|Home | About | Journals | Submit | Contact Us | Français|
Physiological function, disease expression and drug effects vary by time-of-day. Clock disruption in mice results in cardio-metabolic, immunological and neurological dysfunction; circadian misalignment using forced desynchrony increases cardiovascular risk factors in humans. Here we integrated data from remote sensors, physiological and multi-omics analyses to assess the feasibility of detecting time dependent signals - the chronobiome – despite the “noise” attributable to the behavioral differences of free-living human volunteers. The majority (62%) of sensor readouts showed time-specific variability including the expected variation in blood pressure, heart rate, and cortisol. While variance in the multi-omics is dominated by inter-individual differences, temporal patterns are evident in the metabolome (5.4% in plasma, 5.6% in saliva) and in several genera of the oral microbiome. This demonstrates, despite a small sample size and limited sampling, the feasibility of characterizing at scale the human chronobiome “in the wild”. Such reference data at scale are a prerequisite to detect and mechanistically interpret discordant data derived from patients with temporal patterns of disease expression, to develop time-specific therapeutic strategies and to refine existing treatments.
The molecular circadian clock coordinates our body rhythms entrainable by environmental cues, such as light, to the 24hour solar cycle. The master clock, located in the suprachiasmatic nucleus communicates with and is influenced by molecular clocks in peripheral tissues1. The system is highly conserved and tightly regulated by feedback and feed forward transcriptional loops, the elements of which exhibit a high degree of genetic redundancy2. A robust temporal organization is achieved by the functional overlap between many of the molecular circadian clock genes; however, nuanced differences, such as differential responsiveness to photic stimuli3, might impact chronotypes. Studies in model systems have implicated the clock as an integrative network across tissues of particular relevance to metabolism, immune function and vascular homeostasis4.
In humans, many aspects of physiology, including body temperature, blood glucose, catecholamines, insulin and many hormones, including melatonin, cortisol, TSH, ghrelin, leptin and prolactin undergo diurnal variation5, meaning that daily patterns can be discerned. These rhythms lose amplitude and synchrony with age in both humans and mice, and deletion of core clock genes in mice has been associated with accelerated aging6,7. However, more recent studies have suggested that disruption “off target” effects of these transcription factors may account for some of these phenomena8–10. In humans, the incidence or severity of many diseases, such as asthma, myocardial infarction, stroke and depression exhibit diurnal variation4. Similarly, the targets of many drugs oscillate, as do enzymes and transporters relevant to drug metabolism11. Despite this and the long recognized time dependent variation in disposition of many commonly used drugs, there has been little exploitation of chronotherapy in clinical practice12.
Indeed, our understanding of the role of the molecular clock in humans is limited13. The use of forced desynchrony protocols has permitted segregation of clock driven circadian rhythms from diurnal variability secondary to environmental exposures. Endogenous and environmentally driven rhythms often coincide, but may be out of phase, as is the case with blood pressure, where the morning surge associated with increased cardiovascular morbidity does not temporally align with the endogenous peak in blood pressure, revealed by forced desynchrony, that occurs in the evening14. The clinical implications of such divergence are unknown.
Although useful, such studies are performed in highly artificial circumstances in which light and other environmental cues are carefully controlled. In recent years the development of technologies for multiscale “omics” and remote sensors afford new opportunities to explore characterization of the chronobiome of humans free ranging “in the wild”, that is, not sequestered in artificial environments.
Studies in model systems have demonstrated the role of the clock in regulation of the genome15, the epigenome16, the metabolome17, the proteome18 and the microbiome19–21 as well as in the oscillation of temperature22, activity23 and blood pressure24. However, before we can explore how dysfunction in these outputs might relate to expression of human disease, we must establish the ability to discriminate an oscillatory signal from analytical and environmental noise in healthy volunteers and determine the influence on this physiological chronobiome of such variables as gender and age. In this regard, the detectable diurnal variation in body temperature, hormones and blood pressure might serve as “internal standards” for more novel technologies.
Here, we report a pilot study designed to gather preliminary information on the variability in healthy volunteers of the diurnal oscillation of cardiovascular and behavioral phenotypes and of diverse “omics” outputs. Despite the expected intra- and inter-individual variability in behavior, a clear pattern of time dependent oscillation of blood pressure, activity, light exposure, communications and food consumption was detected. Morning- versus evening-dependent differences in both the oral and rectal microbiome abundances were clearly evident, while detection of time-of-day variation in the metabolome, proteome and transcriptome was apparent, but constrained by the number of sampling times and by sample size. We achieved a first level of data integration suggesting multidimensional fingerprints unique to each person.
We enrolled 6 healthy male volunteers (32.3±3.6 years of age, BMI 25.2±3.4kg/m2) after approval by the Institutional Review Board of the University of Pennsylvania (Federalwide Assurance FWA00004028; IRB Registration: IORG0000029) that included an institutional security and privacy information impact assessment and registration (clinicaltrials.gov NCT02249793). Informed consent was obtained from all subjects. This clinical research study was carried out in accordance with relevant guidelines and regulations. The main exclusion criteria consisted of travel across time zones and irregular work hours, e.g. shift work. Volunteers were studied over 4 months to collect data on activity, sleep patterns, light exposure and communication, as well as being deeply phenotyped during two 48hour periods, 2 weeks apart. Biospecimens (plasma, serum, saliva, oral and rectal swabs) were collected from these outpatients in the Center for Human Phenomic Science (CHPS, University of Pennsylvania) at 12 hourly intervals, thus generating a time series of 5 consecutive sample collections during one single 48hour session, i.e. 0hrs=morning, 12hrs=evening, 24hrs=morning, 36hrs=evening, and 48hrs=morning. Plasma and saliva metabolites were analyzed using LC-MS as previously described25, a pre-specified protein panel was run on the SomaLogic platform26, the microbiomic analysis in saliva and from buccal and rectal swabs was conducted as established earlier27,28, and in-house qPCR was used for expression analysis of a small, selected panel of genes.
A triaxial actigraph device (wActiSleep-BT) recorded accelerometer and light sensor data with subsequent wear time analysis and sleep scoring in ActiLife 6.0 software. This achieved quantitative outputs for steps, energy expenditure and metabolic rate as additional outputs in this domain. The HIPAA-compliant Ginger.io platform®, consisting of an android mobile phone application and a web dashboard, was used to monitor cell phone calls and SMS messaging activity in real time. The application gathered communication and mobility data through a background process and transmitted encrypted data to firewall protected linux-based servers with access control lists. Blood pressure monitoring was performed in the ambulatory subjects using clinically validated devices (Spacelab 90207). Intake of food and beverages was collected with the SmartIntake© smartphone application, a validated remote food photography method® 29,30. To facilitate data quality and completeness, the app included Ecological Momentary Assessment (EMA) methodology to remind participants to capture images of the foods and beverages that they consumed. These reminders were text messages that were scheduled for delivery at the personalized meal times of the participants. The responses to EMAs were tracked in near real-time, which allowed us to identify quickly if data collection problems occurred. The app sends participants food/beverage images and accompanying food identifier data (e.g., barcodes, PLU numbers, food descriptions) to a server located at the Pennington Biomedical Research Center where bionutritionists analyze the images to estimate food/beverage intake based on the Food Photography Application© program. This allows the operator to identify a match for each food from the Food and Nutrient Database for Dietary Studies 5.0 and other sources, such as manufacturer’s information and Nutrition Fact Panels, to calculate energy and nutrient intake.
For the bioinformatics analysis, we adopted several packages in R and CircOS for data management, integration and visualization. The web-based version control repository GitHub was used as code development platform (https://github.com/itmat/chronobiome/). In addition to standard descriptive statistics, we applied permutation tests, principal component analysis, principal coordinate analysis, circadian multiresolution analyses, cosinor method, Ingenuity pathway analysis, variance correlation analysis, and a time-versus-subject contribution to variance analysis.
We successfully integrated and analyzed this multidimensional dataset, roughly 2.2 million data points collected from 6 healthy volunteers over the course of 4 months, including two 48-hour sessions of additional deep phenotyping (Fig. 1, Figure S1). This led to several insights relevant to the pursuit of future studies in the field of human chronobiology.
First, we sought to assess the validity of our dataset. We see this accomplished on several levels:
In summary, we were able to detect internally consistent, time dependent patterns in blood pressure, heart rate, cortisol, activity, communication, mobility and light consistent with the physiological and behavioral expectations for this cohort in the natural setting under the conditions of this experiment.
Next, we were interested to assess the comparative contributions of time (the signal) and inter-subject behavioral differences (the noise) to variability in our datasets. There are many sources of variability in these data. By partitioning the total variability, we were able to attribute how much variability was explained by time, the variable of interest in our study (see Supplemental Methods for full details). For the multiomics dataset, the permutation distribution (obtained by permuting the time points in all possible ways) revealed patterns which displayed statistically significant temporal variation between morning (0h, 24h, 48h) and evening (12h, 36h) measurements (permutation p-values: plasma metabolome p=0.009; saliva metabolome p=0.009, saliva microbiome p=0.009). We determined that 5.4% (9/166) of the plasma metabolites, 5.6% (14/250) of the saliva metabolites, 0.5% (6/1141) of the serum proteins, and 3 of the 12 most abundant genera in the oral microbiota underwent time-specific variability (Figs 4, ,55 and and6a).6a). For the metabolites, examples include cortisol in plasma, and ornithine, xanthine and porphobilinogen in saliva. The plasma proteome overall failed to attain significance (p=0.56). Accordingly, variance in protein abundances was driven by inter-subject differences in the majority of cases (99.2%), whereas for some proteins variance was exclusively contributed by inter-subject differences. For the oral microbiota, three genera, Streptococcus, Veillonella, and Actinomyces revealed a predominant time-dependent variance (Figs 5 and and6a).6a). Thus, time-of-day-dependent patterning was detected in the metabolome and the microbiome despite the paucity of sampling times and the behavioral diversity of a small number of free ranging humans. This integrative approach allows us to discern candidate oscillatory variables despite the anticipated inter-individual differences, small sample size and sparse sampling. As expected for cortisol (Fig. 6a), time (33.6%) contributes more variance than inter-subject differences (21.8%), thus demonstrating first proof-of-concept.
In our dataset obtained from remote sensors and wearables, we identified 62% of the variables to show time-specific variability. Here, variables from almost all domains, behavior (activity, mobility), cardiovascular (SDB, DBP, MAP), and environment (light), are represented (Fig. 6a). This includes several food categories - intake of energy, protein, carbohydrates, fat and sodium - where time more than inter-subject differences contributes to variability of intake, despite the unrestricted access to food under the conditions of this study (Fig. 6b bottom insert). By contrast, all readouts for communication depart from this pattern with variance reflecting inter-subject behavior more than time of day. Heart rate has equal time- and inter-subject variance, while, despite the dominant contribution of time to SBP and DBP, variance in pulse pressure mainly reflects inter-subject differences (Fig. 6b).
To parse the relative contributions of time and inter-subject differences to variability in the datasets, we conducted a principal component analysis (Figure S4). As expected, inter-subject differences explain most of the variability observed in the data clustering. Further attribution of variability was not feasible due to our ‘unsupervised’ approach. In an effort to explore which disease categories might be subject to time dependent oscillations, we performed a time-specific pathway analysis of the metabolome and the proteome. Interestingly, the statistically significant categories centered on cancer and inflammation (Figure S5), two conditions subject to clock dependent regulation in mice.
To seek redundancy amongst the parameters tracked by the remote sensors and wearables, we constructed variance correlation matrices where the goodness-of-fit p-values produced from the linear regression analysis informed statistical significance (Fig. 7). This approach visualizes the proportion of variance observed for each variable explained by the variance observed for each other variable. For example, the outputs from the Actigraph’s accelerometers, that is axes 1, 2, and 3 to measure acceleration in three directions, correlate highly with each other as expected (e.g. R2=0.96, Bonferroni corrected p=1.8*10−277 between axis 1 and 2). Clusters of high correlation are evident both within-domain (e.g. between SBP and DBP [R2=72.5, Bonferroni corrected p=3.2*10−112] or between heart rate and SBP [R2=23, Bonferroni corrected p=2*10−21]) and between-domains, as evident between readouts of activity and cardiovascular function. Interestingly, considerable between-subject differences in the variance correlation matrices were noticeable. For example, the relationship between SBP and the oscillatory signal from activity ranged from R2 of 0.22 to 0.67 amongst the 6 volunteers.
Forced desynchrony protocols have provided valuable information on the role of the molecular clock in humans, permitting segregation of rhythmic activities driven by endogenous and environmental factors14,31–34. For example, disruption of endogenous rhythms results in disturbance of cardiovascular homeostasis, including a rise in blood pressure14. An open question is whether it is also possible to interrogate the contribution of discordant clock driven rhythmicity to time dependent expression of disease phenotypes in unrestricted settings, where the “noise” consequent to divergent behavior, therapies and concordant disease might obscure the detection of oscillatory signals of potential mechanistic relevance. A first step towards addressing this question is to perform a pilot study to determine whether oscillatory signals can even be detected in apparently healthy humans, selected for demographic homogeneity, but allowed to free range without environmental restriction. In the present study, we provide the first evidence that many such signals are detectable despite variance, even in a small number of individuals sampled infrequently, illustrating the feasibility of characterizing the chronobiome – the collective of rhythmic phenomena – of humans living “in the wild”.
Here we report the integration of multidimensional data collected via remote sensing, cardiovascular assessments and “omics” analyses. As anticipated, we see a diversity of behavioral patterns in this apparently homogenous population, purposefully standardized for age, gender and health status to increase the likelihood of detecting time dependent variations. Despite this, the small sample size and protocol violations, we see that several clock-determined diurnal readouts, i.e. blood pressure and cortisol, were internally consistent with time-dependent patterns in the volunteers’ physical activity, mobility, communication and environmental cues (ambient light exposure). While the majority of remote sensor readouts showed time-specific variability (62%), we find that inter-subject differences mainly drove variability in communication. This latter observation overlaps with findings from e-mail communications in large university-based cohorts sampled in Europe and the US35. This study described two broad e-mail phenotypes, one restricting use to work hours, the other persistently active during wake hours. As residents in Western societies move increasingly outside the environmental light-dark cycle, our approach might afford new avenues to investigate the health implications of this cultural change. For example, one might parse for synchrony versus asynchrony between outcome variables using circadian phase.
In our cohort, as expected, we observed high correlations between circadian phases of activity, communication, mobility and light, thus suggesting high synchronicity. Notably, time-specific phase shifts can be induced by food intake, for example, carbohydrates in the morning (compared to evening) phase-advance heart rate by three-quarters of an hour36.
Circadian amplitude offers yet another perspective on circadian organization. Healthy volunteers under conditions of forced desychrony variably respond with a reduction in amplitude across clock-determined oscillatory endpoints37; however, the relationship to adverse health effects is less clear. In our cohort, as we would expect, circadian amplitudes of different outputs correlated highly. For example, that between activity and mobility (R2 of 16.4%, Bonferroni corrected-p=4.2*10−14), was similar to the correlation between activity and communication (R2 of 16.4%, Bonferroni corrected-p=3.1*10−14). If and how these relationships change under acute and chronic exposure to stress remains to be seen.
Temporal patterns are discernable in the “omics” data, most pronounced in the metabolome (5.4% in plasma and 5.6% in saliva), and evident in several genera of the oral microbiome. In the case of the plasma proteome and whole blood transcriptome, more frequent analyses in larger cohorts will be necessary comprehensively to discern signal from noise. Our exploratory pathway analysis revealed that metabolomic as well as proteomic pathways associated with cancer and inflammation were enriched in a temporal fashion. Prominent interplay with circadian clocks has been described in mice for both diseases4,38.
These data provide a reference set for the design of larger studies comprehensively to interrogate the chronobiome. For example, we wish to determine how age and gender, two factors that interact with clock-derived outputs in model systems21,39, and seasonal variation40 modulate the human chronobiome. More detailed phenotyping will include additional analytical platforms, for example the breath metabolome41, and characterization of the response to time dependent metabolic42, inflammatory43 and cardiovascular44 perturbations of the chronobiome. Such deep phenotypic characterization will provide a comparator for investigation of chronobiomic divergence of potential mechanistic and therapeutic value in syndromes of time dependent disease expression, such as non-dipping hypertension, nocturnal asthma and depression.
We are indebted to our volunteers who consented to participate in this pilot study as well as to the staff of the Center for Human Phenomic Science (CHPS) including LaVenia Banas, RCN, for excellent clinical support, to Sarah Teegarden, PhD, and Alice Laughlin for excellent technical support, and to Lisa Basel-Brown as bionutritionist. We thank Julia Bernstein, Leanne Kaye, PhD, RD, and Karan Singh, MBA, at Ginger.io, San Francisco, CA for providing access to the Ginger.io mobile and dashboard application. We thank the SmartIntake investigators and staff at the Pennington Biomedical Research Center, Baton Rouge, LA, in particular Corby K. Martin, PhD, FTOS, Rebecca Schulin, and Mandy Cowley. Parts of this study were presented at i) the Chronobiology Symposium “Translation of Circadian Biology: Implications for the Clinic”, Penn Chronobiology Program and ITMAT, University of Pennsylvania Perelman School of Medicine Nov 11, 2015; ii) the Society for Research on Biological Rhythms (SRBR) meeting, Palm Harbor, FL, May 21–25, 2016; and iii) the Gordon Research Conference on Chronobiology, Stow, Vermont, July 16–21, 2017. This publication was made possible by the Clinical Research Program Award, Grant 12CRP11920045, Great Rivers Affiliate, American Heart Association (C.S.); Feodor-Lynen Research Award, Alexander von Humboldt-Foundation, Bonn, Germany (C.S.); CEET Pilot Project funding (C.S.) from parent grant NIH/NIEHS 1P30 ES013508-05; NIH Grant LM009012 (J.H.M.); and NIH/NCRR Grant UL1RR024134 which is now NCATS Grant UL1TR000003 (G.A.F.). Dr. FitzGerald is the McNeil Professor in Translational Medicine and Therapeutics; Dr. Skarke is the McNeil Fellow in Translational Medicine and Therapeutics.
Carsten Skarke, (ITMAT), Literature search, study design, regulatory science, study conduct, data collection, data analysis, data integration, data interpretation, writing, visualizations, figures & tables. Seth Rhoades, (ITMAT), Metabolomics analysis, data interpretation Nicholas Lahens, (ITMAT), Visualizations, statistical analysis, data interpretation. Amy Campbell, (ITMAT), Visualizations, statistical analysis, data interpretation. Kyle Bittinger, (Department of Microbiology), Microbiome, statistical analysis, data interpretation. Aubrey Bailey, (Department of Microbiology), Microbiome analysis. Christian Hoffmann, (Department of Microbiology), Microbiome analysis. Randal S. Olson, (Institute for Biomedical Informatics), Visualizations & statistical analysis. Lihong Chen, (ITMAT), Transcriptomics, data interpretation. Guangrui Yang, (ITMAT), Transcriptomics, data interpretation. Thomas S. Price, (ITMAT), Visualizations & statistical analysis. Jason H. Moore, (Institute for Biomedical Informatics), Visualizations, statistical analysis, data interpretation. Frederik D. Bushman, (Department of Microbiology), Microbiome, statistical analysis, data interpretation. Casey S. Greene, (ITMAT), Visualizations, statistical analysis, data interpretation. Greg R. Grant, (ITMAT), Visualizations, statistical analysis, data interpretation. Aalim M. Weljie, (ITMAT), Metabolomics analysis, data interpretation. Garret A. FitzGerald, (ITMAT), Literature search, study design, regulatory science, data collection, data analysis, data interpretation, writing, figures & tables.
The authors declare that they have no competing interests.
Nicholas F. Lahens and Seth D. Rhoades contributed equally to this work.
Electronic supplementary material
Supplementary information accompanies this paper at 10.1038/s41598-017-17362-6.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Carsten Skarke, Email: ude.nnepu.enicidemnnep@ekraksc.
Garret A. FitzGerald, Email: ude.nnepu@terrag.