Sleep deprivation reduced LPS-induced IgG2b production by up-regulating BMAL1 and CLOCK expression.

Sleep deprivation (SD) weakens the immune system and leads to increased susceptibility to infectious or inflammatory diseases. However, it is still unclear how SD affects humoral immunity. In the present study, sleep disturbance was conducted using an sleep deprivation instrument, and the bacterial endotoxin lipopolysaccharide (LPS) was used to activate the immune response. It was found that SD-pretreatment reduced LPS-induced IgG2b+ B cells and IgG2b isotype antibody production in lymphocytes of spleen. And, SD-pretreatment decreased the proportion of CD4+T cells, production of CD4+T cells derived TGF-ß1 and its contribution in helping IgG2b production. Additionally, BMAL1 and CLOCK were selectively up-regulated in lymphocytes after SD. Importantly, BMAL1 and CLOCK deficiency contributed to TGF-ß1 expression and production of IgG2b+ B cells. Thus, our results provide a novel insight to explain the involvement of BMAL1 and CLOCK under SD stress condition, and their roles in inhibiting TGF-ß1 expression and contributing to reduction of LPS induced IgG2b production.

Improvement in the anti-inflammatory profile with lifelong physical exercise is related to clock genes expression in effector-memory CD4+ T cells in master athletes.

PURPOSE: Ageing is associated with alterations in the immune system as well as with alterations of the circadian rhythm. Immune cells show rhythmicity in execution of their tasks. Chronic inflammation (inflammaging), which is observed in the elderly, is mitigated by lifelong exercise. The aimed this study was to determine the acute effect of a maximal exercise test on clock genes, regulatory proteins and cytokine expression, and evaluate the effect of lifelong exercise on the expression of clock genes in subpopulations of effector-memory (EM) CD4+ and CD8+T cells and the association of these processes with the inflammatory profile. Therefore, this study aimed to investigate the expression of clock genes in subpopulations of effector memory (EM) CD4+ and CD8+ T cells in master athletes and healthy controls and further associate them with systemic inflammatory responses to acute exercise. METHODS: The study population comprised national and international master athletes (n = 18) involved in three sports (athletics, swimming and judo). The control group (n = 8) comprised untrained healthy volunteers who had not participated in any regular and competitive physical exercise in the past 20 years. Anthropometric measurements and blood samples were taken before (Pre), 10 min after (Post) and 1 h after (1 h Post) a maximal cycle ergometer test for the determination of maximum oxygen consumption (VO2 max). The subpopulations of EM CD4+ and CD8+ T cells were purified using fluorescenceactivated cell sorting. RNA extraction of clock genes (CLOCK, BMAL1, PER1, PER2, CRY1, CRY2, REV-ERBα, REV-ERBß, RORa, RORb and RORc) in EM CD4+ and EM CD8+ T cells as well as regulatory proteins (IL-4, IFN-γ, Tbx21, PD-1, Ki67, NF-kB, p53 and p21) in EM CD4+ T cells was performed. The serum concentration of cytokines (IL-8, IL-10, IL-12p70 and IL-17A) was measured. RESULTS: The master athletes showed better physiological parameters than the untrained healthy controls (P < 0.05). The levels of cytokines increased in master athletes at Post compared with those at Pre. The IL-8 level was higher at 1 h Post, whereas the IL-10 and IL-12p70 levels returned to baseline. There was no change in IL-17A levels (P < 0.05). The clock genes were modulated differently in CD4+ T cells after an acute session of exercise in a training status-dependent manner. CONCLUSION: The synchronization of clock genes, immune function and ageing presents new dimensions with interesting challenges. Lifelong athletes showed modified expression patterns of clock genes and cytokine production associated with the physical fitness level. Moreover, the acute bout of exercise altered the clock machinery mainly in CD4+ T cells; however, the clock gene expressions induced by acute exercise were different between the master athletes and control group.

The Effect of Light Exposure at Night (LAN) on Carcinogenesis via Decreased Nocturnal Melatonin Synthesis.

In mammals, a master clock is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, a region that receives input from the retina that is transmitted by the retinohypothalamic tract. The SCN controls the nocturnal synthesis of melatonin by the pineal gland that can influence the activity of the clock's genes and be involved in the inhibition of cancer development. On the other hand, in the literature, some papers highlight that artificial light exposure at night (LAN)-induced circadian disruptions promote cancer. In the present review, we summarize the potential mechanisms by which LAN-evoked disruption of the nocturnal increase in melatonin synthesis counteracts its preventive action on human cancer development and progression. In detail, we discuss: (i) the Warburg effect related to tumor metabolism modification; (ii) genomic instability associated with L1 activity; and (iii) regulation of immunity, including regulatory T cell (Treg) regulation and activity. A better understanding of these processes could significantly contribute to new treatment and prevention strategies against hormone-related cancer types.

Day-night dependence of gene expression and inflammatory responses in the remodeling murine heart post-myocardial infarction.

Diurnal or circadian rhythms are fundamentally important for healthy cardiovascular physiology and play a role in timing of onset and tolerance to myocardial infarction (MI) in patients. Whether time of day of MI triggers different molecular and cellular responses that can influence myocardial remodeling is not known. This study was designed to test whether time of day of MI triggers different gene expression, humoral, and innate inflammatory responses that contribute to cardiac repair after MI. Mice were infarcted by left anterior descending coronary artery ligation (MI model) within a 2-h time window either shortly after lights on or lights off, and the early remodeling responses at 8 h postinfarction were examined. We found that sleep-MI preferentially triggers early expression of genes associated with inflammatory responses, whereas wake-MI triggers more genes associated with metabolic pathways and transcription/translation, by microarray analyses. Homozygous clock mutant mice exhibit altered diurnal gene expression profiles, consistent with their cycling before onset of MI. In the first 8 h, crucial for innate immune responses to MI, there are also significant differences in sleep-MI and wake-MI serum cytokine responses and in neutrophil infiltration to infarcted myocardium. By 1-wk post-MI, there are differences in survivorship between the sleep and wake MI mice that could be explained by the different molecular and cellular responses. Our whole body physiology, tissues, and cells exhibit endogenous daily rhythms, and understanding their role in triggering effective responses after MI could lead to new strategies to benefit patients with cardiovascular disease.

Circadian clock regulates the host response to Salmonella.

Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

Chronic shift-lag alters the circadian clock of NK cells and promotes lung cancer growth in rats.

Prolonged subjection to unstable work or lighting schedules, particularly in rotating shift-workers, is associated with an increased risk of immune-related diseases, including several cancers. Consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as NK cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. To determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle or a chronic shift-lag paradigm consisting of 10 repeated 6-h photic advances occurring every 2 d, followed by 5-7 d of constant darkness. This model resulted in considerable circadian disruption, as assessed by circadian running-wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFN-γ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated in vivo conditions, and promoted lung tumor growth following i.v. injection of MADB106 tumor cells. Together, these findings suggest chronic circadian disruption promotes tumor growth by altering the circadian rhythms of NK cell function.

Circadian variation of the response of T cells to antigen.

Circadian clocks regulate many important aspects of physiology, and their disturbance leads to various medical conditions. Circadian variations have been found in immune system variables, including daily rhythms in circulating WBC numbers and serum concentration of cytokines. However, control of immune functional responses by the circadian clock has remained relatively unexplored. In this study, we show that mouse lymph nodes exhibit rhythmic clock gene expression. T cells from lymph nodes collected over 24 h show a circadian variation in proliferation after stimulation via the TCR, which is blunted in Clock gene mutant mice. The tyrosine kinase ZAP70, which is just downstream of the TCR in the T cell activation pathway and crucial for T cell function, exhibits rhythmic protein expression. Lastly, mice immunized with OVA peptide-loaded dendritic cells in the day show a stronger specific T cell response than mice immunized at night. These data reveal circadian control of the Ag-specific immune response and a novel regulatory mode of T cell proliferation, and may provide clues for more efficient vaccination strategies.

TNFR1 is essential for CD40, but not for lipopolysaccharide-induced sickness behavior and clock gene dysregulation.

Autoimmune and infectious diseases are associated with behavioral changes referred to as sickness behavior syndrome (SBS). In autoimmunity, the generation of anti-self T lymphocytes and autoantibodies critically involves binding of CD40 ligand on T-cells to its receptor CD40 on B-cells, dendritic cells and macrophages. Activation of CD40 leads to production of proinflammatory cytokines and, as shown here, induces SBS. Here we report that these behavioral changes depend on the expression of tumor necrosis factor alpha receptor 1 (TNFR1), but not on interleukin-1 receptor 1 or interleukin-6. Moreover, the intensity of SBS correlates with suppression of E-box controlled clock genes, including Dbp, and upregulation of Bmal1. However, the absence of TNFR1 does not interfere with the development of SBS and dysregulation of clock genes in mice treated with lipopolysaccharide. Thus, our results suggest that TNFR1 mediates SBS and dysregulation of clock genes in autoimmune diseases.

Molecular Interactions Between Components of the Circadian Clock and the Immune System.

The immune system is under control of the circadian clock. Many of the circadian rhythms observed in the immune system originate in direct interactions between components of the circadian clock and components of the immune system. The main means of circadian control over the immune system is by direct control of circadian clock proteins acting as transcription factors driving the expression or repression of immune genes. A second circadian control of immunity lies in the acetylation or methylation of histones to regulate gene transcription or inflammatory proteins. Furthermore, circadian clock proteins can engage in direct physical interactions with components of key inflammatory pathways such as members of the NFκB protein family. This regulation is transcription independent and allows the immune system to also reciprocally exert control over circadian clock function. Thus, the molecular interactions between the circadian clock and the immune system are manifold. We highlight and discuss here the recent findings with respect to the molecular mechanisms that control time-of-day-dependent immunity. This review provides a structured overview focusing on the key circadian clock proteins and discusses their reciprocal interactions with the immune system.

Circadian rhythm reprogramming during lung inflammation.

Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian programme exhibits unique features, including a divergent group of rhythmic genes and metabolites compared with the basal state and a distinct periodicity and phase distribution. At the cellular level, endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex re-organization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.

Antibodies for assessing circadian clock proteins in the rodent suprachiasmatic nucleus.

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.