Semin Immunopathol DOI 10.1007/s00281-013-0414-4

REVIEW

Circadian rhythms in leukocyte trafficking David Druzd & Alba de Juan & Christoph Scheiermann

Received: 30 October 2013 / Accepted: 2 December 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract A broad range of immunological processes oscillates over the course of a day. Recent findings have identified a molecular basis for the circadian clock in the regulation of the immune system. These rhythms manifest themselves in oscillatory behavior of immune cells and proinflammatory mediators, which causes a time-dependent sensitivity in the reaction to pathogens. This rhythmicity impacts disease manifestations and severity and provides an option for therapy that incorporates chronopharmacological considerations. This review will focus on the current knowledge and relevance of rhythmic immune cell trafficking. It will provide an overview of the molecular clock machinery and its interrelations with leukocyte migration and the immune response.

external factors such as light or food, the two most important entrainment cues or Zeitgebers (ZT). These criteria are fulfilled for instance for behavioral activity, core body temperature, and plasma hydrocortisone or melatonin levels, which are therefore all classical markers for circadian rhythms in mammals. In contrast, rhythms observed under nonconstant 12 h light/dark cycle conditions are referred to as diurnal. The aim of this review is to illustrate the impact of circadian rhythms on the migration of leukocytes within the body under physiological and pathological conditions.

Keywords Circadian rhythms . Molecular clock . Leukocyte migration . Immune system . Chronopharmacology

Circadian rhythms influence organisms most notably at the behavioral level, defining the periods of locomotor activity and rest. These broad behavioral patterns are based in specific oscillations at the molecular level, which then drive macroscopic changes. A surprising amount of genes fluctuates over the course of the day with approximately 10 % of genes showing circadian expression patterns in the liver and heart [1, 2]. Still, the dramatic behavioral changes seen between night and day would predict an even greater number of oscillatory regulations. This is likely the case, as rhythmic function is determined at the protein level. In some cases, circadian rhythmicity can even occur independently from transcription (see below). The following short overview of the clock machinery discusses the basic mechanisms in addition to recent new insights into the molecular control of the clock. The reader is encouraged to refer to excellent reviews in which these interrelations are discussed in more detail [3–5].

Introduction Virtually all organisms have developed biological clocks in order to adapt optimally to the rhythmic environmental changes they are being exposed to. Circadian rhythms exhibit a period length of approximately 24 h and can be entrained, i.e., they adjust to different geophysical environments and persist over a range of physiological temperatures. In addition, for a biological rhythm to be circadian in nature, it must oscillate also in a constant environment, independent from This article is a contribution to the special issue on New paradigms in leukocyte trafficking, lessons for therapeutics - Guest Editors: F. W. Luscinskas and B. A. Imhof D. Druzd : A. de Juan : C. Scheiermann (*) Walter-Brendel-Center of Experimental Medicine, Ludwig-Maximilians-Universität München, Marchioninistraße 27, 81377 Munich, Germany e-mail: [email protected]

Molecular clock machinery

Transcriptional-translational control The molecular clock machinery oscillates in virtually all tissues. At the basis of the clock lies a molecular mechanism that

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consists of several transcription factors, which cooperate to form a robust autoregulatory transcriptional-translational feedback loop (TTFL; Fig. 1). The core transcription factors that make up the positive limb of this interlocking loop are BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1, also known as ARNTL or MOP3) and circadian locomotor output cycles kaput (CLOCK) [6]. CLOCK and BMAL1 form a heterodimeric transcription factor complex, which binds canonical enhancer (E)-box response elements in the promoter regions of clockcontrolled genes (CCGs) containing the nucleotide sequence CACGTG. BMAL1-CLOCK induce their own expression by binding the promoter regions within the Clock and Arntl (encoding for BMAL1) genes. In addition, they promote the binding of the transcription factor ROR (retinoid acid receptor-related orphan receptor) to their E-box sequences [7–9]. The central role of BMAL1 is underscored by the fact that Arntl is the only single gene whose deficiency renders the whole organism arrhythmic as Arntl−/− mice exhibit a complete loss of circadian rhythmicity in constant darkness [10].

Binding of the BMAL1-CLOCK transcription factor complex induces the expression of three PER proteins (period circadian protein 1–3), and two CRY flavoproteins (cryptochrome 1 and 2). These proteins make up the negative limb of the feedback loop by forming the heterodimeric PERCRY transcription factor complex. As a result, the PER-CRY complex interferes with binding of the BMAL1-CLOCK complex to target genes, leading to an inhibition of Clock and Arntl transcription [11–13]. When BMAL-CLOCK expression decreases, so do levels of PER-CRY, thus releasing their suppressive effect and allowing a new cycle of Arntl and Clock expression. Apart from inducing expression of the PERCRY complex, BMAL1-CLOCK activates an additional repression cycle, in which REV-ERBα and REV-ERBβ (encoded by Nr1d1 and Nr1d2) play a critical role (Fig. 1). The REV-ERBα/REV-ERBβ heterodimeric complex inhibits Arntl transcription via binding of the Rev-Erb/ROR response elements [14] and thus adds an additional regulatory element to the molecular clock. O’Neill and colleagues recently discovered that circadian oscillations exist also in erythrocytes

NADP+ NADPH

CLOCK

BMAL1

NADH

NAD+

NAMPT PER

SIRT1

CRY

Bmal1 Clock

Cry CLOCK RORE

BMAL1

E-Box

Per

Nucleus

Nr1d1 Nr1d2 REV-ERBα REV-ERBβ

Fig. 1 The molecular clock. After transcription and translation, the core clock components BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1) and CLOCK (circadian locomotor output cycles kaput) form a heterodimeric transcription factor complex that binds to E-box motifs in clock-controlled genes (CCG). This drives their own transcription and initiates negative feedback loops, which consist of period circadian protein (PER) and cryptochrome (CRY) proteins and of REV-ERBα and REV-ERBβ (encoded by Nr1d1 and Nr1d2). The PER and CRY proteins form a transcription factor complex that is able to directly inhibit the BMAL1-CLOCK binding to the E-box

Cytoplasm

elements, whereas the REV-ERBα/REV-ERBβ complex indirectly inhibits BMAL1 transcription via RevErb/ROR response elements (RORE). SIRT1 (sirtuin), whose activity is dependent on the levels of NAD+ (nicotinamide adenine dinucleotide), binds to BMAL1-CLOCK and can deacetylate PER2. NAD+ levels are controlled indirectly by BMAL1-CLOCK, as the complex promotes the activity of NAMPT (nicotinamide phosphoribosyltransferase), leading to higher levels of NAD+. NADH and NADPH can affect the binding of the BMAL1– CLOCK complex to DNA directly

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that do not exhibit a nucleus [15]. Therefore, rhythms can occur transcription-independently without the need of the previously thought essential transcription-translational feedback loop [16]. The complexity in the regulation of the clock indicates that organisms are able to adjust their molecular clock very specifically to their environmental needs and create ideal circumstances for survival.

Entrainment of the organism The main task of the clock is to align the organism with its environment. Due to the opacity of the mammalian body, light, as the main environmental entrainment factor, cannot reach every cell directly. Instead, light is processed in the eye via photosensitive retinal ganglion cells [17], non-image forming cells that transfer the light impulse to the suprachiasmatic nucleus (SCN) via the retinohypothalamic tract. Lesion experiments in the brain have led to the identification of the SCN as essential for rhythmic activity and the master pacemaker of the organism [18, 19]. The SCN is situated directly above the optic chiasm in the anterior hypothalamus. It consists of a pair of nuclei, each of which is made up of approximately 10,000 highly interconnected neurons, which facilitates the rapid establishment of phase coherence once a light stimulus is perceived [20]. Synchronization of peripheral clocks by the SCN The SCN links the organism with the environment. Its key role is to transfer the light-induced phase coherence of its neurons to synchronize all cells of the body. Peripheral clocks can be found in virtually all organs and cells, which are being synchronized via direct and indirect signals that are emitted from the SCN. Feeding-fasting cycles and body temperature are important internal Zeitgebers [21]. Another key pathway for the entrainment of peripheral clocks involves humoral and neuronal output systems. This pathway is mediated by the hypothalamic-pituitary adrenal axis (HPA axis). The paraventricular nucleus of the hypothalamus is controlled by excitatory and inhibitory SCN efferents and is capable of inducing the release of adrenocorticotrophic hormone (ACTH) from the pituitary. As a result, the adrenal gland is producing glucocorticoids, such as hydrocortisone, in a circadian manner [22]. Hydrocortisone binds to the glucocorticoid receptor (GR), which is itself under circadian control of the transcription factor CLOCK [23, 24]. Glucocorticoids play essential roles in the modulation of inflammatory processes, as they upregulate the expression of anti-inflammatory proteins and inhibit the synthesis of proinflammatory molecules [25]. Glucocorticoid synthesis is highly regulated by intestinal epithelial cells (IECs), and IECs show a temporal circadian expression of clock genes. Chambon and colleagues

discovered recently that gut microbiota play a previously unrecognized role in modulating the circadian clock [26]. In IECs, RORα function is dominant during the behavioral activity phase, inducing the expression of toll-like receptors (TLRs) while maintaining low hydrocortisone production. The intestinal microbiota interacts with TLRs, downmodulating expression of RevErbα and ensuing low corticosterone production. In contrast, in the resting phase, REV-ERBα activity is dominant, resulting in enhanced corticosterone production. This indicates the importance of entrainable oscillators like food for the modulation of circadian rhythms due to the interaction between intestinal epithelial cells and the gut microflora.

Rhythms in immune cell trafficking Over the past decades, research has provided ample evidence that integral parts of the immune system oscillate over the course of 24 h. This is reflected in the numbers and activity levels of immune cells in blood and tissues as well as the amount of soluble immunomodulatory factors such as chemokines and cytokines. Many parameters of the immune system have been shown to fluctuate according to whether the species is diurnal (such as humans) or nocturnal (such as rodents) [27, 28] (Table 1). For example, the circulating leukocyte count peaks during the day in rodents but during the night in humans, both during their respective resting phases (Fig. 2). Rhythms in the hematopoietic stem cell niche The hematopoietic stem cell (HSC) niche is located in the bone marrow (BM) in the adult and is the origin for all blood cells. From the BM, HSCs, progenitor cells and mature hematopoietic cell populations disseminate into the circulation. Due to the central role of HSCs in hematopoiesis, the identification of the cellular components that make up and regulate the HSC niche is important and is the intense focus of current research. Recent evidence points to a rhythmic modulation of this specialized microenvironment. The precise geographical location of the niche has remained elusive [29, 30]. Different studies have located HSCs in proximity to the bone [31] and osteoblasts [32], the vasculature and endothelial cells [33] as well as stromal components such as CXCL12-abundant reticular (CAR) cells [34], Nestin + cells [35], sympathetic nerves and associated nonmyelinating Schwann cells [36, 37], Leptin receptor expressing (Lepr+) cells [33], and Prx1 transcription factor expressing cells [38]. New data indicate that the HSC niche is located in an area that is enriched in arterioles and encompasses most if not all of these components [39]. Arterioles are enwrapped by perivascular Nestinperi NG2+ cells expressing

Semin Immunopathol Table 1 Oscillations in immunomodulatory molecules Class of molecule

Molecule

Species

Source

Assay

References

Cytokines

IL2 IL6 IL10 IL12 IFNγ TNFα

Human Mouse Human Mouse Rat Human Rat Human Mouse

Serum Serum; peritoneal macrophages Serum Serum; peritoneal macrophages NK cells Serum NK cells Serum Serum; peritoneal macrophages Muscle endothelial cells Serum; peritoneal macrophages Serum; peritoneal macrophages BM microenviroment

ELISA Multiplex cytokine assay; qPCR ELISA Multiplex cytokine assay; qPCR qPCR; western Blot ELISA qPCR; western Blot ELISA Multiplex cytokine assay; qPCR qPCR Multiplex cytokine assay; qPCR Multiplex cytokine assay; qPCR ELISA; qPCR

[74] [69] [74] [69] [102] [74] [102] [74] [69] [50] [69] [69] [44]

Human Mouse Human Mouse

T cells HSCs T cells BM endothelial cells

FACS FACS FACS qPCR; FACS

[49] [47] [49] [50]

Rat

BM endothelial cells Muscle endothelial cells BM endothelial cells NK Cells NK cells Splenic B cells, macrophages Splenic macrophages

qPCR; FACS Immunofluorescence; qPCR qPCR; FACS qPCR; western Blot qPCR; western Blot qPCR; western Blot; FACS Microarray; qPCR

[50] [50] [50] [102] [102] [63] [62]

Chemokines

GM-CSF CCL2

Chemokine receptors

CCL5 CXCL1 CXCL12 CX3CR1 CXCR4

Adhesion molecules

E-Selectin

Others

P-Selectin ICAM1 VCAM1 Granzyme B Perforin TLR9 Nfkbia (IκBα)

Mouse

IL interleukin, IFNγ interferon gamma, TNFα tumor necrosis factor alpha, CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, CX3CR CX3C chemokine receptor, CXCR4 CXC chemokine receptor 4, ICAM1 intercellular adhesion molecule 1, VCAM1 vascular cell adhesion molecule 1, GM-CSF granulocyte macrophage colony-stimulating factor, TLR9 toll-like-receptor 9, IκBα nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

high levels of the intermediary filament Nestin that are innervated by sympathetic nerve fibers and Schwann cells. In addition, arterioles are found in higher numbers in closer proximity to the endosteum than to the central vein. Sympathetic nerves (and/or their associated Schwann cells) endorse quiescence within niche cells [36, 40], which mediate quiescence in the HSC population [39]. In line with these data, sympathectomy induces an increase in Nestin cell proliferation suggesting an important regulation of the niche size and HSC quiescence by local adrenergic tone [40]. The sympathetic nervous system and its artery-accompanying nerves therefore appear to play a major role in modulating the hematopoietic stem cell niche locally. The SNS is an important regulator of circadian rhythms in peripheral tissues. Levels of locally released nor-adrenaline as well as circulating adrenaline exhibit peak levels at the onset of the activity phase [41]. CAR cell numbers in the murine BM oscillate over the course of a day exhibiting a peak closer to the activity phase (ZT9) [42]. Oscillations in the proliferation rate of hematopoietic stem and progenitor cells (HSPCs)

as well as mature leukocytes in the BM also exist. However, the times of peak and trough have not been clearly defined as values vary dramatically between studies [43]. Together, these data indicate that the HSC niche is located in an arteriole-rich area, which is modulated by the SNS, and that key components of the HSC niche exhibit rhythmic oscillations. Mobilization of hematopoietic cells from the BM In addition to the rhythms in CAR cell numbers in the BM, the release of HSCs from the BM into the circulation follows distinct circadian oscillations as well. HSCs are mobilized into the circulation at the beginning of the resting phase and exhibit peak levels in blood during the day in mice (ZT5) and a trough at night (ZT13) [44]. This is regulated by expression of the chemokine CXCL12 in the bone marrow microenvironment, which is consistent with its role as a major chemoattractant in this organ [45]. CXCL12 levels peak at ZT21 and exhibit a trough at ZT9 [44]. The cell type responsible for these chemokine oscillations are most likely Nestin and CAR cells,

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TLR9

Blood

Spleen

Macrophage

Muscle

Bone Marrow HSC P-selectin, E-selectin, VCAM-1 CXCL12 P-selectin, E-selectin, VCAM-1

CAR cell Nestin+ cell

ICAM1, CCL2

ICAM1, CCL2

Fig. 2 Oscillations in immune cell parameters in different tissues. Oscillations in immune cells between day (behavioral rest phase) and night (behavioral active phase) are shown for different murine tissues. During the active phase, higher numbers of macrophages can be found in the spleen compared to rest. These macrophages express increased levels of Toll-like receptor (TLR) 9. In blood, hematopoietic stem and progenitor cells (HSPCs) as well as leukocyte subsets are more abundant during the resting phase. Red blood cells (RBC) and platelets do not oscillate. In the cremaster muscle, expression of intercellular cell adhesion molecule 1

(ICAM-1) as well as chemokine (C-C motif) ligand 2 (CCL2) is enhanced at the beginning of the active phase, which promotes increased leukocyte recruitment. HSCPs are being mobilized from the bone marrow (BM) into the circulation at the beginning of the resting phase while at the onset of the night, HSPCs as well as neutrophils exhibit enhanced BM homing. This is due to increased expression of the cell adhesion molecules Pselectin, E-selectin, and VCAM-1 on endothelial cells as well as of the chemokine (C-X-C motif) ligand 12 (CXCL12) expressed by CXCL12abundant reticular (CAR) cells and Nestin+ cells

both of which express the highest levels of this chemokine and appear to have overlapping cellular compositions [46]. Nestin cells express the β3-adrenergic receptor on their surface, which sympathetic nerves target to modulate niche cell activity locally via direct adrenergic nerve input. Adrenergic signals lead to degradation of the transcription factor SP1 and rapid downregulation of BM CXCL12 levels [44]. Importantly, CXCR4, the receptor for CXCL12, is also under circadian control on HSPCs indicating that for maximal efficacy receptor-ligand pairs oscillate with the same phase and peak times. Both molecules are regulated by clock genes, as Arntl−/− mice show oscillations in neither CXCR4 nor CXCL12 [44,

47]. Whether this is a direct transcriptional effect or due to lack of general oscillations in these mice is currently unclear. Enforced mobilization of HSCs by granulocyte colonystimulating factor (G-CSF) or plerixafor (AMD3100, a CXCR4 antagonist), agents used in clinic for the mobilization of HSCs, is still rhythmic with similar peaks and troughs as under steady-state conditions. This indicates that administration of these drugs bearing in mind chronopharmacological considerations could enhance transplantation outcomes in poorly mobilizing patients [47, 48]. Similarly to HSCs, all progenitor cell subtypes (CFU-GM, CFU-G, CFU-M, BFU-E and CFU-E) and leukocyte subsets

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are more abundant in blood during the resting phase [47, 49]. T cells fluctuate in a circadian manner, whereas the subtype of T cells affects the phase of oscillation. Central memory and naïve CD4+ and CD8+ cells peak during the resting phase in humans, whereas effector CD8+ cells counts peak during activity [49]. In contrast, platelet and RBC numbers do not oscillate. These data indicate that the whole hierarchy of immune cells, from HSCs and progenitors to mature leukocyte subpopulations exhibit oscillating migration patterns in blood. Hematopoietic cell recruitment to tissues The strong oscillations observed in circulating leukocyte counts suggest that not only their mobilization from the BM is rhythmic but that also their exit from the blood occurs in a cyclical manner. Indeed, oscillations exist in the homing of hematopoietic cells to different organs. These rhythms, however, oscillate with inverted peaks to the ones observed in circulation. Leukocyte recruitment to BM, skeletal muscle, and liver occurs predominantly at the onset of the activity phase (ZT13) with a nadir during the daytime (ZT5) in mice [50, 51]. In the BM, all hematopoietic cells investigated show rhythmic recruitment, including HSCs, progenitor cells, and neutrophils, suggesting that a general rhythmic homing pattern to the BM exists. In contrast, in tissues where under steady-state conditions no infiltration of neutrophils occurs such as is the case for skeletal muscle tissue, oscillations exist instead in the numbers of immigrating monocytes, which as macrophages make up most of this tissue’s leukocyte component [52]. With respect to the mechanism involved, differences manifest themselves in an oscillatory molecular signature of adhesion molecules expressed in the vascular beds. In BM, rhythms in leukocyte recruitment are regulated at the step of leukocyte rolling due to the expression of P-selectin, Eselectin, and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells [50]. These molecules peak at night similar to CXCL12 levels in the BM. In contrast, in skeletal muscle, rhythms occur at the stages of leukocyte adhesion and extravasation, downstream steps within the leukocyte adhesion cascade. This is regulated by rhythmic expression of endothelial cell intercellular adhesion molecule 1 (ICAM-1) and chemokine (C-C motiv) ligand 2 (CCL2). Together, these data describe tissue-specific rhythms similar to what has been observed for liver and heart. While in these latter organs, circadian gene expression is extensive very few genes show oscillations in both tissues even though they are expressed [2]. How this specificity in rhythmic expression is achieved in different organs is currently unknown. Leukocyte recruitment to tissues is under control of the sympathetic nervous system. Adrb2−/− and Adrb3−/− animals that are deficient in the β2- or β3-adrenergic receptors, respectively, show no oscillatory recruitment to either BM or

muscle tissue [50]. In line with these findings, administration of β2- or β3-adrenergic receptor agonists (clenbuterol or BRL37344, respectively) was found to greatly enhance BM engraftment. This leaves the question as to how the same adrenergic stimuli can control opposing physiological phenomena as seen in the homing and release of cells to and from the BM. Clearly, systemic stimulation using adrenergic receptor agonists induces both events as release of HSCs into the circulation is only seen when homing is blocked at the same time with antibodies against P- selectin, E-selectin, and VCAM-1 [44]. Thus, it is likely that the route (neural vs. humoral), the strength of the necessary signals, and/or the involved BM cell type vary in how these events are interpreted. These recent data shows that both rhythmic leukocyte release from the BM and recruitment to tissues contribute to the net effect of oscillatory leukocyte numbers in blood. Sympathetic adrenergic nerves play a key role in orchestrating these events, as do the oscillations of chemokines and adhesion molecules on both hematopoietic and nonhematopoietic cells. Relevance of rhythmic leukocyte trafficking in physiology and pathology The release and the homing of hematopoietic cells are intricately linked. In inflammatory scenarios, neutrophils recruited to sites of inflammation are eventually cleared by tissue-resident macrophages [53]. However, under normal homeostatic conditions, neutrophils are eliminated in spleen, liver, and the BM [54]. Neutrophils age over the course of a day in blood, exhibiting a phenotype that changes from being CD62LhighCXCR4low on nascent cells to CD62LlowCXCR4high on bona fide aged cells in vivo [42, 55], similar to what has been described in vitro. At the time of highest neutrophil homing to the BM (ZT13) aged CD62LlowCXCR4high neutrophils were hardly detected in blood suggesting that these are preferentially recruited to this tissue at that time [42]. Interestingly, in the BM this neutrophil homing process is of importance for the next round of cyclical release of HSPCs. Macrophages phagocytose the aged neutrophil population and modulate the hematopoietic niche by inducing a reduction in the numbers of CAR cells. Macrophages therefore represent novel and key circadian regulators of niche cell activity as they can sense oscillatory neutrophil populations in blood and modulate those numbers indirectly via the HSC niche [56, 57]. A finding that underscores the relevance of rhythms in myeloid cells for leukocyte trafficking was recently made also for monocytes. Chawla and colleagues showed that inflammatory monocytes expressing high levels of Ly6C (Ly6Chigh) oscillate in blood and other tissues whereas the other, Ly6Clow or patrolling monocyte subset, did not [58, 59]. This oscillation was dependent on expression of the circadian gene Arntl in the myeloid lineage and conferred protection against

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infection with Listeria monocytogeneses. Lyz2Cre:Arntl flox/flox mice exhibited greatly reduced survival after infection with this pathogen. What the precise myeloid cell type is that mediates these BMAL1-dependent effects (monocytes, macrophages, or neutrophils) is currently unknown. Due to the described rhythmic nature and interactions of all these populations a contribution by each cell type is possible. For more details on neutrophil and monocyte trafficking, the reader is referred to other reviews in this issue [60, 61]. Leukocytes in tissues and blood exhibit rhythms in cell surface receptor expression. These range from molecules involved in trafficking (adhesion molecules and chemokine receptors of which CXCR4 has been most thoroughly explored [42, 47, 49]) to the expression of pattern-recognition receptors involved in the direct response to invading pathogen. Macrophages in the spleen express the LPS co-receptor CD14 in a circadian manner and show circadian oscillations in TNFα and IL6 secretion after LPS stimulation [62]. Interestingly, these rhythms are not driven by systemic glucocorticoid levels, but rather by a macrophage-intrinsic circadian clock. TLR9, the receptor for bacterial DNA is under direct circadian control in splenic B-cells and macrophages, as BMAL1CLOCK has a binding site in the promoter region of the Tlr9 gene [63]. Importantly, in a vaccination model using a TLR9 ligand as adjuvant, mice immunized at the time of enhanced TLR9 responsiveness showed an improved adaptive immune response weeks later. This suggests that daily oscillations that most likely occur to counter the timing of a specific pathogen interaction can have significant reverberations much later, indicating the significance of short-term, circadian oscillations for a long-term benefit in survival.

Interactions between clock components and immune parameters Components of the molecular clock play important roles in the regulation of the immune system (Table 2). Circadian clock genes have been shown to regulate cytokine secretion and lymphoid cell development. Per2 mutant mice for example, display decreased expression of the pro-inflammatory cytokine IFNγ [64, 65]. As a consequence, these mice are more resistant to LPS-induced endotoxic shock than WT animals [65]. In contrast, Cry1−/−Cry2−/− mice exhibit increased levels of the proinflammatory cytokines IL6, TNFα as well as inducible nitric oxide synthase. This has been attributed to higher levels of cAMP, leading to activation of protein kinase A and activation of NFκB via phosphorylation of the p65 subunit [66]. Similarly to CRY proteins, RORα can suppress the NFκB signaling pathway by reducing p65 translocation. This causes a reduction in TNFα-induced IL6, IL8, and COX2 expression [67]. REV-ERB proteins also play a role in the regulation of cytokine levels. Administration of a

synthetic REV-ERB ligand or genetic knockdown of RevErbα expression is effective at modulating production and release of the proinflammatory cytokine IL6 [68, 69]. REV-ERBs are also able to suppress macrophage gene expression by inhibiting the functions of distal enhancers for macrophage lineage-determining factors such as the transcription factor PU.1 [70]. BMAL1 binds directly to the promoter regions of the chemokines Ccl2 and Ccl8 as well as S100a8. Interestingly, however, the net effect is a suppression of chemokine transcription since BMAL1 recruits the histone methyltransferases EZH2 (enhancer of zeste), EED (extra-sex comb), and SUZ12 (suppressor of zeste) to the genetic loci [58]. This appears to be crucial for downmodulating inflammatory activity as myeloid Arntl-deficient mice exhibited a greatly enhanced inflammatory status [58]. BMAL1 also plays a crucial role in lymphocyte development, as it regulates the development of pre-B cells to mature B-cells. Arntl−/− mice exhibit reduced levels of mature B-cells in blood, spleen, and bone marrow [71]. Recent evidence suggests that B-cells are not the only integral part of adaptive immune responses under circadian control. TH17 cells protect the organism from bacterial and fungal infection and play a decisive role in autoimmune disorders. Hooper and colleagues discovered that the transcription factor nuclear factor interleukin 3 regulated (NFIL3) is a negative regulator of Rorγt and TH17 differentiation and is itself inhibited by RevErbα. As a consequence, NFIL3 expression was higher in Nr1d1−/− mice, leading to reduced levels of TH17 cells [72]. The interactions between clock genes and cytokines are bidirectional. TNFα and IL1β suppress BMAL1-CLOCK binding to E-box containing promoter regions. As a consequence, locomotor activity is reduced and rest periods are prolonged, a phenotype, which can be observed in infectious and autoimmune diseases, as patients often suffer from exhaustion and fatigue [73]. These data indicate an intricate relationship between the molecular clock and immune parameters. Whether these effects are in all cases due to a loss of cellular rhythm or whether direct transcriptional activities are involved is not clear.

Immune diseases exhibiting a rhythmic phenotype Rhythmic oscillations have been reported in human serum under physiological conditions for the cytokines IL2, IL10, TNFα, and GM-CSF [74], all of which can play a decisive role in the pathology of diseases. Accordingly, a number of chronic inflammatory diseases show a diurnal phenotype with a time-of-day dependent aggravation of symptoms (Table 3). Rheumatoid arthritis (RA) is a typical example for a circadian disease with rhythmic oscillations in serum levels of the proinflammatory cytokines IL6 and TNFα peaking in the

Semin Immunopathol Table 2 General and immune phenotypes of molecular clock mutants

Genotype

Phenotype

Refs

Arntl −/− (Mop3, Bmal1)

Impaired and reduced locomotor activity in light-dark cycles; complete loss of circadian rhythmicity in constant darkness No oscillations in CFU-Cs, CXCR4, Cxcl12 or leukocyte adhesion Reduced levels of mature B cells but not other immune cells in peripheral blood, spleen and bone marrow Loss of circadian IL6 expression after LPS-stimulation Increased serum levels of IL1β, IL6, IFNγ, and CCL2; enhanced lethality to L. monocytogeneses; higher macrophage numbers in white adipose tissue Lengthened circadian rhythm; abolishment of persistence of rhythmicity

[10]

Lyz2Cre x Arntl loxP/loxP

Clock−/−

Cry1−/− Cry2−/− Cry1−/−Cry2−/−

Per1−/− Per2−/−

Per1−/− Per2−/− Nr1d1−/− (RevErbα) Nr1d2−/− (RevErbβ) Nr1d1−/−Nr1d2−/−

Reduced nuclear accumulation of p65 and NFκB activation Increased NFκB activation; enhanced levels of IL6, TNFa, Cxcl1 and iNos; heightened sensitivity to LPS Accelerated periodicity of locomotor activity in constant darkness Enhanced levels of IL6 Delayed periodicity of locomotor activity in constant darkness; no immune phenotype described Complete loss of circadian rhythmicity in constant darkness Increased NFκB activation; enhanced levels of IL6, TNFa, Cxcl1 and iNos; heightened sensitivity to LPS Shorter circadian period length in constant darkness Altered rhythms of IFNγ, perforin and granzyme B in splenic NK cells Shorter circadian period length in constant darkness No rhythm and decreased expression of IFNγ in spleen Decreased IFNγ and IL1β levels in serum; increased resistance to LPS-induced endotoxic shock Complete loss of circadian rhythmicity in constant darkness; no immune phenotype described Shorter period length in constant conditions Loss of circadian IL6 expression after LPS-stimulation Normal period length; no immune phenotype described Complete loss of circadian rhythmicity in constant darkness; no immune phenotype described

early morning hours [75]. As a consequence, joint pain and morning stiffness are exacerbated at this time [76]. In contrast to RA, symptoms in asthma worsen during the night. While lung function exhibits fluctuations also in healthy individuals, these oscillations are far more pronounced in patients with nocturnal asthma, showing a significant difference in forced expiratory volume and expiratory flow rate between wakefulness and sleep. This has been associated with increased eosinophil recruitment into the lung and activation of these cells at night [77]. Whether the peak of immune cell trafficking in the lung is similar compared to other organs is currently unclear. With respect to diseases of the cardiovascular system, a morning surge in blood pressure can be observed in humans, which has been associated with a higher susceptibility of sudden cardiac and cerebral events such as myocardial infarction and stroke [78–81]. In addition, leukocyte infiltration aggravates the infarct in a time-dependent manner indicating a critical role for rhythmic leukocyte trafficking for the

[44, 47, 50] [71] [69] [58]

[103] [104] [66] [105] [66] [105] [105] [66] [106] [107] [108] [65] [64] [106] [14] [69] [109] [109]

severity of the disease [82]. Whether also increased intravascular events such as leukocyte adhesion and heterocellular interactions between leukocytes and other free-flowing blood components, which in mice have been observed to occur predominantly at the onset of behavioral activity [50], can rhythmically affect these symptoms in humans is not known. A circadian phenotype is also observed for infectious diseases. Plasmodium chabaudi (rodent malaria) parasites synchronize their bursting from infected erythrocytes every 24 h at approximately midnight. This phenomenon is known as Cinderella syndrome and appears to provide a benefit for the parasite by overwhelming the immune system [83]. Similarly, Plasmodium falciparum and Plasmodium malariae exhibit cell cycle durations of 48 and 72 h and associated “tertian” and “quartan” fevers. Thus, a variety of diseases exhibit striking rhythms in disease occurrence and symptom aggravation. Gaining better knowledge of the underlying molecular events will provide new insights into treatment options.

Semin Immunopathol Table 3 Immune diseases with a rhythmic phenotype Type of disease

Disease

Symptoms

Peak (o’clock)

References

Inflammatory

Rheumatoid arthritis Allergic reactions Asthma

Joint pain; morning stiffness Scratching; erythema Cough; dyspnea; airflow limitation; airways hyper-responsiveness; sudden death Burst of malaria-infected erythrocytes Heart rate >100 bpm. Pain Pain Pain Cognitive impairment; severe headaches; subarachnoid hemorrhages

05:00–08:00 23:00 04:00

[110] [111] [112]

0:00 09:00–12:00 18:00 09:00–13:00 09:00 06:00–10:00

[83] [81] [113] [114, 115] [80] [116]

Cardiovascular

Malaria (Cinderella syndrome) Ventricular tachyarrhythmias Sickle cell vaso-occlusive crisis Myocardial ischemia Myocardial infarction Ischemic and hemorrhagic stroke

Rhythmic alterations and impact on diseases The above-mentioned studies demonstrate that a functioning immune system exhibits oscillatory fluctuations in leukocyte trafficking. As a consequence, disorders and disruptions in the circadian clock can lead to severe changes in immune function and cause diseases. The impact of circadian disruptions on the outcome of diseases is apparent in populations that are being confronted with nonphysiological working habits as seen in shift work (Table 4). A strong correlation between shift work and increased incidence in the development of various types of cancer (colorectal, breast, prostate, and non-Hodgkin lymphoma) can be observed, which may in part be due to a disrupted, non-functioning immunosurveillance system of the body [84–88]. Also cardiovascular diseases are more prevalent in shift workers. Atherosclerotic risk factors are enhanced in this group compared to workers on a normal schedule, which is associated with a higher incidence for atherosclerosis, coronary heart disease, and myocardial infarction [89, 90]. In animal models, rats exposed to chronic jet-lag showed attenuated circadian natural killer (NK) cell cytolytic activity due to altered expression of Arntl, Per2, the cytolytic factors perforin and granzyme B as well as IFNγ. After injection of

Table 4 Shift work and associated diseases

tumor cells, increased lung tumor growth was found in the shifted group underlining the importance of a working clockwork in the detection of cancer cells [91].

Chronopharmacology Circadian rhythms modulate the efficacy of pharmaceutical drugs on many levels, including their toxicity to the host, their sensitivity to the specific target and their half-life. The optimal circadian drug is given only once, is not metabolized and has an intrinsic rhythmic activity that matches that of the target to minimize side-effects. As an alternative, a drug could be given daily at the time of peak target sensitivity with a short half-life to exclude detrimental effects. These aspects are influenced by pharmacokinetics, the processes concerning the liberation, administration, distribution, metabolism, and elimination (LADME) of drugs and by pharmacodynamics, the drugs’ effects. Due to the oscillatory nature of leukocyte trafficking behavior in blood and other tissues, the time-sensitive administration of a drug (chronotherapy) is therefore an important tool to optimize drug efficacy over toxicity. An optimal chronotherapeutic regime could therefore lower the drug amount needed and thus reduce costs significantly.

Increased disease incidence

Occupation

Population

References

Colorectal cancer Breast cancer Prostate cancer Non-Hodgkin lymphoma Peptic ulcer Type 2 diabetes

Nurses in the USA Nurses in the USA Working men in Japan Workers in Finland Workers in Japan Male factory workers in Japan Nurses in the USA Workers in Germany

78,586 115,022 14,052 1,669,272 11,657 2,860 177,184 2,510

[85] [84] [86] [88] [117] [118] [119] [89]

Atherosclerosis and myocardial infarction

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The reasons for time-dependent effects of various drugs lie in the rhythmic differences in the absorption of these xenobiotic compounds (alteration in gastric and intestinal pH), their distribution (changes in blood flow), overall metabolism (variations in expression levels of detoxifying enzymes), and elimination (changes in renal filtration rates). These phenomena are considered as the molecular basis for the timedependence of drug toxicities and efficacies. The metabolism of xenobiotics can be divided into three phases. In phases 1 and 2, compounds are modified by enzymes such as cytochrome P450 and conjugated with charged molecules, creating highly polar compounds. These are then excreted in phase 3 via bile, urine, or feces [92, 93]. The PARdomain basic leucine zipper (PAR bZip) transcription factors DBP (d-site of albumin promoter), thyrotroph embryonic factor (TEF), and hepatocyte leukemia factor (HLF) are transcriptionally regulated by core clock components and accumulate in a highly circadian manner in several peripheral tissues, including liver and kidney [94, 95]. Schibler and colleagues found that besides being prone to epilepsy, PAR bZip triple knockout mice are hypersensitive to xenobiotic compounds as these three proteins control expression of enzymes responsible for xenobiotic detoxification including cytochrome P450 family members [96]. Furthermore, HLF increases the amplitude in the rhythm of multi-drug-resistanceprotein 1 expression, an ATPase-binding cassette transporter responsible for the transport of several drugs and xenobiotics [97]. NFIL3, also a PAR-bZip transcription factor, is an antagonist to DBP, TEF, and HLF with inverse oscillations in mRNA levels. NFIL3 is also expressed in liver and competes with the binding sites of the other PAR-bZip transcription factors leading to a suppression of target genes [97, 98]. The aryl hydrocarbon receptor (AhR) is an additional important transcription factor under circadian control that regulates key detoxifying enzymes such as members of the cytochrome P450 family [99, 100]. Xenobiotics have direct effects on the molecular components of the clock and can thus modulate circadian rhythms. Synthetic glucocorticoids, such as dexamethasone, are used to treat several inflammatory diseases, from rheumatoid arthritis to allergic anaphylactic shock, as well as certain hematological malignancies and asthma. Besides their potent anti-inflammatory and immunosuppressant properties, these compounds, similar to their endogenous analogs, are able to reset circadian gene expression in peripheral tissues, while not affecting neurons of the central clock in the SCN [101]. Thus, pharmacokinetics and pharmacodynamics are critical processes to consider when targeting circadian pathways, as they themselves underlie a strong circadian regulation. Stem cell mobilization and engraftment As indicated by the observed circadian rhythm in HSPC numbers in blood in mice [44], a simple change of collection

time to coincide with the peak of circadian egress may optimize the number of hematopoietic stem and progenitor cells collected in humans. In a retrospective study, mobilizing patients with G-CSF yielded significantly increased CD34+ cell numbers when collected in the afternoon compared to the morning [47]. However, a follow-up single-center, prospective cohort study of 11 allogeneic sibling donors investigating the numbers of CD34+ and CD34+CD38− hematopoietic progenitor/stem cell numbers in peripheral blood failed to show rhythmic changes in cellular yields [48]. These data indicated that endogenous progenitor rhythms may be altered by pharmacodynamic effects and the timing of G-CSF, as well as donor age, medical history, and medications. Still, these findings have important implications for the design of future studies, not only for the mobilization of HSPC but also for their homing and engraftment. Together, the majority of currently available drugs display a constant drug release rate and a long half-life, leading to higher patient compliance but not necessarily to optimal immunomodulatory effects. In addition, current clinical practice mainly relies on staffing convenience rather than kinetic data with respect to circadian oscillations in drug efficacy. As a consequence, the goal is to develop novel drug delivery systems, with the property of modulating drug release rhythmically as well as increase the number of clinical trials that focus on the best time management in the administration of currently available drugs. Simple alterations in therapeutic practices that incorporate the aspect of time may affect clinical outcome dramatically.

Conclusions The immune system is a highly adaptive system that is regulated by environmental factors. The SCN synchronizes an intrinsic circadian oscillation that has so far been characterized in many immune cells via neural and humoral means. This oscillation is an important and previously unrecognized regulator of leukocyte migratory behavior. Recent insight indicates that leukocyte trafficking is regulated in a rhythmic manner at every step of the maturation process. The regulation of the hematopoietic stem cell niche, the mobilization of hematopoietic cells into the circulation, their exit from blood into tissues, expression of immunomodulatory factors, and even their phagocytosis all occur in a rhythmic manner. Components of the molecular clock are intricately regulating immunomodulatory molecules, which themselves modulate behavioral activity. The reason for rhythmic leukocyte trafficking and an oscillatory immune system is most likely a periodic exposure to pathogen with peaks and troughs occurring at specific times. Based on the molecular insights and the rhythmic exacerbations of many acute and chronic inflammatory diseases, incorporating the element of time in the design of

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experimental and clinical studies that target leukocyte and HSPC trafficking seems appropriate. Hence, future research will likely lead to the development of better drug and vaccination strategies by modulating the rhythmic immune system. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (an Emmy-Noether grant to C.S. and a PhD fellowship (SFB 914 TP B09) to A. d. J.).

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