Abstract
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.
Highlights
In mammals, the master or central circadian clock is located in the hypothalamic suprachiasmatic nucleus (SCN) [1] of the brain and is synchronized by the light cycle
More detailed analysis of circadian rhythm by cosinor revealed that the acrophase was significantly altered by LPS in synchronized peritoneal macrophages, indicative of a phase shift (Fig 1B)
Our studies reveal that LPS disrupts circadian rhythms in marophages and blunts the expression of the core clock gene, Bmal1 via a mechanism initiated by TLR4-dependent signaling
Summary
The master or central circadian clock is located in the hypothalamic suprachiasmatic nucleus (SCN) [1] of the brain and is synchronized by the light cycle. Peripheral clocks, interact with the central clock, but are regulated by other signals aside from the light cycle. The CLOCK/BMAL1 heterodimer binds to E-box-sequences in promoter regions to activate transcription of clockregulated genes that include the periods (Per) and cryptochromes (Cry) genes. The increased expression of PER and CRY proteins provide negative feedback by inhibiting CLOCK/BMAL1 activity until their levels are reduced. CLOCK/BMAL1 controls the transcription of ROR and REV-ERB nuclear receptors, which bind to RRE elements in the Bmal promoter and regulate BMAL1 expression. An estimated 43% of protein coding genes and 1000 non-coding RNAs have been proposed to undergo circadian changes in expression [3]
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