The Sleep-Immune Crosstalk in Health and Disease.

Sleep and immunity are bidirectionally linked. Immune system activation alters sleep, and sleep in turn affects the innate and adaptive arm of our body's defense system. Stimulation of the immune system by microbial challenges triggers an inflammatory response, which, depending on its magnitude and time course, can induce an increase in sleep duration and intensity, but also a disruption of sleep. Enhancement of sleep during an infection is assumed to feedback to the immune system to promote host defense. Indeed, sleep affects various immune parameters, is associated with a reduced infection risk, and can improve infection outcome and vaccination responses. The induction of a hormonal constellation that supports immune functions is one likely mechanism underlying the immune-supporting effects of sleep. In the absence of an infectious challenge, sleep appears to promote inflammatory homeostasis through effects on several inflammatory mediators, such as cytokines. This notion is supported by findings that prolonged sleep deficiency (e.g., short sleep duration, sleep disturbance) can lead to chronic, systemic low-grade inflammation and is associated with various diseases that have an inflammatory component, like diabetes, atherosclerosis, and neurodegeneration. Here, we review available data on this regulatory sleep-immune crosstalk, point out methodological challenges, and suggest questions open for future research.

Foundations of circadian medicine.

The circadian clock is an evolutionarily highly conserved endogenous timing program that structures physiology and behavior according to the time of day. Disruption of circadian rhythms is associated with many common pathologies. The emerging field of circadian medicine aims to exploit the mechanisms of circadian physiology and clock-disease interaction for clinical diagnosis, treatment, and prevention. In this Essay, we outline the principle approaches of circadian medicine, highlight the development of the field in selected areas, and point out open questions and challenges. Circadian medicine has unambiguous health benefits over standard care but is rarely utilized. It is time for clock biology to become an integrated part of translational research.

Sleep promotes T-cell migration towards CCL19 via growth hormone and prolactin signaling in humans.

Sleep strongly supports the formation of adaptive immunity, e.g., after vaccination. However, the underlying mechanisms remain largely obscure. Here we show in healthy humans that sleep compared to nocturnal wakefulness specifically promotes the migration of various T-cell subsets towards the chemokine CCL19, which is essential for lymph-node homing and, thus, for the initiation and maintenance of adaptive immune responses. Migration towards the inflammatory chemokine CCL5 remained unaffected. Incubating the cells with plasma from sleeping participants likewise increased CCL19-directed migration, an effect that was dependent on growth hormone and prolactin signaling. These findings show that sleep selectively promotes the lymph node homing potential of T cells by increasing hormonal release, and thus reveal a causal mechanism underlying the supporting effect of sleep on adaptive immunity in humans.

Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

Post-quantum cryptography.

Cryptography is essential for the security of online communication, cars and implanted medical devices. However, many commonly used cryptosystems will be completely broken once large quantum computers exist. Post-quantum cryptography is cryptography under the assumption that the attacker has a large quantum computer; post-quantum cryptosystems strive to remain secure even in this scenario. This relatively young research area has seen some successes in identifying mathematical operations for which quantum algorithms offer little advantage in speed, and then building cryptographic systems around those. The central challenge in post-quantum cryptography is to meet demands for cryptographic usability and flexibility without sacrificing confidence.

Sleep Matters: CD4+ T Cell Memory Formation and the Central Nervous System.

The mechanisms of CD4+ T-cell memory formation in the immune system are debated. With the well-established concept of memory formation in the central nervous system (CNS), we propose that formation of CD4+ T-cell memory depends on the interaction of two different cell systems handling two types of stored information. First, information about antigen (event) and challenge (context) is taken up by antigen-presenting cells, as initial storage. Second, event and context information is transferred to CD4+ T cells. During activation, two categories of CD4+ T cell develop: effector CD4+ T cells, carrying event and context information, enabling them to efficiently focus their response to tissues under attack; and persisting CD4+ T cells, providing context-independent antigen-specific memories and long-term storage. This novel hypothesis is supported by the observation that mammalian sleep can improve both CNS and CD4+ T-cell memory.

Association of stress-related neural activity and baseline interleukin-6 plasma levels in healthy adults.

Several studies suggest a link between acute changes in inflammatory parameters due to an endotoxin or (psychological) stressor and the brain's stress response. The extent to which basal circulating levels of inflammatory markers are associated with the brain's stress response has been hardly investigated so far. In the present study, baseline plasma levels of the cytokine interleukin (IL)-6 were obtained and linked to neural markers of psychosocial stress using a modified version of the Montreal Imaging Stress Task in a sample of N = 65 healthy subjects (N = 39 female). Of three a-priori defined regions of interest - the amygdala, anterior insula, and anterior cingulate cortex - baseline IL-6 was significantly and negatively associated with stress-related neural activation in the right amygdala and left anterior insula. Our results suggest that baseline cytokines might be related to differences in the neural stress response and that this relationship could be inverse to that previously reported for induced acute changes in inflammation markers.

Activated integrins identify functional antigen-specific CD8+ T cells within minutes after antigen stimulation.

Immediate ß2-integrin activation upon T cell receptor stimulation is critical for effective interaction between T cells and their targets and may therefore be used for the rapid identification and isolation of functional T cells. We present a simple and sensitive flow cytometry-based assay to assess antigen-specific T cells using fluorescent intercellular adhesion molecule (ICAM)-1 multimers that specifically bind to activated ß2-integrins. The method is compatible with surface and intracellular staining; it is applicable for monitoring of a broad range of virus-, tumor-, and vaccine-specific CD8+ T cells, and for isolating viable antigen-reacting cells. ICAM-1 binding correlates with peptide-MHC multimer binding but, notably, it identifies the fraction of antigen-specific CD8+ T cells with immediate and high functional capability (i.e., expressing high levels of cytotoxic markers and cytokines). Compared with the currently available methods, staining of activated ß2-integrins presents the unique advantage of requiring activation times of only several minutes, therefore delivering functional information nearly reflecting the in vivo situation. Hence, the ICAM-1 assay is most suitable for rapid and precise monitoring of functional antigen-specific T cell responses, including for patient samples in a variety of clinical settings, as well as for the isolation of functional T cells for adoptive cell-transfer immunotherapies.

Sleep enhances numbers and function of monocytes and improves bacterial infection outcome in mice.

Sleep strongly impacts both humoral and cellular immunity; however, its acute effects on the innate immune defense against pathogens are unclear. Here, we elucidated in mice whether sleep affects the numbers and functions of innate immune cells and their defense against systemic bacterial infection. Sleep significantly increased numbers of classical monocytes in blood and spleen of mice that were allowed to sleep for six hours at the beginning of the normal resting phase compared to mice kept awake for the same time. The sleep-induced effect on classical monocytes was neither caused by alterations in corticosterone nor myelopoiesis, bone marrow egress or death of monocytes and did only partially involve Gαi-protein coupled receptors like chemokine receptor 2 (CCR2), but not the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) or lymphocyte function-associated antigen 1 (LFA-1). Notably, sleep suppressed the expression of the clock gene Arntl in splenic monocytes and the sleep-induced increase in circulating classical monocytes was abrogated in Arntl-deficient animals, indicating that sleep is a prerequisite for clock-gene driven rhythmic trafficking of classical monocytes. Sleep also enhanced the production of reactive oxygen species by monocytes and neutrophils. Moreover, sleep profoundly reduced bacterial load in blood and spleen of mice that were allowed to sleep before systemic bacterial infection and consequently increased survival upon infection. These data provide the first evidence that sleep enhances numbers and function of innate immune cells and therewith strengthens early defense against bacterial pathogens.

Nocturnal sleep uniformly reduces numbers of different T-cell subsets in the blood of healthy men.

In humans, numbers of circulating T cells show a circadian rhythm with peak counts during the night and a steep decline in the morning. Sleep per se appears to counter this rhythm by acutely reducing the total number of T cells. The T-cell population, however, is rather heterogeneous, comprising various subpopulations with different features and functions and also different circadian rhythms. Therefore, we examined here whether sleep likewise differentially affects these subsets. We measured eight different T-cell subsets (naïve, central memory, effector memory, and effector CD4+ and CD8+ T cells) over a 24-h period under conditions of sustained wakefulness compared with a regular sleep-wake cycle in 14 healthy young men. Sleep reduced the number of all T-cell subsets during nighttime with this effect reaching the P < 0.05 level of significance in all but one subpopulation, i.e., effector CD4+ T cells, where it only approached significance. Furthermore, sleep was associated with an increase in growth hormone, prolactin, and aldosterone levels, whereas concentrations of catecholamines tended to be lower than during nocturnal wakefulness. The effect of sleep uniformly decreasing the different T-cell subsets is surprising considering their differential function and circadian rhythms, and even more so, since the sleep-induced decreases in these subsets are probably conveyed by different hormonal mediators. Although the reductions in cell numbers are rather small, they are comparable to changes seen, for example, after vaccination and are, therefore, likely to be of physiological relevance.

Mineralocorticoid receptor signaling reduces numbers of circulating human naïve T cells and increases their CD62L, CCR7, and CXCR4 expression.

The role of mineralocorticoid receptors (MRs) in human T-cell migration is not yet understood. We have recently shown that the MR antagonist spironolactone selectively increases the numbers of circulating naïve and central memory T cells during early sleep, which is the time period in the 24 h cycle hallmarked by predominant MR activation. To investigate whether this effect is specific to spironolactone's blockade of MRs and to study the underlying molecular mechanisms, healthy humans were given the selective MR-agonist fludrocortisone or placebo and numbers of eight T-cell subsets and their CD62L and CXCR4 expression were analyzed. Fludrocortisone selectively reduced counts of naïve CD4(+) , central memory CD4(+), and naïve CD8(+) T cells and increased CXCR4 expression on the naïve subsets. In complementing in vitro studies, fludrocortisone enhanced CXCR4 and CD62L expression, which was counteracted by spironolactone. Incubation of naïve T cells with spironolactone alone reduced CD62L and CCR7 expression. Our results indicate a regulatory influence of MR signaling on human T-cell migration and suggest a role for endogenous aldosterone in the redistribution of T-cell subsets to lymph nodes, involving CD62L, CCR7, and CXCR4. Facilitation of T-cell homing following sleep-dependent aldosterone release might thus essentially contribute to sleep's well-known role in supporting adaptive immunity.

Activated CD4+ T cells enter the splenic T-cell zone and induce autoantibody-producing germinal centers through bystander activation.

CD4(+) T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4(+) T-cell subsets within different organ compartments. Such information is important because there are indications that CD4(+) T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4(+) T cells through the different compartments of the spleen. Resting and recently activated CD4(+) T cells were separated from thoracic duct lymph and activated CD4(+) T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4(+) T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependent T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner.

Emotional memory can be persistently weakened by suppressing cortisol during retrieval.

Cortisol's effects on memory follow an inverted U-shaped function such that memory retrieval is impaired with very low concentrations, presumably due to insufficient activation of high-affine mineralocorticoid receptors (MR), or with very high concentrations, due to predominant low-affine glucocorticoid receptor (GR) activation. Through corresponding changes in re-encoding, the retrieval effect of cortisol might translate into a persistent change of the retrieved memory. We tested whether partial suppression of morning cortisol synthesis by metyrapone, leading to intermediate, circadian nadir-like levels with presumed predominant MR activation, improves retrieval, particularly of emotional memory, and persistently changes the memory. In a randomized, placebo-controlled, double-blind, within-subject cross-over design, 18 men were orally administered metyrapone (1g) vs. placebo at 4:00 AM to suppress the morning cortisol rise. Retrieval of emotional and neutral texts and pictures (learned 3 days earlier) was assessed 4h after substance administration and a second time one week later. Metyrapone suppressed endogenous cortisol release to circadian nadir-equivalent levels at the time of retrieval testing. Contrary to our expectations, metyrapone significantly impaired free recall of emotional texts (p<.05), whereas retrieval of neutral texts or pictures remained unaffected. One week later, participants still showed lower memory for emotional texts in the metyrapone than placebo condition (p<.05). Our finding that suppressing morning cortisol to nadir-like concentrations not only impairs acute retrieval, but also persistently weakens emotional memories corroborates the concept that retrieval effects of cortisol produce persistent memory changes, possibly by affecting re-encoding.

Endogenous glucocorticoid receptor signaling drives rhythmic changes in human T-cell subset numbers and the expression of the chemokine receptor CXCR4.

In humans, numbers of circulating naive T cells strongly decline in the morning, which was suggested to be mediated by cortisol, inducing a CXCR4 up-regulation with a subsequent extravasation of the cells. As a systematic evaluation of this assumption is lacking, we investigated in two human placebo-controlled studies the effects of the glucocorticoid receptor (GR) antagonist mifepristone (200 mg orally at 23:00) and of suppressing endogenous cortisol with metyrapone (1 g orally at 04:00) on temporal changes in CXCR4 expression and numbers of different T-cell subsets using flow cytometry. Mifepristone attenuated, and metyrapone completely blocked, the morning increase in CXCR4 expression on naive T cells. In parallel, both substances also hindered the decline in naive T-cell numbers with this effect, however, being less apparent after mifepristone. We identified, and confirmed in additional in vitro studies, a partial agonistic GR effect of mifepristone at night (i.e., between 02:00 and 03:30) that could explain the lower antagonistic efficacy of the substance on CXCR4 expression and naive T-cell counts. CXCR4 expression emerged to be a most sensitive marker of GR signaling. Our studies jointly show that endogenous cortisol, specifically via GR activation, causes the morning increase in CXCR4 expression and the subsequent extravasation of naive T cells, thus revealing an important immunological function of the morning cortisol rise.

Differential acute effects of sleep on spontaneous and stimulated production of tumor necrosis factor in men.

Tumor necrosis factor (TNF) is considered a key molecule in the regulation of sleep in health and disease. Conversely, sleep compared to sleep deprivation can modulate TNF release, but overall results are conflicting. In this study we focused on the influence of sleep on spontaneous, i.e., unstimulated TNF production, which might be involved in sleep regulation under normal non-infectious conditions, and on lipopolysaccharide (LPS)-stimulated TNF production, which reflects the capacity of the immune system to respond to a pathogen. To this end, we monitored 10 healthy men during a regular sleep-wake cycle and during 24h of wakefulness while blood was sampled repeatedly to analyze circulating TNF levels in serum as well as intracellular TNF production in monocytes spontaneously and after stimulation with LPS employing whole blood cell cultures. In addition we assessed numbers of monocyte subsets and levels of various hormones in blood. In comparison with nocturnal wakefulness, sleep acutely decreased serum TNF levels, with no parallel decrease in spontaneous monocytic TNF production, but was associated with a striking nighttime increase in the percentage of TNF producing monocytes after stimulation with LPS. The following day circulating TNF showed a reverse pattern with higher levels after regular sleep than after the nocturnal vigil. The mechanisms mediating the differential effects of sleep on circulating TNF (acutely decreased) vs. stimulated monocytic TNF production (acutely increased) remain unclear, although explorative correlational analyses pointed to a regulatory involvement of cortisol, norepinephrine and prolactin. The acute enhancing effect of sleep on LPS stimulated monocytic TNF production adds to the notion that nocturnal sleep favors immune defense to a microbial challenge.

Effects of an interleukin-1 receptor antagonist on human sleep, sleep-associated memory consolidation, and blood monocytes.

Pro-inflammatory cytokines like interleukin-1 beta (IL-1) are major players in the interaction between the immune system and the central nervous system. Various animal studies report a sleep-promoting effect of IL-1 leading to enhanced slow wave sleep (SWS). Moreover, this cytokine was shown to affect hippocampus-dependent memory. However, the role of IL-1 in human sleep and memory is not yet understood. We administered the synthetic IL-1 receptor antagonist anakinra (IL-1ra) in healthy humans (100mg, subcutaneously, before sleep; n=16) to investigate the role of IL-1 signaling in sleep regulation and sleep-dependent declarative memory consolidation. Inasmuch monocytes have been considered a model for central nervous microglia, we monitored cytokine production in classical and non-classical blood monocytes to gain clues about how central nervous effects of IL-1ra are conveyed. Contrary to our expectation, IL-1ra increased EEG slow wave activity during SWS and non-rapid eye movement (NonREM) sleep, indicating a deepening of sleep, while sleep-associated memory consolidation remained unchanged. Moreover, IL-1ra slightly increased prolactin and reduced cortisol levels during sleep. Production of IL-1 by classical monocytes was diminished after IL-1ra. The discrepancy to findings in animal studies might reflect species differences and underlines the importance of studying cytokine effects in humans.

Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors.

Sleep after vaccination boosts immunological memory.

Sleep regulates immune functions. We asked whether sleep can influence immunological memory formation. Twenty-seven healthy men were vaccinated against hepatitis A three times, at weeks 0, 8, and 16 with conditions of sleep versus wakefulness in the following night. Sleep was recorded polysomnographically, and hormone levels were assessed throughout the night. Vaccination-induced Th cell and Ab responses were repeatedly monitored for 1 y. Compared with the wake condition, sleep after vaccination doubled the frequency of Ag-specific Th cells and increased the fraction of Th1 cytokine-producing cells in this population. Moreover, sleep markedly increased Ag-specific IgG1. The effects were followed up for 1 y and were associated with high sleep slow-wave activity during the postvaccination night as well as with accompanying levels of immunoregulatory hormones (i.e., increased growth hormone and prolactin but decreased cortisol release). Our findings provide novel evidence that sleep promotes human Th1 immune responses, implicating a critical role for slow-wave sleep in this process. The proinflammatory milieu induced during this sleep stage apparently acts as adjuvant that facilitates the transfer of antigenic information from APCs to Ag-specific Th cells. Like the nervous system, the immune system takes advantage of the offline conditions during sleep to foster adaptive immune responses resulting in improved immunological memory.

Detecting and exploiting the circadian clock in rheumatoid arthritis.

Over the past four decades, research on 24-h rhythms has yielded numerous remarkable findings, revealing their genetic, molecular, and physiological significance for immunity and various diseases. Thus, circadian rhythms are of fundamental importance to mammals, as their disruption and misalignment have been associated with many diseases and the abnormal functioning of many physiological processes. In this article, we provide a brief overview of the molecular regulation of 24-h rhythms, their importance for immunity, the deleterious effects of misalignment, the link between such pathological rhythms and rheumatoid arthritis (RA), and the potential exploitation of chronobiological rhythms for the chronotherapy of inflammatory autoimmune diseases, using RA as an example.