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Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.

ABSTRACT: Neutrophil extracellular traps (NETs) were first reported in 2004, and since their discovery, there has been an increasing interest in NETs, how they are formed, their role in controlling infections, and their contribution to disease pathogenesis. Despite this rapid expansion of our understanding of NETs, many details remain unclear including the role of reactive oxygen species (ROS) in the formation of NETs. Further, to study NETs, investigators typically require a large number of cells purified via a lengthy purification regimen. Here, we report a microfluidic device used to quantify both ROS and NET production over time in response to various stimulants, including live bacteria. This device enables ROS and NET analysis using a process that purifies primary human neutrophils in less than 10 minutes and requires only a few microliters of whole blood. Using this device we demonstrate the ability to identify distinct capabilities of neutrophil subsets (including ROS production and NET formation), the ability to use different stimulants/inhibitors, and the ability to effectively use samples stored for up to 8 hours. This device permits the study of ROS and NETs in a user-friendly format and has potential for widespread applications in the study of human disease.

SUBMITTER: Moussavi-Harami SF

PROVIDER: S-EPMC4776335 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.

Moussavi-Harami S F SF Mladinich K M KM Sackmann E K EK Shelef M A MA Starnes T W TW Guckenberger D J DJ Huttenlocher A A Beebe D J DJ

Integrative biology : quantitative biosciences from nano to macro 20160125 2

Neutrophil extracellular traps (NETs) were first reported in 2004, and since their discovery, there has been an increasing interest in NETs, how they are formed, their role in controlling infections, and their contribution to disease pathogenesis. Despite this rapid expansion of our understanding of NETs, many details remain unclear including the role of reactive oxygen species (ROS) in the formation of NETs. Further, to study NETs, investigators typically require a large number of cells purifie ...[more]

PMID: 26805445

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Project description:Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.

Project description:Biological production and decay of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O[Formula: see text]) likely have significant effects on the cycling of trace metals and carbon in marine systems. In this study, extracellular production rates of H2O2 and O[Formula: see text] were determined for five species of marine diatoms in the presence and absence of light. Production of both ROS was measured in parallel by suspending cells on filters and measuring the ROS downstream using chemiluminescence probes. In addition, the ability of these organisms to break down O[Formula: see text] and H2O2 was examined by measuring recovery of O[Formula: see text] and H2O2 added to the influent medium. O[Formula: see text] production rates ranged from undetectable to 7.3 × 10(-16) mol cell(-1) h(-1), while H2O2 production rates ranged from undetectable to 3.4 × 10(-16) mol cell(-1) h(-1). Results suggest that extracellular ROS production occurs through a variety of pathways even amongst organisms of the same genus. Thalassiosira spp. produced more O[Formula: see text] in light than dark, even when the organisms were killed, indicating that O[Formula: see text] is produced via a passive photochemical process on the cell surface. The ratio of H2O2 to O[Formula: see text] production rates was consistent with production of H2O2 solely through dismutation of O[Formula: see text] for T. oceanica, while T. pseudonana made much more H2O2 than O[Formula: see text]. T. weissflogii only produced H2O2 when stressed or killed. P. tricornutum cells did not make cell-associated ROS, but did secrete H2O2-producing substances into the growth medium. In all organisms, recovery rates for killed cultures (94-100% H2O2; 10-80% O[Formula: see text]) were consistently higher than those for live cultures (65-95% H2O2; 10-50% O[Formula: see text]). While recovery rates for killed cultures in H2O2 indicate that nearly all H2O2 was degraded by active cell processes, O[Formula: see text] decay appeared to occur via a combination of active and passive processes. Overall, this study shows that the rates and pathways for ROS production and decay vary greatly among diatom species, even between those that are closely related, and as a function of light conditions.

Dataset Information

Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.

Publications

Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.

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