Melatonin,clock genes and mitochondria in sepsis

After the characterization of the central pacemaker in the suprachiasmatic nucleus, the expression of clock genes was identified in several peripheral tissues including the immune system. The hierarchical control from the central clock to peripheral clocks extends to other functions including endocrine, metabolic, immune, and mitochondrial responses. Increasing evidence links the disruption of the clock genes expression with multiple diseases and aging. Chronodisruption is associated with alterations of the immune system, immunosenescence, impairment of energy metabolism, and reduction of pineal and extrapineal melatonin production. Regarding sepsis, a condition coursing with an exaggerated response of innate immunity, experimental and clinical data showed an alteration of circadian rhythms that reflects the loss of the normal oscillation of the clock. Moreover, recent data point to that some mediators of the immune system affects the normal function of the clock. Under specific conditions, this control disappears reactivating the immune response. So, it seems that clock gene disruption favors the innate immune response, which in turn induces the expression of proinflammatory mediators, causing a further alteration of the clock. Here, the clock control of the mitochondrial function turns off, leading to a bioenergetic decay and formation of reactive oxygen species that, in turn, activate the inflammasome. This arm of the innate immunity is responsible for the huge increase of interleukin-1β and entrance into a vicious cycle that could lead to the death of the patient. The broken clock is recovered by melatonin administration, that is accompanied by the normalization of the innate immunity and mitochondrial homeostasis. Thus, this review emphasizes the connection between clock genes, innate immunity and mitochondria in health and sepsis, and the role of melatonin to maintain clock homeostasis.     

Endocrine-dependent expression of circadian clock genes in insects

Current models state that insect peripheral oscillators are directly responsive to light, while mammalian peripheral clock genes are coordinated by a master clock in the brain via intermediate factors, possibly hormonal. We show that the expression levels of two circadian clock genes, period (per) and Par Domain Protein 1 (Pdp1) in the peripheral tissue of an insect model species, the linden bug Pyrrhocoris apterus, are inversely affected by contrasting photoperiods. The effect of photoperiod on per and Pdp1 mRNA levels was found to be mediated by the corpus allatum, an endocrine gland producing juvenile hormone. Our results provide the first experimental evidence for the effect of an endocrine gland on circadian clock gene expression in insects. Received 31 October 2007; received after revision 7 January 2008; accepted 9 January 2008 D. Dolezel, L. Zdechovanova: These authors contributed equally to this work.     

The absence of melanopsin alters retinal clock function and dopamine regulation by light

The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn ₄ ^?/? ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn ₄ ^?/? mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.     

Nucleolar localization and circadian regulation of Per2S,a novel splicing variant of the Period 2 gene

In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.     

Maternal eating behavior is a major synchronizer of fetal and postnatal peripheral clocks in mice

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring.     

Indoleamines and the UV-light-sensitive photoperiodic responses of the melanocyte network: a biological calendar?

The pineal, serotoninergic and pigmented neurons are associated with light-dependent sleep/arousal, serving as a biological clock with a circadian rhythm. This rhythm is maintained by melatonin which serves to recognise the dark phase. The neural network that responds to seasonal variations in day/night length has not been identified. The present study demonstrates that melanocytes in human skin respond to changes in the duration of UV exposure, and can serve as a biological calendar. These responses are mediated by two indoleamines, serotonin and melatonin. Higher melatonin levels correspond to long nights and long days (short UV pulse), while high serotonin levels in the presence of melatonin reflect short nights and long days (long UV exposure). This response recapitulates the sleep/arousal patterns in animals exposed to large variations in day/night cycle that cause changes in coat colour from pure white in winter to complete repigmentation in summer.     

Strain differences in the pattern and intensity of wheel running activity in laboratory rats

Wheel running activity rhythms of three inbred rat strains, ACI/Ztm, BH/Ztm, and LEW/Ztm, were compared in order to evaluate the effect of genetic differences on circadian rhythm parameters. Significant strain differences were found in the general pattern of the activity rhythms and their characteristic periodicities as well as in the amount and duration of wheel running activity and the timing of activity onsets and offsets. The results suggest that genetic differences exist in the coupling of the multiple circadian oscillators that generate the overall pattern of wheel running activity.     

Strain differences in the pattern and intensity of wheel running activity in laboratory rats

Summary Wheel running activity rhythms of three inbred rat strains, ACI/Ztm, BH/Ztm, and LEW/Ztm, were compared in order to evaluate the effect of genetic differences on circadian rhythm parameters. Significant strain differences were found in the general pattern of the activity rhythms and their characteristic periodicities as well as in the amount and duration of wheel running activity and the timing of activity onsets and offsets. The results suggest that genetic differences exist in the coupling of the multiple circadian oscillators that generate the overall pattern of wheel running activity.     

The design of intracellular oscillators that interact with metabolism

Biological oscillations such as circadian rhythms are ubiquitous in nature and have attracted significant attention because of their intriguing dynamics and important biological roles. Understanding of such sophisticated and robust oscillators requires more than the traditional reductionist approach. Recently, a synthetic approach similar to the design of engineering machinery has provided a valuable alternative for testing hypothetical operating principles. These man-made genetic circuits and gene-metabolic circuits are designed based on physical concepts, guided by mathematical models, and constrained by biological and chemical reality. While still primitive compared with the natural circuits, the designed gene-metabolic oscillator has begun to show hallmarks of circadian rhythms such as temperature compensation and close interaction with metabolism. Received 29 December 2005; received after revision 12 February 2006; accepted 16 March 2006     

Entrainment of human circadian rhythms by artificial bright light cycles

Artificial bright light cycles (LD 8:16) of about 5000 lux during the light period were applied to two subjects in a temporal isolation unit, who had shown free-running circadian rhythms in sleep-wakefulness and rectal temperature. The circadian rhythms were successfully entrained by the artificial light cycle, but the phase relation of the rhythms to the light cycle was substantially different between the two subjects. The result indicated that the artificial bright lights are able to reset human circadian rhythms.     

Entrainment of human circadian rhythms by artificial bright light cycles

Summary Artificial bright light cycles (LD 816) of about 5000 lux during the light period were applied to two subjects in a temporal isolation unit, who had shown free-running circadian rhythms in sleep-wakefulness and rectal temperature. The circadian rhythms were successfully entrained by the artificial light cycle, but the phase relation of the rhythms to the light cycle was substantially different between the two subjects. The result indicated that the artificial bright lights are able to reset human circadian rhythms.     

UV guided dendritic growth patterns and the networking of melanocytes

Whole skin organ cultures of vitiliginous, skin show that the marginal melanocytes are highly sensitive to a pulse of UV exposure (210–380 nm) during the G₂ phase of the cell cycle, as seen by prominent dendricity. Melanocytes are highly dendritic in the epidermis overlying rapidly growing tumors, as well as within proliferative lesions such as basal cell carcinomas and aggressive seborrheic keratosis. In the organ cultures the dendrites extend towards the source of UV, i.e. the surface, while the main body lies along the basement membrane. The epidermal melanocytes overlying tumors lie, almost vertically, dendrites aligned towards the underlying tumor on one side and the surface on the other. Within tumors dendritic elongation is guided by mitotic and PCNA positive (S-phase) tumor cells, which are a source of ultraweak UV emissions in the range of 210–330 nm. These observations indicate that ultraweak biophoton emissions from neighbouring cells can simulate environmental cues and contribute to the plasticity of networks such as the melanocytes or the visual pathways.     

Structures and molecules involved in generation and regulation of biological rhythms in vertebrates and invertebrates

Melatonin from the retina and the pineal gland functions in neuroendocrine hierarchies. Photoreceptors — eyes and extraretinal — detect light. Oscillators — pineal and suprachiasmatic nuclei — act as pacemakers. Driven neuroendocrine rhythms carry temporal hormone signals throughout the body. Light controls melatonin: light sets the phase of the melatonin rhythm and determines the duration of melatonin synthesis. By these means, circadian rhythms (e.g. in locomotor activity and body temperature) and seasonal rhythms (e.g. in reproduction) are controlled.     

Impaired nuclear translocation of glucocorticoid receptors: novel findings from psoriatic epidermal keratinocytes

Psoriasis is a chronic proliferative skin disease and is usually treated with topical glucocorticoids, which act through the glucocorticoid receptor (GR), a component of the physiological systems essential for immune responses, differentiation, and homeostasis. To investigate the possible role of GR in the pathogenesis of psoriasis, normal and psoriatic lesional skin were recruited. Firstly, the immunolocalization of GR in the skin and cultured epidermal keratinocytes were determined by immunofluorescence. In normal skin and cultured human epidermal keratinocytes, intracellular GR is localized in the nuclei, while in psoriatic skin and cultured keratinocytes, GR is in the cytoplasm. Next, we investigated possible factors associated with the cytoplasmic distribution. We found that VEGF and IFN-γ led to impaired nuclear translocation of GR through p53 and microtubule-inhibitor, vincristine, and inhibited nuclear uptake of GR in normal keratinocytes. In addition to dexamethasone, interleukin (IL)-13 was also able to transfer GR into nuclei of psoriatic keratinocytes. Furthermore, discontinuation of dexamethasone induced cytoplasmic retention of GR in normal keratinocytes. In contrast, energy depletion of normal epidermal keratinocytes did not change the nuclear distribution of GR. To confirm our findings in vivo, an imiquimod-induced psoriasis-like skin mouse model was included. IL-13 ameliorated (but vincristine exacerbated) the skin lesions on the mouse. Taken together, our findings define that impaired nuclear translocation of GR is associated with VEGF, IFN-γ, p53, and microtubule. Therapeutic strategies designed to accumulate GR in the nucleus, such as IL-13, may be beneficial for the therapy of psoriasis.     

An ancient cytokine,astakine, mediates circadian regulation of invertebrate hematopoiesis

Invertebrate circulating hemocytes are key players in the innate immune defense and their continuous renewal from hematopoietic tissues is tightly regulated in crustaceans by astakine, a new family of cytokines sharing a prokineticin (PROK) domain. In vertebrates, brain PROKs function as transmitters of circadian rhythms and we present evidence that hemocyte release from hematopoietic tissues in crayfish is under circadian regulation, a direct result of rhythmic expression of astakine. We demonstrate that the observed variation in astakine expression has an impact on innate immunity assessed as susceptibility to a pathogenic Pseudomonas species. These findings enlighten the importance of comparing immune responses at fixed times not to neglect circadian regulation of innate immunity. Moreover, our results entail an evolutionary conserved function for prokineticins as mediators of circadian rhythm, and for the first time show a role for this domain in circadian regulation of hematopoiesis that may have implications also in vertebrates.