Microglia inflammatory responses are controlled by an intrinsic circadian clock
Introduction
Circadian rhythms have evolved in response to the consistent 24 h light cycle and allow animals to anticipate predictable daily events such as food availability, fluctuations in predation, and sleep opportunity (Hut and Beersma, 2011). Importantly, these activities are associated with time of day variations in risk for encountering pathogens, infection, and tissue damage to the host (Curtis et al., 2014). Thus, it follows that several aspects of the immune system are regulated by the circadian system and disruption of the circadian system is linked to inflammatory pathologies including cancer, metabolic disorder, and premature aging (Evans and Davidson, 2013, Fonken and Nelson, 2014).
In mammals, circadian rhythms are initiated in the suprachiasmatic nuclei (SCN) of the hypothalamus. Within SCN neurons, rhythms are driven by an autoregulatory feedback loop of transcriptional activators and repressors (Reppert and Weaver, 2002). The transcriptional activators, circadian locomotor output cycles kaput (CLOCK) and brain and muscle arnt-like protein 1 (BMAL1), form heterodimers that induce expression of the period (Per) and cryptochrome (Cry) genes through E-box enhancers. Per and Cry proteins accumulate in the cytoplasm and upon reaching critical levels form a complex that translocates back to the nucleus to interact with clock and bmal1 to inhibit their own transcription. This process takes approximately 24 h. While the SCN is the master circadian oscillator in mammals, the molecular machinery necessary for generating circadian rhythms is expressed in many tissues and cells throughout the body (Mohawk et al., 2012). Indeed, circadian clocks persist in several immune cells including macrophages/monocytes (Boivin et al., 2003, Hayashi et al., 2007, Keller et al., 2009, Silver et al., 2012a), T cells (Bollinger et al., 2011), NK cells (Arjona and Sarkar, 2006), dendritic cells (Silver et al., 2012a), and B cells (Silver et al., 2012a).
In addition to driving circadian rhythms, clock genes are involved in regulating immunological activities. For example, the circadian clock gene Rev-erb represses macrophage gene expression (Lam et al., 2013) and targets inflammatory function of macrophages through the direct regulation of Ccl2 (Sato et al., 2014). Bmal1 controls rhythmic trafficking of inflammatory monocytes to sites of inflammation (Nguyen et al., 2013). Additionally, the CLOCK:BMAL1 heterodimer binds E-boxes in the TLR9 promoter (Silver et al., 2012b) and clock protein complexes with the NF-κB subunit p65 (RELA), leading to enhanced transcriptional activity of the NF-κB complex (Spengler et al., 2012). The relationship between circadian clock genes and immune function also appears bi-directional with immune activation altering circadian rhythms (O’Callaghan et al., 2012).
Circadian differences in immune regulation have important physiological consequences in mammals. For example, outcome following global cerebral ischemia varies depending on the time of day at which the ischemic event occurs (Weil et al., 2009). Furthermore, mortality following bacterial challenge varies depending on the time of immunostimulation. Halsberg et al. first demonstrated that a dose of Escherichia coli endotoxin that is non-lethal in most mice when given during the dark (active) period of the day, is highly lethal when administered 8–12 h earlier (Halberg et al., 1960). Subsequent studies revealed that lipopolysaccharide (LPS) produces similar responses, with peak mortality in rodents occurring when LPS is administered during the light phase (Marpegan et al., 2009, Spengler et al., 2012).
Peripheral immune cells mediate LPS lethality. However, peripherally administered LPS also impacts centrally mediated processes. For example, there are diurnal variations in LPS induced alterations in sleep and body temperature (Morrow and Opp, 2005). Importantly, circadian regulation of sickness behaviors (outside of the context of sleep) and neuroinflammatory responses has not been well characterized. Peripheral macrophages are under circadian control (Keller et al., 2009) and there is evidence that microglia express clock genes (Nakazato et al., 2011, Hayashi et al., 2013). Furthermore, circadian system disruptions exacerbate inflammatory responses in both the periphery (Castanon-Cervantes et al., 2010) and CNS (Fonken and Nelson, 2013). Thus, we hypothesized that there are temporal differences in coping with an immune challenge and that circadian variations in the sickness response are mediated by microglia.
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Animals
Male Sprague–Dawley rats (60–90 days old; Harlan Sprague–Dawley, Inc, Indianapolis, IN, USA) were pair-housed (unless otherwise specified) with food and water available ad libitum at an ambient temperature of 22 ± 2 °C. Rats were given at least two weeks to acclimate to colony conditions before experimentation began. All rats were maintained on a 12:12 light cycle with lights on either at 0700 or 1700 h. All experimental procedures were conducted in accordance with the University of Colorado
Daily variations in sickness behavior are associated with rhythmic changes in hippocampal cytokine expression
To test whether there are circadian differences in sickness responses to a sub-septic immune stimulation, rats were injected with 100 μg/kg LPS (E. coli serotype 0111:B4; Sigma) either during the middle of the light phase (ZT6) or during the dark phase (ZT16; n = 4/group). Rats injected with LPS during the dark (active) phase displayed a blunted sickness responses compared to those injected during the light (rest) phase. There were no time-of-day differences in baseline social exploration between
Discussion
The circadian system is an integral part of homeostatic mechanisms in mammals. Daily variations in inflammatory challenges have likely led to temporal regulation of immune functions (Curtis et al., 2014). Here, we demonstrate that there is circadian regulation of inflammatory processes in the CNS. Microglia possess circadian clock mechanisms and display rhythmic fluctuations in basal inflammatory gene expression as well as inflammatory potential. Time-of-day differences in microglia priming
Conflict of interest
The authors declare no competing financial interests. All authors concur with the submission of this manuscript and none of the data have been previously reported or are under consideration for publication elsewhere.
Acknowledgments
We thank Robert Spencer for helpful discussion and John D’Angelo for excellent animal care. This research was supported by NIH grant MH096224-01 to S.F.M. and 1F32AG048672-01 to L.K.F.
References (55)
- et al.
Evidence supporting a circadian control of natural killer cell function
Brain Behav. Immun.
(2006) - et al.
Circadian expression of clock genes in human oral mucosa and skin: association with specific cell-cycle phases
Am. J. Pathol.
(2001) - et al.
Circadian clock genes oscillate in human peripheral blood mononuclear cells
Blood
(2003) - et al.
Circadian clock proteins and immunity
Immunity
(2014) - et al.
Health consequences of circadian disruption in humans and animal models
Prog. Mol. Biol. Transl. Sci.
(2013) - et al.
Mice exposed to dim light at night exaggerate inflammatory responses to lipopolysaccharide
Brain Behav. Immun.
(2013) - et al.
Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: immunophenotypic and functional characteristics
J. Neurosci. Methods
(2006) - et al.
Aging sensitizes rapidly isolated hippocampal microglia to LPS ex vivo
J. Neuroimmunol.
(2010) - et al.
Prior exposure to glucocorticoids sensitizes the neuroinflammatory and peripheral inflammatory responses to E. coli lipopolysaccharide
Brain Behav. Immun.
(2010) - et al.
Glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses
Brain Behav. Immun.
(2012)