Elsevier

Brain, Behavior, and Immunity

Volume 45, March 2015, Pages 171-179
Brain, Behavior, and Immunity

Microglia inflammatory responses are controlled by an intrinsic circadian clock

https://doi.org/10.1016/j.bbi.2014.11.009Get rights and content

Highlights

  • There are temporal differences in sickness behavior in rats.

  • Changes in sickness behavior are reflected in hippocampal cytokine and microglial responses.

  • Microglia show dramatic differences in immune activation throughout the day, with peak activation during the light phase.

  • Microglia rhythms are entrained by, but oscillate independent of, glucocorticoids.

  • These results suggest time-of-day should be considered when planning procedures that induce neuroinflammation.

Abstract

The circadian system regulates many physiological functions including inflammatory responses. For example, mortality caused by lipopolysaccharide (LPS) injection varies depending on the time of immunostimulation in mammals. The effects of more subtle challenges on the immune system and cellular mechanisms underlying circadian differences in neuroinflammatory responses are not well understood. Here we show that adult male Sprague–Dawley rats injected with a sub-septic dose of LPS during the light phase displayed elevated sickness behaviors and hippocampal cytokine production compared to rats injected during the dark phase. Microglia are the primary central nervous system (CNS) immune cell type and may mediate diurnal differences in sickness response, thus we explored whether microglia demonstrate temporal variations in inflammatory factors. Hippocampal microglia isolated from adult rats rhythmically expressed inflammatory factors and circadian clock genes. Microglia displayed robust rhythms of TNFα, IL1β and IL6 mRNA, with peak cytokine gene expression occurring during the middle of the light phase. Microglia isolated during the light phase were also more reactive to immune stimulation; such that, ex vivo LPS treatment induced an exaggerated cytokine response in light phase-isolated microglia. Treating microglia with corticosterone ex vivo induced expression of the circadian clock gene Per1. However, microglia isolated from adrenalectomized rats maintained temporal differences in clock and inflammatory gene expression. This suggests circadian clock gene expression in microglia is entrained by, but oscillates in the absence of, glucocorticoids. Taken together, these findings demonstrate that microglia possess a circadian clock that influences inflammatory responses. These results indicate time-of-day is an important factor to consider when planning inflammatory interventions such as surgeries or immunotherapies.

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.

Section snippets

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)

  • M.G. Frank et al.

    Stress-induced glucocorticoids as a neuroendocrine alarm signal of danger

    Brain Behav. Immun.

    (2013)
  • M.G. Frank et al.

    Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus

    Psychoneuroendocrinology

    (2014)
  • E.M. Gibson et al.

    Aging in the circadian system: considerations for health, disease prevention and longevity

    Exp. Gerontol.

    (2009)
  • A. Kalsbeek et al.

    Circadian rhythms in the hypothalamo-pituitary–adrenal (HPA) axis

    Mol. Cell. Endocrinol.

    (2012)
  • R.W. Logan et al.

    Role of sympathetic nervous system in the entrainment of circadian natural-killer cell function

    Brain Behav. Immun.

    (2011)
  • J.D. Morrow et al.

    Diurnal variation of lipopolysaccharide-induced alterations in sleep and body temperature of interleukin-6-deficient mice

    Brain Behav. Immun.

    (2005)
  • R. Nakazato et al.

    Selective upregulation of Per1 mRNA expression by ATP through activation of P2X7 purinergic receptors expressed in microglial cells

    J. Pharmacol. Sci.

    (2011)
  • S. Panda et al.

    Coordinated transcription of key pathways in the mouse by the circadian clock

    Cell

    (2002)
  • P. Parnet et al.

    Expression of type I and type II interleukin-1 receptors in mouse brain

    Brain Res. Mol. Brain Res.

    (1994)
  • A.C. Silver et al.

    Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells

    Brain Behav. Immun.

    (2012)
  • A.C. Silver et al.

    The circadian clock controls toll-like receptor 9-mediated innate and adaptive immunity

    Immunity

    (2012)
  • M. Takata et al.

    Daily expression of mRNAs for the mammalian Clock genes Per2 and clock in mouse suprachiasmatic nuclei and liver and human peripheral blood mononuclear cells

    Jpn. J. Pharmacol.

    (2002)
  • Z.M. Weil et al.

    Time-of-day determines neuronal damage and mortality after cardiac arrest

    Neurobiol. Dis.

    (2009)
  • A. Balsalobre et al.

    Resetting of circadian time in peripheral tissues by glucocorticoid signaling

    Science

    (2000)
  • T. Bollinger et al.

    Circadian clocks in mouse and human CD4+ T cells

    PLoS ONE

    (2011)
  • O. Castanon-Cervantes et al.

    Dysregulation of inflammatory responses by chronic circadian disruption

    J. Immunol.

    (2010)
  • B.L. Conway-Campbell et al.

    Glucocorticoid ultradian rhythmicity directs cyclical gene pulsing of the clock gene period 1 in rat hippocampus

    J. Neuroendocrinol.

    (2010)
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