Keith C. Summa

Disruption of the Circadian Clock in Mice Increases Intestinal Permeability and Promotes Alcohol-Induced Hepatic Pathology and Inflammation

The circadian clock orchestrates temporal patterns of physiology and behavior relative to the environmental light:dark cycle by generating and organizing transcriptional and biochemical rhythms in cells and tissues throughout the body. Circadian clock genes have been shown to regulate the physiology and function of the gastrointestinal tract. Disruption of the intestinal epithelial barrier enables the translocation of proinflammatory bacterial products, such as endotoxin, across the intestinal wall and into systemic circulation; a process that has been linked to pathologic inflammatory states associated with metabolic, hepatic, cardiovascular and neurodegenerative diseases – many of which are commonly reported in shift workers. Here we report, for the first time, that circadian disorganization, using independent genetic and environmental strategies, increases permeability of the intestinal epithelial barrier (i.e., gut leakiness) in mice. Utilizing chronic alcohol consumption as a well-established model of induced intestinal hyperpermeability, we also found that both genetic and environmental circadian disruption promote alcohol-induced gut leakiness, endotoxemia and steatohepatitis, possibly through a mechanism involving the tight junction protein occludin. Circadian organization thus appears critical for the maintenance of intestinal barrier integrity, especially in the context of injurious agents, such as alcohol. Circadian disruption may therefore represent a previously unrecognized risk factor underlying the susceptibility to or development of alcoholic liver disease, as well as other conditions associated with intestinal hyperpermeability and an endotoxin-triggered inflammatory state.

Environmental Perturbation of the Circadian Clock Disrupts Pregnancy in the Mouse

Background The circadian clock has been linked to reproduction at many levels in mammals. Epidemiological studies of female shift workers have reported increased rates of reproductive abnormalities and adverse pregnancy outcomes, although whether the cause is circadian disruption or another factor associated with shift work is unknown. Here we test whether environmental disruption of circadian rhythms, using repeated shifts of the light:dark (LD) cycle, adversely affects reproductive success in mice. Methodology/Principal Findings Young adult female C57BL/6J (B6) mice were paired with B6 males until copulation was verified by visual identification of vaginal plug formation. Females were then randomly assigned to one of three groups: control, phase-delay or phase-advance. Controls remained on a constant 12-hr light:12-hr dark cycle, whereas phase-delayed and phase-advanced mice were subjected to 6-hr delays or advances in the LD cycle every 5–6 days, respectively. The number of copulations resulting in term pregnancies was determined. Control females had a full-term pregnancy success rate of 90% (11/12), which fell to 50% (9/18; p<0.1) in the phase-delay group and 22% (4/18; p<0.01) in the phase-advance group. Conclusions/Significance Repeated shifting of the LD cycle, which disrupts endogenous circadian timekeeping, dramatically reduces pregnancy success in mice. Advances of the LD cycle have a greater negative impact on pregnancy outcomes and, in non-pregnant female mice, require longer for circadian re-entrainment, suggesting that the magnitude or duration of circadian misalignment may be related to the severity of the adverse impact on pregnancy. These results explicitly link disruptions of circadian entrainment to adverse pregnancy outcomes in mammals, which may have important implications for the reproductive health of female shift workers, women with circadian rhythm sleep disorders and/or women with disturbed circadian rhythms for other reasons.

Chronobiology and Obesity: Interactions between Circadian Rhythms and Energy Regulation

Recent advances in the understanding of the molecular, genetic, neural, and physiologic basis for the generation and organization of circadian clocks in mammals have revealed profound bidirectional interactions between the circadian clock system and pathways critical for the regulation of metabolism and energy balance. The discovery that mice harboring a mutation in the core circadian gene circadian locomotor output cycles kaput (Clock) develop obesity and evidence of the metabolic syndrome represented a seminal moment for the field, clearly establishing a link between circadian rhythms, energy balance, and metabolism at the genetic level. Subsequent studies have characterized in great detail the depth and magnitude of the circadian clocks crucial role in regulating body weight and other metabolic processes. Dietary nutrients have been shown to influence circadian rhythms at both molecular and behavioral levels; and many nuclear hormone receptors, which bind nutrients as well as other circulating ligands, have been observed to exhibit robust circadian rhythms of expression in peripheral metabolic tissues. Furthermore, the daily timing of food intake has itself been shown to affect body weight regulation in mammals, likely through, at least in part, regulation of the temporal expression patterns of metabolic genes. Taken together, these and other related findings have transformed our understanding of the important role of time, on a 24-h scale, in the complex physiologic processes of energy balance and coordinated regulation of metabolism. This research has implications for human metabolic disease and may provide unique and novel insights into the development of new therapeutic strategies to control and combat the epidemic of obesity.

The Circadian Clock Mutation Promotes Intestinal Dysbiosis.

BACKGROUND Circadian rhythm disruption is a prevalent feature of modern day society that is associated with an increase in pro-inflammatory diseases, and there is a clear need for a better understanding of the mechanism(s) underlying this phenomenon. We have previously demonstrated that both environmental and genetic circadian rhythm disruption causes intestinal hyperpermeability and exacerbates alcohol-induced intestinal hyperpermeability and liver pathology. The intestinal microbiota can influence intestinal barrier integrity and impact immune system function; thus, in this study, we sought to determine whether genetic alteration of the core circadian clock gene, Clock, altered the intestinal microbiota community. METHODS Male Clock(Δ19) -mutant mice (mice homozygous for a dominant-negative-mutant allele) or littermate wild-type mice were fed 1 of 3 experimental diets: (i) a standard chow diet, (ii) an alcohol-containing diet, or (iii) an alcohol-control diet in which the alcohol calories were replaced with dextrose. Stool microbiota was assessed with 16S ribosomal RNA gene amplicon sequencing. RESULTS The fecal microbial community of Clock-mutant mice had lower taxonomic diversity, relative to wild-type mice, and the Clock(Δ19) mutation was associated with intestinal dysbiosis when mice were fed either the alcohol-containing or the control diet. We found that alcohol consumption significantly altered the intestinal microbiota in both wild-type and Clock-mutant mice. CONCLUSIONS Our data support a model by which circadian rhythm disruption by the Clock(Δ19) mutation perturbs normal intestinal microbial communities, and this trend was exacerbated in the context of a secondary dietary intestinal stressor.

Identification of causal genes, networks, and transcriptional regulators of REM sleep and wake

STUDY OBJECTIVE Sleep-wake traits are well-known to be under substantial genetic control, but the specific genes and gene networks underlying primary sleep-wake traits have largely eluded identification using conventional approaches, especially in mammals. Thus, the aim of this study was to use systems genetics and statistical approaches to uncover the genetic networks underlying 2 primary sleep traits in the mouse: 24-h duration of REM sleep and wake. DESIGN Genome-wide RNA expression data from 3 tissues (anterior cortex, hypothalamus, thalamus/midbrain) were used in conjunction with high-density genotyping to identify candidate causal genes and networks mediating the effects of 2 QTL regulating the 24-h duration of REM sleep and one regulating the 24-h duration of wake. SETTING Basic sleep research laboratory. PATIENTS OR PARTICIPANTS Male [C57BL/6J × (BALB/cByJ × C57BL/6J*) F1] N(2) mice (n = 283). INTERVENTIONS None. MEASUREMENTS AND RESULTS The genetic variation of a mouse N2 mapping cross was leveraged against sleep-state phenotypic variation as well as quantitative gene expression measurement in key brain regions using integrative genomics approaches to uncover multiple causal sleep-state regulatory genes, including several surprising novel candidates, which interact as components of networks that modulate REM sleep and wake. In particular, it was discovered that a core network module, consisting of 20 genes, involved in the regulation of REM sleep duration is conserved across the cortex, hypothalamus, and thalamus. A novel application of a formal causal inference test was also used to identify those genes directly regulating sleep via control of expression. CONCLUSION Systems genetics approaches reveal novel candidate genes, complex networks and specific transcriptional regulators of REM sleep and wake duration in mammals.

A Systems Approach Identifies Networks and Genes Linking Sleep and Stress: Implications for Neuropsychiatric Disorders

Sleep dysfunction and stress susceptibility are comorbid complex traits that often precede and predispose patients to a variety of neuropsychiatric diseases. Here, we demonstrate multilevel organizations of genetic landscape, candidate genes, and molecular networks associated with 328 stress and sleep traits in a chronically stressed population of 338 (C57BL/6J × A/J) F2 mice. We constructed striatal gene co-expression networks, revealing functionally and cell-type-specific gene co-regulations important for stress and sleep. Using a composite ranking system, we identified network modules most relevant for 15 independent phenotypic categories, highlighting a mitochondria/synaptic module that links sleep and stress. The key network regulators of this module are overrepresented with genes implicated in neuropsychiatric diseases. Our work suggests that the interplay among sleep, stress, and neuropathology emerges from genetic influences on gene expression and their collective organization through complex molecular networks, providing a framework for interrogating the mechanisms underlying sleep, stress susceptibility, and related neuropsychiatric disorders.

Environmental Disruption of Circadian Rhythm Predisposes Mice to Osteoarthritis-Like Changes in Knee Joint

Circadian rhythm dysfunction is linked to many diseases, yet pathophysiological roles in articular cartilage homeostasis and degenerative joint disease including osteoarthritis (OA) remains to be investigated in vivo. Here, we tested whether environmental or genetic disruption of circadian homeostasis predisposes to OA‐like pathological changes. Male mice were examined for circadian locomotor activity upon changes in the light:dark (LD) cycle or genetic disruption of circadian rhythms. Wild‐type (WT) mice were maintained on a constant 12 h:12 h LD cycle (12:12 LD) or exposed to weekly 12 h phase shifts. Alternatively, male circadian mutant mice (ClockΔ19 or Csnk1etau mutants) were compared with age‐matched WT littermates that were maintained on a constant 12:12 LD cycle. Disruption of circadian rhythms promoted osteoarthritic changes by suppressing proteoglycan accumulation, upregulating matrix‐degrading enzymes and downregulating anabolic mediators in the mouse knee joint. Mechanistically, these effects involved activation of the PKCδ‐ERK‐RUNX2/NFκB and β‐catenin signaling pathways, stimulation of MMP‐13 and ADAMTS‐5, as well as suppression of the anabolic mediators SOX9 and TIMP‐3 in articular chondrocytes of phase‐shifted mice. Genetic disruption of circadian homeostasis does not predispose to OA‐like pathological changes in joints. Our results, for the first time, provide compelling in vivo evidence that environmental disruption of circadian rhythms is a risk factor for the development of OA‐like pathological changes in the mouse knee joint. J. Cell. Physiol. 230: 2174–2183, 2015.

Osteoarthritis-like pathologic changes in the knee joint induced by environmental disruption of circadian rhythms is potentiated by a high-fat diet

A variety of environmental factors contribute to progressive development of osteoarthritis (OA). Environmental factors that upset circadian rhythms have been linked to various diseases. Our recent work establishes chronic environmental circadian disruption - analogous to rotating shiftwork-associated disruption of circadian rhythms in humans - as a novel risk factor for the development of OA. Evidence suggests shift workers are prone to obesity and also show altered eating habits (i.e., increased preference for high-fat containing food). In the present study, we investigated the impact of chronic circadian rhythm disruption in combination with a high-fat diet (HFD) on progression of OA in a mouse model. Our study demonstrates that when mice with chronically circadian rhythms were fed a HFD, there was a significant proteoglycan (PG) loss and fibrillation in knee joint as well as increased activation of the expression of the catabolic mediators involved in cartilage homeostasis. Our results, for the first time, provide the evidence that environmental disruption of circadian rhythms plus HFD potentiate OA-like pathological changes in the mouse joints. Thus, our findings may open new perspectives on the interactions of chronic circadian rhythms disruption with diet in the development of OA and may have potential clinical implications.

Chronic Alcohol Exposure and the Circadian Clock Mutation Exert Tissue-Specific Effects on Gene Expression in Mouse Hippocampus, Liver, and Proximal Colon.

BACKGROUND Chronic alcohol exposure exerts numerous adverse effects, although the specific mechanisms underlying these negative effects on different tissues are not completely understood. Alcohol also affects core properties of the circadian clock system, and it has been shown that disruption of circadian rhythms confers vulnerability to alcohol-induced pathology of the gastrointestinal barrier and liver. Despite these findings, little is known of the molecular interactions between alcohol and the circadian clock system, especially regarding implications for tissue-specific susceptibility to alcohol pathologies. The aim of this study was to identify changes in expression of genes relevant to alcohol pathologies and circadian clock function in different tissues in response to chronic alcohol intake. METHODS Wild-type and circadian Clock(Δ19) mutant mice were subjected to a 10-week chronic alcohol protocol, after which hippocampal, liver, and proximal colon tissues were harvested for gene expression analysis using a custom-designed multiplex magnetic bead hybridization assay that provided quantitative assessment of 80 mRNA targets of interest, including 5 housekeeping genes and a predetermined set of 75 genes relevant for alcohol pathology and circadian clock function. RESULTS Significant alterations in expression levels attributable to genotype, alcohol, and/or a genotype by alcohol interaction were observed in all 3 tissues, with distinct patterns of expression changes observed in each. Of particular interest was the finding that a high proportion of genes involved in inflammation and metabolism on the array was significantly affected by alcohol and the Clock(Δ19) mutation in the hippocampus, suggesting a suite of molecular changes that may contribute to pathological change. CONCLUSIONS These results reveal the tissue-specific nature of gene expression responses to chronic alcohol exposure and the Clock(Δ19) mutation and identify specific expression profiles that may contribute to tissue-specific vulnerability to alcohol-induced injury in the brain, colon, and liver.

Induction of Osteoarthritis‐like Pathologic Changes by Chronic Alcohol Consumption in an Experimental Mouse Model

Osteoarthritis (OA) is characterized by slow and progressive deterioration of articular cartilage. OA likely arises from a combination of systemic factors (genetics, age, environment) and local factors (abnormal joint loading, overuse, or trauma) working in concert to create a condition with definable morphologic and clinical characteristics. Several risk factors for OA have been identified previously, including genetic predisposition, obesity, diabetes, hypertension, hyperuricemia, history of trauma, and aging (1). However, due in large part to an inability to control for confounding factors, the underlying pathogenesis and causative features responsible for initiation and progression of the disease remain largely unknown. There is a growing body of evidence indicating that progression of OA is correlated with up-regulation of inflammatory processes (2). Oxidative stress elicited by reactive oxygen species (ROS) further disturbs cartilage homeostasis and promotes catabolism via induction of cell death, breakdown of matrix proteoglycans (PGs), up-regulation of latent matrixdegrading enzyme production, inhibition of extracellular matrix synthesis, and oxidation of intracellular and extracellular molecules (3). Thus, environmental factors that promote oxidative stress and inflammatory states could potentially act as a risk factor for OA. Alcohol consumption could be one potential risk factor, because 1) chronic alcohol consumption, highly common in Western and industrial societies, generates ROS, leading to systemic and tissue oxidative stress in humans and rodents (4), and 2) alcohol is capable of inducing proinflammatory states in multiple organs, e.g., the liver, heart, central nervous system, and pancreas (4,5). There have been several previous studies in which investigators attempted to elucidate a relationship between alcohol consumption and inflammatory arthritis such as rheumatoid arthritis, with conflicting results (6,7). However, despite recent evidence demonstrating the importance of oxidative stress and proinflammatory states in the development and progression of degenerative joint disease (8), the impact of alcohol consumption on OA has not yet been studied. In the present study, we obtained evidence suggesting that chronic alcohol exposure may increase susceptibility to the development and/or progression of OA. Using a validated in vivo model of chronic alcohol treatment, we found that chronic alcohol consumption increases PG loss in both the knee and the shoulder joints of mice and stimulates multiple inflammatory, catabolic, and antianabolic mediators involved in cartilage. In our experimental protocol, young adult (ages 7–9 weeks) male C57BL/6 mice were provided ad libitum access to an alcohol diet (i.e., the Nanji diet), containing 4.5% (volume/volume) ethanol (29% ethanol-derived calories) or an isocaloric alcohol-free control diet for 8 weeks (n 5 14–16 per group). All animal protocols and practices were reviewed and approved in advance by the Institutional Animal Care and Use Committees at Rush University and Northwestern University. Following 8 weeks on the alcohol-containing diet or the control diet, mice were killed and joint sections were collected, fixed, paraffin embedded, and stained with Safranin O to assess cartilage structure and matrix PG content. The alcohol diet used in this study is a slight modification of the wellvalidated Lieber-DeCarli diet (9) in which the fat source comes from fish oil, and consumption of this diet by BL6 mice has been shown to produce blood alcohol levels that are in the low-to-moderate range (10). Our group and others have successfully used this alcohol diet to induce a variety of alcohol-related pathologies, including colon cancer, intestinal hyperpermeability, endotoxemia, and liver disease, in rodents (10–12). Serum alcohol levels at the time of death in the alcohol-fed mice were ;3 mg/dl, and these mice did not exhibit any overt behavioral abnormalities during the experimental protocol. Histologic examination of knee joints of control diet–fed mice demonstrated normal architecture of articular cartilage, with intense Safranin O staining. In contrast, knee joints of alcohol-fed mice displayed OA-like characteristics, with increased PG loss as indicated by reduced Safranin O staining and mild fibrillation (Figure 1A). These results were quantified using the Osteoarthritis Research Society International (OARSI) semiquantitative scoring system (13). Scores were significantly higher in alcohol-fed mice than in control mice (mean 6 SD 1.3 6 0.67 versus 0.3 6 0.27; P , 0.05) (Figure 1A), indicating more severe arthritic changes in the knee joints of alcohol-fed mice. Similar results were observed in shoulder joints (Figure 1B), with decreased PG content as shown by Safranin O staining, and an irregular cartilage surface, in the shoulders of alcohol-fed mice. As in the knee, pathologic changes in the shoulder, quantified using the OARSI scoring system, were significantly greater in alcohol-fed mice compared to control mice (0.75 6 0.28 versus 0.12 6 0.25; P , 0.05) (Figure 1B). Interestingly, the intervertebral discs of alcohol-fed mice exhibited no pathologic changes compared to control mice (Figure 1C). Our results demonstrate a pathologic effect of alcohol on specific catabolic and antianabolic mediators in the knee joints, which may increase susceptibility to OA development. We found that levels of the phosphorylated forms of protein kinase Cd (PKCd), NF-kB, and ERK-1/2 were significantly increased in the knee joints of alcohol-fed mice compared to control mice (P , 0.05) (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary. wiley.com/doi/10.1002/art.39090/abstract), suggesting that chronic alcohol consumption stimulates these catabolic signaling pathways, which may result in subsequent production of cartilagedestructive enzymes. Levels of the hypertrophic marker RUNX-2, as well as the key cartilage-destructive enzymes matrix metalloprotease 13 and ADAMTS-5, were also significantly increased in the knee joints of alcohol-fed mice (P , 0.05) (Supplementary Figure 1).