Dr Silke Kiessling

Chemostat systems can be used to cultivate complex intestinal microbial communities ex vivo. Here, we present a protocol to transfer bacteria from human fecal material into chemostat systems as well as settings to simulate infant or adult colonic conditions. We describe the experimental setup, media design, donor selection, 16S rRNA amplicon sequencing, and circadian analysis of bacterial abundance. This protocol enables the investigation of changes in microbial community composition and bacteria-derived metabolites upon exposure to different dietary components. For complete details on the use and execution of this protocol, please refer to Heppner et al.1 [Display omitted] •Details of chemostat setup for continuous ex vivo cultivation of intestinal microbiota•Characterization of nutrient-microbiota interactions ex vivo•Versatile applications to simulate different intestinal conditions•Circadian analysis to determine rhythmic changes in microbiota composition and function Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Chemostat systems can be used to cultivate complex intestinal microbial communities ex vivo. Here, we present a protocol to transfer bacteria from human fecal material into chemostat systems as well as settings to simulate infant or adult colonic conditions. We describe the experimental setup, media design, donor selection, 16S rRNA amplicon sequencing, and circadian analysis of bacterial abundance. This protocol enables the investigation of changes in microbial community composition and bacteria-derived metabolites upon exposure to different dietary components.

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

The expression of clock genes has been observed to be impaired in biopsies from patients with inflammatory bowel disease (IBD). Disruption of circadian rhythms, which occurs in shift workers, has been linked to an increased risk of gastrointestinal diseases, including IBD. The peripheral circadian clock in intestinal epithelial cells (IECs) was previously shown to balance gastrointestinal homeostasis by regulating the microbiome. Here, we demonstrated that the intestinal clock is disrupted in an IBD-relevant mouse model (IL-10-/-). A lack of the intestinal clock gene (Bmal1) in intestinal epithelial cells (IECs) in a chemically and a novel genetically induced colitis model (DSS, Bmal1IEC-/-xIL-10-/-) promoted colitis and dramatically reduced survival rates. Germ-free Bmal1IEC-/- mice colonized with disease-associated microbiota from IL-10-/- mice exhibited increased inflammatory responses, highlighting the importance of the local intestinal clock for microbiota-induced IBD development. Targeting the intestinal clock directly by timed restricted feeding (RF) in IL-10-/- mice restored intestinal clock functions, including immune cell recruitment and microbial rhythmicity; improved inflammatory responses; dramatically enhanced survival rates and rescued the histopathological phenotype. In contrast, RF failed to improve IBD symptoms in Bmal1IEC-/-xIL-10-/- mice, demonstrating the significance of the intestinal clock in determining the beneficial effect of RF. Overall, we provide evidence that intestinal clock dysfunction triggers host immune imbalance and promotes the development and progression of IBD-like colitis. Enhancing intestinal clock function by RF modulates the pathogenesis of IBD and thus could become a novel strategy to ameliorate symptoms in IBD patients.

The genotype/phenotype dichotomy is being slowly replaced by a more complex relationship whereby the majority of phenotypes arise from interactions between one’s genotype and the environment in which one lives. Interestingly, it seems that not only our lives, but also our ancestors’ lives, determine how we look. This newly recognized form of inheritance is known as (epi)genetic, as it involves an additional layer of information on top of the one encoded by the genes. Its discovery has constituted one of the biggest paradigm shifts in biology in recent years. Understanding epigenetic factors may help explain the pathogenesis of several complex human diseases (such as diabetes, obesity and cancer) and provide alternative paths for disease prevention, management and therapy. This book introduces the reader to the importance of the environment for our own health and the health of our descendants, sheds light on the current knowledge on epigenetic inheritance and opens a window to future developments in the field.

MicroRNAs (miRs) are important regulators of a wide range of biological processes. Antagomir studies suggest an implication of miR-132 in the functionality of the mammalian circadian clock. miR-212 and miR-132 are tandemly processed from the same transcript and share the same seed region. We found the clock modulator miR-132 and miR-212 to be expressed rhythmically in the central circadian clock. Consequently, mRNAs implicated in circadian functions may likely be targeted by both miRs. To further characterize the circadian role we generated mice with stable deletion of the miR-132/212 locus and compared the circadian behavior of mutant and wild-type control animals on two genetic backgrounds frequently used in chronobiological research, C57BL/6N and 129/Sv. Surprisingly, the wheel-running activity phenotype of miR mutant mice was highly background specific. A prolonged circadian free-running period in constant darkness was found in 129/Sv, but not in C57BL/6N miR-132/212 knockout mice. In contrast, in C57BL/6N, but not in 129/Sv miRNA132/212 knockout mice a lengthened free-running period was observed in constant light conditions. Furthermore, miR-132/212 knockout mice on 129/Sv background exhibited enhanced photic phase shifts of locomotor activity accompanied by reduced light induction of Period gene transcription in the SCN. This phenotype was absent in miRNA-132/212 knockout mice on a C57BL/6N background. Together, our results reveal a strain and light regimen-specific function of miR-132/212 in the circadian clock machinery suggesting that miR-132 and miR-212 act as background-dependent circadian rhythm modulators.

In mammals, the master clock of the suprachiasmatic nuclei (SCN) and subordinate clocks found throughout the body coordinate circadian rhythms of behavior and physiology. We characterize the clock of the adrenal, an important endocrine gland that synchronizes physiological and metabolic rhythms. Clock gene expression was detected in the outer adrenal cortex prefiguring a role of the clock in regulating gluco- and mineral corticoid biogenesis. In Per2/Cry1 double mutant mice, which lack a circadian clock, hypothalamus/pituitary/adrenal axis regulation was defective. Organ culture and tissue transplantation suggest that the adrenal pacemaker gates glucocorticoid production in response to adrenocorticotropin (ACTH). In vivo the adrenal circadian clock can be entrained by light. Transcriptome profiling identified rhythmically expressed genes located at diverse nodes of steroid biogenesis that may mediate gating of the ACTH response by the adrenal clock.

Jet lag encompasses a range of psycho- and physiopathological symptoms that arise from temporal misalignment of the endogenous circadian clock with external time. Repeated jet lag exposure, encountered by business travelers and airline personnel as well as shift workers, has been correlated with immune deficiency, mood disorders, elevated cancer risk, and anatomical anomalies of the forebrain. Here, we have characterized the molecular response of the mouse circadian system in an established experimental paradigm for jet lag whereby mice entrained to a 12-hour light/12-hour dark cycle undergo light phase advancement by 6 hours. Unexpectedly, strong heterogeneity of entrainment kinetics was found not only between different organs, but also within the molecular clockwork of each tissue. Manipulation of the adrenal circadian clock, in particular phase-shifting of adrenal glucocorticoid rhythms, regulated the speed of behavioral reentrainment. Blocking adrenal corticosterone either prolonged or shortened jet lag, depending on the time of administration. This key role of adrenal glucocorticoid phasing for resetting of the circadian system provides what we believe to be a novel mechanism-based approach for possible therapies for jet lag and jet lag-associated diseases.

Objective: Impaired clock genes expression has been observed in biopsy samples from patients with inflammatory bowel disease (IBD). Disruption of circadian rhythms, which occurs in shift workers, has been linked to an increased risk of gastrointestinal diseases, including IBD. The intestinal clock balances gastrointestinal homeostasis by regulating the microbiome. Here we characterize intestinal immune functions in mice lacking the intestinal clock and IBD-relevant mouse model under different feeding conditions to describe the functional impact of the intestinal clock in the development of gastrointestinal inflammation. Design: Tissues and fecal samples from intestinal clock-deficient mice (Bmal1IEC-/-) and mouse models for colitis (IL-10-/-, Bmal1IEC-/-xIL-10-/-, dextran sulfate sodium (DSS) administration) under ad libitum and restricted feeding (RF) conditions were used to determine the causal role of the intestinal clock for colitis. Results: In IL-10-/- mice, inflammation correlated with disrupted colon clock genes expression. Genetic loss of intestinal clock functions promoted DSS and IBD inflammatory phenotypes and dramatically reduces survival, and colonization with disease-associated microbiota in germ-free Bmal1IEC-/- hosts increased their inflammatory responses, demonstrating the causal role of colonic clock disruption and the severity of IBD. RF in IL-10-/- mice restored the colon clock and related immune functions, improved the inflammatory responses and rescued the histopathological phenotype. In contrast, RF failed to improve IBD symptoms in Bmal1IEC-/-xIL-10-/- demonstrating the significance of the colonic clock to gate the effect of RF. Conclusion: We provide evidence that inflammation-associated intestinal clock dysfunction triggers host immune imbalance and promotes the development and progression of IBD-like colitis. Enhancing intestinal clock function by RF modulates the pathogenesis of IBD and thus could become a novel strategy to ameliorate the symptoms in IBD patients. Competing Interest Statement The authors have declared no competing interest.

The brain's master circadian pacemaker resides within the hypothalamic suprachiasmatic nucleus (SCN). SCN clock neurons are entrained to the day/night cycle via the retinohypothalamic tract and the SCN provides temporal information to the central nervous system and to peripheral organs that function as secondary oscillators. The SCN clock-cell network is thought to be the hypothalamic link between the retina and descending autonomic circuits to peripheral organs such as the adrenal gland, thereby entraining those organs to the day/night cycle. However, there are at least three different routes or mechanisms by which retinal signals transmitted to the hypothalamus may be conveyed to peripheral organs: 1) via retinal input to SCN clock neurons; 2) via retinal input to non-clock neurons in the SCN; or 3) via retinal input to hypothalamic regions neighboring the SCN. It is very well documented that light-induced responses of the SCN clock (i.e., clock gene expression, neural activity, and behavioral phase shifts) occur primarily during the subjective night. Thus to determine the role of the SCN clock in transmitting photic signals to descending autonomic circuits, we compared the phase dependency of light-evoked responses in the SCN and a peripheral oscillator, the adrenal gland. We observed light-evoked clock gene expression in the mouse adrenal throughout the subjective day and subjective night. Light also induced adrenal corticosterone secretion during both the subjective day and subjective night. The irradiance threshold for light-evoked adrenal responses was greater during the subjective day compared to the subjective night. These results suggest that retinohypothalamic signals may be relayed to the adrenal clock during the subjective day by a retinal pathway or cellular mechanism that is independent of an effect of light on the SCN neural clock network and thus may be important for the temporal integration of physiology and metabolism.

Circadian disruption, e.g. through shift work, causes microbial dysbiosis and increases the risk of metabolic diseases. Microbial rhythmicity in mice depends on a functional intestinal clock and frequent jetlag as well as high-caloric energy intake induces loss of these oscillations. Similarly, arrhythmic microbiota was found in obese and T2D populations. However, the interplay between the intestinal circadian clock, the microbiome, diet and host metabolism is poorly understood. In intestinal-specific Bmal1 knockout mice (Bmal1IEC-/- ) we demonstrate the relevance of the intestinal clock in microbiome oscillations and host and microbial nutrient metabolism. Microbiota transfer from Bmal1IEC-/- mice into germ-free recipients led to obesity, reflected by increased bodyweight and fat mass. Western diet-fed Bmal1IEC-/- mice increased bodyweight likely through mechanisms involving the intestinal clock-control of lipid and hexose transporters. Additionally, we identified dietary fiber as novel link between circadian microbial rhythmicity, intestinal clock functioning and host physiology. Thus, revealing the potential of fiber-rich diet intervention as a non-invasive strategy targeting microbial oscillations in metabolic disease prevention.

Various psychiatric disorders, including schizophrenia, are comorbid with sleep and circadian rhythm disruptions. To understand the links between circadian rhythms and schizophrenia, we analyzed wheel-running behavior of Sandy (Sdy) mice, which have a loss-of-function mutation in the schizophrenia risk gene Dtnbpl, and exhibit several behavioral features of schizophrenia. While rhythms of Sdy mice were mainly normal under light-dark conditions (LD) or in constant darkness (DD), they had a significantly longer free-running period under constant light (LL) compared to wild-type (WT) littermates. The mutant mice also had a higher subjective day/subjective night ratio of activity under LL, indicating lower amplitude, and a lower precision of their onsets of activity under all three lighting conditions. These observations are reminiscent of the circadian disruptions observed in schizophrenia patients. This prompted us to assess schizophrenia-relevant behavioral abnormalities in Sdy mice following alteration of the circadian rhythms by presentation of constant light. Spontaneous locomotor activity, prepulse inhibition (PPI) of acoustic startle and anxiety-like behavior were assessed under baseline LD conditions, then in LL, and then again in LD. Under LL, the Sdy mice showed significantly increased spontaneous locomotion as well as deficits in PPI compared to WT mice. Strikingly, these behavioral deficits persisted even after the mice were returned in LD conditions. While LL led to an increase in anxiety-like behavior in WT animals that was fully reversed after 3 weeks in LD, this effect was not observed in the Sdy mutants. Overall, these results suggest that Dtnbp1 deficiency may lead to increased vulnerability to schizophrenia under environmental conditions where circadian rhythms are altered. (C) 2015 Elsevier B.V. All rights reserved.

Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.

Objective: Internal clocks time behavior and physiology, including the gut microbiome in a circadian (~24 h) manner. Mismatch between internal and external time, e.g. during shift work, disrupts circadian system coordination promoting the development of obesity and type 2 diabetes (T2D). Conversely, body weight changes induce microbiota dysbiosis. The relationship between circadian disruption and microbiota dysbiosis in metabolic diseases, however, remains largely unknown. Methods: Core and accessory clock gene expression in different gastrointestinal (GI) tissues were determined by qPCR in two different models of circadian disruption - mice with Bmal1 deficiency in the circadian pacemaker, the suprachiasmatic nucleus (Bmal1SCNfl/-), and wild-type mice exposed to simulated shift work (SSW). Body composition and energy balance were evaluated by nuclear magnetic resonance (NMR), bomb calorimetry, food intake and running-wheel activity. Intestinal permeability was measured in an Ussing chamber. Microbiota composition and functionality were evaluated by 16S rRNA gene amplicon sequencing, PICRUST2.0 analysis and targeted metabolomics. Finally, microbiota transfer was conducted to evaluate the functional impact of SSW-associated microbiota on the hosts physiology. Results: Both chronodisruption models show desynchronization within and between peripheral clocks in GI tissues and reduced microbial rhythmicity, in particular in taxa involved in short-chain fatty acid (SCFA) fermentation and lipid metabolism. In Bmal1SCNfl/- mice, loss of rhythmicity in microbial functioning associates with previously shown increased body weight, dysfunctional glucose homeostasis and adiposity. Similarly, we observe an increase in body weight in SSW mice. Germ-free colonization experiments with SSW-associated microbiota mechanistically link body weight gain to microbial changes. Moreover, alterations in expression of peripheral clock genes as well as clock-controlled genes (CCGs) relevant for metabolic functioning of the host were observed in recipients, indicating a bidirectional relationship between microbiota rhythmicity and peripheral clock regulation. Conclusions: Collectively, our data suggest that loss of rhythmicity in bacteria taxa and their products, which likely originates in desynchronization of intestinal clocks, promotes metabolic abnormalities during shift work. Competing Interest Statement The authors have declared no competing interest.

Initiation of drug use during adolescence is a strong predictor of both the incidence and severity of addiction throughout the lifetime. Intriguingly, adolescence is a period of dynamic refinement in the organization of neuronal connectivity, in particular medial prefrontal cortex (mPFC) dopamine circuitry. The guidance cue receptor, DCC (deleted in colorectal cancer), is highly expressed by dopamine neurons and orchestrates their innervation to the mPFC during adolescence. Furthermore, we have shown that amphetamine in adolescence regulates DCC expression in dopamine neurons. Drugs in adolescence may therefore induce their enduring behavioral effects via DCC-mediated disruption in mPFC dopamine development. In this study, we investigated the impact of repeated exposure to amphetamine during adolescence on both the development of mPFC dopamine connectivity and on salience attribution to drug context in adulthood. We compare these effects to those induced by adult exposure to an identical amphetamine regimen. Finally, we determine whether DCC signaling within dopamine neurons is necessary for these events. Exposure to amphetamine in adolescence, but not in adulthood, leads to an increase in the span of dopamine innervation to the mPFC, but a reduction of presynaptic sites present on these axons. Amphetamine treatment in adolescence, but not in adulthood, also produces an increase in salience attribution to a previously drug-paired context in adulthood. Remarkably, DCC signaling within dopamine neurons is required for both of these effects. Drugs of abuse in adolescence may therefore induce their detrimental behavioral consequences by disrupting mesocortical dopamine development through alterations in the DCC signaling cascade.

Lifestyle, obesity, and the gut microbiome are important risk factors for metabolic disorders. We demonstrate in 1,976 subjects of a German population cohort (KORA) that specific microbiota members show 24-h oscillations in their relative abundance and identified 13 taxa with disrupted rhythmicity in type 2 diabetes (T2D). Cross-validated prediction models based on this signature similarly classified T2D. In an independent cohort (FoCus), disruption of microbial oscillation and the model for T2D classification was confirmed in 1,363 subjects. This arrhythmic risk signature was able to predict T2D in 699 KORA subjects 5 years after initial sampling, being most effective in combination with BMI. Shotgun metagenomic analysis functionally linked 26 metabolic pathways to the diurnal oscillation of gut bacteria. Thus, a cohort-specific risk pattern of arrhythmic taxa enables classification and prediction of T2D, suggesting a functional link between circadian rhythms and the microbiome in metabolic diseases. [Display omitted] •Human gut microbiome exhibits diurnal rhythmicity across populations and individuals•Obese and T2D individuals show disrupted circadian rhythms in the gut microbiome•Arrhytmic bacterial signatures contribute to risk classification and prediction of T2D•These risk signatures show regional differences in applicability across three cohorts Reitmeier et al. show that specific gut microbes exhibit rhythmic oscillations in relative abundance and identified taxa with disrupted rhythmicity in individuals with type 2 diabetes (T2D). This arrhythmic signature contributed to the classification and prediction of T2D, suggesting functional links between circadian rhythmicity and the microbiome in metabolic diseases.

The immune system is deeply interconnected with the endogenous 24-h oscillators of the circadian system. Indeed, the connection between these two physiological systems occurs at multiple levels and in both directions. On one hand, various aspects of the immune system show daily rhythms, which appear to be essential for healthy immune maintenance and proper immune response. On the other hand, immune responses cause changes in circadian rhythms, disrupting their delicate balance and manifesting in disease. Indeed, immune challenges cause various time-, gene-, and tissue-specific effects on circadian-regulated factors. This article reviews the possible mediators of the cross talk between the circadian clock and the immune system, in particular the inflammatory pathways. The rhythmic expression of cytokines and their receptors, as well as other rhythmically regulated humoral factors such as glucocorticoids, melatonin, leptin, or prostaglandins, could gate the effects of the immune response on the circadian system. In addition, systemic cues such as body temperature and neuronal connections between the brain and peripheral tissues may underlie the immune-circadian communication.

Magnesium (Mg 2+ ) plays pleiotropic roles in cellular biology, and it is essentially required for all living organisms. Although previous studies demonstrated intracellular Mg 2+ levels were regulated by the complex of phosphatase of regenerating liver 2 (PRL2) and Mg 2+ transporter of cyclin M (CNNMs), physiological functions of PRL2 in whole animals remain unclear. Interestingly, Mg 2+ was recently identified as a regulator of circadian rhythm–dependent metabolism; however, no mechanism was found to explain the clock-dependent Mg 2+ oscillation. Herein, we report PRL2 as a missing link between sex and metabolism, as well as clock genes and daily cycles of Mg 2+ fluxes. Our results unveil that PRL2-null animals displayed sex-dependent alterations in body composition, and expression of PRLs and CNNMs were sex- and circadian time–dependently regulated in brown adipose tissues. Consistently, PRL2-KO mice showed sex-dependent alterations in thermogenesis and in circadian energy metabolism. These physiological changes were associated with an increased rate of uncoupled respiration with lower intracellular Mg 2+ in PRL2-KO cells. Moreover, PRL2 deficiency causes inhibition of the ATP citrate lyase axis, which is involved in fatty acid synthesis. Overall, our findings support that sex- and circadian-dependent PRL2 expression alter intracellular Mg 2+ levels, which accordingly controls energy metabolism status. PRL2 regulates overall cellular energy metabolism by controlling Mg2 2+ fluxes in gender- and circadian rhythm-dependent manner.

Objective Internal clocks time behavior and physiology, including the gut microbiome, in a circadian (∼24 h) manner. Mismatch between internal and external time, e.g. during shift work, disrupts circadian system coordination promoting the development of obesity and type 2 diabetes (T2D). Conversely, body weight changes induce microbiota dysbiosis. The relationship between circadian disruption and microbiota dysbiosis in metabolic diseases, however, remains largely unknown. Methods Core and accessory clock gene expression in different gastrointestinal (GI) tissues were determined by qPCR in two different models of circadian disruption - mice with Bmal1 deficiency in the circadian pacemaker, the suprachiasmatic nucleus (Bmal1SCNfl/-), and wild-type mice exposed to simulated shift work (SSW). Body composition and energy balance were evaluated by nuclear magnetic resonance (NMR), bomb calorimetry, food intake and running-wheel activity. Intestinal permeability was measured in an Ussing chamber. Microbiota composition and functionality were evaluated by 16S rRNA gene amplicon sequencing, PICRUST2.0 analysis and targeted metabolomics. Finally, microbiota transfer was conducted to evaluate the functional impact of SSW-associated microbiota on the host's physiology. Results Both chronodisruption models show desynchronization within and between peripheral clocks in GI tissues and reduced microbial rhythmicity, in particular in taxa involved in short-chain fatty acid (SCFA) fermentation and lipid metabolism. In Bmal1SCNfl/- mice, loss of rhythmicity in microbial functioning associates with previously shown increased body weight, dysfunctional glucose homeostasis and adiposity. Similarly, we observe an increase in body weight in SSW mice. Germ-free colonization experiments with SSW-associated microbiota mechanistically link body weight gain to microbial changes. Moreover, alterations in expression of peripheral clock genes as well as clock-controlled genes (CCGs) relevant for metabolic functioning of the host were observed in recipients, indicating a bidirectional relationship between microbiota rhythmicity and peripheral clock regulation. Conclusions Collectively, our data suggest that loss of rhythmicity in bacteria taxa and their products, which likely originates in desynchronization of intestinal clocks, promotes metabolic abnormalities during shift work.

Abstract Objective Impaired clock genes expression has been observed in biopsy samples from patients with inflammatory bowel disease (IBD). Disruption of circadian rhythms, which occurs in shift workers, has been linked to an increased risk of gastrointestinal diseases, including IBD. The intestinal clock balances gastrointestinal homeostasis by regulating the microbiome. Here we characterize intestinal immune functions in mice lacking the intestinal clock and IBD-relevant mouse model under different feeding conditions to describe the functional impact of the intestinal clock in the development of gastrointestinal inflammation. Design Tissues and fecal samples from intestinal clock-deficient mice (Bmal1IEC-/-) and mouse models for colitis (IL-10-/-, Bmal1IEC-/-xIL-10-/-, dextran sulfate sodium (DSS) administration) under ad libitum and restricted feeding (RF) conditions were used to determine the causal role of the intestinal clock for colitis. Results In IL-10-/- mice, inflammation correlated with disrupted colon clock genes expression. Genetic loss of intestinal clock functions promoted DSS and IBD inflammatory phenotypes and dramatically reduces survival, and colonization with disease-associated microbiota in germ- free Bmal1IEC-/- hosts increased their inflammatory responses, demonstrating the causal role of colonic clock disruption and the severity of IBD. RF in IL-10-/- mice restored the colon clock and related immune functions, improved the inflammatory responses and rescued the histopathological phenotype. In contrast, RF failed to improve IBD symptoms in Bmal1IEC-/- xIL-10-/- demonstrating the significance of the colonic clock to gate the effect of RF. Conclusion We provide evidence that inflammation-associated intestinal clock dysfunction triggers host immune imbalance and promotes the development and progression of IBD-like colitis. Enhancing intestinal clock function by RF modulates the pathogenesis of IBD and thus could become a novel strategy to ameliorate the symptoms in IBD patients. Competing Interest Statement The authors have declared no competing interest.

The intracellular parasite Leishmania uses neutrophils and macrophages as host cells upon infection. These immune cells harbour their own intrinsic circadian clocks, known to influence many aspects of their functions. Therefore, we tested whether the host circadian clocks regulate the magnitude of Leishmania major infection in mice. The extent of parasitic infection varied over 24 h in bone marrow-derived macrophages in vitro and in two different in vivo models, footpad and peritoneal cavity infection. In vivo this was paralleled by time of day-dependent neutrophil and macrophage infiltration to the infection site and rhythmic chemokine expression. Thus, rhythmic parasitic infection observed in vivo was likely initiated by the circadian expression of chemoattractants and the subsequent rhythmic infiltration of neutrophils and macrophages. Importantly, all rhythms were abolished in clock-deficient macrophages and when mice lacking the circadian clock in immune cells were infected. Therefore we demonstrated a critical role for the circadian clocks in immune cells in modulating the magnitude of Leishmania infection. To our knowledge this is the first report showing that the circadian clock controls infection by protozoan parasites in mammals. Understanding the timed regulation of host-parasite interactions will allow developing better prophylactic and therapeutic strategies to fight off vector-borne diseases.

Targeted sequencing of 16S rRNA genes enables the analysis of microbiomes. Here, we describe a protocol for the collection, storage, and preparation of fecal samples. We describe how we cluster similar sequences and assign bacterial taxonomies. Using diversity analysis and machine learning, we can extract disease-associated features. We also describe a circadian analysis to identify the presence or absence of rhythms in taxonomies. Differences in rhythmicity between cohorts can contribute to determining disease-associated bacterial signatures. For complete details on the use and execution of this protocol, please refer to Reitmeier et al. (2020).

Endogenous circadian (∼24 h) clocks regulate key physiological and cognitive processes via rhythmic expression of clock genes. The main circadian pacemaker is the hypothalamic suprachiasmatic nucleus (SCN). Mood disorders, including bipolar disorder (BD), are commonly associated with disturbed circadian rhythms. Dopamine (DA) contributes to mania in BD and has direct impact on clock gene expression. Therefore, we hypothesized that high levels of DA during episodes of mania contribute to disturbed circadian rhythms in BD. The mood stabilizer valproic acid (VPA) also affects circadian rhythms. Thus, we further hypothesized that VPA normalizes circadian disturbances caused by elevated levels of DA. To test these hypotheses, we examined locomotor rhythms and circadian gene cycling in mice with reduced expression of the dopamine transporter (DAT-KD mice), which results in elevated DA levels and mania-like behavior. We found that elevated DA signaling lengthened the circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants. In contrast, we found that VPA shortened circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants, hippocampal cell lines, and human fibroblasts from BD patients. Thus, DA and VPA have opposing effects on circadian period. To test whether the impact of VPA on circadian rhythms contributes to its behavioral effects, we fed VPA to DAT-deficient Drosophila with and without functioning circadian clocks. Consistent with our hypothesis, we found that VPA had potent activity-suppressing effects in hyperactive DAT-deficient flies with intact circadian clocks. However, these effects were attenuated in DAT-deficient flies in which circadian clocks were disrupted, suggesting that VPA functions partly through the circadian clock to suppress activity. Here, we provide in vivo and in vitro evidence across species that elevated DA signaling lengthens the circadian period, an effect remediated by VPA treatment. Hence, VPA may exert beneficial effects on mood by normalizing lengthened circadian rhythm period in subjects with elevated DA resulting from reduced DAT. [Display omitted] •Dopamine lengthens the period of behavioral and SCN circadian rhythms in mice.•The mood stabilizer valproic acid (VPA) shortens the period of circadian rhythms.•VPA normalizes circadian rhythms in fibroblasts from bipolar patients.•In Drosophila lacking circadian clocks, effects of VPA on hyperactivity are attenuated.

Diurnal (i.e., 24-hour) oscillations of the gut microbiome have been described in various species including mice and humans. However, the driving force behind these rhythms remains less clear. In this study, we differentiate between endogenous and exogenous time cues driving microbial rhythms. Our results demonstrate that fecal microbial oscillations are maintained in mice kept in the absence of light, supporting a role of the host’s circadian system rather than representing a diurnal response to environmental changes. Intestinal epithelial cell-specific ablation of the core clock gene Bmal1 disrupts rhythmicity of microbiota. Targeted metabolomics functionally link intestinal clock-controlled bacteria to microbial-derived products, in particular branched-chain fatty acids and secondary bile acids. Microbiota transfer from intestinal clock-deficient mice into germ-free mice altered intestinal gene expression, enhanced lymphoid organ weights and suppressed immune cell recruitment. These results highlight the importance of functional intestinal clocks for microbiota composition and function, which is required to balance the host’s gastrointestinal homeostasis.