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Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood.

ABSTRACT: Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, including the oxidative burst from neutrophils and monocytes from individual donors. With the exception of neutrophils, all cell types tested exhibited oxygen consumption that could be ascribed to oxidative phosphorylation with each having a distinct bioenergetic profile and distribution of respiratory chain proteins. In marked contrast, neutrophils were essentially unresponsive to mitochondrial respiratory inhibitors indicating that they have a minimal requirement for oxidative phosphorylation. In monocytes and neutrophils, we demonstrate the stimulation of the oxidative burst using phorbol 12-myristate 13-acetate and its validation in normal human subjects. Taken together, these data suggest that selection of cell type from blood cells is critical for assessing bioenergetic dysfunction and redox biology in translational research.

SUBMITTER: Chacko BK

PROVIDER: S-EPMC3674307 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood.

Chacko Balu K BK Kramer Philip A PA Ravi Saranya S Johnson Michelle S MS Hardy Robert W RW Ballinger Scott W SW Darley-Usmar Victor M VM

Laboratory investigation; a journal of technical methods and pathology 20130325 6

Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, inc ...[more]

PMID: 23528848

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Project description:BackgroundNeutrophils to lymphocytes ratio (NLR) and platelets to lymphocytes ratio (PLR) are both inflammatory ratios that can be easily calculated from a simple blood count. They are frequently reported and tested as prognostic factors in several medical disciplines. Pregnancy involves special reference values for laboratory assays.ObjectiveThe aim of this study was to define pregnancy-related reference values for NLR and PLR according to trimester, background morbidity and according to the patient's age.Study designA retrospective analysis of a large cohort undergoing community-based pregnancy surveillance between the years 2011-2016. Data were analyzed according to high-risk patient versus normal-risk patient.ResultsA total of 11,415 patients were included. Mean PLR and NLR values were 136.3±44.3, 2.6±1, respectively during the first trimester, 144.6±47.1, 4.0±1.4 respectively during the second trimester and 118.1±42.0, 3.5±1.2 respectively during the third trimester. No difference was detected between the high-risk and the normal population (P-values 0.3, 0.5 and 0.4 for PLR in each trimester respectively and 0.3, 0.4, 0.6 for NLR in each trimester, respectively). No differences were detected among parity categories. The correlation between patient's age and either PLR and NLR was a weak positive correlation (though statistically significant). Both PLR and NLR reached a maximum value during the second trimester. The differences between mean NLR and PLR between trimesters were significant (P <0.01 for all differences tested). PLR rises in the presence of anemia, reaching statistical significance (P-value for PLR in each trimester was <0.01). NLR showed an opposite trend (P-values for NLR were 0.4, 0.005 and 0.06 in each trimester, respectively).ConclusionsIn our cohort, there were generally no differences between the high-risk and the normal population, excluding patients with a fibroid uterus or inflammatory bowel disease who presented a significantly elevated PLR through all trimesters. Both PLR and NLR reached a maximum value during the second trimester and were positively correlated with age. We anticipate that the population-based data will assist in providing accurate reference values for future research testing NLR and PLR measures during pregnancy.

Project description:Metabolic control of cellular function is significant in the context of inflammation-induced metabolic dysregulation in immune cells. Generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide are one of the critical events that modulate the immune response in neutrophils. When activated, neutrophil NADPH oxidases consume large quantities of oxygen to rapidly generate ROS, a process that is referred to as the oxidative burst. These ROS are required for the efficient removal of phagocytized cellular debris and pathogens. In chronic inflammatory diseases, neutrophils are exposed to increased levels of oxidants and pro-inflammatory cytokines that can further prime oxidative burst responses and generate lipid oxidation products such as 4-hydroxynonenal (4-HNE). In this study we hypothesized that since 4-HNE can target glycolysis then this could modify the oxidative burst. To address this the oxidative burst was determined in freshly isolated healthy subject neutrophils using 13-phorbol myristate acetate (PMA) and the extracellular flux analyzer. Neutrophils pretreated with 4-HNE exhibited a significant decrease in the oxidative burst response and phagocytosis. Mass spectrometric analysis of alkyne-HNE treated neutrophils followed by click chemistry detected modification of a number of cytoskeletal, metabolic, redox and signaling proteins that are critical for the NADPH oxidase mediated oxidative burst. These modifications were confirmed using a candidate immunoblot approach for critical proteins of the active NADPH oxidase enzyme complex (Nox2 gp91phox subunit and Rac1 of the NADPH oxidase) and glyceraldehyde phosphate dehydrogenase, a critical enzyme in the metabolic regulation of oxidative burst. Taken together, these data suggest that 4-HNE-induces a pleiotropic mechanism to inhibit neutrophil function. These mechanisms may contribute to the immune dysregulation associated with chronic pathological conditions where 4-HNE is generated.

Dataset Information

Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood.

Publications

Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood.

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