We first assessed the influence of sleep on the immune system. To do so we extracted data on sleeping times for different mammalian species from the published literature and matched these data where possible with white blood cell counts reported by the International Species Information System (ISIS [24
]; [see Additional File 1
]). We use white blood cells as a proxy for immune system investment as they are central to all immune responses and are a measure of immunocompetence [25
]. White blood cells originate in bone marrow and are derived from the same hematopoietic stem cells that produce red blood cells and platelets [25
]. As these latter cells have no direct immunological function, we use them as natural controls to test the specificity of any relationship between sleep and the immune system. If a key selective advantage of sleep is that it allows greater investment in the immune system, then species that sleep for longer should have increased numbers of immune cells in circulation, but there should be no similar relationship with control cells. After matching species values from each database we were able to analyze data for 26 mammalian species while controlling for confounding factors (body size and activity period; see the Methods for details).
As expected if sleep enhances immune defences, species that engaged in more sleep had higher numbers of white blood cells circulating in peripheral blood (coefficient = 0.00976, s.e. = 0.00171, t22 = 5.71, P < 0.001; Figure ). Across our dataset, a 14 hour increase in sleeping times corresponded to an additional 30 million white blood cells in each millilitre of blood (a 615% increase). Crucially, no similar patterns were evident with either red blood cells or platelets (red blood cells: coefficient = -0.028, s.e. = 0.113, t24 = -0.24, P > 0.8, platelets: coefficient = -0.00602, s.e. = 0.00576, t21 = -1.04, P > 0.3).
Figure 2 Sleep, immune defences and parasitism. Interspecific evidence that sleep protects against parasitic infection. (a) The number of white blood cells in peripheral blood increases among species with longer sleep durations. The fitted line is derived from (more ...)
We also tested predictions using phylogenetically independent contrasts to account for the lack of statistical independence in species data [27
]. These analyses examine evolutionary change and showed that when lineages evolved longer sleep durations, they also increased their white blood cell counts (coefficient = 0.0153, s.e. = 0.00250, t23
= 6.11, P
< 0.001). Again, this relationship was specific to immune cells (for red blood cells: coefficient = -0.200, s.e. = 0.0123, t23
= -1.63, P
> 0.11, for platelets: coefficient = -0.00463, s.e. = 0.00628, t21
= -0.74, P
> 0.45), leading to an increase in the ratio of immune cells to other blood cell types when species evolved longer sleeping durations (coefficient = 0.0639, s.e. = 0.0108, t20
= 5.93, P
Total white blood cell counts are a compound measure of the abundance of different cell types, each of which fulfils specialized immunological roles [25
]. We therefore also investigated whether the correlated evolution of sleep and white blood cell counts was limited to specific cell types. Neutrophils constitute the largest component of the innate immune system, representing 47% of white blood cells in our sample, and are seen as a first line of defence that responds rapidly upon detection of invading pathogens [25
]. Analysis of independent contrasts indicated that higher numbers of neutrophils in the bloodstream have evolved in association with elevated sleep durations (bootstrapped coefficient = 0.0948, s.e. = 0.0223, n = 25, P < 0.001). Lymphocytes, which account for 44% of the white cell count in our sample and are mediators of the acquired immune response [25
], were also present in greater numbers when evolutionary increases in sleeping durations occurred (bootstrapped coefficient = 0.0103, s.e. = 0.00385, n = 25, P = 0.004). Finally, eosinophils and basophils, which are relatively minor components of the white blood cell count (5% and 1%, respectively) and act primarily against macroparasites [25
], showed the predicted association with sleep (eosinophils: bootstrapped coefficient = 0.0246, s.e. = 0.0125, n = 25, P < 0.035; basophils: coefficient = 0.0676, s.e. = 0.0198, t24 = 3.42, P = 0.002). Only monocytes, accounting for 5% of the total white cell count [25
], failed to show a significant association with sleep durations (bootstrapped coefficient = 0.0478, s.e. = 0.0323, n = 25, P > 0.14). Thus, evolutionary increases in the abundance of four of the five immune cell types have occurred in association with longer sleep times, suggesting that increased sleep may allow a generalized heightening of immune defences.
Next, we addressed hypotheses regarding the role of the two main sleep stages (NREM and REM) in immune system investment. In clinical studies, increased duration and intensity of NREM sleep during illness are associated with an improved prognosis, but occur at the expense of REM sleep [11
]. From this, it is argued that immunological benefits of sleep occur while brain function is down-regulated during the NREM phase [11
]. Our results did not support this suggestion, however, as we found that evolutionary increases in both NREM and REM sleep occur in parallel with elevated white blood cell counts (NREM: coefficient = 0.0853, s.e. = 0.0249, t17
= 3.42; P
= 0.003; REM: bootstrapped coefficient = 0.0633, s.e. = 0.0112, n = 19, P
< 0.001). Thus, evolutionary increases in sleep are associated with increased investment in the immune system regardless of its specific form.
Finally, we assessed whether a role for sleep in enhancing immune defences could translate into improved resistance against parasitic infections. We were able to match sleep times with parasitism for 12 mammalian species from the Global Mammal Parasite Database
], which details the diversity and prevalence of microparasites (viruses, bacteria and fungi) and macroparasites (helminths, protozoa and arthropods) that infect wild populations of mammals [see Additional File 1
]. If sleep is effective in protecting against infection, then species that engage in more sleep should have fewer parasites (measured as a combination of species richness and prevalence, see Methods). After correcting for differences arising from sampling effort [29
], we found this predicted relationship (coefficient = -3.554, s.e. = 0.888, t10
= -4.00, P
= 0.003; Figure ). This analysis suggests that across the 10 hour range of sleep durations present in the dataset there is a 24-fold decline in levels of parasitism. A significant negative relationship was also evident in analyses of independent contrasts (bootstrapped coefficient = -3.48, s.e. = 1.51, n = 11, P
= 0.006). Thus, as species evolved longer sleep durations and enhanced their immune systems, they become less parasitized.
Our results are consistent with parasite resistance having played an important role in the evolution of sleep, and suggest therefore that sleep is of greater immunological significance than is currently recognized. It had been noted that the physiological links between sleep and immunity that have been highlighted by experimental studies (e.g. in rats [12
]) could arise from a negative impact of sleep deprivation on the brain, leading to an impaired coordination of immune system activity [11
]. However, our findings reveal strong relationships between sleep and immune defences in the absence of sleep deprivation, and thus point to a more direct constitutive role for sleep in promoting immunocompetence.
We suggest that sleep fuels the immune system. While awake, animals must be ready to meet multiple demands on a limited energy supply, including the need to search for food, acquire mates, and provide parental care. When asleep, animals largely avoid these energetic costs, and can thus allocate resources to the immune system (sensu
]). Unlike the energy conservation hypothesis of sleep [7
], this reallocation hypothesis predicts little or no overall energy savings during sleep. This appears to be true: in humans, for example, energy savings from eight hours of sleep would be expended within one hour of waking (63 kilocalories [32
]). Direct estimates of the energetic cost of maintaining immunity are not currently available. However, numerous studies have suggested that these costs are large enough to generate trade-offs with key life history traits, such as growth and reproduction (e.g. [33
]). Increased energy requirements when the immune system is upregulated also point toward a substantial metabolic cost associated with immune defence. Even during mild antigenic challenge, basal metabolic rate can be increased by as much as 15 to 30% [14
]. Thus, a generalised elevation of immune defences may come at considerable energetic cost.
The energetic costs of immune system maintenance and routine functioning take multiple forms (see [14
]). These include the relatively short lifespan and so high turnover rate of granulocytes (every 2 to 3 days [25
]), the cost of sustaining the hypermetabolic rate of immune cells [38
], and repairing the immunopathological damage that results when cytotoxic compounds are released by immune cells responding to antigens [39
]. Thus, an influence of sleep need not be confined to investing in greater numbers of immune cells. Indeed, both antibody responses and natural killer cell activity are reduced following sleep deprivation [19
], showing that sleep could have a far broader influence on immunocompetence.
Our results and interpretation are consistent with experimental studies showing that animals sleep for extended periods when mounting an immune response [13
]. If evolved increases in sleep allow animals to channel more energy into their immune defences and so protect against the development of acute infections, then short term increases in sleep may help provide the additional energy required for an acute phase response to an already established infection [13
]. The possibility also exists that evolutionary and facultative changes in sleep share a common underlying mechanism. Short term increases in sleep appear to be triggered by immunomodulatory cytokines that are released by white blood cells during immune reactions [17
]. If larger numbers of white blood cells produce a greater immune response to antigenic challenge, and hence a greater release of sleep promoting cytokines, this could potentially drive evolutionary increases in sleep durations.
Our finding that both sleep phases are associated with immune investment appears to be in conflict with observations that REM sleep is reduced during acute infection (e.g. [13
]). It should be noted, however, that the advantage gained through evolutionary increases in normal sleep can also differ from the selective advantage of modulating sleep phases during the course of an infection. For example, REM sleep is partly characterized by a loss of thermoregulation [41
], and thus it is argued that REM sleep is inhibited during an acute phases response to infection as it would prevent animals from maintaining an elevated body temperature that impedes further microbial proliferation [42
]. While this explanation is plausible, it cannot be applied to evolutionary changes in normal REM sleep durations and immune investment, which occur in the absence of an acute phase response. As data become available on sleep architecture during the acute phase response of different species, comparative studies may be able to assess the benefits of REM sleep suppression directly. Similarly, analysis of the increased intensity of sleep that occurs during infection, as identified through an elevation in slow wave activity [22
], could reveal an additional role of the 'quality' of sleep.
It is commonly suggested that sleep may serve multiple functions (e.g. [1
]). While our analyses yielded no evidence that sleep influenced cell production in the other physiological systems we assessed, they do not eliminate the possibility that sleep could have an additional function(s) elsewhere in the body. In particular, it has been argued that sleep is 'primarily for the brain' [43
], which is a view that has both intuitive merit and considerable experimental support (see review [44
]). However, from an evolutionary perspective, phylogenetically controlled analyses have yet to produce support for a key expectation of this hypothesis, namely that species with greater cognitive abilities should require more sleep. At best, a recent study has suggested that REM sleep durations may increase with the brain size of mammals ([3
] but see [4
]). Since REM sleep usually accounts for less than 20% of total sleep durations [our dataset; see Additional File 1
], these comparative findings cannot explain why different mammalian species sleep for as long as they do. Instead, it may be that sleep quality is of greater importance to brain function than the overall duration of sleep per se
, which is a possibility that could be assessed when sufficient data have accumulated. In the absence of these data, the presence of characteristic patterns of brain activity in mammals during NREM and REM sleep, alongside the cognitive 'black-out' that is experienced while sleeping, imply that it does perform some important function for the brain. The nature of this function remains hotly debated [1
An important implication of our findings is that ecological factors that impact sleep could indirectly affect immune defences. By sleeping regularly in 'safe' sites such as burrows or dens, for example, individuals may be better protected from predators, and thus able to sleep for longer durations [2
]. Conversely, herbivorous species with large foraging requirements could have less time available for sleep than species living on an energy-rich carnivorous diet [2
]. Trade-offs between time invested in sleep and alternative activities may occur at key life history stages, such as during periods of reproductive competition [6
] or parental care [46
]. Current evidence links these activities to reduced immunocompetence [47
], which could be due to reductions in the time available for sleep. However, field studies should also ascertain the relationship between sleeping behaviour and an animal's exposure to parasites, which could reveal important ecological relationships between sleep and parasitic infection that have the potential to influence the analyses we report here [48