Abstract 17936: Endotoxin Disrupts Circadian Rhythms in Macrophages via Reactive Oxygen Species

Introduction: The circadian clock helps to regulate the expression of a subset 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 is strongly regulated by a macrophage-intrinsic circadian clock. Hypothesis: We hypothesized that the circadian pattern of gene expression might be influenced by inflammatory stimuli and that loss of circadian function in inflammatory cells can predispose to cardiovascular disease. Methods and Results: To investigate circadian rhythms in inflammatory cells, peritoneal macrophage were isolated from Per2-luciferase transgenic mice. Using Cosinor analysis, we found that LPS significantly altered the circadian period in peritoneal macrophages from Per2-luc mice while real-time PCR data suggested that the pattern of expression of the core circadian genes (Bmal1 and Per2) was disrupted. The ability of LPS to disrupt circadian rhythms was reduced by inhibitors of TLR4. 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 mitigated LPS-induced changes in circadian periodicity. In gain of function experiments, we found that overexpression of Nox5, a source of ROS, could significant disrupt circadian function in U2OS cells whereas iNOS, 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. Conclusions: 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.