Journal Article

Endotoxin Disrupts Circadian Rhythms in Macrophages via Reactive Oxygen Species

Wang, Yusi, Pati, Paramita, Chen, Feng, Rudic, R. Daniel, Fulton, David J. R., Xu, Yiming, Huo, Yuqing, Stepp, David W.

2016

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Analysis / Animals / Apolipoproteins / Atherosclerosis / Biology / Biology and Life Sciences / Blood pressure / BMAL1 protein / Cardiovascular diseases / Cardiovascular system / Cell Adhesion - drug effects / Cell Movement - drug effects / Circadian rhythm / Circadian Rhythm - drug effects / Circadian Rhythm - genetics / Circadian rhythms / Cytokines / Diabetes / Endotoxins / Endotoxins - toxicity / Gene expression / Gene Expression Regulation - drug effects / Genetic engineering / Heart diseases / Immune system / Immunology / Inflammation / Inhibition / Laboratory animals / Lipopolysaccharides / Lipopolysaccharides - pharmacology / Lipoproteins, LDL - metabolism / Macrophages / Macrophages, Peritoneal - cytology / Macrophages, Peritoneal - drug effects / Macrophages, Peritoneal - metabolism / Male / Medicine and Health Sciences / Mice / Mice, Knockout / NG-Nitroarginine methyl ester / Nitric Oxide - metabolism / Nitric-oxide synthase / Oscillations / Oxygen / Periodic variations / Periodicity / Peritoneum / Physical Sciences / Physiological aspects / Physiology / Polyethylene glycol / Proteins / Reactive oxygen species / Reactive Oxygen Species - metabolism / Real-Time Polymerase Chain Reaction / Research and Analysis Methods / Rodents / Signaling / Stem cells / Stimuli / Superoxide / Tempol / TLR4 protein / Toll-Like Receptor 4 - metabolism / Toll-like receptors / Transcription / Transcription Factors - metabolism / Transgenic mice