Time of day-dependent variations of immune system parameters are ubiquitous phenomena in immunology. The circadian clock has been attributed with coordinating these variations on multiple levels; however, their molecular basis is little understood. Here, we systematically investigated the link between the circadian clock and rhythmic immune functions. We show that spleen, lymph nodes, and peritoneal macrophages of mice contain intrinsic circadian clockworks that operate autonomously even ex vivo. These clocks regulate circadian rhythms in inflammatory innate immune functions: Isolated spleen cells stimulated with bacterial endotoxin at different circadian times display circadian rhythms in TNF-␣ and IL-6 secretion. Interestingly, we found that these rhythms are not driven by systemic glucocorticoid variations nor are they due to the detected circadian fluctuation in the cellular constitution of the spleen. Rather, a local circadian clock operative in splenic macrophages likely governs these oscillations as indicated by endotoxin stimulation experiments in rhythmic primary cell cultures. On the molecular level, we show that >8% of the macrophage transcriptome oscillates in a circadian fashion, including many important regulators for pathogen recognition and cytokine secretion. As such, understanding the cross-talk between the circadian clock and the immune system provides insights into the timing mechanism of physiological and pathophysiological immune functions.adrenalectomy ͉ LPS ͉ IL-6 ͉ microarray ͉ TNF-␣ A 24-h periodicity in the environment has led to the evolution of molecular circadian clocks in organisms ranging from cyanobacteria to humans. Circadian rhythms display a near 24-h period and persist even in the absence of external timing information. In mammals, a small hypothalamic region, the suprachiasmatic nucleus (SCN), has been identified as the master pacemaker regulating circadian rhythms in physiology, metabolism, and behavior (1). Recent evidence shows that also peripheral organs such as liver, heart, kidney, skin, and even cultured cell lines contain circadian oscillators. Although the SCN probably sets the phase of these peripheral clocks (by as yet unknown means), recent reports implicate peripheral clocks in the regulation of local physiology (2-4). The fundamental mechanism of rhythm generation is cell autonomous and highly conserved in SCN and peripheral cells: Interlocked transcriptional/translational feedback loops involving clock genes, such as Per1-3, Cry1-2, Clock, Bmal1, and Rev-Erb␣ create oscillations on the molecular level (reviewed in ref. 2).In the immune system, many functions and parameters have been described to be time-of-day dependent, e.g., lymphocyte proliferation (5), natural killer (NK) cell activity (6), humoral immune response (7), rhythms in absolute and relative numbers of circulating white blood cells and their subsets (8), cytokine levels (9), and serum cortisol (10) (reviewed in ref. 11). In addition, time-of-day variation in susceptibility to infection (12), cour...

In mammals, the circadian pacemaker, which controls daily rhythms, is located in the suprachiasmatic nucleus (SCN). Circadian oscillations are generated in individual SCN neurons by a molecular regulatory network. Cells oscillate with periods ranging from 20 to 28 h, but at the tissue level, SCN neurons display significant synchrony, suggesting a robust intercellular coupling in which neurotransmitters are assumed to play a crucial role. We present a dynamical model for the coupling of a population of circadian oscillators in the SCN. The cellular oscillator, a three-variable model, describes the core negative feedback loop of the circadian clock. The coupling mechanism is incorporated through the global level of neurotransmitter concentration. Global coupling is efficient to synchronize a population of 10,000 cells. Synchronized cells can be entrained by a 24-h light-dark cycle. Simulations of the interaction between two populations representing two regions of the SCN show that the driven population can be phase-leading. Experimentally testable predictions are: 1), phases of individual cells are governed by their intrinsic periods; and 2), efficient synchronization is achieved when the average neurotransmitter concentration would dampen individual oscillators. However, due to the global neurotransmitter oscillation, cells are effectively synchronized.

Circadian clock oscillator properties that are crucial for synchronization with the environment (entrainment) are studied in experiment and theory.The ratio between stimulus (zeitgeber) strength and oscillator amplitude, and the rigidity of the oscillatory system (relaxation rate upon perturbation) determine entrainment properties. Coupling among oscillators affects both qualities resulting in increased amplitude and rigidity.Uncoupled lung clocks entrain to extreme zeitgeber cycles, whereas the coupled oscillator system in the suprachiasmatic nucleus (SCN) does not; however, when coupling in the SCN is inhibited, larger ranges of entrainment can be achieved.

PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients' behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbt S and dbt L ) in Drosophila.[ These cell-autonomous oscillations are thought to be established by feedback loops involving transcription of clock genes and their subsequent autoregulatory transcriptional repression. In mammals, the transcription factor heterodimer CLOCK-BMAL1 activates the expression of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes via E-box enhancer elements in their promoters. PER and CRY proteins are believed to form complexes that translocate in the nucleus to inhibit their own transcription by directly interacting with the CLOCK-BMAL1 complex.Critical to the properties of this oscillator is the delay between the production of PER and CRY proteins and their autorepression. Post-translational events such as complex formation among clock proteins, nuclear import and export, regulated degradation, modulation of transcriptional activity, and chromatin modification have all been implicated in the generation of this delay (for a review, see Harms et al. 2004). In many cases, phosphorylation of clock proteins is the key step that both initiates these events and regulates their correct timing. In cyanobacteria, even the core of the circadian oscillator seems to be based on rhythmic phosphorylation and dephosphorylation of clock proteins rather than on a transcriptional-translational feedback loop (Nakajima et al.

The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period - a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations.