Conceived and designed the experiments: WMAvL ML MS MH TPH HA. Performed the experiments: WMAvL ML PK HL. Analyzed the data: WMAvL ML PK RL TPH HA. Contributed reagents/materials/analysis tools: WMAvL ML HL RL MS MH TPH HA. Wrote the paper: WMAvL ML PK HL RL MS MH TPH HA.
The authors have declared that no competing interests exist.
Sleep restriction, leading to deprivation of sleep, is common in modern 24-h societies and is associated with the development of health problems including cardiovascular diseases. Our objective was to investigate the immunological effects of prolonged sleep restriction and subsequent recovery sleep, by simulating a working week and following recovery weekend in a laboratory environment.
After 2 baseline nights of 8 hours time in bed (TIB), 13 healthy young men had only 4 hours TIB per night for 5 nights, followed by 2 recovery nights with 8 hours TIB. 6 control subjects had 8 hours TIB per night throughout the experiment. Heart rate, blood pressure, salivary cortisol and serum C-reactive protein (CRP) were measured after the baseline (BL), sleep restriction (SR) and recovery (REC) period. Peripheral blood mononuclear cells (PBMC) were collected at these time points, counted and stimulated with PHA. Cell proliferation was analyzed by thymidine incorporation and cytokine production by ELISA and RT-PCR. CRP was increased after SR (145% of BL;
5 nights of sleep restriction increased lymphocyte activation and the production of proinflammatory cytokines including IL-1β IL-6 and IL-17; they remained elevated after 2 nights of recovery sleep, accompanied by increased heart rate and serum CRP, 2 important risk factors for cardiovascular diseases. Therefore, long-term sleep restriction may lead to persistent changes in the immune system and the increased production of IL-17 together with CRP may increase the risk of developing cardiovascular diseases.
Sleep is generally considered to be a restorative process, having beneficial effects on immune functions. Partial loss of sleep is common among people who experience environmental or psychological stress such as travelling across time zones, having to do shift work, and in those individuals with psychiatric or physical disorders. Sleep restriction is becoming increasingly prevalent, especially among employed middle-aged populations
Sufficient sleep is vital for cardiovascular health and reduced sleep duration is specifically associated with increased cardiovascular morbidity
In the present study, we simulated the accumulation of sleep loss during five working days followed by two days of weekend recovery sleep, and measured the changes in immunological parameters at these time points. We hypothesize that, in addition to the previously reported adverse effects on cognitive functioning and metabolism
Nineteen healthy men, aged 19–29 (mean [SD] age 23.1 [2.5] years), with a regular sleep-wake schedule and habitual sleep duration of 7–9 h participated in the study. Physical screening included blood tests (triglycerides, cholesterol, creatinine, haemoglobin, haematocrit, MCV, MCH, MCHC, leukocytes, erythrocytes, TSH, ASAT, ALAT) and polysomnography (
The experimental group (n = 13) spent 8 h in bed for the first two nights (BL; from 11 PM to 7 AM), followed by 5 nights where they rested for only 4 h in bed (SR; from 3 AM to 7 AM) and, finally, again 3 nights of 8 h in bed (REC). The control group (n = 6) spent 8 h in bed (11 PM to 7 AM) throughout the entire experiment. Napping during daytime was not allowed and this was monitored by continuous EEG recordings and a continuously present investigator. Meals were standardized, provided at fixed times and finished by all participants throughout the experiment: breakfast at 8 AM (600 kcal), lunch at 12.30 PM (800 kcal), dinner at 6 PM (700 kcal); snacks at 3.30 PM (300 kcal) and 9.30 PM (200 kcal). In addition, participants in the experimental group ate a piece of fruit at 12.15 AM. Participants were not allowed to leave the building, but could stay in a living room where there was a television and a personal computer. Illumination in the sleeping room and in the test room ranged from 150 to 400 lux, and in the living room from 350 to 600 lux. The temperature ranged from 19 to 23°C.
Blood samples were taken from participants at 7.30 AM and analyzed by Medix Laboratories, Espoo, Finland for high sensitivity C-reactive protein (hs-CRP) using immunoturbidimetry. Morning peak values of cortisol were assessed using saliva samples that were collected 10 times a day and their cortisol levels were measured using a commercial kit assay (Salivary Cortisol, LIA, IBL, Hamburg, Germany). The measurement range was 0.43–110 nmol/L with assay repeatability values of 5% (within series) and 8% (between series).
ECG- based (WinAcq, AbsoluteAliens, Finland) heart rate together with continuous systolic and diastolic blood pressure (Portapres, Finapres Medical Systems, the Netherlands) was measured between 8 AM and 9 AM during a 10 minute period of rest.
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by density gradient centrifugation as earlier described
Isolated PBMC were washed twice with phosphate buffered saline (PBS) and proliferation of PBMC was performed in complete RPMI 1640 containing 5% heat-inactivated human AB serum on 96-well U-bottomed plates (Costar Corning Incorporated, Corning, NY, USA) as earlier described
Separated and washed PBMC were stimulated with PHA (45 µg/mL) in complete RPMI 1640 medium on 24-well plates (Costar) (3×106 cells/1.5 mL/well). The total number of cells per stimulation was 6×106. Cell pellets were collected after 6 h of incubation and used for RNA isolation. Cell culture supernatants were obtained after 24 h of incubation and stored at −70°C before measurement of cytokine protein levels.
Total RNA isolation, synthesis of cDNA and real-time quantitative PCR with an AbiPrism 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) were performed as described earlier
Cytokine protein analysis was made with the Luminex bead system (Bio-Plex 200 System, Bio-Rad Laboratories, Hercules, CA, USA) by labelled cytokine capture antibody pairs (Bio-Rad Laboratories).
Data were expressed as percentages of each individual participant's baseline values unless otherwise specified. We have compared sleep restriction and recovery values to baseline values by applying paired t-tests for normally distributed differences and Wilcoxon signed ranks tests for differences that were not normally distributed. The normality of differences was checked using Kolmogorov-Smirnov goodness of fit test. A
The cell distribution of peripheral blood describes the general immune status of these individuals. The total number of T cells (
Number of T cells, B cells, and natural killer (NK) cells in PBMC, expressed as percentage of participant's individual baseline values. SR = sleep restriction, REC = recovery, Con = control group, Exp = sleep restriction group. Data are presented as mean values ±SEM. *
C-reactive protein (CRP) is an important risk factor for many diseases, including stroke and hypertension
Proliferation of PBMC after stimulation with phytohaemagglutinin (PHA, 45 µg/mL) and high sensitivity C-reactive protein (hs-CRP) concentrations in plasma. SR = sleep restriction, REC = recovery, Con = control group, Exp = sleep restriction group. Data are expressed as percentages of participant's individual baseline values (mean±SEM). *
Proliferation of PHA activated PBMC reflects the immunological capability of peripheral blood cells. Proliferation of PBMC in the experimental group was significantly increased after sleep restriction compared to baseline (to 233% [
Cytokine profiles of activated PBMC reflect the dominating immune responses. The amounts of proinflammatory cytokines IL-1β and IL-6 increased significantly at the mRNA level (to 137% [
Cytokine mRNA and protein expression of phytohaemagglutinin (PHA, 45 µg/mL) activated PBMC. SR = sleep restriction, REC = recovery, Con = control group, Exp = sleep restriction group. Data are expressed as percentages of participant's individual baseline values (mean±SEM). *
Chronic sleep deprivation is becoming increasingly common in modern, 24-h societies due to voluntary sleep restriction and increasing work demands
NK cells are phagocytes of innate immunity that quickly recognize, engulf and destroy intracellular pathogens whereas T cells and B cells orchestrate adaptive immunity through cellular and humoral responses. We observed a decrease in NK cell numbers as well as an increase in the number of B cells after five nights of sleep restriction. On the other hand, there were no changes in the number of T cells or their CD4+ and CD8+ subtypes. Sleep deprivation as well as stress factors have been shown to decrease the number and function of NK cells, often associated with increased susceptibility to infections
In our study, peripheral T cells showed highly elevated proliferation responses to PHA. This suggests that T cells in sleep deprived people, compared to people with normal sleep, are primed and after non-specific stimulation they respond more efficiently. A similar effect was observed in a recent study where stressed mice survived as well as or even better than non-stressed mice during a primary pneumococcus infection, but their survival was strongly reduced during secondary infection
CRP is widely used as a general marker for inflammation
In the present study, five days of sleep restriction were associated with increased IL-17 production both at the mRNA and the protein levels from PHA activated PBMC, and the amount of IL-17 remained elevated after the recovery period. IL-17 is a relatively newly-discovered member of the proinflammatory cytokines. It plays a key role in sustaining tissue damage in several tissues such as brain, joints, heart, lung and intestine, sometimes promoting the development of autoimmune diseases
Schematic flow chart showing the proposed mechanism explaining how sleep restriction may ultimately evoke increased cardiovascular morbidity.
In conclusion, we identified how prolonged sleep restriction can change immune cell functions, and may lead to an increased risk to develop cardiovascular diseases. Several immunological changes that occurred after five days of sleep restriction did recover after two nights of normal sleep, but the elevated level of serum hs-CRP that was accompanied by increased production of proinflammatory cytokines, especially IL-17, did not return to normal. In summary, these results indicate that immunological changes that take place after multiple nights of short sleep cannot be restored completely by sleeping normally for a few nights, and long-term sleep restriction may lead to an increased risk of developing cardiovascular diseases.
Pre-study screening results
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Overview of the Results of All Variables with Paired T-tests: Comparison to Baseline
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We thank Sirkku Hopeakangas for help with collection of blood samples, Marja-Leena Haavisto for patient recruitment, and Sirpa Hyttinen for help with cortisol analysis.