Influence of sleep deprivation and circadian misalignment on cortisol, inflammatory markers, and cytokine balance

Abstract

Cortisol and inflammatory proteins are released into the blood in response to stressors and chronic elevations of blood cortisol and inflammatory proteins may contribute to ongoing disease processes and could be useful biomarkers of disease. How chronic circadian misalignment influences cortisol and inflammatory proteins, however, is largely unknown and this was the focus of the current study. Specifically, we examined the influence of weeks of chronic circadian misalignment on cortisol, stress ratings, and pro- and anti-inflammatory proteins in humans. We also compared the effects of acute total sleep deprivation and chronic circadian misalignment on cortisol levels. Healthy, drug free females and males (N=17) aged 20-41 participated. After 3weeks of maintaining consistent sleep-wake schedules at home, six laboratory baseline days and nights, a 40-h constant routine (CR, total sleep deprivation) to examine circadian rhythms for melatonin and cortisol, participants were scheduled to a 25-day laboratory entrainment protocol that resulted in sleep and circadian disruption for eight of the participants. A second constant routine was conducted to reassess melatonin and cortisol rhythms on days 34-35. Plasma cortisol levels were also measured during sampling windows every week and trapezoidal area under the curve (AUC) was used to estimate 24-h cortisol levels. Inflammatory proteins were assessed at baseline and near the end of the entrainment protocol. Acute total sleep deprivation significantly increased cortisol levels (p<0.0001), whereas chronic circadian misalignment significantly reduced cortisol levels (p<0.05). Participants who exhibited normal circadian phase relationships with the wakefulness-sleep schedule showed little change in cortisol levels. Stress ratings increased during acute sleep deprivation (p<0.0001), whereas stress ratings remained low across weeks of study for both the misaligned and synchronized control group. Circadian misalignment significantly increased plasma tumor necrosis factor-alpha (TNF-α), interleukin 10 (IL-10) and C-reactive protein (CRP) (p<0.05). Little change was observed for the TNF-α/IL-10 ratio during circadian misalignment, whereas the TNF-α/IL-10 ratio and CRP levels decreased in the synchronized control group across weeks of circadian entrainment. The current findings demonstrate that total sleep deprivation and chronic circadian misalignment modulate cortisol levels and that chronic circadian misalignment increases plasma concentrations of pro- and anti-inflammatory proteins.

Keywords: C-reactive protein; Circadian clock; Cortisol; Cytokines; Inflammation; Interleukin-10; Sleep loss; Tumor necrosis factor alpha.

Fig. 1. Protocol figures

Data are plotted to a relative clock hour with wake time arbitrarily assigned a value of 0800h on baseline Day 1 and all other times referenced to this value. Black bars represent scheduled sleep. Day 1-6 are baseline days with scheduled 8-h sleep episodes at the participants' habitual bedtime. Days 7-8 is a 40-h constant routine 1 (CR1) with 8-h scheduled recovery sleep. Days 9-33 are experimental conditions and Days 34-35 is CR 2. (a) imposed maintenance of a 24.0-h day for 25 days, and (b) imposed 24.6-h day for 25 days with lights out and lights on delayed by 36 min each day. Blood sampling segments denoted by shading on Days 5-8, 12-14, 19-21, 26-28 and 34-35. Cortisol assessed at each blood sampling segment and inflammatory proteins assessed at baseline Days 5-6 and experimental Days 26-27. Circadian period findings from 28-h forced desynchrony Days 36-49 previously reported in (Wright et al., 2001, 2006).

Fig. 2. Plasma cortisol levels every 30 min across a standard 8-h sleep – 16-h wakefulness day and sleep deprivation (n=17)

Two consecutive 24-h episodes of plasma cortisol plotted overlying each other beginning with the sleep episode on Day 6 and ending after 40-h of wakefulness of the constant routine on Days 7-8. Scheduled waketime arbitrarily assigned a value of 0800h (relative clock hour). Box represents scheduled sleep during baseline and nighttime sleep deprivation 24-h later. * denotes p< 0.049

Fig 3. Stress ratings (a) bi-hourly during total sleep deprivation (n=17) and (b) average daily for synchronized (n=9) and not-synchronized (n=8) participants

(a) Stress ratings were relatively stable across the first 24-h of wakefulness and were higher during the day of sleep deprivation. Dashed line indicates end of standard 16-h waking day and beginning of sleep deprivation. (b) Average daily stress ratings were similar for the synchronized and not-synchronize participants across the protocol. Note that day 8 shows average stress ratings during sleep deprivation for hours awake 25-40 on constant routine 1.

Fig. 4. Individual participant cortisol rhythm plots aligned to scheduled sleep-wakefulness time on baseline Days 5-6 (a & b) and on entrainment protocol Days 26-27 (c & d)

Scheduled waketime arbitrarily assigned a value of 0800h (relative clock hour). Box represents scheduled sleep.

Fig. 5. Cortisol (a & b) and melatonin (c & d) levels every 30 min during constant routines and aligned to melatonin onset (n=14)

Average cortisol levels in (a) synchronized (n=8) and (b) non-synchronized (n=6). Average melatonin levels in (c) synchronized and (d) not-synchronized participants. Dashed lines represent timing of the melatonin onset.

Fig. 6. Twenty-four hour cortisol area under the curve assessments

Not-synchronized (n=5, 7, 7, 6; respectively for days of study shown) compared to the synchronized participants (n=7, 7, 6, 8; respectively for days of study shown). Lines denote significant differences in cortisol AUC between synchronized and not-synchronized groups (independent t-test; p<0.05); * denote significant reduction in cortisol AUC for the not-synchronized participants (single sample t-test difference from zero change; p< 0.05). Not-synchronized group difference from zero change on day 12-14 showed non-significant trend p=0.052, and synchronized group difference from zero change on day 34-35 showed non-significant trend p=0.053.

Fig. 7. Hourly pro- and anti- inflammatory proteins on baseline days 5-6 and experimental days 26-27

Average samples during scheduled sleep (black box) and scheduled wakefulness for baseline days (filled symbols) and entrainment protocol days (open symbols) for synchronized (left panels) and not-synchronized (right panels) groups. Sample sizes were n=6, 5, 5, and 6 for synchronized, and n=7, 7, 7, and 6 for not-synchronized groups for TNF, IL-10, TNF/IL-10 ration and CRP, respectively. * denote significant differences between days within group for that time of day (p<0.0239).