Arginine-vasopressin-expressing neurons in the murine suprachiasmatic nucleus exhibit a circadian rhythm in network coherence in vivo.

The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits, exhibit profound heterogenicity of function over time and space.

Long-term circadian disruption shortens life span and dampens blood pressure diurnal rhythms in stroke-prone spontaneously hypertensive rats.

Environmental cues such as light and timing of food intake influence molecular clocks that produce circadian rhythmicity of many biological functions. The master circadian clock is entrained by light input and synchronizes with peripheral clocks in every organ of the body. Careers that require rotating shift work schedules predispose workers to a constant desynchronization of these biological clocks and are associated with increased risk of cardiovascular disease. We used a stroke-prone spontaneously hypertensive rat model exposed to a known biological desynchronizer, chronic environmental circadian disruption (ECD), to test the hypothesis that it would accelerate the time to stroke onset. We then investigated whether time-restricted feeding could delay stroke onset and evaluated its usefulness as a countermeasure when combined with the constant disruption of the light cycle. We found that phase advancing of the light schedule accelerated stroke onset. Restricting food access time to 5 h/day regardless of lighting profoundly delayed stroke onset in both standard 12-h:12-h light/dark or ECD-lighting conditions compared with ad libitum feeding; however, acceleration by ECD versus control lighting conditions was still observed. Since hypertension is a precursor to stroke in this model, we assessed blood pressure in a small cohort longitudinally using telemetry. Mean daily systolic and diastolic blood pressure increased in a similar manner across rats in control and ECD conditions, thus hypertension was not grossly accelerated to cause earlier strokes. However, we observed intermittent dampening of rhythms after each shift of the light cycle reminiscent of a relapsing-remitting nondipping state. Our results suggest that constant disruption of environmental rhythms may be associated with an increased risk of cardiovascular complications in the presence of cardiovascular risk factors.NEW & NOTEWORTHY This stroke-prone spontaneously hypertensive rat model significantly delayed stroke onset with the timed food restriction intervention. Blood pressure recordings in this same model were continuous through the 3 mo and showed dampened systolic rhythms after each shift in the lighting schedule.

Environmental circadian disruption suppresses rhythms in kidney function and accelerates excretion of renal injury markers in urine of male hypertensive rats.

Nontraditional work schedules, such as shift work, have been associated with numerous health issues, including cardiovascular and metabolic disease. These work schedules can chronically misalign environmental timing cues with internal circadian clock systems in the brain and in peripheral organs, leading to dysfunction of those systems and their associated biological processes. Environmental circadian disruption in the kidney may be an important factor in the increased incidence of hypertension and adverse health outcomes in human shift workers. The relationship between renal rhythmicity and injury resilience is not well understood, especially in the context of environmental, rather than genetic, manipulations of the circadian system. We conducted a longitudinal study to determine whether chronic shifting of the light cycle that mimics shift work schedules would disrupt output rhythms of the kidney and accelerate kidney injury in salt-loaded male spontaneously hypertensive, stroke-prone rats. We observed that chronic shifting of the light-dark (LD) cycle misaligned and decreased the amplitude of urinary volume rhythms as the kidney phase-shifted to match each new lighting cycle. This schedule also accelerated glomerular and tubular injury marker excretion, as quantified by nephrin and KIM-1 compared with rats kept in a static LD cycle. These data suggest that disrupted rhythms in the kidney may decrease resilience and contribute to disease development in systems dependent on renal and cardiovascular functions.

mTOR signaling in VIP neurons regulates circadian clock synchrony and olfaction.

Mammalian/mechanistic target of rapamycin (mTOR) signaling controls cell growth, proliferation, and metabolism in dividing cells. Less is known regarding its function in postmitotic neurons in the adult brain. Here we created a conditional mTOR knockout mouse model to address this question. Using the Cre-LoxP system, the mTOR gene was specifically knocked out in cells expressing Vip (vasoactive intestinal peptide), which represent a major population of interneurons widely distributed in the neocortex, suprachiasmatic nucleus (SCN), olfactory bulb (OB), and other brain regions. Using a combination of biochemical, behavioral, and imaging approaches, we found that mice lacking mTOR in VIP neurons displayed erratic circadian behavior and weakened synchronization among cells in the SCN, the master circadian pacemaker in mammals. Furthermore, we have discovered a critical role for mTOR signaling in mediating olfaction. Odor stimulated mTOR activation in the OB, anterior olfactory nucleus, as well as piriform cortex. Odor-evoked c-Fos responses along the olfactory pathway were abolished in mice lacking mTOR in VIP neurons, which is consistent with reduced olfactory sensitivity in these animals. Together, these results demonstrate that mTOR is a key regulator of SCN circadian clock synchrony and olfaction.

Shell neurons of the master circadian clock coordinate the phase of tissue clocks throughout the brain and body.

BACKGROUND: Daily rhythms in mammals are programmed by a master clock in the suprachiasmatic nucleus (SCN). The SCN contains two main compartments (shell and core), but the role of each region in system-level coordination remains ill defined. Herein, we use a functional assay to investigate how downstream tissues interpret region-specific outputs by using in vivo exposure to long day photoperiods to temporally dissociate the SCN. We then analyze resulting changes in the rhythms of clocks located throughout the brain and body to examine whether they maintain phase synchrony with the SCN shell or core. RESULTS: Nearly all of the 17 tissues examined in the brain and body maintain phase synchrony with the SCN shell, but not the SCN core, which indicates that downstream oscillators are set by cues controlled specifically by the SCN shell. Interestingly, we also found that SCN dissociation diminished the amplitude of rhythms in core clock gene and protein expression in brain tissues by 50-75 %, which suggests that light-driven changes in the functional organization of the SCN markedly influence the strength of rhythms in downstream tissues. CONCLUSIONS: Overall, our results reveal that body clocks receive time-of-day cues specifically from the SCN shell, which may be an adaptive design principle that serves to maintain system-level phase relationships in a changing environment. Further, we demonstrate that lighting conditions alter the amplitude of the molecular clock in downstream tissues, which uncovers a new form of plasticity that may contribute to seasonal changes in physiology and behavior.

Neural correlates of individual differences in circadian behaviour.

Daily rhythms in mammals are controlled by the circadian system, which is a collection of biological clocks regulated by a central pacemaker within the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Changes in SCN function have pronounced consequences for behaviour and physiology; however, few studies have examined whether individual differences in circadian behaviour reflect changes in SCN function. Here, PERIOD2::LUCIFERASE mice were exposed to a behavioural assay to characterize individual differences in baseline entrainment, rate of re-entrainment and free-running rhythms. SCN slices were then collected for ex vivo bioluminescence imaging to gain insight into how the properties of the SCN clock influence individual differences in behavioural rhythms. First, individual differences in the timing of locomotor activity rhythms were positively correlated with the timing of SCN rhythms. Second, slower adjustment during simulated jetlag was associated with a larger degree of phase heterogeneity among SCN neurons. Collectively, these findings highlight the role of the SCN network in determining individual differences in circadian behaviour. Furthermore, these results reveal novel ways that the network organization of the SCN influences plasticity at the behavioural level, and lend insight into potential interventions designed to modulate the rate of resynchronization during transmeridian travel and shift work.

Endogenous circadian regulation of pro-inflammatory cytokines and chemokines in the presence of bacterial lipopolysaccharide in humans.

Various aspects of immune response exhibit 24-h variations suggesting that infection susceptibility and treatment efficacy may vary by time of day. Whether these 24-h variations are endogenous or evoked by changes in environmental or behavioral conditions is not known. We assessed the endogenous circadian control and environmental and behavioral influences on ex-vivo lipopolysaccharide stimulation of whole blood in thirteen healthy participants under 48h of baseline conditions with standard sleep-wake schedules and 40-50h of constant environmental and behavioral (constant routine; CR) conditions. Significant 24-h rhythms were observed under baseline conditions in Monocyte Chemotactic Protein, Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin 8 but not Tumor Necrosis Factor alpha whereas significant 24-h rhythms were observed in all four immune factors under CR conditions. The rhythm amplitudes, expressed as a percentage of mean, were comparable between immune factors and across conditions. In contrast, the acrophase time (time of the fitted peak) was different between immune factors, and included daytime and nighttime peaks and changes across behavioral conditions. These results suggest that the endogenous circadian system underpins the temporal organization of immune responses in humans with additional effects of external environmental and behavioral cycles. These findings have implications for understanding the adverse effects of recurrent circadian disruption and sleep curtailment on immune function.

Suprachiasmatic astrocytes modulate the circadian clock in response to TNF-α.

The immune and the circadian systems interact in a bidirectional fashion. The master circadian oscillator, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, responds to peripheral and local immune stimuli, such as proinflammatory cytokines and bacterial endotoxin. Astrocytes exert several immune functions in the CNS, and there is growing evidence that points toward a role of these cells in the regulation of circadian rhythms. The aim of this work was to assess the response of SCN astrocytes to immune stimuli, particularly to the proinflammatory cytokine TNF-α. TNF-α applied to cultures of SCN astrocytes from Per2(luc) knockin mice altered both the phase and amplitude of PER2 expression rhythms, in a phase-dependent manner. Furthermore, conditioned media from SCN astrocyte cultures transiently challenged with TNF-α induced an increase in Per1 expression in NIH 3T3 cells, which was blocked by TNF-α antagonism. In addition, these conditioned media could induce phase shifts in SCN PER2 rhythms and, when administered intracerebroventricularly, induced phase delays in behavioral circadian rhythms and SCN activation in control mice, but not in TNFR-1 mutants. In summary, our results show that TNF-α modulates the molecular clock of SCN astrocytes in vitro, and also that, in response to this molecule, SCN astrocytes can modulate clock gene expression in other cells and tissues, and induce phase shifts in a circadian behavioral output in vivo. These findings suggest a role for astroglial cells in the alteration of circadian timing by immune activation.

Environmental circadian disruption re-writes liver circadian proteomes.

Circadian gene expression is fundamental to the establishment and functions of the circadian clock, a cell-autonomous and evolutionary-conserved timing system. Yet, how it is affected by environmental-circadian disruption (ECD) such as shiftwork and jetlag are ill-defined. Here, we provided a comprehensive and comparative description of male liver circadian gene expression, encompassing transcriptomes, whole-cell proteomes and nuclear proteomes, under normal and after ECD conditions. Under both conditions, post-translation, rather than transcription, is the dominant contributor to circadian functional outputs. After ECD, post-transcriptional and post-translational processes are the major contributors to whole-cell or nuclear circadian proteome, respectively. Furthermore, ECD re-writes the rhythmicity of 64% transcriptome, 98% whole-cell proteome and 95% nuclear proteome. The re-writing, which is associated with changes of circadian regulatory cis-elements, RNA-processing and protein localization, diminishes circadian regulation of fat and carbohydrate metabolism and persists after one week of ECD-recovery.

Mixed-methods analysis of satisfaction during a 12-session mindfulness-based intervention for women with a substance use disorder and trauma symptomatology.

Satisfaction with an intervention influences the uptake of behavior changes and the long-term efficacy of the intervention. Therefore, it is crucial to assess satisfaction by participant profile when creating and adapting behavior interventions for minoritized populations. Qualitative and quantitative data on participant trauma symptom severity and intervention satisfaction were collected through self-report surveys from 54 women. The sample was 59.3% Hispanic, with an average age of 33.21 (SD = 10.42), who were in residential treatment for substance use disorders (SUDs) and participated in a 12-session mindfulness-based intervention. Qualitative responses were coded using thematic analysis, and an integrative mixed-methods approach was used to compare qualitative theme frequency between high-trauma (N = 28) and low-trauma (N = 26) groups at session 2 and session 11. High- and low-trauma groups were determined by interquartile ranges (bottom 25% = low; top 75% = high). In session 2, the low-trauma group reported significantly higher satisfaction (M = 4.20, SD = 0.55) than the high-trauma group (M = 3.77, SD = 0.89); t(43) = 1.90, p = 0.03. In session 11, there was no significant difference between groups. The mixed-methods analysis revealed that "trouble focusing" appeared more frequently in the high-trauma group than in the low-trauma group during session 2, but the theme was not present in either group at session 11, suggesting that this might pose an initial barrier for individuals with high trauma but subsides as the intervention progresses. This speaks to the importance of retention strategies tailored for participants with SUDs and high trauma while they adjust to the intervention. Assessing initial challenges with satisfaction may help facilitators intervene to increase participant satisfaction.

Aging differentially affects the re-entrainment response of central and peripheral circadian oscillators.

Aging produces a decline in the amplitude and precision of 24 h behavioral, endocrine, and metabolic rhythms, which are regulated in mammals by a central circadian pacemaker within the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. Disruption of the circadian system, as experienced during transmeridian travel, can lead to adverse health consequences, particularly in the elderly. To test the hypothesis that age-related changes in the response to simulated jet lag will reflect altered circadian function, we examined re-entrainment of central and peripheral oscillators from young and old PER2::luciferase mice. As in previous studies, locomotor activity rhythms in older mice required more days to re-entrain following a shift than younger mice. At the tissue level, effects of age on baseline entrainment were evident, with older mice displaying earlier phases for the majority of peripheral oscillators studied and later phases for cells within most SCN subregions. Following a 6 h advance of the light:dark cycle, old mice displayed slower rates of re-entrainment for peripheral tissues but a larger, more rapid SCN response compared to younger mice. Thus, aging alters the circadian timing system in a manner that differentially affects the re-entrainment responses of central and peripheral circadian clocks. This pattern of results suggests that a major consequence of aging is a decrease in pacemaker amplitude, which would slow re-entrainment of peripheral oscillators and reduce SCN resistance to external perturbation.

A Journey in the Brain's Clock: In Vivo Veritas?

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the circadian pacemaker that coordinates mammalian rhythms in tune with the day-night cycle. Understanding the determinants of the intrinsic rhythmicity of this biological clock, its outputs, and resetting by environmental cues, has been a longstanding goal of the field. Integrated techniques of neurophysiology, including lesion studies and in vivo multi-unit electrophysiology, have been key to characterizing the rhythmic nature and outputs of the SCN in animal models. In parallel, reduced ex vivo and in vitro approaches have permitted us to unravel molecular, cellular, and multicellular mechanisms underlying the pacemaker properties of the SCN. New questions have emerged in recent years that will require combining investigation at a cell resolution within the physiological context of the living animal: What is the role of specific cell subpopulations in the SCN neural network? How do they integrate various external and internal inputs? What are the circuits involved in controlling other body rhythms? Here, we review what we have already learned about the SCN from in vivo studies, and how the recent development of new genetically encoded tools and cutting-edge imaging technology in neuroscience offers chronobiologists the opportunity to meet these challenges.

Environmental circadian disruption re-programs liver circadian gene expression.

Circadian gene expression is fundamental to the establishment and functions of the circadian clock, a cell-autonomous and evolutionary-conserved timing system. Yet, how it is affected by environmental-circadian disruption (ECD) such as shiftwork and jetlag, which impact millions of people worldwide, are ill-defined. Here, we provided the first comprehensive description of liver circadian gene expression under normal and after ECD conditions. We found that post-transcription and post-translation processes are dominant contributors to whole-cell or nuclear circadian proteome, respectively. Furthermore, rhythmicity of 64% transcriptome, 98% whole-cell proteome and 95% nuclear proteome is re-written by ECD. The re-writing, which is associated with changes of circadian cis-regulatory elements, RNA-processing and protein trafficking, diminishes circadian regulation of fat and carbohydrate metabolism and persists after one week of ECD-recovery.

What is well-being? A scoping review of the conceptual and operational definitions of occupational well-being.

Well-being is a multifaceted construct that is used across disciplines to portray a state of wellness, health, and happiness. While aspects of well-being seem universal, how it is depicted in the literature has substantial variation. The aim of this scoping review was to identify conceptual and operational definitions of well-being within the field of occupational health. Broad search terms were used related to well-being and scale/assessment. Inclusion criteria were (1) peer-reviewed articles, (2) published in English, (3) included a measure of well-being in the methods and results section of the article, and (4) empirical paper. The searches resulted in 4394 articles, 3733 articles were excluded by reading the abstract, 661 articles received a full review, and 273 articles were excluded after a full review, leaving 388 articles that met our inclusion criteria and were used to extract well-being assessment information. Many studies did not define well-being or link their conceptual definition to the operational assessment tool being used. There were 158 assessments of well-being represented across studies. Results highlight the lack of a consistent definitions of well-being and standardized measurements.

Dysregulation of inflammatory responses by chronic circadian disruption.

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.

The circadian clock mediates the response to oxidative stress in a cone photoreceptor‒like (661W) cell line via regulation of glutathione peroxidase activity.

Background: The mammalian retina contains an autonomous circadian clock that controls many physiological functions within this tissue. Our previous studies have indicated that disruption of this circadian clock by removing Bmal1 from the retina affects the visual function, retinal circuitry, and cone photoreceptor viability during aging. In the present study, we employed a mouse-derived cone photoreceptor‒like cell, 661W, to investigate which molecular mechanisms of the circadian clock may modulate cone photoreceptor viability during aging. Methods: Bmal1 knockout (BKO) cells were generated from 661W cells using the CRISPR/Cas9 gene editing tool. Deletion of Bmal1 from 661W was verified by western blot and monitoring Per2-luc bioluminescence circadian rhythms. To investigate the effect of Bmal1 removal on an oxidative stress challenge, cells were treated with hydrogen peroxide (H 2O 2,1 mM) for two hours and then cell viability was assessed. Cells were also cultured and harvested for gene expression analysis and antioxidant assay. Results: Our data indicated that 661W cells contain a functional circadian clock that mediates the response to an oxidative stress challenge in vitro and that such a response is no longer present in the BKO cell. We also hypothesized that the effect was due to the circadian regulation of the intracellular antioxidant defense mechanism. Our results revealed that in 661W cells, the antioxidant defense mechanism showed time dependent variation , whereas in BKO cells, there was an overall reduction in this antioxidant defense mechanism, and it no longer showed time dependent variation. Conclusions: Our work supported the notion that the presence of a functional circadian clock and its ability to modulate the response to an oxidative stress is the underlying mechanism that may protect cones during aging.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

TIP150 interacts with and targets MCAK at the microtubule plus ends.

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins-called plus-end tracking proteins (+TIPs)-bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150-MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.

Collection of Mouse Brain Slices for Bioluminescence Imaging of Circadian Clock Networks.

Circadian rhythms in cellular function can be monitored in real time with bioluminescence imaging. In this approach, bioluminescence is produced by an enzymatic reaction, which can be used to report dynamic changes in gene or protein expression in living cells. Bioluminescence imaging in circadian experiments typically uses an ex vivo slice preparation, with the most commonly studied structure being the master clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Here we describe procedures for dissecting and collecting SCN slices for bioluminescence imaging experiments.

Shift Work Predicts Increases in Lipopolysaccharide-Binding Protein, Interleukin-10, and Leukocyte Counts in a Cross-Sectional Study of Healthy Volunteers Carrying Low-Grade Systemic Inflammation.

The disruption of inflammatory responses is a potential mechanism behind the harmful effects of shift work and is associated with increased risk of hypertension, stroke, obesity, diabetes, and cancer. These responses are linked to the proliferation of leukocytes in shift workers, suggesting a systemic signal as a potential mediator. The purpose of this study was to assess the relationship between systemic inflammation, leukocyte counts, and systemic endotoxemia in samples from a diverse cohort of day workers and shift workers. Participants (normothermic and normotensive) were healthy volunteers, non-smoking, and drug- and medication-free. The following outcomes were measured: C-reactive protein, TNF-α, IL-6, IL-1ß, IL-10, leukocyte counts (monocytes, lymphocytes, and neutrophils), and lipopolysaccharide-binding protein (LBP). Risk factors that increase systemic inflammation, such as blood pressure, sleep loss, and cortisol, were also assessed. The results indicated that shift workers slept significantly less than day workers and had significantly increased concentrations of all of the cytokines measured as well as plasma cortisol. Regression models found that after controlling for covariates, shift-work exposure predicted the significant increase observed in IL-10, leukocyte counts, and LBP. Our results suggest that acute increases in low-grade systemic endotoxemia are unresolved during chronic shift-work exposure. This ongoing immune challenge may underlie the disrupted inflammatory responses characteristic of shift-work-related pathologies. Systemic endotoxemia may represent a novel target to investigate the early effects of exposure to shift-work schedules.