Ravi Shankar Keshari

Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils†

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013.

Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition.

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.

Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation.

High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release.

Increased myeloperoxidase enzyme activity in plasma is an indicator of inflammation and onset of sepsis

INTRODUCTION Circulating lipopolysaccharides released from bacteria may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO enzyme activity in plasma of critically ill patients and to check the hypothesis that these concentrations in plasma would be higher in sepsis and systemic inflammatory conditions, as neutrophils release their contents before proliferating in response to stress. MATERIAL AND METHODS Blood samples were collected from 105 critically ill patients admitted to the intensive care unit, consisting of those with systemic inflammatory response syndrome (n = 42), sepsis (n = 37), and septic shock (n = 26). Plasma MPO enzyme activity was determined by o-dianisidine-H(2)O(2) method, modified for 96-well plates. RESULTS The plasma MPO enzyme activity in sepsis patients was significantly higher than that in the control group (mean, 2.4 ± 1.8 in sepsis and 1.86 ± 1.2 nmol per milligram protein per 10 minutes in systemic inflammatory response syndrome vs 0.32 ± 0.11 nmol per milligram protein per 10 minutes in healthy controls). Mean plasma lactate levels in sepsis (7.8 ± 1.2 mmol/L) and shock patients (9.5 ± 1.2 mmol/L) and cytokines like tumor necrosis factor-α, interleukin-8, and interleukin-1β were simultaneously evaluated to establish onset of inflammation and sepsis. These results show that neutrophil activation occurring during inflammation and sepsis could be detected by plasma MPO concentration. CONCLUSION The plasma MPO concentrations may be a marker of the neutrophil proliferation and severity of inflammation.

Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential

Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro‐organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP‐1 cells, and assessment of NO‐mediated free radical formation by using a consistent NO donor, DETA‐NONOate. NO‐mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO‐mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO‐ or PMA‐mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL‐1β and IL‐8, while with THP‐1 cells, release of IL‐1β, IL‐8, and TNFα was observed. This study demonstrates that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of proinflammatory cytokines.

Functional and molecular characterization of NOS isoforms in rat neutrophil precursor cells

Previous studies from this laboratory have demonstrated importance of neutrophil‐derived nitric oxide (NO) in free radical generation, characterized nitric oxide synthase (NOS) isoforms, and have reported subcellular distribution of NOS in rat peripheral neutrophils. Maximum number of neutrophils are added per day to the circulation from bone marrow, thus neutrophils might add substantial amount of NO in the bone marrow. NO generating ability and NOS isoforms characteristics in bone marrow neutrophil precursor cells is, however, still unexplored. This study was, therefore, undertaken to investigate NO generation ability and the molecular/biochemical characteristics of NOS isoforms in neutrophil precursor cells. The neutrophil precursors were separated on Percoll density gradient and characterized by Giemsa staining, CD markers, and by their size and granularity at various stages of maturation as Bands 1, 2, and 3. Mature neutrophils were efficient in free radical generation and phagocytosis, whereas immature cells had more mitochondria and myeloperoxidase. Amount of NO augmented from immature to mature neutrophils as assessed by fluorescent probe DAF‐2DA and Griess reagent. Measurement of NOS enzyme activity further confirmed the functional status of NOS in these cells. NOS isoforms were differentially expressed during neutrophil maturation as confirmed by enzyme activity, Western blotting, flowcytometry, and RT‐PCR. Expression of nNOS was predominantly stable in all the stages of neutrophil maturation. iNOS expression was, however, consistently augmented during maturation, whereas eNOS expression was downregulated with neutrophil maturation. Furthermore, all NOS isoforms proteins were distributed in cytosol as well as nucleus as assessed by confocal microscopy. This study for the first time report biochemical and molecular characteristics of NOS isoforms in rat neutrophil precursor cells.

Nitric oxide-mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

Previous reports from this laboratory and others demonstrated NO‐mediated biphasic modulation of NADPH oxidase and attenuation of neutrophil reactive oxygen species generation, whereas recently we reported augmentation in DCF fluorescence following NO treatment. These discrepancies seem to be due to utilization of different probes/methods to assess effect of NO on reactive oxygen and nitrogen species (ROS/RNS, reactive species) generation. This study aims to look into this and evaluate NO‐mediated enzymatic reactive species formation by using multiple probes, human neutrophils/HL60 cells and various interventions. Addition of NO donor, SNP or SNAP (100 nM–1 mM) to PMNs suspension, exhibited a concentration‐ and time‐dependent augmentation in DCF fluorescence, but reduced DHE fluorescence. Collective generation of reactive species was confirmed by enhanced DMPO‐nitrone adduct, dityrosine and rhodamine‐123 and quenching of scopoletin. NO also enhanced bacterial killing, without altering phagocytosis. Addition of NO to HL‐60 cells lacking functional NADPH oxidase enhanced reactive species formation, indicating importance of other enzyme(s) too. NO‐dependent ROS/RNS generation was substantially reduced by NADPH oxidase inhibitor (DPI), MPO inhibitor (ABAH), or NOS inhibitor (7‐NI). However, 7‐NI reduced MPO activity, warranting reappraisal of those reports, which implied NOS in reactive species formation. The results obtained demonstrated NO‐mediated reactive species augmentation in human PMNs. Furthermore, superoxide scavenging by NO seems to be the key process in the decrease of DHE fluorescence and suggest usefulness of DCF as the most appropriate probe to measure the NO‐mediated modulation of reactive oxygen species in particular in various pathological situations.

Role of active nitrogen molecules in progression of septic shock.

Active nitrogen molecules are formed as a result of cell metabolism. They are essential for cell metabolism, but when produced in excess, they contribute to the pathogenesis of several disease processes. These nitrogen molecules play an important role in vascular instability of septic shock. This study was planned to detect the role of active nitrogen molecules in the progression of septic shock.