Circadian Clock Proteins and Immunity

Immunity Review Circadian Clock Proteins and Immunity Anne M. Curtis,1,* Marina M. Bellet,2,3 Paolo Sassone-Corsi,2 and Luke A.J. O’Neill1,* 1School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland for Epigenetics and Metabolism, Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA 3Present address: Department of Experimental Medicine, University of Perugia, 06132 Perugia, Italy *Correspondence: [email protected] (A.M.C.), [email protected] (L.A.J.O.) http://dx.doi.org/10.1016/j.immuni.2014.02.002 2Center Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. A circadian-clock-controlled immune system might allow an organism to anticipate daily changes in activity and feeding and the associated risk of infection or tissue damage to the host. Responses to bacteria have been shown to vary depending on time of infection, with mice being more at risk of sepsis when challenged ahead of their activity phase. Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBa, control fundamental aspects of the immune response. Examples include the BMAL1:CLOCK heterodimer regulating toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBa suppressing the induction of interleukin-6. Understanding the daily rhythm of the immune system could have implications for vaccinations and how we manage infectious and inflammatory diseases. Introduction Organisms require the ability to tell the time of day in order to anticipate and respond to changes in the external environment imposed by solar time. This need is illustrated in the physiological changes required to accommodate the daily pattern of rest, activity, and feeding. Circadian rhythms are these daily changes or oscillations in physiology and are due to numerous genes whose expression peaks and troughs approximately 12 hr apart to undergo a full cycle within 24 hr. The full breadth of control by the molecular clock on the genome was revealed by R. Zhang et al. (2013, Keystone Symposia on Molecular Clockworks, conference) who harvested multiple tissues at 2 hr intervals and found that more than a third of the mammalian genome is under clock control. The master clock resides in the suprachiasmatic nucleus (SCN) of the brain and is oriented to the external environment via the retinohypothalamic tract receiving light information from the eye. The clock is cell autonomous and in its simplest form consists as a transcription-translation oscillator loop. At the core of this oscillator lies the heterodimeric partnership of the basic-helix-loop-helix PER-ARNT-SIM (PAS) domain proteins, BMAL1 (also known as ARNTL) and CLOCK, that bind E-box sites and induce the expression of the repressors Period (PER) and Cryptochrome (CRY), which in time translocate back into the nucleus and inhibit their own expression by interfering with the BMAL:CLOCK complex (Figure 1). As PER and CRY proteins are gradually degraded, the repression on BMAL1 and CLOCK is relieved and the cycle begins afresh with another 24 hr cycle. This machinery lies at the heart of the circadian clock in the SCN. However, an important finding was the existence of the same clockwork machinery in peripheral cells (Schibler and Sassone-Corsi, 2002) including fibroblasts cultured in vitro by what is now known as the serum shock model (Balsalobre et al., 1998). The serum shock model synchronizes cells so that circadian gene expression and outputs of the clock can be analyzed within an in vitro system. Since then, the molecular clock has been characterized in immune cells (Boivin et al., 2003) including 178 Immunity 40, February 20, 2014 ª2014 Elsevier Inc. macrophages (Keller et al., 2009), T cells (Bollinger et al., 2011), dendritic cells, and B cells (Silver et al., 2012a). Here we discuss recent findings indicating that certain clock proteins, notably BMAL1, CLOCK, and REV-ERBa, can indeed impact immune cell function, host defense, and inflammation. The emerging picture is that time of day is critical to the nature of the immune response and that dysregulation may lead to inflammatory diseases or immunodeficiency. New therapeutic options are presenting themselves that might provide a new prospect for correcting pathologies associated with aberrant immunity and inflammation. The Anatomy of the Molecular Clock Molecularly, the core oscillator is comprised of at least three interlocking feedback loops. As stated above, the central loop consists of the BMAL1:CLOCK heterodimer driving expression of the repressors PER1-3 and CRY1-2, which feedback and limit their own expression (Figure 1). This loop provides oscillations in Per and Cry transcription and also oscillations in the activity of the BMAL:CLOCK heterodimer. The second loop consists of the nuclear receptors RARrelated orphan receptor (ROR) (a,b,g) and REV-ERB (a,b) (also known as NR1D1 and NR1D2), which through E-box activation by BMAL1:CLOCK can then translocate back into the nucleus to bind to receptor-related orphan receptor response elements (ROREs) in the promoter of Bmal1 (Figure 1). RORs activate whereas REV-ERBs repress the expression of BMAL1 (Preitner et al., 2002; Sato et al., 2004). The third loop is comprised of the transcriptional activator albumin D-box binding protein (DBP), regulated through its E-box (loop 1) and the repressor nuclear factor interleukin 3 (NFIL3; also known as E4BP4), regulated via a RORE (loop 2). These two factors then synergistically regulate the expression of D-box genes including that of Per. This tripartite system forms the foundation of the molecular oscillator and controls the circadian network of clock-controlled genes. In addition to this network of transcriptional control, posttranslational modifications provide a further layer of regulation Immunity Review Figure 1. The Molecular Clock Consists of at Least Three Interlocking Feedback Loops Loop 1 consists of the core clock proteins BMAL1 and CLOCK binding to E-box elements within the genes encoding the repressor proteins PER, CRY, REV-ERBa RORa and DBP. After a period of time, PER and CRY can translocate back into the nucleus and repress their own expression by interfering with the BMAL1:CLOCK complex on the gene promoter. The expression of these proteins is regulated further by posttranslational modifications such as phosphorylation of PER by CKIε, which marks it for proteosomal degradation. Loop 2 consists of the alternate regulation by REV-ERBa and RORa on RORE promoter elements, which includes Bmal1 and Nfil3. Loop 3 consists of the alternate regulation by NFIL3 and DBP on D box promoter elements. The clock products from each of these loops can shuttle back to the nucleus and either repress or reactivate these loops. The transcription factors within each of these loops can also regulate clock-controlled genes (CCGs). These are genes that have a circadian profile of expression but do not feedback to affect the core molecular clock. If some of these CCGs are themselves transcription factors, they may confer a circadian profile of expression on their target genes. Therefore, the core clock components and CCGs have the capacity to regulate transcription of a wide variety of cellular programs that is independent of their function within the molecular clock. onto this network, with various kinases and phosphatases regulating the precision and function of the clock along with histone modifications and the epigenetic code. These areas have been reviewed extensively elsewhere (Aguilar-Arnal and SassoneCorsi, 2013; Bellet and Sassone-Corsi, 2010; Feng and Lazar, 2012). Although not entirely understood, the SCN clock keeps peripheral clocks in harmony via the hypothalamus pituitary adrenal (HPA) axis and the autonomic nervous system (ANS) and their respective hormones glucocorticoids and the catecholamines (epinephrine and norepinephrine), which act as synchronizing messengers or ‘‘zeitgebers’’ to the peripheral clock (Curtis et al., 2007; Kalsbeek et al., 2012). Circulating glucocorticoids, catecholamines, and other hormones such as prolactin, melatonin, and growth hormone, all of which affect the immune system, peak at certain times of the day. The control by the SCN on these autonomic and endocrine outputs keeps peripheral clocks, including that of immune cells, in phase with each other and allows for the coordination of a temporal program of physiology across many tissues at certain times of the day (Guo et al., 2006). The Molecular Clock Controls Antibacterial Host Defense, Sepsis, and Inflammation Pioneering studies in the 1960s and 1970s showed a striking circadian variation in survival when mice were treated with lethal doses of bacteria or bacterial products (Halberg et al., 1960; Shackelford and Feigin, 1973). These studies showed enhanced lethality toward the end of the resting phase, approximately 2 hr before onset of activity. Mice are nocturnal species and therefore the onset of activity occurs when lights are turned off in a mouse facility. This is denoted by zeitgeber time (ZT), relating to the time at which the lights are turned on and off in the animal facility. ZT0 denotes lights on and start of inactive phase and ZT12 denotes lights off and start of activity phase (Figure 2). Numbers of leuko- cytes in the mouse circulation have a strong circadian variability and reach their highest number at ZT5 and are recruited in greater numbers into tissues at ZT13, just after the animal’s transition to activity (Scheiermann et al., 2012). These changes coincide with the sensitivity to lipopolysaccharide (LPS) in which the observed phenotype of increased cytokine release at ZT12 (Gibbs et al., 2012) could be in part due to rising leukocyte numbers at this transition point. Host defense response is also altered according to circadian time. Bellet et al. (2013) reported that, after intragastric infection of wild-type mice with the pathogen Salmonella enterica Typhimurium at different circadian times, ZT4 (day) and ZT16 (night), the degree of intestinal inflammation and cytokine gene expression, as well as bacterial colonization, change depending on both time of infection and time of sacrifice (Bellet et al., 2013). The ability to clear the bacteria 72 hr postinfection (hpi) from the colon was greater in mice infected during the night (ZT16) than during the day (ZT4), but Clock mutant mice had lower numbers of bacteria at both time points. The authors found that the growth of Salmonella and its ability to compete with other microorganisms in the gut is dependent on the circadian regulation of antimicrobial peptides. They found that the antimicrobial peptide lipocalin-2, to which Salmonella is resistant, is higher during the day than at night. This suppresses the resident microbiota, allowing for the higher outgrowth of Salmonella during the day versus the night. In addition, a circadian variation has been found in the ability of mouse macrophages to ingest particles with the increase observed ahead of the transition to activity (Hayashi et al., 2007). This may also enhance bacterial clearance at the beginning of the active phase. The induction of proinflammatory cytokines and chemokine ligands is far greater when mice are challenged with LPS at ZT12 versus ZT0. This includes a greater induction of interleukin-6 (IL6), IL-12(p40), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 from macrophages challenged at Immunity 40, February 20, 2014 ª2014 Elsevier Inc. 179 Immunity Review Figure 2. Circadian Effects on Immune Function The data so far indicate a partitioning of the immune system into two states: (1) a state of anticipation and enhanced immune activity (red dotted line) and (2) an opposing period that may relate to repair and regeneration of the immune system (green dotted line). Data indicate that around the time when mice transition into activity (ZT12), the immune system is poised and anticipates the higher risk of infection, but with that comes enhanced susceptibility to sepsis. This is evidenced by enhanced clearance of bacteria at ZT8 and ZT10. This period also correlates with enhanced lethality from high doses of LPS observed at ZT10, greater induction of proinflammatory cytokines at ZT12, and enhanced numbers of leukocytes at ZT13. The opposing state of regeneration and repair is mainly speculative and requires further investigation. It does coincide, however, with the period of reduced induction of proinflammatory cytokines along with reduced clearance and lethality from bacteria. Whether similar processes occur in humans at the same states of transition have yet to be clearly determined. ZT12 versus ZT0 (Gibbs et al., 2012). Again these differences go some way to explaining the boost in host defense as mice transition into activity, but equally the greater risk of sepsis resulting from an overactive response. Keller et al. (2009) harvested peritoneal macrophages from mice every 4 hr for 24 hr and performed microarray analysis. They found that 8% of the macrophage transcriptome was cycling with a circadian variation, and when they analyzed genes involved in the toll-like receptor 4 (TLR4) pathway, they found circadian control on genes involved in all aspects of the TLR4 response. This indicates further that the entire TLR4 pathway in macrophages is under tight circadian control and suggests again that the circadian clock prepares the immune cell for an integrated response at times of greatest risk. Therefore, the temporal increase in chemoattractants, leukocyte trafficking, proinflammatory cytokines, and phagocytic ability in the hours approaching the commencement of activity is interpreted as being indicative of clock-controlled enhanced sensitivity and immunosurveillance ahead of activity and feeding when the risk of infection would be highest. Conversely, perturbations or desynchronization of the clock (by modulating light input) can adversely affect immune function. Under constant dark conditions (Marpegán et al., 2009) or with circadian desynchronization by experimental jet lag (Castanon-Cervantes et al., 2010), a challenge of LPS in mice leads to an uncoordinated and more severe inflammatory response and increased mortality. One molecular feature of circadian desynchronization by jet lag is a sustained reduction in Bmal1 transcript (CastanonCervantes et al., 2010). This mirrors a ZT12 macrophage, in which the circadian phase of Bmal1 mRNA is at its lowest (Keller et al., 2009). Macrophages subjected to jet lag and ZT12 macrophages have a heightened inflammatory response once activated by LPS, because both have low expression of BMAL1 prior to activation. This suggests that BMAL1 (discussed in more detail later) may be one of the central gatekeepers governing the circadian inflammatory response (Figure 3). The effects of the central clock in the SCN and its rhythmic control on autonomic and endocrine regulators are important determinants of immune modulation (Logan and Sarkar, 2012). The 180 Immunity 40, February 20, 2014 ª2014 Elsevier Inc. SCN produces diffusible signals as well as axonal projections to both the paraventricular nucleus (PVN) and arcuate nucleus (ARC) in the hypothalamus of the brain. For example, the PVN governs noradrenergic input to the spleen and releases norepinephrine to modulate the activity of natural killer (NK) cells (Logan et al., 2011). Also, the enhanced recruitment of neutrophils to skeletal tissues at ZT13 is dependent on circadian noradrenergic input to those skeletal tissues, producing rhythmic expression of the adhesion molecule ICAM-1 (Scheiermann et al., 2012). The glucocorticoids are under clock control. The SCN and adrenal clock drive rhythms in glucocorticoid secretion from the adrenal glands. The SCN controls the release of adrenocorticotropin (ACTH) from the pituitary gland and ACTH in turn stimulates release of glucocorticoids from the adrenal gland (Dickmeis et al., 2013). The adrenal clock then controls sensitivity of the adrenal gland to ACTH (Oster et al., 2006). Glucocorticoids exhibit broad anti-inflammatory properties and can control cytokine production and leukocyte trafficking (Coutinho and Chapman, 2011). Glucocorticoids peak as the mice transition to the active phase, and may possibly be required at that time because of the heightened risk of infection and injury. A comprehensive analysis of the role and targets of glucocorticoids in the circadian control of innate immunity has yet to be performed. Importantly, lack of endocrine rhythms does not ablate the rhythms in immune cell function because mice lacking the adrenal glands and splenocytes in culture still maintain rhythms of cytokine release over a number of days (Keller et al., 2009). This suggests that the intracellular immune clock also plays a role in determining the circadian rhythm of immune function. In simple terms, the circadian system may partition the immune system into two states over the circadian day (Figure 2), one being a state of heightened alert as the animal prepares to transition to activity and the risk of infection or injury is greatest. This state would require an increase in leukocyte numbers along with increased sensitivity of immune cells to infectious agents or danger signals. The second state would occur when the animal rests and the risk of infection and injury is lessened. This state may provide an opportunity for resolution of inflammation and repair of tissues. Immunity Review Figure 3. BMAL1 Is the Central Mediator on the Circadian Control of the Immune System and Promotes an Anti-inflammatory State BMAL1 directly represses Ccl2 expression, leading to lower numbers of the Ly6Chi inflammatory monocytes in circulation and lower recruitment of these inflammatory monocytes into inflamed tissues. BMAL1 sequesters CLOCK and prevents it from acetylating and activating p65, which leads to lower amounts of transcriptionally active NF-kB, leading to less induction of specific genes such as cytokines and regulators of survival and proliferation. BMAL1 drives the expression of Nr1d1 (encoding REV-ERBa) that can inhibit Il6 and Ccl2 expression. BMAL1 drives the expression of Rora that can increase the expression of IkB, a major negative regulator of NF-kB. This would have the effect of retarding the NF-kB complex as a nonactive form in the cytoplasm, preventing it from translocating into the nucleus and activating a range of genes including cytokines. It is no surprise that the immune system would be under circadian control given that a clock system would enable a precise, integrated, and cohesive response across a number of immune cells. The molecular clock could act as a ‘‘ground controller’’ for immune cells, orchestrating a response, dependent on the integration of a range of inputs, including neural, hormonal, and local factors along with the circadian phase of gene expression within the cell. There is a daily rhythm to life, and with that comes daily challenges for which an organism must prepare for and respond to. For example, the clock system prepares an organism to anticipate the onset of activity and feeding, but with that comes the increased threat of infection associated with foraging and feeding. Given the intense energy demands for mounting an immune response, a clock might allow the body to utilize energy differently across tissues during periods of rest versus periods of activity. If the immune system needs to be ‘‘poised for attack’’ just as the animal enters the activity and feeding phase, a clock might then allow for the enhancement of immune function, with resolution and repair of tissues occurring at alternative times (Figure 2). Specific Clock Proteins in Immune Cell Function More recently, roles for the specific clock proteins in the immune response have been studied. Mice with clock gene manipulations have unveiled the important contribution of the core clock genes on the immune response. In this section we will discuss the core clock components, paying particular attention to BMAL1, CLOCK, REV-ERBa, and RORa and the emerging data surrounding their impact on the immune system. BMAL1 BMAL1 is the central clock component and is the only single clock gene knockout in which the mouse loses all rhythmic behavioral activity (Bunger et al., 2000). Global deletion of Bmal1 causes a range of underlying pathologies under steadystate conditions related to accelerated aging (Kondratov et al., 2006), thus making them unsuitable for investigating immune function. By using a mouse model in which Bmal1 was selectively deleted from myeloid cells, Nguyen et al. (2013) investigated the role of myeloid BMAL1 in the clearance of the gram-positive bacterium Listeria monocytogenes. Ly6Chi monocytes provide the first line of defense against this bacterium and the authors had detected a diurnal variation in the absolute numbers of these specific monocytes in blood and in spleen under basal conditions and enhanced recruitment of these monocytes to an inflamed peritoneum at ZT8 versus ZT0. Improved bacterial clearance was observed at ZT8 versus ZT0 and this was likely to be due to the higher recruitment of Ly6Chi monocytes at sites of infection and a higher production of the proinflammatory cytokines, IL-1b, IL-6, tumor necrosis factor-a (TNF-a), and interferon-g (IFN-g) and the chemokine ligand CCL2. The rhythmic oscillation in the numbers of Ly6Chi monocytes in circulation across 24 hr and enhanced recruitment of these cells into inflamed tissue was entirely dependent on BMAL1 in myeloid cells as shown by the fact that the circadian control over Ly6Chi cell number was abolished in the myeloid BMAL1-depleted mice. The authors found that BMAL1 binds to E-boxes in the promoters of Ccl2 (encoding chemokine ligand 2; Ccl2), Ccl8, and S100a8 (encoding S100 calcium binding protein A8) and recruits with it members of the polycomb repressor complex (PRC2) epigenetically marking histones for repression. The authors went on to show that BMAL1 reduces Ccl2 transcription and attenuates Ly6Chi monocyte numbers and inflammation at an inflamed site. Myeloid deletion of Bmal1 also exacerbates metabolic disease driven by a high-fat diet. The authors speculated that this could be due to BMAL1 limiting Ly6Chi monocytosis and recruitment into metabolically stressed tissues such as the fat pads. The absence of BMAL1 in these cells leads to chronic inflammation, insulin resistance, and hyperglycemia. BMAL1 therefore functions as an anti-inflammatory molecule in monocytes in part because of its repression on Ccl2. This anti-inflammatory effect of BMAL1 was confirmed previously in another study (Gibbs et al., 2012). Peritoneal macrophages lacking BMAL1 produced higher amounts of IL-6 in response to LPS at ZT0 in comparison to wild-type peritoneal macrophages. In circulating monocytes and in peritoneal macrophages, Bmal1 mRNA is high at ZT0 and low at ZT12 and this could impact the response to LPS or infection at these two times. However, it is not the absolute amount of Bmal1 mRNA but the binding of BMAL1 to gene promoters that will determine the magnitude of response. ChIP-seq analysis conducted on mouse livers harvested every 4 hr across the 24 hr revealed that BMAL1 binding to gene promoters rises from circadian time (CT, see Box 1) 0 to CT8 and this then falls from CT12 to CT20 (Koike et al., 2012). When the animals transition to activity, the reduced binding of BMAL1 to promoters may allow for a more robust inflammatory response, with better clearance of a pathogen. However, excessive inflammation leading to sepsis can occur when BMAL1 is absent in myeloid cells. Nguyen et al. (2013) demonstrated this: a nonlethal dose of Listeria in wild-type mice caused massive lethality in mice lacking myeloid BMAL1 when challenged at both ZT0 and ZT8. Immunity 40, February 20, 2014 ª2014 Elsevier Inc. 181 Immunity Review Box 1. Glossary of Terms Used in Circadian Biology Circadian time (CT) Corresponds to time without the zeitgeber of light. The lights remain off from CT0 onward. Diurnal Daily rhythms with the zeitgeber of light. Period length The time to complete a full cycle. Zeitgeber An environmental cue that synchronizes the endogenous rhythm of the clock in an organism with the Earth’s 24 hr cycle. Examples include light, food, and temperature. Zeitgeber time (ZT) Corresponds to the time (hours) after onset of light. In an animal facility with 12 hr of light and dark, lights go on at ZT0 and off at ZT12. Apart from Ccl2, what other immune genes might be regulated by BMAL1? Silver et al. (2012b) observed that the expression of TLR9, the pattern recognition receptor that recognizes bacterial and viral DNA, has a circadian pattern of expression. It is controlled directly by CLOCK:BMAL1, which binds E-boxes in the TLR9 promoter. TLR9 expression in spleen cells was far greater in mice at ZT19 versus ZT7. This daily variation in TLR9 led to a more severe response in a model of cecal ligation puncture when performed at ZT19 versus ZT7. The control of BMAL1:CLOCK on TLR9 expression has a strong impact on the adaptive response, as indicated by the fact that immunization of mice with CpG as an adjuvant showed maximum responsiveness to vaccination at ZT19 versus ZT7, coincident with enhanced clock controlled expression of TLR9 (Silver et al., 2012b). This single challenge with CpG at ZT19 resulted in a more marked response to antigen (OVA) 4 weeks after the initial immunization. This illustrates that the circadian control of TLR9 has long-term effects in modulating the magnitude of the adaptive immune response, suggesting that time of vaccination could have a significant impact on efficacy. If the phase of TLR9 expression is similar in humans, the optional time to vaccinate humans may also be during the middle of the night or early morning. CLOCK The BMAL1 binding partner CLOCK has also been shown to impact immune signals. Unlike transcription of Bmal1, Clock mRNA does not alter significantly throughout the day in macrophages (Keller et al., 2009). However, CLOCK has intrinsic histone acetyl transferase (HAT) activity capable of acetylating lysine residues on histones and it is this HAT activity of CLOCK that is essential for circadian gene expression (Doi et al., 2006). CLOCK can also acetylate the glucocorticoid receptor and this suppresses binding of the glucocorticoid receptor to its target genes (Nader et al., 2009). Although not yet investigated, this may have strong implications regarding the effect of CLOCK on the immune system. The daily variations observed in lethality from sepsis correlate with activation of the NF-kB transcriptional complex and CLOCK converges directly on this key immune transcription. CLOCK is found in protein complexes with the NF-kB subunit p65 (RELA) and overexpression of CLOCK leads to enhanced phosphorylation and acetylation of p65, leading to enhanced transcriptional activity of the NF-kB complex (Spengler et al., 2012). BMAL1 182 Immunity 40, February 20, 2014 ª2014 Elsevier Inc. attenuates the effect of CLOCK on NF-kB most probably by sequestering CLOCK. This is likely to be another mechanism by which BMAL1 limits inflammation. Clock mutant mouse embryonic fibroblasts (MEFs) (Bellet et al., 2012) and bonemarrow-derived macrophages (BMDMs) (Bellet et al., 2013) are less responsive to LPS or TNF-a in terms of NF-kB activation, and the inflamed intestine of Clock mutant mice showed reduced expression of many proinflammatory, metabolic, and circadian genes and reduced the number of genes involved in the immune response oscillating in a circadian manner, compared to wildtype mice (Bellet et al., 2013). Given that Salmonella and other pathogens exploit host defenses to their own advantage (Raffatellu et al., 2009), Clock mutant mice also have reduced colonization of Salmonella in the gut (Bellet et al., 2013). However, the absence of CLOCK may not be beneficial against infection with more susceptible microorganisms. Therefore, conditional knockouts of CLOCK in discrete immune compartments will provide a clearer picture on the function of CLOCK in the immune system. The Nuclear Receptors REV-ERBa and RORa There is also compelling evidence for REV-ERBa (also known as NR1D1) and RORa affecting immunity. A synthetic agonist for REV-ERBa limits the release of IL-6 from macrophages (Gibbs et al., 2012). This agonist was also shown to reduce Cxcl11, Ccl2, Cxcl6, and Il19 mRNA but not Il8 mRNA. The mechanism behind this pharmacological effect has not yet been described, but it may be due to the ability of REV-ERBa to recruit the repressor complex NCoR to selected proinflammatory genes including Il6. REV-ERBa also has effects on enhancer-derived RNAs (eRNAs). eRNAs are short RNA strands produced from an enhancer site and play a role in the transcription of an adjacent gene. Binding of REV-ERBa proteins that bring in histone deacetylases repress the production of these eRNAs and thus lead to the inactivation of the adjacent gene (Lam et al., 2013). Target genes here include the metalloproteinase Mmp9 and the chemokine receptor Cx3cr1, providing another mechanism for the anti-inflammatory effects of REVERBa activation. In a macrophage, BMAL1 induces the transcription of Nr1d1 (Figure 1), thus suggesting that some of the anti-inflammatory effects of BMAL1 may be through its direct regulation on REV-ERBa. In support of this, a recent study showed that REV-ERBa also represses Ccl2 by binding to an RORE sequence in the promoter of Ccl2 (Sato et al., 2014). As stated above, BMAL1 represses Ccl2 by binding to an Ebox in the Ccl2 promoter (Nguyen et al., 2013). This indicates that BMAL1 and REV-ERBa cooperate across the circadian cycle to repress Ccl2 transcription under inflammatory conditions. The effect of REV-ERBa is possibly cell type specific. REV-ERBa has been shown to drive Th17 cell differentiation by repression of Nfil3 transcription (Figure 1, loop 2), which in turn will allow RORgt to induce IL17 production (Yu et al., 2013). In adaptive immunity, REV-ERBa may actually promote inflammation. BMAL1 also controls the expression of RORa, and mice deficient in RORa (the neurological mutant mouse Staggerer) are susceptible to LPS lethality (Stapleton et al., 2005). RORa has been shown to induce the transcription of IkBa (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha) and limit NF-kB translocation to the nucleus. Therefore, Immunity Review another mechanism whereby BMAL1 might be anti-inflammatory could be via induction of RORa, which in turn will induce IkBa and limit NF-kB (Delerive et al., 2001). Therefore, both nuclear receptors REV-ERBa and RORa, which are components of the molecular clock, also have strong anti-inflammatory capability. PER and CRY Two other clock proteins, PER and CRY, also modulate inflammation. The PER2 mutant leads to the loss of a daily rhythm in IFN-g (Arjona and Sarkar, 2006). The oscillation in mortality from LPS is abolished in PER2-deficient mice, with Per2 / mice being protected against LPS-induced lethality at all time points analyzed (Liu et al., 2006). The serum concentrations of IFN-g and IL-1b were lowered dramatically in the Per2 / mice with LPS injection, but amounts of TNF-a, IL-6, and IL-10 were normal. The impaired IFN-g was attributed to defective natural killer (NK) cell function. It was also observed that Per2 mutants have reduced amounts of Tlr9 mRNA in macrophages and activation of TLR9 by CpG produced less TNF-a and IL-12 from these macrophages (Silver et al., 2012b). Therefore, PER2 may function to upregulate proinflammatory cytokines at certain stages of the circadian cycle. Per2 mRNA rhythms are antiphase to Bmal1 and Per2 mRNA peaks in macrophages just after the animals enter the active phase (Keller et al., 2009). PER2 is the negative component of the feedback loop that inhibits BMAL1:CLOCK activity as the animal enters the active phase (Figure 1). Therefore, PER2 may promote inflammation in part by repressing the activity of BMAL1. PER2 abundance also inhibits the function of REV-ERBa (Preitner et al., 2002) and repressing REV-ERBa could lead also to greater inflammation from the mechanisms discussed above (Figure 3). The other components of the negative feedback loop are the cryptochromes (Figure 1). Absence of the cryptochrome CRY1 and CRY2 in fibroblasts and BMDMs leads to increased Il6, Tnfa, Cxcl1, and Inos mRNA at baseline. Upon LPS stimulation, IL-6 and TNF-a expression are significantly higher in the BMDMs lacking Cry1 and Cry2. In fibroblasts this was due to CRY1 binding adenylate cyclase and limiting cAMP production. In the absence of CRY1, elevated cAMP increases PKA activity, leading to enhanced phosphorylation of p65 and NF-kB activation (Narasimamurthy et al., 2012). Cry1 / Cry2 / mice have heightened inflammatory joint disease and enhanced production of TNF-a in a model of collagen-induced arthritis (Hashiramoto et al., 2010). Treatment of these mice with anti-TNF-a antibody reduced the severity of the disease in the Cry1 / Cry2 / mice, and ectopic expression of Cry1 in mouse embryonic fibroblasts from Cry1 / Cry2 / mice significantly reduced activation of the TNF-a promoter luciferase construct. This may again be due to the effects of Cry1 to limit PKA-induced phosphorylation of p65 and NF-kB activation in fibroblasts (Narasimamurthy et al., 2012). Inflammation Disrupts the Molecular Clock As discussed above, the clock can impact the inflammatory process in a number of ways. In a reciprocal fashion, inflammation induced by agents such as LPS, TNF-a, and IFN-g (Cavadini et al., 2007; Kwak et al., 2008; Lundkvist et al., 2002; Marpegán et al., 2005; Okada et al., 2008) or acute bacterial infection (Bellet et al., 2013) can affect the circadian clock. The oscilla- tions in the clock genes can be disrupted with inflammation and infection, with effects on the expression of core clock genes and clock-controlled genes, including clock-controlled metabolic genes being reported (Bellet et al., 2013). Rodent studies indicate that LPS transiently suppresses clock gene expression and oscillation in the SCN (Okada et al., 2008) and phase-shift locomotor activity (Marpegán et al., 2005, 2009). One possible explanation for this is that TNF-a and IL-1b can inhibit the ability of BMAL1:CLOCK to induce activation of E-box-dependent genes in the SCN and liver (Cavadini et al., 2007). Also, the NF-kB subunit RelB interacts directly with BMAL1 to repress the circadian gene Dbp (Bellet et al., 2012). The mRNA expression of Bmal1 was repressed in the spleens across the full circadian day in mice subjected to collagen-induced arthritis versus controls (Hashiramoto et al., 2010). The constitutively low expression of BMAL1 in this model of arthritis could promote further inflammation from immune cells by the mechanisms stated above (Figure 3). The effect of cytokines on the circadian system is important given the increased use of biotherapeutics such as anti-TNF-a for chronic inflammatory conditions such as arthritis. Anti-TNF-a, by relieving the repression on the BMAL1:CLOCK heterodimer, may restore the function of the clock heterodimer and its ability to repress inflammatory genes (Figure 3). This could be another mechanism of action for this biological therapy in chronic diseases such as rheumatoid arthritis (RA). Molecular Clocks in Human Health and Disease The recent work identifying the roles for clock proteins such as BMAL1 and REV-ERBa in immunoregulation may provide new insights into the pathogenesis of infectious and inflammatory diseases. It is well known that inflammatory diseases such as asthma (Barnes et al., 1980; Kraft et al., 1996), RA (Haus et al., 2012), and atherosclerosis (Paschos and FitzGerald, 2010) have strong circadian components with exacerbations at night and into the early morning hours. There is also an intimate link between circadian rhythms and cancer, a disease associated with aberrant inflammation (Sahar and Sassone-Corsi, 2009). In healthy humans, the proinflammatory cytokines TNF-a and IL-6 peak in serum at 3 am and 6 am, respectively (Cutolo and Straub, 2008). In RA patients, the peak of both cytokines shift forward to the early morning with peak amounts of IL-6 10-fold higher in the serum of patients with RA versus controls (Cutolo and Straub, 2008). In addition, oscillations in clock proteins are lost in synovial fibroblasts cultured from patients with RA (Kouri et al., 2013). These data suggest severe circadian disruption in RA. The irregularity in the amount and timing of these cytokines might be responsible for the symptoms of stiffness and pain for RA patients in the morning hours. Therefore, the underlying basis of many inflammatory conditions could depend on a perturbed clockwork system both systemically and locally. Chronic disruption of the external cues of light to the molecular clock, as imposed by shift work and airline travel, augments the inflammatory response (Castanon-Cervantes et al., 2010) and increases susceptibility to many metabolic diseases with inflammatory features, such as atherosclerosis, obesity, and diabetes (Antunes et al., 2010). Our clock and output rhythms deteriorate and have a reduced amplitude with advancing age (Froy and Chapnik, 2007). Dampened circadian rhythms also occur with Immunity 40, February 20, 2014 ª2014 Elsevier Inc. 183 Immunity Review mice allowed ad lib access to high-fat diet (Kohsaka et al., 2007) and coincident metabolic disease. Therefore, the modern 24 hr society, in terms of our ad hoc pattern of activity, continuous caloric consumption, and high exposure to ambient light, can run at odds with our internal clock. It has also been concluded that ‘‘shift work that involves circadian disruption is probably carcinogenic to humans’’ (Straif et al., 2007) and can lead to higher incidence of cardiovascular disease and obesity (Karlsson et al., 2001; Stevens, 2009). Such studies on shift work might, however, be confounded by other variables such as gender, age, and weight. Mice that undergo a model of jet lag (akin to one transatlantic flight every week for 4 weeks) followed by recovery for 1 week, however, produce three times as much IL-6 upon challenge with LPS (Castanon-Cervantes et al., 2010). Conversely, recovery from sepsis induced in rats by caecal ligation and puncture is impaired when the regular 12:12 light:dark cycle is replaced by constant conditions (either constant light or constant darkness) after surgery (Carlson and Chiu, 2008). These observations highlight the possibility that recovery of patients from sepsis could be improved by simple measures such as reducing nocturnal light and noise and improving quality of sleep in intensive care units (ICUs) (Hrushesky and Wood, 1997; Herdegen, 2002). Concluding Remarks and Future Directions The picture that is fast emerging is that time of day is critical in terms of the type of immune response generated by an organism. The type of immune response can be broadly partitioned into two states, one of heightened alert and the other of rest and repair. The last decade provided a number of descriptive insights regarding the rhythmic changes that occur in immune parameters, and this decade has provided some of the molecular mechanisms underpinning these descriptions. What is intriguing is that the key components of the molecular clock, whose expression and activity will change across the circadian day, have direct relationships with important components of the immune system. We have detailed the data that we know so far connecting the core clock components, most notably BMAL1, CLOCK, and REV-ERBa and RORa, with important regulators of immune function and inflammation. BMAL1 appears to be a central mediator connecting both the clock and immune system together, its role being to limit inflammation. However, we are only at the beginning of our journey to understand the tight coupling of the circadian and immune systems and how disturbances to this relationship can lead to pathology and disease. There are still a number of questions to answer, among them: (1) What are the main signals that entrain or perturb the immune cell clock under different situations such as infection or inflammation? (2) What role do the clock proteins play in each type of immune cell? (3) What role does the clock play in the homeostatic functioning of the immune system? (4) Is the response of the human immune clock partitioned in the same way as the mouse immune clock? (5) Are inflammatory diseases in essence diseases of clock dysfunction? One question to investigate in this last regard is whether the anti-inflammatory effects of BMAL1 and REVERBa are somehow disrupted in diseases such as RA, giving rise to an inflammatory phenotype. 184 Immunity 40, February 20, 2014 ª2014 Elsevier Inc. Given that the molecular clock may fundamentally regulate many if not all aspects of our immune system, an understanding of how the clock and immune function intersect may reveal much-needed therapeutic opportunities for some of our most common chronic diseases. These insights might also provide rationale for chronotherapy, the dosing of medications with reference to 24 hr rhythms of disease activity, for existing and upcoming treatments of immune diseases. ACKNOWLEDGEMENTS This work was supported by a grant from the European Research Council (ERC), number 268155_MicroInnateto, to L.A.J.O. REFERENCES Aguilar-Arnal, L., and Sassone-Corsi, P. (2013). The circadian epigenome: how metabolism talks to chromatin remodeling. Curr. Opin. Cell Biol. 25, 170–176. Antunes, L.C., Levandovski, R., Dantas, G., Caumo, W., and Hidalgo, M.P. (2010). Obesity and shift work: chronobiological aspects. Nutr. Res. Rev. 23, 155–168. Arjona, A., and Sarkar, D.K. (2006). 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