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Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements.

ABSTRACT: Metabolic modulation of macrophage activation has emerged as a promising strategy lately in immunotherapeutics. However, macrophages have a broad spectrum of functions and thus, understanding the exact metabolic changes that drive a particular immune response, is of major importance. In our previous work, we have reported a key role of nitric oxide (NO^●) in two(2)-signal activated macrophages [M(2-signals)]. Further characterization using metabolic analysis in intact cells, showed that the basal and maximal respiration levels of M(2-signals) were comparable, with cells being unresponsive to the injections-inducd mitochondrial stress. Here, we show that excessive NO^● secretion by the M(2-signals) macrophages, interferes with the oxygen (O₂) consumption measurements on cells using the seahorse metabolic analyzer. This is attributed mainly to the consumption of ambient oxygen by NO^● to form NO₂^- and/or NO₃^- but also to the reduction of O₂ to superoxide anion (O₂^●-) by stray electrons from the electron transport chain, leading to the formation of peroxynitrite (ONOO^-). We found that reactive species-donors in the absence of cells, produce comparable oxygen consumption rates (OCR) with M(2-signals) macrophages. Furthermore, inhibition of NO^● production, partly recovered the respiration of activated macrophages, while external addition of NO^● in non-activated macrophages downregulated their OCR levels. Our findings are crucial for the accurate metabolic characterization of cells, especially in cases where reactive nitrogen or oxygen species are produced in excess.

SUBMITTER: Christopoulos PF

PROVIDER: S-EPMC8301004 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements.

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