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.     

Sex and dosing-time dependencies in irinotecan-induced circadian disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.     

Circadian disruption alters mouse lung clock gene expression and lung mechanics

Most aspects of human physiology and behavior exhibit 24-h rhythms driven by a master circadian clock in the brain, which synchronizes peripheral clocks. Lung function and ventilation are subject to circadian regulation and exhibit circadian oscillations. Sleep disruption, which causes circadian disruption, is common in those with chronic lung disease, and in the general population; however, little is known about the effect on the lung of circadian disruption. We tested the hypothesis circadian disruption alters expression of clock genes in the lung and that this is associated with altered lung mechanics. Female and male mice were maintained on a 12:12-h light/dark cycle (control) or exposed for 4 wk to a shifting light regimen mimicking chronic jet lag (CJL). Airway resistance (Rn), tissue damping (G), and tissue elastance (H) did not differ between control and CJL females. Rn at positive end-expiratory pressure (PEEP) of 2 and 3 cmH(2)O was lower in CJL males compared with controls. G, H, and G/H did not differ between CJL and control males. Among CJL females, expression of clock genes, Bmal1 and Rev-erb alpha, was decreased; expression of their repressors, Per2 and Cry 2, was increased. Among CJL males, expression of Clock was decreased; Per 2 and Rev-erb alpha expression was increased. We conclude circadian disruption alters lung mechanics and clock gene expression and does so in a sexually dimorphic manner.     

Circadian Clock and Breast Cancer: A Molecular Link

The circadian clock controls a large array of behavioral and physiological systems of fundamental importance to most organisms. Consequently, abnormal functioning of the clock results in severe dysfunctions and pathologies. Although epidemiological studies show a clear correlation between disruption of circadian rhythms and incidence of breast cancer, a molecular interpretation of how clock-related mechanisms may link to tumor development remains elusive. Here we speculate on the molecular pathways that may couple the circadian machinery to breast cancer.     

Sex and Dosing-Time Dependencies in Irinotecan-Induced Circadian Disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50?mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers. (Author correspondence: francis.levi@inserm.fr)     

Global Co‐Distribution of Light at Night (LAN) and Cancers of Prostate,Colon, and Lung in Men

The incidence rates of cancers in men differ by countries of the world. We compared the incidence rates of three of the most common cancers (prostate, lung, and colon) in men residing in 164 different countries with the population‐weighted light at night (LAN) exposure and with several developmental and environmental indicators, including per capita income, percent urban population, and electricity consumption. The estimate of per capita LAN exposure was a novel aspect of this study. Both ordinary least squares (OLS) and spatial error (SE) regression models were used in the analysis. We found a significant positive association between population exposure to LAN and incidence rates of prostate cancer, but no such association with lung cancer or colon cancer. The prostate cancer result is consistent with a biological theory and a limited number of previous studies of circadian disruption and risk. The LAN‐prostate cancer connection is postulated to be due to suppression of melatonin and/or disruption of clock gene function. An analysis holding other variables at average values across the 164 countries yielded a risk of prostate cancer in the highest LAN‐exposed countries 110% higher than in the lowest LAN exposed countries. This observed association is a necessary condition for a potentially large effect of LAN on risk of prostate cancer. However, it is not sufficient due to potential confounding by factors that increase the risk of prostate cancer and are also associated with LAN among the studied countries.     

The circadian system of Trypanosoma cruzi-infected mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.     

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.     

Impaired light detection of the circadian clock in a zebrafish melanoma model

The circadian clock controls the timing of the cell cycle in healthy tissues and clock disruption is known to increase tumourigenesis. Melanoma is one of the most rapidly increasing forms of cancer and the precise molecular circadian changes that occur in a melanoma tumor are unknown. Using a melanoma zebrafish model, we have explored the molecular changes that occur to the circadian clock within tumors. We have found disruptions in melanoma clock gene expression due to a major impairment to the light input pathway, with a parallel loss of light-dependent activation of DNA repair genes. Furthermore, the timing of mitosis in tumors is perturbed, as well as the regulation of certain key cell cycle regulators, such that cells divide arhythmically. The inability to co-ordinate DNA damage repair and cell division is likely to promote further tumourigenesis and accelerate melanoma development.     

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.

Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.     

Adverse effects of chronic circadian desynchronization in animals in a "challenging" environment

Continuous disruption of circadian rhythms, as seen in human shift workers, has been associated with the development of a number of adverse mental and physiological conditions. However, scientific evidence linking circadian disruption to overall health, particularly in animal models, is not well documented. In this study, we have demonstrated that exposing C57BL/6J mice to 12-h phase shifts every 5 days for 3 mo had no effect on body weight or intestinal physiology. However, when animals were further challenged with dextran sodium sulfate to induce colitis, chronic shifting of the light-dark cycle led to a dramatic increase in the progression of the colitis as indicated by reduced body weight, abnormal intestinal histopathology, and an exacerbated inflammatory response. These data indicate that circadian disruption is an important predisposing factor that may provoke the onset or worsening of various disease states such as inflammatory disorders. This study provides further evidence for continued investigations using animal models of circadian disruption to examine the consequences of circadian disruption on health when organisms are faced with a "challenging" environment.     

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.     

Development of dilated cardiomyopathy in Bmal1-deficient mice

Circadian rhythms are approximate 24-h oscillations in physiology and behavior. Circadian rhythm disruption has been associated with increased incidence of hypertension, coronary artery disease, dyslipidemia, and other cardiovascular pathologies in both humans and animal models. Mice lacking the core circadian clock gene, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein (Bmal1), are behaviorally arrhythmic, die prematurely, and display a wide range of organ pathologies. However, data are lacking on the role of Bmal1 on the structural and functional integrity of cardiac muscle. In the present study, we demonstrate that Bmal1(-/-) mice develop dilated cardiomyopathy with age, characterized by thinning of the myocardial walls, dilation of the left ventricle, and decreased cardiac performance. Shortly after birth the Bmal1(-/-) mice exhibit a transient increase in myocardial weight, followed by regression and later onset of dilation and failure. Ex vivo working heart preparations revealed systolic ventricular dysfunction at the onset of dilation and failure, preceded by downregulation of both myosin heavy chain isoform mRNAs. We observed structural disorganization at the level of the sarcomere with a shift in titin isoform composition toward the stiffer N2B isoform. However, passive tension generation in single cardiomyocytes was not increased. Collectively, these findings suggest that the loss of the circadian clock gene, Bmal1, gives rise to the development of an age-associated dilated cardiomyopathy, which is associated with shifts in titin isoform composition, altered myosin heavy chain gene expression, and disruption of sarcomere structure.     

Shift work,circadian gene variants and risk of breast cancer

Circadian (clock) genes have been linked with several functions relevant to cancer, and epidemiologic research has suggested relationships with breast cancer risk for variants in NPAS2, CLOCK, CRY2 and TIMELESS. Increased breast cancer risk has also been observed among shift workers, suggesting potential interactions in relationships of circadian genes with breast cancer. Relationships with breast cancer of 100 SNPs in 14 clock-related genes, as well as potential interactions with shift work history, were investigated in a case–control study (1042 cases, 1051 controls). Odds ratios in an additive genetic model for European-ancestry participants (645 cases, 806 controls) were calculated, using a two-step correction for multiple testing: within each gene through permutation testing (10,000 permutations), and correcting for the false discovery rate across genes. Interactions of genotypes with ethnicity and shift work (<2 years vs ≥2 years) were evaluated individually. Following permutation analysis, two SNPs (rs3816360 in ARNTL and rs11113179 in CRY1) displayed significant associations with breast cancer and one SNP (rs3027188 in PER1) was marginally significant; however, none were significant following adjustment for the false discovery rate. No significant interaction with shift work history was detected. If shift work causes circadian disruption, this was not reflected in associations between clock gene variants and breast cancer risk in this study. Larger studies are needed to assess interactions with longer durations (>30 years) of shift work that have been associated with breast cancer.