the cells. SA liposomes had a complex impact on the activity of neutrophils: they inhibited the effects of zymozan and caused a prominent respiratory burst independently of the presence of the additional stimulation. The time before the maximal intensity of chemiluminescence peak after incubation with SA-liposomes ranged from 1 to 3 h and significantly differed from the time before the maximal intensity of chemoluminescence peak after incubation with zymozan or PMA (30–40 min). Apocynin, a NADPH-oxidase inhibitor, suppressed the respiratory burst induced by SA liposomes, indicating that NADPH oxidase has a role in ROS production caused by SA liposomes. To assess characteristics of the respiratory burst, lucigenin-dependent chemiluminescence sensitive to superoxide anion radical ( $${\text{O}}_{2}^{{ \bullet -}}$$ ) was registered, and it was found that $${\text{O}}_{2}^{{ \bullet -}}$$ is not produced after stimulation of neutrophils by SA liposomes. On the basis of these findings we conclude that SA liposomes are a polyfunctional factor that can affect the formation of NETs, suppress the action of zymozan, and stimulate ROS production by neutrophils.

Targeting Allosteric Site of AKT by 5,7-Dimethoxy-1,4-Phenanthrenequinone Suppresses Neutrophilic Inflammation

Background: Acute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown. Methods: Human neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI. Findings: CLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice. Interpretation: Our results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation. Funding Statement: This research was financial supported by the grants from the Ministry of Science Technology (MOST 106-2320-B-255-003-MY3 and MOST 104-2320-B-255-004-MY3), Ministry of Education (EMRPD1G0231 and EMRPD1H0381), and Chang Gung Memorial Hospital (CMRPF1F0011~3, CMRPF1F0061~3, CMRPF1G0241~3, and BMRP450), Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The authors declare that no competing interests exist. Ethics Approval Statement: The procedure of neutrophil isolation was approved by the Institutional Review Board at Chang Gung Memorial Hospital. ALI was induced by intra-tracheal spray of 2 mg/kg LPS (Escherichia coli 0111:B4) in seven to eight weeks old C57BL/6 male mice, according to the guidelines and approved by Institutional Animal Care and Use Committee of Chang Gung University, Taiwan.

Primary Human Neutrophils Exhibit a Unique HIV-Directed Antibody-Dependent Phagocytosis Profile

The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with the antibodies (Abs) mediating function via the “constant” immunoglobulin region, the crystallizable fragment (Fc). These data have supported a focus on the induction of Abs by vaccines that trigger antiviral activities by relevant leukocytes via Fc receptors (FcRs). Neutrophils are phagocytes that comprise > 50% of leukocytes and display unique FcRs. We sought to compare the Ab-dependent cellular phagocytosis (ADCP) activity of human neutrophils to the commonly assayed THP-1 cell line. HIV-specific Abs were employed to elicit ADCP of beads coated with HIV envelope protein. Overall, trends were noted among neutrophil donors and the ADCP profile was different from that of THP-1 cells. mAb ELISA titers correlated with ADCP by THP-1 cells but not neutrophils. Monoclonal (m)Abs were also tested with primary monocytes. Donor-to-donor variation was high, and hindered the analysis of this dataset, but it was, in itself, an important finding. This study illustrates the concept that the assessment of FcR-mediated Ab activity with a frequently used cell line such as THP-1 is not necessarily indicative of relevant Ab functionality in vivo, and this calls for in-depth study of the properties of the HIV antibodies best-suited to eliciting antiviral activities by primary cells.

Aluminum oxide nanowires as safe and effective adjuvants for next-generation vaccines

Improving efficiency of an adjuvant, material that enhances the body’s immune response to an antigen, has become vital for the development of safer, cheaper, and more effective next-generation vaccines. Commercial vaccines typically use aluminum salt-based adjuvant particles, most commonly aluminum oxyhydroxide (AlOOH) and aluminum hydroxide (Al(OH) 3 ) based, often referred to as “alum”. Despite their broad use, their adjuvant properties are rather moderate. This is even worse in the case of aluminum oxide (Al 2 O 3 )-based adjuvant. While being more robust and less cytotoxic, Al 2 O 3 is a significantly less effective adjuvant than above-mentioned Al compounds and is consequently not commonly used. Here, we report on the remarkably enhanced adjuvant properties of Al 2 O 3 when produced in the form of nanowires (NWs). Based on recent advances in understanding neutrophil activation by inert nanoscaffolds, we have created ultra-long Al 2 O 3 NWs with a high aspect ratio of ∼1000. These NWs showed strong humoral immune response with no damaging effect on the microvasculature. Since only the change of shape of Al adjuvants is responsible for the excellent adjuvant properties, our finding holds great promise for rapid implementation as safer and more effective adjuvant alternative for human vaccines. The mechanism behind human blood-derived neutrophil activation with Al 2 O 3 NWs was found to be sequestering of Al 2 O 3 NWs by neutrophils via formation of neutrophil extracellular traps (NETs).