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.

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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-1b and IL-8, while with THP-1 cells, release of IL-1b, IL-8, and TNFa 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. ' 2011 International Society for Advancement of Cytometry Key terms nitric oxide; neutrophil extracellular traps; NADPH-oxidase; myeloperoxidase; inflammatory cytokine; mitochondrial and nuclear DNA

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Sepsis is a condition caused by infection followed by unregulated inflammatory response which may lead to the organ dysfunction. During such condition, over-production of oxidants is one of the factors which contribute cellular toxicity and ultimately organ failure and mortality. Antioxidants having free radicals scavenging activity exert protective role in various diseases. This study has been designed to evaluate the levels of oxidative and antioxidative activity in sepsis patients and their correlation with the severity of the sepsis. A total of 100 sepsis patients and 50 healthy controls subjects were enrolled in this study from the period October 2016 to June 2017. The investigation included measurements of oxidative enzyme, myeloperoxidase (MPO), antioxidant enzymes including superoxide dismutase activity (SOD) and catalase activity (CAT) and cytokines (TNF-α, IL-8 and IFN-γ). Furthermore, the level of these activities was correlated with severity of sepsis. Augmented levels of oxidants were found in sepsis as demonstrated by DMPO nitrone adduct formation and plasma MPO level activity (1.37 ± 0.51 in sepsis vs 0.405 ± 0.16 in control subjects). Cytokines were also found to be increased in sepsis patients. However, plasma SOD and CAT activities were significantly attenuated (P < .001) in the sepsis patients compared with controls subjects. Moreover, inverse relation between antioxidant enzymes (SOD and CAT) and organ failure assessment (SOFA), physiological score (APACHE II), organ toxicity specific markers have been observed as demonstrated by Pearson's correlation coefficient. This study suggests that imbalance between oxidant and antioxidant plays key role in the severity of sepsis.

Altogether results obtained demonstrate the role of iNOS in NO and ROS/RNS generation, after phagocytosis of coated latex beads by human polymorphonuclear neutrophils. These studies imply functional importance of iNOS and its interaction with Rac2 in pathogen killing by the neutrophils.

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