Dataset Information

Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages.

ABSTRACT: Reactive oxygen and nitrogen species (ROS and RNS) produced by macrophages are essential for protecting a human body against bacteria and viruses. Micrometer-sized electrodes coated with Pt black have previously been used for selective and sensitive detection of ROS and RNS in biological systems. To determine ROS and RNS inside macrophages, one needs smaller (i.e., nanometer-sized) sensors. In this article, the methodologies have been extended to the fabrication and characterization of Pt/Pt black nanoelectrodes. Electrodes with the metal surface flush with glass insulator, most suitable for quantitative voltammetric experiments, were fabricated by electrodeposition of Pt black inside an etched nanocavity under the atomic force microscope control. Despite a nanometer-scale radius, the true surface area of Pt electrodes was sufficiently large to yield stable and reproducible responses to ROS and RNS in vitro. The prepared nanoprobes were used to penetrate cells and detect ROS and RNS inside macrophages. Weak and very short leaks of ROS/RNS from the vacuoles into the cytoplasm were detected, which a macrophage is equipped to clean within a couple of seconds, while higher intensity oxidative bursts due to the emptying of vacuoles outside persist on the time scale of tens of seconds.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3406879 | biostudies-other | 2012 Jul

REPOSITORIES: biostudies-other

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Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages.

Wang Yixian Y Noël Jean-Marc JM Velmurugan Jeyavel J Nogala Wojciech W Mirkin Michael V MV Lu Cong C Guille Collignon Manon M Lemaître Frédéric F Amatore Christian C

Proceedings of the National Academy of Sciences of the United States of America 20120521 29

Reactive oxygen and nitrogen species (ROS and RNS) produced by macrophages are essential for protecting a human body against bacteria and viruses. Micrometer-sized electrodes coated with Pt black have previously been used for selective and sensitive detection of ROS and RNS in biological systems. To determine ROS and RNS inside macrophages, one needs smaller (i.e., nanometer-sized) sensors. In this article, the methodologies have been extended to the fabrication and characterization of Pt/Pt bla ...[more]

PMID: 22615353

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Project description:The circadian clock is a transcriptional network that functions to regulate the expression of genes important in the anticipation of changes in cellular and organ function. Recent studies have revealed that the recognition of pathogens and subsequent initiation of inflammatory responses are strongly regulated by a macrophage-intrinsic circadian clock. We hypothesized that the circadian pattern of gene expression might be influenced by inflammatory stimuli and that loss of circadian function in immune cells can promote pro-inflammatory behavior. To investigate circadian rhythms in inflammatory cells, peritoneal macrophages were isolated from mPer2luciferase transgenic mice and circadian oscillations were studied in response to stimuli. Using Cosinor analysis, we found that LPS significantly altered the circadian period in peritoneal macrophages from mPer2luciferase mice while qPCR data suggested that the pattern of expression of the core circadian gene (Bmal1) was disrupted. Inhibition of TLR4 offered protection from the LPS-induced impairment in rhythm, suggesting a role for toll-like receptor signaling. To explore the mechanisms involved, we inhibited LPS-stimulated NO and superoxide. Inhibition of NO synthesis with L-NAME had no effect on circadian rhythms. In contrast, inhibition of superoxide with Tempol or PEG-SOD ameliorated the LPS-induced changes in circadian periodicity. In gain of function experiments, we found that overexpression of NOX5, a source of ROS, could significantly disrupt circadian function in a circadian reporter cell line (U2OS) whereas iNOS overexpression, a source of NO, was ineffective. To assess whether alteration of circadian rhythms influences macrophage function, peritoneal macrophages were isolated from Bmal1-KO and Per-TKO mice. Compared to WT macrophages, macrophages from circadian knockout mice exhibited altered balance between NO and ROS release, increased uptake of oxLDL and increased adhesion and migration. These results suggest that pro-inflammatory stimuli can disrupt circadian rhythms in macrophages and that impaired circadian rhythms may contribute to cardiovascular diseases by altering macrophage behavior.

Dataset Information

Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages.

Publications

Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages.

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