Circadian Control of the Response of Macrophages to Plasmodium Spp.-Infected Red Blood Cells.

Malaria is a serious vector-borne disease characterized by periodic episodes of high fever and strong immune responses that are coordinated with the daily synchronized parasite replication cycle inside RBCs. As immune cells harbor an autonomous circadian clock that controls various aspects of the immune response, we sought to determine whether the intensity of the immune response to Plasmodium spp., the parasite causing malaria, depends on time of infection. To do this, we developed a culture model in which mouse bone marrow-derived macrophages are stimulated with RBCs infected with Plasmodium berghei ANKA (iRBCs). Lysed iRBCs, but not intact iRBCs or uninfected RBCs, triggered an inflammatory immune response in bone marrow-derived macrophages. By stimulating at four different circadian time points (16, 22, 28, or 34 h postsynchronization of the cells' clock), 24-h rhythms in reactive oxygen species and cytokines/chemokines were found. Furthermore, the analysis of the macrophage proteome and phosphoproteome revealed global changes in response to iRBCs that varied according to circadian time. This included many proteins and signaling pathways known to be involved in the response to Plasmodium infection. In summary, our findings show that the circadian clock within macrophages determines the magnitude of the inflammatory response upon stimulation with ruptured iRBCs, along with changes of the cell proteome and phosphoproteome.

Watch your clock: it matters for immunotherapy.

Does time of day matter for cancer immunotherapy? Whereas the concept of optimizing the time of treatment is well documented for chemotherapy, whether it applies to immunotherapy, a revolutionizing treatment exploiting the power of immune cells to control tumors, has recently been addressed in a study published in Cell.

The influence of circadian rhythms on CD8+ T cell activation upon vaccination: A mathematical modeling perspective.

Circadian rhythms have been implicated in the modulation of many physiological processes, including those associated with the immune system. For example, these rhythms influence CD8+ T cell responses within the adaptive immune system. The mechanism underlying this immune-circadian interaction, however, remains unclear, particularly in the context of vaccination. Here, we devise a molecularly-explicit gene regulatory network model of early signaling in the naïve CD8+ T cell activation pathway, comprised of three axes (or subsystems) labeled ZAP70, LAT and CD28, to elucidate the molecular details of this immune-circadian mechanism and its relation to vaccination. This is done by coupling the model to a periodic forcing function to identify the molecular players targeted by circadian rhythms, and analyzing how these rhythms subsequently affect CD8+ T cell activation under differing levels of T cell receptor (TCR) phosphorylation, which we designate as vaccine load. By performing both bifurcation and parameter sensitivity analyses on the model at the single cell and ensemble levels, we find that applying periodic forcing on molecular targets within the ZAP70 axis is sufficient to create a day-night discrepancy in CD8+ T cell activation in a manner that is dependent on the bistable switch inherent in CD8+ T cell early signaling. We also demonstrate that the resulting CD8+ T cell activation is dependent on the strength of the periodic coupling as well as on the level of TCR phosphorylation. Our results show that this day-night discrepancy is not transmitted to certain downstream molecules within the LAT subsystem, such as mTORC1, suggesting a secondary, independent circadian regulation on that protein complex. We also corroborate experimental results by showing that the circadian regulation of CD8+ T cell primarily acts at a baseline, pre-vaccination state, playing a facilitating role in priming CD8+ T cells to vaccine inputs according to the time of day. By applying an ensemble level analysis using bifurcation theory and by including several hypothesized molecular targets of this circadian rhythm, we further demonstrate an increased variability between CD8+ T cells (due to heterogeneity) induced by its circadian regulation, which may allow an ensemble of CD8+ T cells to activate at a lower vaccine load, improving its sensitivity. This modeling study thus provides insights into the immune targets of the circadian clock, and proposes an interaction between vaccine load and the influence of circadian rhythms on CD8+ T cell activation.

The environment and the internal clocks: The study of their relationships from prehistoric to modern times.

The origin of biological rhythms goes back to the very beginning of life. They are observed in the animal and plant world at all levels of organization, from cells to ecosystems. As early as the 18th century, plant scientists were the first to explain the relationship between flowering cycles and environmental cycles, emphasizing the importance of daily light-dark cycles and the seasons. Our temporal structure is controlled by external and internal rhythmic signals. Light is the main synchronizer of the circadian system, as daily exposure to light entrains our clock over 24 hours, the endogenous period of the circadian system being close to, but not exactly, 24 hours. In 1960, a seminal scientific meeting, the Cold Spring Harbor Symposium on Biological Rhythms, brought together all the biological rhythms scientists of the time, a number of whom are considered the founders of modern chronobiology. All aspects of biological rhythms were addressed, from the properties of circadian rhythms to their practical and ecological aspects. Birth of chronobiology dates from this period, with the definition of its vocabulary and specificities in metabolism, photoperiodism, animal physiology, etc. At around the same time, and right up to the present day, research has focused on melatonin, the circadian neurohormone of the pineal gland, with data on its pattern, metabolism, control by light and clinical applications. However, light has a double face, as it has positive effects as a circadian clock entraining agent, but also deleterious effects, as it can lead to chronodisruption when exposed chronically at night, which can increase the risk of cancer and other diseases. Finally, research over the past few decades has unraveled the anatomical location of circadian clocks and their cellular and molecular mechanisms. This recent research has in turn allowed us to explain how circadian rhythms control physiology and health.

The circadian system: A neglected player in neurodevelopmental disorders.

Patients with neurodevelopmental disorders, such as autism spectrum disorder, often display abnormal circadian rhythms. The role of the circadian system in these disorders has gained considerable attention over the last decades. Yet, it remains largely unknown how these disruptions occur and to what extent they contribute to the disorders' development. In this review, we examine circadian system dysregulation as observed in patients and animal models of neurodevelopmental disorders. Second, we explore whether circadian rhythm disruptions constitute a risk factor for neurodevelopmental disorders from studies in humans and model organisms. Lastly, we focus on the impact of psychiatric medications on circadian rhythms and the potential benefits of chronotherapy. The literature reveals that patients with neurodevelopmental disorders display altered sleep-wake cycles and melatonin rhythms/levels in a heterogeneous manner, and model organisms used to study these disorders appear to support that circadian dysfunction may be an inherent characteristic of neurodevelopmental disorders. Furthermore, the pre-clinical and clinical evidence indicates that circadian disruption at the environmental and genetic levels may contribute to the behavioural changes observed in these disorders. Finally, studies suggest that psychiatric medications, particularly those prescribed for attention-deficit/hyperactivity disorder and schizophrenia, can have direct effects on the circadian system and that chronotherapy may be leveraged to offset some of these side effects. This review highlights that circadian system dysfunction is likely a core pathological feature of neurodevelopmental disorders and that further research is required to elucidate this relationship.

Circadian Regulation of Leishmania Parasite Internalisation in Macrophages and Downstream Cellular Events.

Leishmania spp. parasites use macrophages as a host cell during infection. As a result, macrophages have a dual role: clearing the parasite as well as acting as host cells. Recently, studies have shown that macrophages harbour circadian clocks, which affect many of their functions such as phagocytosis, receptor expression and cytokine release. Interestingly, Leishmania major infection in hosts was also shown to be under circadian control. Therefore, we decided to investigate what underlies the rhythms of L. major infection within macrophages. Using a culture model of infection of bone marrow-derived macrophages with L. major promastigotes, we show that the parasites are internalised into macrophages with a 24-h variation dependent on a functional circadian clock in the cells. This was associated with a variation in the number of parasites per macrophage. The cell surface expression of parasite receptors was not controlled by the cells' circadian clock. In contrast, the expression of the components of the endocytic pathway, EEA1 and LC3b, varied according to the time of infection. This was paralleled by variations in parasite-induced ROS production as well as cytokine tumour necrosis factor α. In summary, we have uncovered a time-dependent regulation of the internalisation of L. major promastigotes in macrophages, controlled by the circadian clock in these cells, as well as subsequent cellular events in the endocytic pathway, intracellular signalling and cytokine production.

Time of day and circadian disruption influence host response and parasite growth in a mouse model of cerebral malaria.

Malaria is a disease caused by infection with parasite Plasmodium spp. We studied the circadian regulation of host responses to the parasite, in a mouse model of cerebral malaria. The course of the disease was markedly affected by time of infection, with decreased parasitemia and increased inflammation upon infection in the middle of the night. At this time, there were fewer reticulocytes, which are target cells of the parasites. We next investigated the effects of desynchronization of host clocks on the infection: after 10 weeks of recurrent jet lags, mice showed decreased parasite growth and lack of parasite load rhythmicity, paralleled by a loss of glucose rhythm. Accordingly, disrupting host metabolic rhythms impacted parasite load rhythmicity. In summary, our findings of a circadian modulation of malaria parasite growth and infection shed light on aspects of the disease relevant to human malaria and could contribute to new therapeutic or prophylactic measures.

Clockwork intruders: Do parasites manipulate their hosts' circadian rhythms?

Most organisms have developed circadian clocks to adapt to 24-hour cycles in the environment. These clocks have become crucial for modulating and synchronizing complex behavioral and biological processes. A number of parasites seem to have evolved to take advantage of their hosts' circadian rhythms to favor their own infection and survival. Some species, such as Microphallus sp. and Trypanosoma cruzi, can alter the patterns of locomotor behavior of infected intermediate hosts, which can promote transmission to a subsequent primary host. Some fungi of the genera Ophiocordyceps and Entomophthora, as well as hairworms (Nematomorpha), elicit complex behaviors that promote their host's death at a time and place that optimizes continuation of the parasite's life-cycle. At least in some cases, a proposed mechanism might involve a change in the expression of clock-controlled genes. Lastly, some disease-causing protozoan parasites of the genera Trypanosoma, Plasmodium, and Leishmania induce changes in the circadian rhythms of their primary hosts upon infection. Some of these changes may be attributed to circadian alterations resulting from the host's inflammatory response to the infection or other unexplored responses or adaptations to the illness. Thus, a distinction must be made between manipulation of the parasite and response of the host when studying these alterations in the future. Parasitic manipulation of circadian rhythms, which vastly modulates behavior and physiology, is an essential issue that has been relatively understudied. A deeper understanding of this phenomenon could lead to the development of novel therapeutic approaches for the diseases that these parasites convey.

Peripheral clock gene oscillations are perturbed in neonatal and adult rat offspring raised under adverse limited bedding conditions.

Circadian (24-h) rhythms in the suprachiasmatic nucleus (SCN) are established in utero in rodents, but rhythmicity of peripheral circadian clocks appears later in postnatal development. Since peripheral oscillators can be influenced by maternal feeding and behavior, we investigated whether exposure to the adverse environmental conditions of limited bedding (LB) during postnatal life would alter rhythmicity in the SCN, adrenal gland and liver in neonatal (postnatal day PND10), juvenile (PND28) and adult rats. We also examined locomotor activity in adults. Limited bedding increased nursing time and slightly increased fragmentation of maternal behavior. Exposure to LB reduced the amplitude of Per2 in the SCN on PND10. Adrenal clock gene expression (Bmal1, Per2, Cry1, Rev-erbα, Dbp) and corticosterone secretion were rhythmic at all ages in NB offspring, whereas rhythmicity of Bmal1, Cry1 and corticosterone was abolished in neonatal LB pups. Circadian gene expression in the adrenal and liver was well established by PND28. In adults, liver expression of several circadian genes was increased at specific daytimes by LB and the microstructure of locomotor behavior was altered. Thus, changes in maternal care and behavior might provide important signals to the maturing peripheral oscillators and modify, in particular their output functions in the long-term.

Behavioral and cellular responses to circadian disruption and prenatal immune activation in mice.

Most individuals with neurodevelopmental disorders (NDDs), including schizophrenia and autism spectrum disorders, experience disruptions in sleep and circadian rhythms. Epidemiological studies indicate that exposure to prenatal infection increases the risk of developing NDDs. We studied how environmental circadian disruption contributes to NDDs using maternal immune activation (MIA) in mice, which models prenatal infection. Pregnant dams were injected with viral mimetic poly IC (or saline) at E9.5. Adult poly IC- and saline-exposed offspring were subjected to 4 weeks of each of the following: standard lighting (LD1), constant light (LL) and standard lighting again (LD2). Behavioral tests were conducted in the last 12 days of each condition. Poly IC exposure led to significant behavioral differences, including reduced sociability (males only) and deficits in prepulse inhibition. Interestingly, poly IC exposure led to reduced sociability specifically when males were tested after LL exposure. Mice were exposed again to either LD or LL for 4 weeks and microglia were characterized. Notably, poly IC exposure led to increased microglial morphology index and density in dentate gyrus, an effect attenuated by LL exposure. Our findings highlight interactions between circadian disruption and prenatal infection, which has implications in informing the development of circadian-based therapies for individuals with NDDs.

Regulation of Cytotoxic CD8+ T Cells by the Circadian Clock.

Most aspects of physiology, including immunity, present 24-h variations called circadian rhythms. In this review, we examine the literature on the circadian regulation of CD8+ T cells, which are important to fight intracellular infections and tumors. CD8+ T cells express circadian clock genes, and ∼6% of their transcriptome presents circadian oscillations. CD8+ T cell counts present 24-h rhythms in the blood and in secondary lymphoid organs, which depend on the clock in these cells as well as on hormonal rhythms. Moreover, the strength of the response of these cells to Ag presentation varies according to time of day, a rhythm dependent on the CD8+ T cell clock. The relevance of CD8+ T cell circadian rhythms is shown by the daily variations in the fight of intracellular infections. Such a circadian regulation also has implications for cancer, as well as the optimization of vaccination and immunotherapy.

The Effect of Night Shifts on 24-h Rhythms in the Urinary Metabolome of Police Officers on a Rotating Work Schedule.

Shift workers face an increased risk of metabolic health problems, but the direct metabolic response to working nights is not fully understood. The aim of this study was to investigate the effect of night shifts on the 24-h urinary metabolome of shift workers. Eleven police officers working rotating shifts completed two 24-h laboratory visits that took place before and after they worked 7 consecutive nights. Sleep and meals were scheduled on a day schedule in the first visit and then on a night schedule (i.e., sleep and meals shifted by approximately 12 h) in the second visit. Targeted metabolomic analysis was performed on urine samples collected throughout these laboratory visits. Differential rhythmicity analysis was used to compare 24-h rhythms in urinary metabolites in both conditions. Our results show that on the day schedule, 24-h rhythms are present in the urinary levels of the majority of metabolites, but that this is significantly reduced on the night schedule, partly due to loss of organic acid rhythmicity. Furthermore, misalignment of 24-h metabolite rhythms with the shifted behavioral cycles in the night schedule was observed in more than half of the metabolites that were rhythmic in both conditions (all acylcarnitines). These results show that working nights alters the daily rhythms of the urinary metabolome in rotating shift workers, with the most notable impact observed for acylcarnitines and organic acids, 2 metabolite classes involved in mitochondrial function. Further research is warranted to study how these changes relate to the increased metabolic risks associated with shift work.

Deubiquitinases: key regulators of the circadian clock.

All living organisms experience daily environmental cycles and have consequently evolved to synchronize and adapt to this changing environment. Biological processes such as hormonal secretion, body temperature, and sleep follow daily cycles called circadian rhythms that are driven by a molecular clock running in most cells and tissues of the body. This clock is composed of transcriptional-translational negative feedback loops involving clock genes and proteins. This molecular mechanism functions with a period of ∼24 h, and it promotes daily rhythms in the expression of numerous genes. For this robust mechanism to function, the abundance and activity of clock proteins need to be tightly regulated. One of the mechanisms by which this can be achieved is ubiquitination. Indeed, many ubiquitin ligases can tag core clock proteins to target them for proteasomal degradation. However, deubiquitinases can reverse this process by removing or modifying these ubiquitin signals and are thus important enzymes in clock protein homeostasis and regulation. Recent studies on the mammalian and Drosophila clock mechanisms have identified a number of deubiquitinases able to stabilize core clock proteins, change their cellular localization or even regulate their activity. In this review, we aim to discuss the fundamental roles of ubiquitination and deubiquitination in the circadian clock by presenting all deubiquitinases found to be involved in circadian rhythms with the aim to give a global view of recent advances in this emerging field.

Exposure to Circadian Disruption During Adolescence Interacts With a Genetic Risk Factor to Modify Schizophrenia-relevant Behaviors in a Sex-dependent Manner.

DTNBP1 is a gene associated with schizophrenia. Postmortem studies found a reduced expression of DTNBP1 in regions associated with schizophrenia in patients' brains. Sandy (Sdy) mice have a loss-of-function mutation in Dtnbp1 gene, resulting in behavioral deficits and brain changes similar to those seen in patients with schizophrenia. We previously showed that exposing adult Sdy mice to circadian disruption led to an exacerbation of schizophrenia-relevant behaviors. Here we asked whether the interaction between this genetic risk factor and circadian disruption occurs during adolescence, a period when environmental insults can promote schizophrenia symptoms, and whether sex affects this interaction. Starting at postnatal day 21, wild-type (WT) and Sdy males and females were housed for 4 weeks either in a 12 h light:12 h dark (LD 12:12) cycle or under chronic jetlag (CJL). Then, after 2 weeks in LD 12:12, behavioral assessments were conducted, including elevated plus maze (EPM), novel object recognition (NOR), social interaction, and prepulse inhibition (PPI) of acoustic startle. NOR and social novelty tests showed that, surprisingly, CJL during adolescence had opposite effects on WT and Sdy males, that is, behavioral deficits in WT males while rescuing preexisting deficits in Sdy mice. CJL led to decreased sociability in WT and Sdy mice while decreasing PPI only in females. Sdy mice showed decreased anxiety-like behavior compared with wild-type (WT), which was further accentuated by CJL in males. Thus, circadian disruption during adolescence, on its own or in association with Dtnbp1 mutation, can influence cognition, sociability, sensorimotor gating, and anxiety-like behaviors in a sex-dependent manner.

Chronic Exposure to Dim Light at Night or Irregular Lighting Conditions Impact Circadian Behavior, Motor Coordination, and Neuronal Morphology.

Mistimed exposure to light has been demonstrated to negatively affect multiple aspects of physiology and behavior. Here we analyzed the effects of chronic exposure to abnormal lighting conditions in mice. We exposed mice for 1 year to either: a standard light/dark cycle, a "light-pollution" condition in which low levels of light were present in the dark phase of the circadian cycle (dim light at night, DLAN), or altered light cycles in which the length of the weekday and weekend light phase differed by 6 h ("social jetlag"). Mice exhibited several circadian activity phenotypes, as well as changes in motor function, associated particularly with the DLAN condition. Our data suggest that these phenotypes might be due to changes outside the core clock. Dendritic spine changes in other brain regions raise the possibility that these phenotypes are mediated by changes in neuronal coordination outside of the clock. Given the prevalence of artificial light exposure in the modern world, further work is required to establish whether these negative effects are observed in humans as well.

The involvement of host circadian clocks in the regulation of the immune response to parasitic infections in mammals.

Circadian rhythms are recurring variations of physiology with a period of ~24 h, generated by circadian clocks located throughout the body. Studies have shown a circadian regulation of many aspects of immunity. Immune cells have intrinsic clock mechanisms, and innate and adaptive immune responses - such as leukocyte migration, magnitude of inflammation, cytokine production and cell differentiation - are under circadian control. This circadian regulation has consequences for infections including parasitic infections. In the context of Leishmania infection, the circadian clock within host immune cells modulates the magnitude of the infection and the inflammatory response triggered by the parasite. As for malaria, rhythms within the immune system were shown to impact the developmental cycles of Plasmodium parasites within red blood cells. Further, host circadian rhythms impact infections by multicellular parasites; for example, infection with helminth Trichuris muris shows different kinetics of worm expulsion depending on time of day of infection, a variation that depends on the dendritic cell clock. Although the research on the circadian control of immunity in the context of parasitic infections is in its infancy, the research reviewed here suggests a crucial involvement of host circadian rhythms in immunity on the development and progression of parasitic infections.

Bone adaptation to mechanical loading in mice is affected by circadian rhythms.

Physical forces are critical for successful function of many organs including bone. Interestingly, the timing of exercise during the day alters physiology and gene expression in many organs due to circadian rhythms. Circadian clocks in tissues, such as bone, express circadian clock genes that target tissue-specific genes, resulting in tissue-specific rhythmic gene expression (clock-controlled genes). We hypothesized that the adaptive response of bone to mechanical loading is regulated by circadian rhythms. First, mice were sham loaded and sacrificed 8 h later, which amounted to tissues being collected at zeitgeber time (ZT)2, 6, 10, 14, 18, and 22. Cortical bone of the tibiae collected from these mice displayed diurnal expression of core clock genes and key osteocyte and osteoblast-related genes, such as the Wnt-signaling inhibitors Sost and Dkk1, indicating these are clock-controlled genes. Serum bone turnover markers did not display rhythmicity. Second, mice underwent a single bout of in vivo loading at either ZT2 or ZT14 and were sacrificed 1, 8, or 24 h after loading. Loading at ZT2 resulted in Sost upregulation, while loading at ZT14 led to Sost and Dkk1 downregulation. Third, mice underwent daily in vivo tibial loading over 2 weeks administered either in the morning, (ZT2, resting phase) or evening (ZT14, active phase). In vivo microCT was performed at days 0, 5, 10, and 15 and conventional histomorphometry was performed at day 15. All outcome measures indicated a robust response to loading, but only microCT-based time-lapse morphometry showed that loading at ZT14 resulted in a greater endocortical bone formation response compared to mice loaded at ZT2. The decreased Sost and Dkk1 expression coincident with the modest, but significant time-of-day specific increase in adaptive bone formation, suggests that circadian clocks influence bone mechanoresponse.

Circadian rhythms in adaptive immunity and vaccination.

Adaptive immunity allows an organism to respond in a specific manner to pathogens and other non-self-agents. Also, cells of the adaptive immune system, such as T and B lymphocytes, can mediate a memory of an encounter with a pathogen, allowing a more efficient response to a future infection. As for other aspects of physiology and of the immune system, the adaptive immune system is regulated by circadian clocks. Consequently, the development, differentiation, and trafficking between tissues of adaptive immune cells have been shown to display daily rhythms. Also, the response of T cells to stimuli (e.g., antigen presentation to T cells by dendritic cells) varies according to a circadian rhythm, due to T cell-intrinsic mechanisms as well as cues from other tissues. The circadian control of adaptive immune response has implications for our understanding of the fight against pathogens as well as auto-immune diseases, but also for vaccination, a preventive measure based on the development of immune memory.

Behavioral phenotyping of mice lacking the deubiquitinase USP2.

Ubiquitin specific peptidase 2 (USP2) is a deubiquitinating enzyme expressed almost ubiquitously in the body, including in multiple brain regions. We previously showed that mice lacking USP2 present altered locomotor activity rhythms and response of the clock to light. However, the possible implication of USP2 in regulating other behaviors has yet to be tested. To address this, we ran a battery of behavioral tests on Usp2 KO mice. Firstly, we confirmed our prior findings of increased daily activity and reduced activity fragmentation in Usp2 KO mice. Further, mice lacking USP2 showed impaired motor coordination and equilibrium, a decrease in anxiety-like behavior, a deficit in working memory and in sensorimotor gating. On the other hand, no effects of Usp2 gene deletion were found on spatial memory. Hence, our data uncover the implication of USP2 in different behaviors and expands the range of the known functions of this deubiquitinase.