Synchronizing our clocks as we age: the influence of the brain-gut-immune axis on the sleep-wake cycle across the lifespan

Abstract

The microbes that colonize the small and large intestines, known as the gut microbiome, play an integral role in optimal brain development and function. The gut microbiome is a vital component of the bidirectional communication pathway between the brain, immune system, and gut, also known as the brain-gut-immune axis. To date, there has been minimal investigation into the implications of improper development of the gut microbiome and the brain-gut-immune axis on the sleep-wake cycle, particularly during sensitive periods of physical and neurological development, such as childhood, adolescence, and senescence. Therefore, this review will explore the current literature surrounding the overlapping developmental periods of the gut microbiome, brain, and immune system from birth through to senescence, while highlighting how the brain-gut-immune axis affects the maturation and organization of the sleep-wake cycle. We also examine how a dysfunction to either the microbiome or the sleep-wake cycle negatively affects the bidirectional relationship between the brain and gut, and subsequently the overall health and functionality of this complex system. Additionally, this review integrates therapeutic studies to demonstrate when dietary manipulations, such as supplementation with probiotics and prebiotics, can modulate the gut microbiome to enhance the health of the brain-gut-immune axis and optimize our sleep-wake cycle.

microbiome, adolescence, senescence, circadian, neurodevelopment

Introduction

It is now well recognized that the microbes that colonize the small and large intestines, known as the gut microbiome, play an integral role in optimal brain development and function across the lifespan. The gut microbiome plays a fundamental role in the regulation of cellular and molecular processes including neuroinflammation, blood brain barrier (BBB) permeability, the immune response, microglial activation, as well as intestinal motility, and permeability [1]. Furthermore, there is also increasing evidence that the gut microbiota plays a vital role in the regulation of sleep [2]. However, there has been a minimal investigation into the concurrent development of the gut microbiome and changes in the sleep-wake cycle across the lifespan, with particular gaps in the literature regarding adolescence and senescence. Therefore, the purpose of this review is to (1) examine the interactions between the developmental windows of the brain, gut microbiome, and immune system (brain-gut-immune axis) from birth into old age, (2) demonstrate the bidirectional effect that the brain-gut-immune axis has on the development and organization of the sleep-wake cycle, and (3) highlight current gaps in the literature and important future directions.

The Brain-Gut-Immune Axis

The gut-brain axis is a bidirectional biochemical communication pathway between the enteric nervous system (which is a major component of the autonomic nervous system, also known as the “mini-brain” that controls the gastrointestinal (GI) tract), and central nervous system (CNS). The gut microbiota is defined as the collection of microorganisms, including bacteria, fungi, viruses, and archaea that reside within the GI tract [3]. Although the gut microbiota and gut microbiome are often used interchangeably, the microbiome accounts for the microorganisms, as well as their collective genome [3]. Recent evidence indicates that the bidirectional communication between the gut microbiota and the brain is facilitated by various mechanisms, including the vagus nerve [4], the immune system, the production of neurotransmitters by microbes [5, 6], and the endocrine system (summarized in Figure 1) [7, 8].

Figure 1.

The role of the HPA axis in functioning of the brain-gut-immune axis. The gut microbiota influences the gut-brain axis via the primary direct route, the vagus nerve, as well as, through the indirect route; via the production of short-chain fatty acids (SCFAs), immune system mediators (TNF-α, IL-6, IL-1β), and neurotransmitters such as GABA, tryptophan, and serotonin. These bacterial products travel through the circulation activating specific areas of the brain and influencing HPA axis functioning. ACTH, adrenocorticotropic hormone; GABA, γ-aminobutyric acid; NTS, nucleus tractus; HPA, hypothalamic pituitary adrenal axis; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor alpha; IL-6, interleukin 6; IL-1β, interleukin 1 beta.

The vagus nerve as a key interface between the microbiota, gut, and brain

The primary communication pathway between the gut microbiota and the brain is the vagus nerve. The vagus nerve is the 10th cranial nerve and is the most direct route whereby signals from the microbiota reach the brain [9]. In the intestines, the vagus nerve functions to regulate smooth muscle contraction as well as glandular secretion [10]. It is comprised of two divisions, the afferent and efferent pathways, by which it permits bidirectional communication between the gut and the brain [11].

Afferent projections make up 80% of all vagal nerve fibers, which are generally involved in relaying sensory information from the visceral organs, such as the gut, up to the brain [10]. The afferent division is involved in the regulation of the hypothalamic pituitary adrenal (HPA) axis [12]. The GI vagal afferent fibers innervate the intestinal wall without crossing the epithelial layer [13]. As such, these afferent fibers are in indirect contact with the gut luminal microbiota, detecting chemical signals via diffusion of bacterial compounds or metabolites [13]. Enteroendocrine cells (EEC) make up 1% of total intestinal epithelial cells, but play a fundamental role in the regulation of GI motility [14], intestinal permeability, secretion, and food intake through their indirect communication with vagal afferent fibers [15]. Specialized EECs within the gut known as enterochromaffin cells are also involved in the secretion of neurotransmitters including serotonin [16]. In the presence of nutrients (luminal triglycerides, carbohydrates, and proteins) EECs release compounds/metabolites [16]. Specifically, vagal afferent fiber receptors interact with EEC-secreted neurotransmitters including serotonin and somatostatin, or gut hormones such as cholecystokinin, peptide YY, and glucagon-like peptide 1 and 2 [17]. Furthermore, depending on the hormone that is released, various brain pathways, such as the nucleus tractus solitarii in the medulla [18], and the suprachiasmatic nucleus (SCN) within the hypothalamus [19, 20], are activated. The microbiota also influences the secretion of particular compounds and metabolites by EECs through toll-like receptors (TLRs), which play a critical signaling role in the innate immune system [21]. EECs express TLRs, which recognize inflammatory lipopolysaccharide (LPS) endotoxins produced by bacteria or short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber and resistant starch [22, 23]. For example, TLR4 is expressed by vagal afferent fibers and is further involved in the detection of LPS which then activates relevant areas of the brain [24].

SCFAs, such as butyrate and propionate are produced by the fermentation of dietary ingested proteins and carbohydrates in the GI tract by specific gut microbes [25] and can communicate with the afferent vagus nerve terminals through direct mechanisms [23, 26]. Specifically, free SCFAs can travel via the vagus nerve to the brain and can cross the BBB and activate specific receptors [11, 23, 27, 28]. Butyric acid and propionic acid in particular have been found to affect dopamine and noradrenalin synthesis through tyrosine hydroxylase gene expression [29]. In particular, the administration of sodium butyrate induces histone acetylation, a critical process that modulates gene expression and chromatin remodeling within the hippocampus and frontal cortex, and was shown to have an antidepressant-like effect in mice [28]. Interestingly, SCFAs originating in the gut influence the maturation and function of microglia in the brain and the CNS [30]. In a study conducted by Erny et al. [30], germ-free (GF) mice that were deficient in the SCFA receptor Free Fatty Acid Receptor 2, exhibited microglia malformations and dendrite length alterations. Furthermore, GF mouse studies also demonstrate that long-term administration of SCFAs can reverse microglia abnormalities [30]. Therefore, it is possible that SCFAs are involved in the regulation of the CNS and hypothalamic pathways. Other microbiota-derived molecules, such as peptidoglycan (PGN) have also been found to cross the BBB, and influence the physical development of the brain by modulating gene expression [31].

In contrast to the afferent pathway, the efferent pathway of the vagus nerve accounts for 10% of the vagal nerve fibers, and transmits signals from the brain to the gut [32]. The efferent pathway is involved in mediating inflammatory pathways, in particular the cholinergic anti-inflammatory pathway (CAIP) [33]. As the CAIP regulates the innate immune response to pathogens and injury by inhibiting pro-inflammatory cytokine release, it plays an important role in regulating the intestinal immune response and homeostasis as it modulates inflammation in affected regions [34]. The efferent pathway is also regulated by activation of the HPA axis [11]. Activation of the HPA axis results in the release of cortisol/corticosterone and affects immune cell activity in the gut and systemically [35]. Therefore, the immune system also contributes to the communication between the gut, commensal microbes within the intestines, and the brain, providing a connection between the HPA axis and the gut.

Although there has been significant research on the vagus nerve in relation to the gut-brain axis and its contribution to the regulation of digestive function and satiety [8, 36], there has been little investigation into its role in other aspects of biological rhythms such as the sleep-wake cycle, particularly during sensitive periods of development.

Circadian Rhythms

Regulation of the sleep-wake cycle—role of the SCN

Originating from the Latin word for circa “around” and diem “day,” circadian rhythms are biological and physiological processes that cycle in synchronization with the solar day [37]. The most prominent circadian rhythm is the sleep-wake cycle [38]. The sleep-wake cycle is regulated by the primary clock within the SCN located in the anterior ventral hypothalamus [38, 39]. The appropriate regulation of the sleep-wake cycle relies on a transcription-translation feedback loop of clock gene expression [19]. During the wake, the clock genes Period (per1 and per2) and Cryptochrome (Cry1 and Cry2) are transcribed by the dimerization of circadian locomotor output cycles kaput (CLOCK) and Brain and muscle ARNT-like protein 1 (BMAL1) [19, 40]. During sleep, Period and Cryptochrome inhibit the CLOCK:BMAL1 heterodimerization via translocation from the cytoplasm to the nucleus thereby repressing their own transcription and creating a negative feedback loop [19, 40]. In a study conducted by Wisor et al. [41], double knockout of Cry1 and Cry2 in mice resulted in increased non-rapid eye movement (NREM) sleep and a loss of sleep consolidation suggesting a lack of circadian rhythmicity and disrupted regulation of sleep. Moreover, knockouts of per1 and per2 further demonstrate disruption to circadian rhythms, however, NREM and rapid eye movement (REM) length remained similar in these mice [42].

Neuropeptides, neurotransmitters, and cytokines that are synthesized by SCN neurons, in response to environmental input (e.g. light) via photosensitive retinal cells, are involved in the coordination of tissue-specific oscillators that subsequently regulate peripheral outputs (overt rhythms) [19, 20]. The two-process model of sleep regulation explains how sleep is controlled by the interaction of two separate processes; the circadian and the homeostatic [43]. Circadian regulation of sleep, known as Process C is driven by the tissue-specific oscillators that are controlled by the SCN and are attuned by the light-dark cycle [44]. Conversely, the homeostatic regulation of sleep, known as Process S is driven by the length of time an individual has been awake [43]. Specifically, sleep pressure increases (Process S) the longer a person has been awake and decreases the longer they have been asleep [43]. The flip-flop model, also known as the “flip-flop switch” is another model that demonstrates the transition from sleep to wakefulness based on the activation and inhibition of certain brain regions and neurotransmitters, whereby Process C and S consolidate the sleep bouts [45]. Specifically, neurons within the lateral hypothalamus, that release orexin, are hypothesized to stabilize this transition of sleep to awake [46]. Activation of the ventrolateral preoptic nucleus promotes sleep by inhibiting arousal, through the activation of the TMN (histaminergic tuberomammillary nucleus) histaminergic neurons, serotonergic, dopaminergic, and noradrenaline signaling in the arousal brainstem regions [45]. Wakefulness is promoted by neurons within the basal forebrain, upper brainstem, and posterior hypothalamus that produce specific neurotransmitters [47]. Serotonin, γ-aminobutyric acid (GABA), norepinephrine, and melatonin are important neurotransmitters and hormones that regulate the sleep-wake cycle, with serotonin and norepinephrine generally peaking during the wake cycle, and melatonin peaking at night [39]. Furthermore, as serotonin is the precursor for melatonin, low serotonin levels have been associated with reduced melatonin levels, thereby resulting in a disruption to sleep-wake cycles and an increased risk for depression and sleeping problems [38, 48, 49].

Sleep structure

Sleep is known to be a critical factor in the general maintenance of physiological processes, such as immune function [50] and metabolism [51] in all animal species, including humans [37]. Sleep is characterized by a reversible suspended consciousness, reduction in responsiveness to environmental stimuli, and behavioral inactivity [37]. Nocturnal and diurnal mammals demonstrate alternating bouts of rapid eye REM sleep and NREM sleep [52]. In nocturnal mammals, sleep bouts are usually fragmented and involve the alternation of these two categories, with these bouts occurring more so in the day than at night. Whereas, in diurnal humans, sleep structure is different whereby, sleep encompasses one primary bout in which; NREM is of greatest frequency in the beginning of the cycle, REM is more common at the end of the cycle, each sleep bout lasts approximately 8-hours, and full sleep cycles occur roughly every 90–110 min [37].

Feeding rhythms and the sleep-wake cycle

The foods we eat and when we choose to eat them are major regulating factors of the sleep-wake cycle [38, 53]. The timing and composition of meals are one of many “zeitgebers”—environmental time cues, that can entrain peripheral systems, with animal studies demonstrating that food availability influencing circadian rhythms [54–56]; however, the brain network that controls this process has not yet been identified. Food intake has the ability to uncouple the SCN’s control of peripheral clocks in the liver, adipocytes, pancreas, and GI tract [57, 58]. Production of hormones including melatonin and cortisol play an important role in the sleep-wake cycle and are considered to be nutrient sensitive, oscillate on a circadian basis, and are therefore regulated by feeding and fasting cycles [59]. During periods of arousal, cortisol peaks, preparing the body for energy expenditure by inhibiting the production of the nocturnal hormone, melatonin [60]. Furthermore, during early morning, early afternoon, and late afternoon—i.e. breakfast, lunch, and dinner—ghrelin is released to regulate energy homeostasis by signaling the body to increase appetite and food intake [61]. Adiponectin, a regulator of lipid and glucose metabolism is similarly secreted in the morning and dissipates in the early evening [57]. Insulin is also produced in the afternoon and together, adiponectin and insulin, play an important role in preventing fat accumulation and increasing glucose breakdown [62, 63]. Importantly, melatonin oscillators are the major regulator of the sleep-wake cycle with its secretion peaking during periods of darkness [64]. Moreover, orexin, a key neurotransmitter involved in arousal and feeding behavior is controlled by the SCN and regulates glucose levels by stimulating glucose production in the liver [65] and its uptake into muscle [66, 67].

Considering this, the times at which food intake and cessation occurs, as well as when sleep and wake occur, can have a bidirectional impact on their oscillators and appropriate functioning. Furthermore, poor sleep patterns, jet lag, and shift-work have all been found to disrupt the circadian regulation of the sleep-wake cycle and the feeding-fasting cycle [54, 68]. Rodent studies have demonstrated that feeding during periods of rest led to alterations in the physiology of peripheral circadian clocks including the liver and subsequently demonstrated altered rhythmic expression of clock genes in the hypothalamus, as well as, desynchronization of hepatic and muscle clock, and metabolic gene expression [66, 69]. Food intake at unnatural times has been shown to increase insulin resistance and hypertension due to the intestinal rhythms of nutrient absorption being low during the resting phase [70, 71]. Furthermore, mice that were fed during their wake periods gained less weight than those fed during their usual sleep periods, demonstrating a metabolic influence of feeding schedules [72]. Therefore, because there is a complex interaction between diet, sleep, and the microbiome, brain development over the lifespan can only be understood by examining these factors in combination.

Gut Microbiome Development and the Sleep-Wake Cycle

Infancy

The brain-gut-immune axis in infancy

The early postnatal period is permeated with critical developmental windows characterized by increased sensitivity and heighted plasticity that are associated with rapid changes in microbial and neuronal organization [73]. During this critical period, systems are highly responsive to environmental stimuli including nutrition [74], social contact, and stressors [75]. These environmental influences assist in the appropriate development of brain, intestinal, and immune systems and may induce long-term influences on behavior [3, 76]. These factors or cues can act to optimize the development of the entire system, such as the window of microbiota plasticity that has been identified acutely after birth [3]. Conversely, the disruption or dysregulation of the microbiota at certain developmental windows has the potential to negatively impact appropriate functioning and regulation of the entire system [3]. Interestingly, current research suggests that sensitive periods for development of the microbiota-gut-brain axis may actually coincide with sensitive periods of neurodevelopment and maturation of the sleep-wake cycle [77]. Given that neuronal development involves a complex balance of both genetic and environmental factors, disruption of these processes also has the potential to alter developmental trajectories and predispose individuals to neurodevelopmental disorders later in life [78, 79]. Preclinical studies have recently highlighted the influence of the early gut microbiota on neurodevelopment, during sensitive periods, and its ability to induce long-lasting effects on neuronal function [77, 80, 81] (see Figure 2).

Figure 2.

Illustrative representation of the development of the brain, immune system, gut microbiota, and sleep-wake cycle in parallel from the prenatal phase through adolescence to senescence, indicating vulnerable periods/critical windows of development in which there is heightened plasticity that can alter the trajectory of development and increase risk of pathologies.

Sleep in infancy and the developing brain-gut-immune axis

The appropriate development of the gut microbiota and sleep patterns are key determinants of an individual’s health across their lifespan. Very rapidly after birth, the microbiota is colonized [82]. The initial colonization and composition of the gut microbiota and sleep patterns are largely dependent on various factors, including; mode of delivery (cesarean vs. vaginal birth) and feeding (formula vs. breast milk), which can fundamentally modify the microbiome profile and the sleep-wake cycle of an individual later in life [82]. For example, in the first years of life, circadian rhythms are being formed and sleep patterns are developing [83, 84]. In this early period, sleep quality, duration, and timing have a large impact on the development of adverse health outcomes, mental wellbeing, growth, and adiposity later in life [83, 85, 86]. An infant’s sleep duration is maximal during this time [87], as they spend up to 16 h of their day sleeping with more rapid cycling between sleep stages [37]. This significant time dedication to sleep in infancy is thought to facilitate many of the critical neurodevelopmental processes. At this time point, newborns present with a microbiome that is low in diversity and dominated by anaerobic bacteria from phylum such as, Proteobacteria and Actinobacteria [88]. Studies of genetically heritable gut microbiota suggest that only a small proportion of gut bacteria is heritable, with environmental factors playing a more dominant role in microbiota heterogeneity [89]. Bacterial colonization patterns in the gut at this time influence the function of the BBB and gut epithelial barrier, including the development of the immune system [90].

Of importance for this review, appropriate bacterial colonization, immune function, and neurodevelopment during the first year of life are contributing factors to the development of sleep-wake behavior including developmental sleep problems [91]. By 3 months of age, the sleep-wake rhythm is thought to be “established” [92]. During this time the synchronization of sleep and wakefulness to light/dark phases, REM and NREM, and the alteration and frequency between these phases during sleep are being consolidated [47, 93]. Therefore, any disruptions to the gut-brain-immune axis during this time may cause developmental issues within the SCN, predisposing infants to sleep and mental health disorders later in life [74].

Breastfeeding in particular plays a large role in shaping the gut microbiota and immune system postnatally. Maternal breastmilk assists in promoting the colonization and maturation of the infant microbiota, through an increase in symbiotic and commensal gut microbes [94, 95], and further assists in the development of the immune system [96, 97]. Interestingly, breastmilk has also been found to influence sleep patterns and sleep-wake cycle development [98]. Specifically, tryptophan in breastmilk stimulates the production of melatonin by the pineal gland [98]. Cubero et al. [98], demonstrated that breastfed infants had greater sleep times overall and longer sleep bouts compared to formula-fed infants. Furthermore, nucleotides such as purine adenosine 5′ monophosphate (5′GMP) and pyrimidine-uridine 5′ monophosphate (5′UMP) in breastmilk are controlled by circadian rhythms and peak at night [99]. Formula enriched with 5′GMP, 5′UMP, and tryptophan improved sleep parameters in infants including sleep duration, assumed sleep, and wake periods [100, 101], indicating that not only the type of nutrition is important during this time, but the timing also plays an important role in developing the sleep/wake cycle. Given that the gut microbiota is also involved in the production of tryptophan [102] and the proper development and function of brain structures involved in maintaining the sleep-wake cycle [76, 90], we postulate that it plays additional roles in the development of sleep patterns.

During the neonatal period, the intestinal immune system undergoes important changes. After birth, fetal-derived T cells, myeloid cells, and innate lymphoid cells (ILCs) are replaced progressively overtime by other cells that are produced in the thymus and bone marrow [103]. At this time of gut microbial colonization, the microbiota induces the production and proliferation of regulatory T cells (T_regs), ILCs, and isolated lymphoid follicles, that are involved in anti-microbial IgA production [103]. These immune mediators are also under circadian control [104]. T cells and ILCs peak during early sleep while anti-inflammatory cytokines peak during wakefulness [50, 105]. Both clinical and preclinical studies have found negative effects associated with antibiotic administration during the neonatal period, as exhibited by an increased vulnerability to allergies originating in the gut (e.g. food intolerances) and lungs (e.g. asthma) later in life [103, 106]. Furthermore, when GF mice were exposed to microbiota prior to weaning, but not after weaning, they exhibited reduced vulnerability to inflammatory allergies later in life [103, 107]. Therefore, development of the gut microbiota, brain, and immune system are largely vulnerable to disruption in the early stages of life, particularly during weaning.

Of note, sleep deprivation is a significant adverse early life experience that has also been associated with gut microbiome-induced inflammation, which can result in an increase in opportunistic pathogenic bacteria [108]. Short sleep durations (sleep fragmentation) are associated with tight junction impairments between intestinal epithelial cells, that permit LPS and PGN to enter systemic circulation, increasing immune system activation, and metabolic dysfunction [38, 109, 110]. Specifically, minimal sleep with frequent awakenings during infancy has been associated with an increased risk of asthma and obesity later in life [38, 111, 112].

The introduction of solid foods and the overlapping relationship to the development of the gut microbiome and feeding-fasting rhythms

At weaning, a surge in microbiota colonization occurs as a result of the introduction of solid food and more dietary variation [103]. The microbiota enters an unstable sensitive phase because of this sudden change, activating an inflammatory response known as the “weaning reaction” [103]. The weaning reaction is known as the first extreme immune response to the colonizing gut microbiota post-birth [103]. It is a restricted window of plasticity that is characterized by high levels of pro-inflammatory cytokines, such as TNF-α (tumor necrosis factor alpha), that gradually decline in the following weeks [103]. This weaning reaction overlaps with and influences the appropriate development of the immune system, a key pathway in the gut-brain axis. Importantly, perturbation at this time, by early life stress, environmental factors, poor sleep, inadequate nutrition, or antibiotic use increases an individual’s susceptibility to inflammatory pathologies later in life [103, 106, 113]. Moreover, the type of nutrients consumed and the timing they were consumed in relation to sleeping times influence sleep patterns [47, 114]. Disruptions to development during the weaning reaction may lead to pathological imprinting on the immune system that predisposes risk for inflammatory disorders when exposed to later immune challenges [3]. This has been demonstrated in GF studies, where recolonization with a “normal” microbiota postweaning is effective at restoring immune, brain function, and behavioral deficits, but recolonization at later timepoints is not [77, 80, 115]. Therefore, within the weaning reaction window of plasticity, maturation of the microbiota is important for the healthy development of the immune system [103].

In humans, a similar window of plasticity has been identified when children transition from breastfeeding or formula to solid foods. This induces a shift from the simple environment of Bifidobacteria microbes that mainly function to metabolize human milk oligosaccharides from breastmilk to a diverse microbiota rich in Bacteroides that aid in the metabolism of starches found in a more complex diet [94]. Although the introduction to solid foods induces changes in microbial communities, it is the gradual cessation of breastfeeding that causes the greatest effect to the microbiomes maturation and subsequently flora diversity [94]. After 2–3 years of life, a complex adult-like microbiota is established [90] and the microbiota is dominated by Bacteroides, Prevotella, Ruminococcus, Clostridium, and Veillonella bacteria [116]. Consumption of omega-3 polyunsaturated fatty acids (PUFAs) during this time has been shown to play a role in the development of the CNS, the immune system, and sleep behavior [47, 117]. The fact that PUFAs increase bacterial diversity, reduce pathogenic bacterial species [118], promote an anti-inflammatory state, and are associated with healthier sleep patterns [119], provides further evidence that the microbiota-gut-brain axis plays an important role in the sleep-wake cycle.

Childhood and adolescence

The developing brain-gut-immune axis in childhood and adolescence

During childhood and adolescence, the brain and gut microbiota are sensitive to environmental stimuli as they are undergoing critical development. Refinement and elimination of synapses characterize this phase of neurodevelopment as neural circuits begin to be strengthened and consolidated to adult levels [110]. However, minimal literature is currently available regarding the state and growth of the microbiota at this time point. Some studies have suggested that postweaning the microbiota becomes stable, however, other studies have proposed that continuous changes occur in the microbiota throughout childhood and into adolescence [97]. We do know that in early childhood there is a gradual increase in anaerobic species and a decrease in the number of aerobes and facultative anaerobes [120]. The gut microbiota is also vulnerable to environmental insults, such as the use of antibiotics and sleep deprivation which can result in dysbiosis and have negative impacts on neurodevelopment [76]. Rodent studies have demonstrated that early-life vancomycin administration in male rat pups resulted in long-lasting alterations to CNS pathways associated with heightened visceral pain sensitivity [121]. Modulation of the microbiome by dietary manipulations of fat, fiber, and protein content during the juvenile-adolescence period has structural effects on white matter integrity within cortical regions measured by diffusion tensor imaging, and these changes were associated with populations of Roseburia, a butyrate-producing Gram-positive Firmicute bacteria [122]. Moreover, colonization of the gut microbiome of GF adolescent mice with fecal microbiome transplants (FMTs) from ADHD patients resulted in altered white matter integrity within the hippocampus and internal volume 28 days after the transplant, as well as ADHD/anxiety-like behavioral deficits [123]. However, how the gut microbiome develops in childhood and adolescence, as well as how it influences the brain-gut-immune axis during these times are largely understudied.

The sleep-wake cycle and developing brain-gut-immune axis in childhood and adolescence

As sleep is essential for healthy immune, gut microbiome, and neurodevelopment in childhood and adolescence, alterations to sleep patterns and quality may negatively impact development. Typically, total sleep duration in childhood (6 to 12 years old) [124] is 9.2 h and 8.1 h in adolescents (13 to 18 years old) [125]. Sleep duration shortens throughout childhood and adolescence, whereby a progressively later bedtime known as an evening chronotype is favored due to a delay in the sleep phase of the sleep-wake cycle [125, 126]. This gradual shift toward an evening chronotype typically peaks earlier in females, and reverses when changing back to a morning chronotype into adulthood [127]. Moreover, in childhood wake times tend to be more stable, whereas, in adolescence, wake times become progressively later with adolescents typically “sleeping in” on the weekends [128]. Adolescents commonly lack sleep during the school week and in an attempt to catch up will have extended sleep durations on weekends, causing disordered circadian clocks called “social jetlag” [57]. Social jetlag has been associated with increased risk for obesity and the preference for consumption of more sugary beverages in adolescent populations [129].

This sensitive period is also critical for neurodevelopment and is typically associated with the onset of many neuropsychiatric conditions [130]. Poor sleep quality is similarly associated with reduced socioemotional health and poorer mental health outcomes, such as anxiety and depression [131]. Cortisol levels also increase throughout adolescence, and females experience higher cortisol levels in the morning than males [132]. This may be due to changes in sex hormones during puberty [132] as well as significant shifts in gut microbiome development [90]. Adolescent neurodevelopment involves further synaptic pruning, thinning of cortical gray matter, increased myelination, and the consolidation of neuronal connections, all of which result in a period of heightened plasticity particularly within the prefrontal cortex (PFC) [76]. Remarkably, studies have hypothesized the involvement of the gut microbiota in the development of PFC circuitry [133]. The gut microbiome is proposed to play a critical role in the appropriate and dynamic regulation of myelin-related genes during brain development, with implications for cortical myelination at an ultrastructural level. GF mice were shown to have hyper-myelination within the PFC, along with upregulated expression of genes involved in myelination, suggesting that the gut microbiome is vital for the refinement of PFC white matter, which was reversed following microbial colonization [134]. Importantly, the PFC is highly vulnerable to the effects of sleep loss [135], while interference of the sleep-wake cycle has been associated with changes to the structure, function, and diversity of the gut microbiota [136]. Specifically, an individual’s sleep patterns may be affected by alteration to intestinal permeability, immune system activation, inflammation, energy harvest, and the diversity of the gut microbiota [2]. Puberty itself has been associated with alterations to the sleep-wake cycle, driven by changes in the gut microbiota composition, refinement of neurotransmitter levels, as well as alterations to behavioral patterns [137, 138].

Feeding-fasting rhythms and the developing gut-microbiota-brain-immune axis in childhood and adolescence

The composition and functionality of the gut microbiota have been found to fluctuate during the 24 h light-dark cycle and are largely influenced by feeding and fasting [139]. Moreover, the sleep-wake cycle and gut microbiota both have the ability to regulate metabolism [38, 140]. During childhood and adolescence, colonization by beneficial (e.g. bifidobacteria) or pathogenic (e.g. Escherichia coli) bacteria in the gut may modulate the development of the HPA axis along different trajectories, with either beneficial or detrimental consequences in relation to the sleep-wake cycle [90]. Furthermore, since the gut microbiota is involved in the production of serotonin, GABA, and norepinephrine, alterations to the gut microbiome may lead to sleep-wake cycle impairments [38]. General dysbiosis of the gut microbiota during adolescence, as a result of sleep loss or reduced sleep quality, leads to improper functioning and development of the gut immune system and gut barrier, resulting in dysregulation of microbiota-gut-brain signaling [90]. Specifically, the microenvironment of the gut, as well as its permeability, microbiome diversity, visceral sensation, brain-gut interactions, immune regulation, and neuroendocrine function are dependent upon and modulated by diet [141]. Diet can therefore produce beneficial or detrimental effects on the host’s health [142]. Malnutrition during childhood delays microbiota development, which is only partially restored by dietary intervention [74]. Moreover, the quality of diet consumed during this critical window is essential in the healthy development of the gut microbiota, brain, and sleep-wake cycle. Diets high in saturated fats have been associated with a lower abundance of anti-inflammatory microbial metabolites (i.e. SCFAs), an increase in pathogenic bacteria, increased intestinal permeability, and subsequently disruptions to the sleep-wake cycle [38, 143]. Disruption to microbial metabolites also alters host metabolism causing diet-induced obesity [143]. Importantly, gastric vagal mechanoreceptors have been found to have their own circadian rhythm and may regulate food intake during the day, which when disrupted due to sleep fragmentation alters food intake [144]. Sleep fragmentation has also been associated with increased food intake, obesity, and gut dysbiosis [145] (see Figure 3). Considering feeding-fasting varies in a circadian manner, altering the time at which these occur also can alter host metabolism [143]. Diet-induced dysbiosis is also associated with increased hunger cues and cravings, and reduced physical activity that further perpetuates alterations to the gut microbiota [38]. This is similarly found in those that experience sleep loss [38]. Specifically, the gut microbiota is involved in the regulation of fat storage, with dysbiosis being associated with insulin, glucose, and hepatic lipid metabolism impairments [146]. This may be regulated through gut microbiota production of certain neurotransmitters (serotonin and GABA) or SCFAs, which modify appetite through the vagus nerve or immune-neuroendocrine pathways [146]. Given that adolescents often engage in poor food choices—particularly over-consuming palatable high-fat and high-sugar junk foods, and lack adequate sleep during this critical period of microbiota and neurodevelopment [147], they may consequently be at risk for gut dysfunction, microbiome imbalance, increased HPA axis activation, and a range of other comorbidities [74, 141, 142].

Figure 3.

Factors that influence the bidirectional relationship between the brain-gut-immune axis and sleep-wake cycle. (A) Alterations to clock gene function [250–253], sleep-wake cycles, and their consequential factors induce changes to microbiome composition, resulting in decreased SCFA production and increased LPS translocation into the systemic circulation. (B) These triggers increased immune cell activation resulting in increased pro-inflammatory cytokines. (C) Changes in the expression of pro-inflammatory immune factors and microbial diversity consequently induces gut dysbiosis allowing for increased gut epithelial permeability and overactive translocation of molecules inside the gut lumen and out into the systemic circulation. The summation of these changes can reduce hormones involved in the sleep/wake cycle including; GABA, 5-HT, norepinephrine, and melatonin. This in turn alters vagal nerve function and the SCN and creates a stress/immune response in which cortisol is elevated and pro-inflammatory cytokines are elevated, altering HPA axis functioning. Therefore, the sleep-wake cycle is disrupted, resulting in sleeping disorders and altering the appropriate functioning of bodily systems and metabolic processes which can further exacerbate gut microbiome dysfunction.

Adulthood

Brain-gut-immune axis in adulthood

In adulthood, the gut microbiota becomes stable but more diverse [148]. Specifically, there are an increase in methane-producing (methanogenic) bacteria [149], and a gradual decrease in Bifidobacterium [97]. Within the brain, further synaptic pruning and refinement occur whereby connections are strengthened and neural circuits are consolidated [110]. Rodent studies have demonstrated that the BBB and gut microbiota have a life-long connection as BBB and gut barrier permeability dysfunctions can be restored by recolonization even in adulthood [150]. When the microbiome has been depleted in adult mice, alterations in gene expression, neuronal activity, and dendritic spine organization have been observed [151]. It has been suggested that the adult gut microbiota is more resilient to environmental factors, such as stress and antibiotic administration, and can therefore easily restore itself and prevent dysbiosis [152]. However, even short-term antibiotic administration in adult mice has been associated with the up-regulation of BDNF expression in the hippocampus and surprisingly a decrease in anxiety [133, 153]. Although there does not appear to be a window of vulnerability, changes in the gut microbiota can induce changes to the adult brain and sleep behavior [76, 154].

Sleep-wake cycle and the gut microbiome in adulthood

Appropriate sleep-wake cycles in adulthood, as well as the functionality of the brain-gut-immune axis, are largely dependent on early life experiences and environmental factors [3, 76]. Compared to infancy, sleep duration in adulthood is significantly reduced (approximately 8 h a day) and is comprised of only 2 hours of REM [37, 126]. To understand the bidirectional relationship between the adult brain-gut-immune axis and the sleep/wake cycle, studies have capitalized on sleep deprivation, shift work, and jetlag paradigms.

In a study conducted by Gao et al. [155], adult mice that were subjected to sleep deprivation demonstrated reduced melatonin secretion, increased norepinephrine, elevated levels of CRP and pro-inflammatory cytokines (TNF-α and IL-6 [interleukin 6] reduced levels of anti-inflammatory cytokines, and altered gut mucosal structure. Sleep deprivation has also been associated with lower bacterial diversity, as well as specific reductions in Akkermansia and Bacteroides [155]. Ma et al. [156], demonstrated reduced Akkermansia, in sleep-deprived adult male rats which have been associated with stress-related disorders [157], while Polidarova et al. [158], similarly found circadian disruptions to sleep in adult rats created a pro-inflammatory state in the colon.

Peptidoglycan recognition proteins (PGRPs), pathogen detecting innate immune proteins, also increase following sleep deprivation in the hypothalamus and brain stem of adult rats, suggesting that PGRPs may potentially be involved in the regulation of the sleep-wake cycle [159]. Disruption to the regular oscillations of the gut microbiota due to inflammation may also further impact the SCN whereby the gut microbiota can impact the sleep-wake cycle by the circadian clock [160]. Adults that experience sleep disorders or changes to their usual sleep-wake cycle are similarly found to develop psychological stress, which in turn perpetuates intestinal dysbiosis [136]. Interestingly, supplementation with melatonin has been found to increase beneficial bacteria (Lactobacilli spp.) in stressed mice, while also restoring appropriate sleep-wake patterns, and improving GI health and gut microbiota dysbiosis in patients with inflammatory bowel disease [155, 161]. Leaky gut is also a potential complication for those that experience chronic stress and sleep deprivation, given that persistent stress and reduced sleep times disrupt the intestinal barrier leading to systemic circulation of LPS [162, 163]. Probiotic intervention of Lactobacillus, Bifidobacterium, and Enterococcus can reduce pro-inflammatory cytokine levels and reduce the damage caused by LPS systemically [164]. Considering melatonin inhibits pro-inflammatory downstream processes by LPS [165], it is possible that the supplementation with certain gut bacteria may assist in melatonin production, thereby reducing this inflammatory process.

Appropriate rhythmic release of neurotransmitters plays an important role in the regulation of the sleep-wake cycle. Certain bacteria, such as those of the genera Escherichia or Streptococcus synthesize 5-hydroxytryptamine (5-HT/Serotonin), while bacteria of the Escherichia and Bacillus can synthesize GABA. Depletion of the gut microbiome in adult male mice altered levels of these neurotransmitters and hormones responsible for regulating the sleep-wake cycle [166]. Ogawa et al. [166], found compositional alterations to the microbiota along with reductions in serotonin and vitamin B6, which is important in the synthesis of neurotransmitters such as serotonin, GABA, and dopamine. Furthermore, mice with antibiotic-induced microbiome depletion had reduced NREM during their sleep phase and increased NREM and REM during their wake phase [166]. This has been attributed to alterations in gut bacterial composition and depleted vitamin B6, both of which may alter dopaminergic and serotonergic neurotransmission, which are important for the regulation of arousal [166]. Considering a majority of serotonin is synthesized in the gut by EECs and gut bacteria, this provides further evidence for the role of the gut microbiome in the bidirectional relationship between the gut-brain-immune axis and the sleep/wake cycle.

Exciting new literature is beginning to emerge regarding the role of space travel on the sleep-wake cycle and the microbiome in adulthood. Numerous studies have demonstrated that astronauts experience significant modifications to their sleep-wake rhythms [167, 168], specifically exhibiting reduced latency to first REM episode, slow-wave sleep redistribution, as well as shorter and more fragmented sleep bouts [169]. These disruptions were hypothesized to be due to altered circadian zeitgebers, deficiencies in Process S, or a desynchrony of gene expression in the circadian feedback loop [170]. While a rodent study found that clock gene expression in peripheral tissue was asynchronous in mice who experienced a 37-day spaceflight [171], a study in drosophila failed to find disruptions in the circadian clock system [172]. Remarkably, a research group from Harvard Medical School was able to entrain participants to 24.65 solar day-night cycles [173], precisely what is required for synchronization to the Mars circadian day. Given the strong evidence for space-travel-induced disruptions to the sleep-wake cycle further studies are needed to untangle these discrepancies and determine the underlying cause. Coinciding with interest in the sleep-wake cycle of astronauts, researchers have also begun to examine the impact of space flight on the microbiome. Given the strong link between diet and microbiome composition, many speculated that the microbiome would be diminished during space travel, and treatment with probiotics would be necessary [174, 175]. Interestingly, however, a study of long-term space travel (approximately 6-month missions) actually identified an increase in Shannon alpha diversity and richness of the GI microbiome [176]. This was consistent with mouse models of hypergravity that also identified an increase in microbiota enrichment [177]. As we approach the new frontier of commercialized space travel via companies such as Space X and Virgin Galactic, understanding the relationship between microgravity, sleep, and the microbiome becomes even more important.

The association between disrupted sleep-wake cycles, the gut microbiome, and feeding-fasting rhythms in adulthood

Circadian rhythm misalignment in adulthood is commonly induced by sleep loss, shift work, evening chronotypes, and jet lag [68]. Individuals with altered sleep-wake cycles due to jet lag or shift work are at an increased risk for metabolic disease, obesity, cancer, and type II diabetes [38, 178]. This may be due to the disruption of feeding-fasting patterns which thereby alter the composition of the gut microbiota [38]. Individuals that engage in shiftwork commonly report poor sleep quality, shorter sleep durations, and day-time fatigue. These sleep-related disturbances place significant stress on the body, increase systemic inflammation, and subsequently alter HPA axis functioning, gut microbiota composition, and GI permeability [179]. Gut microbiome composition alterations and sleep-wake cycle disturbances have been associated with metabolic syndromes independently, however, the exact mechanisms remain unknown. Adult mice with disrupted circadian clocks were found to have increased GI permeability and alterations to gut microbiota composition that were associated with fatty liver disease [180]. Shiftwork and feeding during dark periods have been associated with altered microbiota composition and metabolic disorders [69, 179]. Shift workers typically consume food at unnatural times of day and this is hypothesized to contribute to their increased risk for metabolic disorders [69]. Food absorption, gastric emptying, and digestion are all regulated by clock genes that exhibit their own circadian rhythms [181]. Moreover, adult mice that were fed a high-fat diet during their sleep phase, emulating shift-workers and jet lag, exhibited impaired diurnal rhythmicity, weight gain, microbiota dysbiosis, and increased glucose intolerance [69, 182]. Food absorption, gastric emptying, and digestion are all regulated by clock genes that exhibit their own circadian rhythms [181]. Moreover, adult mice that were fed a high fat diet during their sleep phase, emulating shift-workers and jet lag, exhibited impaired diurnal rhythmicity, weight gain, microbiota dysbiosis, and increased glucose intolerance [182] . Astonishingly, treatment with broad-spectrum antibiotics reverted the weight gain and altered the glucose intolerance [182]. However, it remains unknown how the SCN interacts with the peripheral clocks to signal downstream processes that result in microbiota dysbiosis.

Senescence

The aging brain-gut-immune axis

The aging process is characterized by the decline of pathophysiological function including; reduced intestinal motility and alterations to digestion, such as changes in salivary and hydrochloric acid production [183, 184], increased BBB permeability [185], and reduced functionality of the immune system, also known as immune-senescence [186]. Combined, these physiological changes induce chronic low-grade inflammation, termed “inflammaging” [185, 187, 188], and reduced functionality of the immune system, also known as immune-senescence [186]. Combined, these physiological changes induce chronic low-grade inflammation, termed “inflammaging” [187, 188]. As described in Figure 3, the bidirectional relationship between the gut microbiota and other pathways may therefore be compromised. Throughout the lifespan, including in the senior years, the gut microbiome goes through dynamic changes as it integrates and responds to environmental stimuli [188, 189]. However, as functioning of the immune system declines with age this delicate balance is compromised [188]. Age-related dysbiosis is consistently reported in the literature, whereby aging is characterized by a reduction in Bifidobacterium and Clostridium and an increase in Proteobacteria and Enterobacteriaceae [190]; opportunistic bacteria that induce pathology by overtaking commensal bacteria [188, 189, 191, 192]. The immune system plays an important role in regulating these changes to the gut microbiome by altering the architecture of the gut microbiome, allowing for commensal bacteria to flourish and diminish more harmful bacteria [188]. However, as functioning of the immune system declines with age, this delicate balance is compromised [188]. Age-related dysbiosis is consistently reported in the literature, whereby aging is characterized by a reduction in Bifidobacterium and Clostridium and an increase in Proteobacteria and Enterobacteriaceae [190]; opportunistic bacteria that induce pathology by overtaking commensal bacteria [191, 192]. Furthermore, aging is associated with a gradual reduction in SCFA production [193] and a reduction to structural integrity of the GI barrier. Aging gradually slows down intestinal motility, reduces epithelial barrier formation, and the immune defense at the GI mucosa [194], potentially allowing for the translocation of microbes into the systemic circulation, exacerbating low-grade inflammation in the periphery and within the intestines [188, 193] and a reduction to structural integrity of the GI barrier. Aging gradually slows down intestinal motility, reduces epithelial barrier formation, and the immune defense at the GI mucosa [194], potentially allowing for the translocation of microbes into the systemic circulation, exacerbating low-grade inflammation in the periphery and within the intestines [188].

Aging disrupts the delicate balance of pro- and anti-inflammatory cytokines [195], resulting in an increase of pro-inflammatory cytokines, including IL-6, CRP, and TNF-α [196, 197]. This chronic inflammatory state, predisposes older individuals to wide array of age-associated diseases, including neurodegenerative disorders such as Alzheimer’s [188], cardiovascular disease [198], osteoarthritis, and chronic pain [196, 197, 199]. This chronic inflammatory state, predisposes older individuals to wide array of age-associated diseases, including neurodegenerative disorders such as Alzheimer’s [188], cardiovascular disease [198], osteoarthritis, and chronic pain [199]. It is widely hypothesized that the gut microbiome has a regulatory role in the general decline in function of the immune system in older age and this chronic inflammatory state [190, 200]. In the brain, alterations to neurotransmitters levels [201], HPA axis functioning [202], cognition, and vasculature occur [203]. Changes in serotonin, dopamine, and BDNF, as well as overactive HPA axis activity is hypothesized to be associated with changes in gut microbiota composition as well as immune dysregulation [204]. Recent studies have further demonstrated the involvement of the gut microbiome in healthy aging and immune system regulation. Boehme et al. [205], found that FMTs from young mice (yFMT) into aged mice attenuated this pro-inflammatory state peripherally and in the brain, enhancing memory and cognition as well as increasing microbial metabolites including GABA. Other than immune system dysregulation, sleep deprivation, circadian disturbances, and gut dysbiosis have also been identified as factors that initiate and maintain inflammaging [190, 200, 206]. In the brain, alterations to neurotransmitters levels [201], HPA axis functioning [202], cognition, and vasculature occur [203]. Changes in serotonin, dopamine, and BDNF, as well as overactive HPA axis activity, are hypothesized to be associated with changes in gut microbiota composition as well as immune dysregulation [204]. Recent studies have further demonstrated the involvement of the gut microbiome in healthy aging and immune system regulation. Boehme et al, [205] found that yFMT into aged mice attenuated this pro-inflammatory state peripherally and in the brain, enhancing memory and cognition as well as increasing microbial metabolites including GABA. Other than immune system dysregulation, sleep deprivation, circadian disturbances, and gut dysbiosis have also been identified as factors that initiate and maintain inflammaging [206].

The breakdown of the sleep-wake cycle and the gut microbiome in later life

The organization of the sleep-wake cycle and sleep architecture undergo critical changes in the later years of life [207]. Aging is associated with decline in wake-promoting neurons in the cortex [208]. Wake-promoting neurons include orexinergic, noradrenergic, cholinergic, histaminergic, and serotonergic, all of which fire action potentials during waking and are at a reduced action potential firing frequency during sleep [208, 209]. With increasing age, REM sleep decreases [210] and sensitivity to sleep-wake cycle changes, such as shiftwork, jet lag [211], and stress hormones are increased [212]. Homeostatic and circadian processes are also altered in aging, independently and together as a sleep regulatory process [207]. Homeostatic control of sleep is altered in aging as individuals experience lighter NREM stages of sleep [207]. The loss of orexin neurons in the lateral hypothalamus is believed to disrupt this sleep-wake regulation [213]. Compared to younger individuals, the aging demographic demonstrate an earlier chronotype, whereby they go to bed earlier and wake up 1.5 hours earlier [214]. Aging also associated with alterations to circadian timing whereby aging promotes an earlier circadian rhythm of melatonin and cortisol secretion [214]. However, the peak level of melatonin in older individuals is later in the sleep phase [215]. These changes in circadian clock function may be due to the breakdown of SCN networks, reducing the clock signals that are transferred to peripheral oscillators [216]. Furthermore, increased age promotes sleep latency, number of wakings in the night, and reductions in total sleep duration, causing altered sleep quality and efficiency [217]. How exactly the gradual decline in these sleep-wake processes affect the aging gut microbiome is currently completely unexplored. The organization of the sleep-wake cycle and sleep architecture undergo critical changes in the later years of life [207]. Aging is associated with decline in wake-promoting neurons in the cortex [208]. Wake-promoting neurons include orexinergic, noradrenergic, cholinergic, histaminergic, and serotonergic, all of which fire action potentials during waking and are at a reduced action potential firing frequency during sleep [208, 209]. With increasing age, REM sleep decreases [210] and sensitivity to sleep-wake cycle changes, such as shiftwork, jet lag [211], and stress hormones are increased [212]. Homeostatic and circadian processes are also altered in aging, independently and together as a sleep regulatory process [207]. Homeostatic control of sleep is altered in aging as individuals experience lighter NREM stages of sleep [207]. The loss of orexin neurons in the lateral hypothalamus is believed to disrupt this sleep-wake regulation [213]. Compared to younger individuals, the aging demographic demonstrate an earlier chronotype, whereby they go to bed earlier and wake up 1.5 h earlier [214]. Aging also associated with alterations to circadian timing whereby aging promotes an earlier circadian rhythm of melatonin and cortisol secretion [214]. However, the peak level of melatonin in older individuals is later in the sleep phase [215]. These changes in circadian clock function may be due to the breakdown SCN networks, reducing the clock signals that are transferred to peripheral oscillators [216]. Furthermore, increased age promotes sleep latency, number of waking’s in the night, and reductions in total sleep duration, causing altered sleep quality and efficiency [217]. How exactly the gradual decline in these sleep-wake processes affect the aging gut microbiome are currently completely unexplored.

Alterations to the sleep/wake cycle caused by aging may further perpetuate gut dysbiosis and promote an inflammatory state. Specifically, melatonin gradually decreases over the lifespan and may therefore be associated with reduced sleep efficacy and quality in older individuals [218]. Melatonin supplementation has been found to increase beneficial bacteria [219]. Therefore, reductions in melatonin later in life may be due to disruptions in the delicate balance between beneficial and pathogenic bacteria. This highlights a possible safe therapeutic approach that could be implemented to attenuate some sleep problems that are common in older individuals. However, further studies are required to understand the aging gut microbiota composition and its association with the sleep/wake cycle.

Age-related dysbiosis has been linked to inflammaging and immunosenescence. A pro-inflammatory state within the gut and systemically is associated with gut dysbiosis, alterations to HPA-axis functioning, and changes in neurotransmitter production/signaling in structures of the brain involved in regulating the sleep-wake cycle [90]. Given that “leaky gut” is prevalent in aging populations, reductions of Bifidobacterium and increased age-related inflammation perpetuate damage to the intestinal barrier which leads to systemic circulation of LPS [162, 163]. As melatonin levels are reduced at this period of time, melatonin’s ability to inhibit LPS’ pro-inflammatory downstream processes are compromised [165], potentially causing further alterations to the SCN and peripheral clocks. Specifically, when the microbiome of aged mice was transferred into young mice, the researchers identified increased leakage of microbial products, LPS, intestinal inflammation and chronic low-grade inflammation, and consequently increased T-cell activation.

Inflammaging may also be responsible for the gradual decline in energy demand and reduction in metabolic flexibility (cells and tissues ability to alter between fasting to energy storage after feeding) [217, 220]. Specifically, with age, peripheral clocks in the liver and pancreas that are involved in the regulation of glucose homeostasis and lipid metabolism gradually reduce in amplitude [221]. Dysbiosis caused by inflammation is also associated with alterations to serotonergic and GABAergic signaling [222]. The appropriate cyclical release of serotonin during the sleep phase is imperative for the alternation of NREM and REM sleep bouts [223]. Reductions in serotonin may therefore alter the transitions in NREM and REM sleep in older individuals, potentially mediating their sleep efficacy and being a driver of reduced sleep duration commonly seen in aging [207]. Tryptophan gradually decreases with aging and this loss in tryptophan is accelerated by the age-related dysbiosis of certain gut bacteria that are responsible for its synthesis. This age-related reduction in tryptophan further perpetuates dysfunction in sleep-wake rhythms. The precursor to serotonin, tryptophan, is particularly important in adaptive responses, with the dysregulation of the kynurenine arm of the tryptophan metabolic pathway being associated with cognitive impairment, stress reactivity, and GI disorders [222, 224, 225]. The appropriate cyclical release of serotonin during the sleep phase is imperative for the alternation of NREM and REM sleep bouts [223]. Reductions in serotonin may therefore alter the transitions in NREM and REM sleep in older individuals, potentially mediating their sleep efficacy and being a driver of reduced sleep duration commonly seen in aging [207]. Tryptophan gradually decreases with aging and this loss in tryptophan is accelerated by the age-related dysbiosis of certain gut bacteria that are responsible for its synthesis. This age-related reduction in tryptophan further perpetuates dysfunction in sleep-wake rhythms. The precursor to serotonin, tryptophan, is particularly important in adaptive responses, with the dysregulation of the kynurenine arm of the tryptophan metabolic pathway being associated with cognitive impairment, stress reactivity, and GI disorders [224, 225]. Clinical studies involving elderly participants found that consumption of tryptophan-enriched cereal improved sleep efficacy and duration, increased 5-HT levels, and improved anxiety and depression symptoms [226].

Gradual decline in the functionality of the immune system causing chronic low-grade inflammation has also been associated with reductions in certain SCFAs, including butyrate. Butyrate contains anti-inflammatory properties and assists in maintaining the intestinal epithelial barrier [227]. The elderly gut microbiome, however, has reduced capacity to produce SCFAs likely the result of reduced butyrate-producing gut bacteria, such as the Roseburia groups [191]. Decreases in the anti-inflammatory protective functionality of SCFA-producing bacteria may further perpetuate inflammaging [227] causing clock gene disruptions, alterations to the SCN networks, gut-brain axis connections, and induce insomnia symptoms in aged people. The loss of orexin neurons in the lateral hypothalamus [213] and loss of GABA terminals in the SCN, decrease the amplitude and synchrony of circadian rhythms [228]. Recently, it has been suggested that SCFAs act as a link between the gut microbiomes influence on the brain structures associated with the sleep-wake cycle. Gradual decline in the functionality of the immune system causing chronic low-grade inflammation has also been associated with reductions in certain SCFAs, including butyrate. Butyrate contains anti-inflammatory properties and assists in maintaining the intestinal epithelial barrier [227]. The elderly gut microbiome, however, has reduced capacity to produce SCFAs likely the result of reduced butyrate-producing gut bacteria, such as the Roseburia groups [191]. Decreases in the anti-inflammatory protective functionality of SCFA-producing bacteria may further perpetuate inflammaging [227] causing clock gene disruptions, alterations to the SCN networks, gut-brain axis connections, and induce insomnia symptoms in aged people. The loss of orexin neurons in the lateral hypothalamus [213] and loss of GABA terminals in the SCN, decrease the amplitude and synchrony of circadian rhythms [228]. Recently, it has been suggested that SCFAs act as a link between the gut microbiomes influence on the brain structures associated with the sleep-wake cycle [68, 229, 230], however, this has not been investigated in elderly populations.

Sex Differences in the Microbiome and Sleep-Wake Cycle Across the Lifespan

Although research regarding sex differences in the microbiome, particularly as they relate to functioning of the sleep-wake cycle and circadian gene function across the lifespan are limited, we have attempted to provide a summary of the current literature. Baby boys show slight delays in the development of coherent sleeping rhythms relative to girls of the same age [231], with girls spending more total time asleep than boys [232]. There are no substantial sex differences in the microbiomes of children [233], which may be due to dormancy of sex-specific development and small influences of gonadal hormones at this time [234]. Similarly, the microbiomes of male and female mice were indistinguishable at weaning, but sex-specific variations in microbiome composition were evident at puberty and pronounced in adulthood [235]. Microbiome composition is at least in part responsible for testosterone levels as transfer of gut microbiota from adult males to immature females elevated circulating testosterone and protected females from developing type 1 diabetes [235], while castration during puberty eliminates sex differences in the microbiome of mice [236]. Interestingly, probiotic administration during puberty has been shown to have sex-specific efficacy in mitigating emotional dysfunction (depressive-like behaviors in females and anxiety-like behaviors in males) that were associated with inflammatory and stress-related processes in mice [237]. It is therefore plausible that hormone-supported expansion of certain microbial lineages may serve as a positive feedback mechanism that contributes to sexual dimorphisms, specifically in adolescence and early adulthood [236]. Similarly, the changes in chronotype that occur in adolescence are likely driven by pubertal hormone changes. Puberty begins earlier in girls and this coincides with their earlier chronotype shift as well as their peak delay in sleep onset [238, 239]. Although there is scarce literature on the role of sex hormones on the regulation of clock genes, we do know that androgen receptor expression is higher in the SCN of adult males [240]. and male sleep quality and quantity is largely influenced by testosterone levels [241]; while estrogen receptor expression in the SCN is greater in adult females [242], and female sleep quality and quantity is regulated by ovarian hormones [243, 244]. In addition, shift work was associated with alterations to peripheral clock gene expression and increased 17-B-estradiol levels in female Italian nurses [245]. One would therefore hypothesize that CLOCK gene expression is also largely influenced by circulating levels of gonadal hormones.

In support of this idea, the tendency for women to go to bed earlier than men and get more sleep than men, disappears at the onset of menopause [246]. Moreover, as testosterone levels progressively decline with age, sleep impairments increase, manifesting as reduced sleep efficiency, increased time to sleep onset, and a decrease in the number of REM episodes [241]. There are also corresponding sex-dependent changes in the microbiome during this time. The gut microbiome of premenopausal women differs from postmenopausal women, with postmenopausal women exhibiting microbiomes more similar to men; with the ratio of Firmicutes/Bacteroidetes, and the relative abundance of SCFA-producing bacteria, being highest in premenopausal women and lower in postmenopausal women and men [247]. Although limited literature suggests that treating menopause-induced estrogen deficiency with low doses of estrogen and progesterone is beneficial and increases the diversity of the gut microbiome [248], future research into the microbiome and circadian rhythm response to hormone-replacement therapies is desperately needed.

Conclusion and Future Directions

The relationship between the brain, gut microbiome, immune system, and the sleep-wake cycles is extremely complex, and we are only beginning to scratch the surface of understanding the innate intricacies. What is more, this multifaceted network is not static—significantly changing and adapting as we age. We know that sleep plays a vital role in healthy development and successful aging and we are starting to appreciate that a healthy and diverse microbiome is necessary for optimal brain health and function, across the lifespan. Therefore, as we move toward personalized therapeutics, such as FMTs or probiotic interventions, we will not be merely targeting an individual’s genome, but also their microbiome potentially assisting with “healthy ageing.” And to add further complexity, these personalized strategies will significantly change with age; a young person’s medicine, may be an old person’s poison [249]. Furthermore, given what we know about chronopharmacology and the efficacy of the immune system following healthy versus dysfunctional sleep, future research must strive to optimize not only sleep-wake cycles but also the administration regiments of potential therapeutics; should they be delivered when arousal systems peak, when microbiomes show their greatest activity, or when the body is in a state of rest? This is unchartered territory but has the potential to revolutionize the way we sleep, and the way we manage and treat sleep-related disorders. However, there is currently a large gap in our understanding of the mechanisms driving age-dependent changes in the bidirectional relationship between the gut microbiome and the sleep-wake cycle. For example, given the significant sexual differentiation that occurs throughout development, which is accompanied by substantial changes in circulating sex hormones, biological sex is an important variable that should be incorporated into future studies regarding the brain-gut-immune axis and maturation of the sleep-wake cycle. Nevertheless, we believe that future studies will demonstrate that the gut microbiome has been the missing link in understanding and optimizing maturation of the brain, immune system, and the sleep-wake cycle.

Funding

The authors would like to acknowledge the NHMRC Investigator Grant - AP1173565 - to RM.

Disclosure Statement

Financial disclosure: The authors report no financial disclosures related to this manuscript or the work presented.

Nonfinancial disclosure: The authors report no conflicts of interest related to this manuscript or the work presented.