Chronic sleep deprivation is becoming increasingly common in modern, 24-h societies due to voluntary sleep restriction and increasing work demands 
. Sleep loss results in tiredness and impaired cognitive performance 
, but it also affects immune functions, leading to an increased number of infections 
. Moreover, it has been shown both epidemiologically and experimentally that reduced sleep duration is associated with an increased risk of developing diabetes 
, obesity 
, and cardiovascular diseases 
. An accelerated heart rate is a sign of stress in the cardiovascular system 
. In the present study, heart rate was significantly increased at the end of the experiment. This could be viewed as an alarm reaction in the circulatory system that might, at least partially, be a consequence of reduced parasympathetic tone and/or increased sympathetic tone in the autonomic nervous system (ANS). Serum cortisol and blood pressure remained unaffected throughout the current experiment, probably due to the fact that chronic changes in those parameters require more time to develop. Indeed, it has been shown that a more extended period of six nights of sleep restricted to 4 h per night results in elevated evening cortisol concentrations 
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 
. It should be noted, however, that the number of both NK cells and B-cells returned to baseline level during the two nights of recovery sleep in the present study. This suggests that the differences in the numbers of these cells in the circulation are probably due to reversible redistribution of these cells between lymphoid organs and periphery. In addition to cell numbers, the function of the immune cells plays a key role in successful immunity.
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 
. This was assumed to be due to a temporary immune-enhancing effect that later converted to a diminished adaptive immune response. In more extreme cases, chronic sleep loss in rats has resulted in severe inflammation in body tissues, culminating in lethal bacterial invasion of the bloodstream 
. Therefore, over-activation of effector cells may enhance immunity and help the individual to survive through extraordinary conditions in the short term, but prolonging this situation leads to inflammation, and tissue injury.
CRP is widely used as a general marker for inflammation 
. In the present study, serum hs-CRP concentrations were elevated immediately after sleep restriction and, since this peptide has an in vivo half life of 19 h 
, this elevation sustained after two days of recovery sleep. Similarly, a previous study has shown that both total (88 hours) as well as partial (4.2 hours during 10 consecutive nights) sleep restriction significantly increased serum concentrations of hs-CRP 
. Elevated serum CRP is a risk factor for cardiovascular diseases and predicts future cardiovascular events and even mortality in apparently healthy people 
. CRP co-localizes with modified low density lipoprotein (LDL) in human atherosclerotic plaques, and it has been shown to increase platelet adhesion to endothelial cells. Therefore, in addition to acting as a biomarker, CRP plays a causal role in the development of atherosclerosis and thrombosis 
. Animal studies also support the proinflammatory and pro-atherogenic role of CRP, because administration of human CRP or over-expression of CRP in apolipoprotein E -deficient mice promotes the development of spontaneous atherosclerosis 
. Previously it has been reported that synthesis of CRP in the liver is controlled by proinflammatory cytokines, including IL-6, TNF-α and IL-1 
. In our study, the production of IL-6 and IL-1β was clearly increased by PHA activation of PBMC after sleep restriction and remained elevated after recovery sleep, whereas the production of TNF-α was slightly reduced, but recovered after two days of recovery sleep. Cytokines IL-1 and IL-6 play a crucial role in immune defenses and their secretion also regulates sleep-wake rhythms and sleep patterns, respectively 
. In line with the results of our study, it was recently shown that IL-6 increased during prolonged sleep restriction when subjects slept only 4 hours per night for 12 days 
. Independently of high cholesterol or myocardial damage markers, IL-6 predicts future adverse cardiovascular events and reflects increased inflammatory activity in plaques and is therefore a strong marker of increased risk for mortality in coronary artery diseases 
. IL-1β is a proinflammatory cytokine; the processing of its inactive form (pro-IL-1β) to the active form is triggered by microbial products and metabolic stress, leading to increased lymphocyte activation and destruction of local tissues. Thus, secretion of biologically active IL-1β protein may be induced when sleep deprived people are infected (i.e. enhanced susceptibility to viral and bacterial infections), a phenomenon which is frequently associated with chronic sleep deprivation.
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 
. Inflammation is an important component in all stages of atherosclerosis and interestingly, IL-17 has recently been reported to stimulate expression of CRP in hepatocytes and in coronary artery smooth muscle cells 
. On the basis of these results, we propose a hypothesis for the sleep restriction-induced development of cardiovascular diseases (). Prolonged sleep restriction results in activation of the synthesis of proinflammatory cytokines IL-1β and IL-6 which in turn increases the expression of IL-17. These cytokines play an important role in the induction of CRP which may facilitate, directly or indirectly, the formation of atherosclerotic plaques leading to an increased risk for cardiovascular diseases. This detrimental pathway may be even further activated by simultaneous exposure to a microbial infection. However, understanding of the mechanisms of IL-17 in bridging innate and adaptive immunity is still in its infancy, and therefore the specific role of IL-17 in the development of cardiovascular diseases needs to be studied in more detail.
From sleep restriction to cardiovascular diseases.
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.