Interactions between the body's daily clock and gut bacteria

The microbiota and the immune system have co‐evolved to maintain homeostasis and help protect our bodies from pathogens.1, 2 Approximately 1000 microbial species reside in the human intestines, encoding a metagenome of trillions of genes, which are over 100 times greater than the human genome, with millions of unique genes.3, 4, 5, 6 The microbiota influence many important aspects of host physiology, including modulating development, maturation and functions of the innate and adaptive immune systems. This has been highlighted in studies with gnotobiotic (mice with a defined microbial community) or germ‐free (GF; no microbiota) animals, which have a more naïve immune system, particularly in mucosa‐associated lymphoid tissues.7 The microbial composition can be modulated by a number of factors, including age, diet and host health (Figure 1). Disruption of the balance between the immune system and the microbiota promotes dysbiosis and increases susceptibility to various health issues including cancers, infections, autoimmune diseases and metabolic disorders.8, 9, 10, 11 Furthermore, microbiota can also modulate responses to immunotherapy, for example anti‐PD1 treatment in cancer.12, 13

Circadian rhythms, referring to daily oscillations in gene activation and repression, as well as biological and physiological processes, can be induced by light, hormones, metabolic cues and the microbiota. The circadian rhythm influences immunity, microbial dynamics and host metabolism. Circadian rhythms are controlled by highly regulated transcription/translation feedback loops (Figure 2) and are reviewed in more detail elsewhere.14 Light is the main inducer of circadian rhythms via activation of the suprachiasmatic nucleus (SCN).15, 16 The SCN comprises ~20,000 specialized neurons within the hypothalamus, which control and co‐ordinate circadian rhythms in exercise, hormones, body temperature and eating. However, additional peripheral rhythms are needed for fine‐tuning the circadian clock, enhancing responses to environmental cues, for example food intake, body temperature and the microbiota. These peripheral rhythms differ from central circadian rhythms in that individual circadian clock components differentially modulate both types of rhythms. Furthermore, peripheral rhythms are subject to different influences which reset individual rhythms and control their outputs. Genes controlling these peripheral rhythms in different tissues also control individual cellular physiology.17, 18 Thus, peripheral rhythms temporally control many aspects of metabolism, including glucose homeostasis, lipogenesis and xenobiotic detoxification.19, 20, 21, 22, 23 These evolutionarily conserved, cell‐autonomous, biological clocks enable organisms to both anticipate, and adapt to, important environmental changes, aiding in their survival. In circadian studies, zeitgeber (ZT) measurements identify the time from the start of the rhythm to the end of the daily oscillation. In most cases, ZT times coincide with the number of hours after light exposure. For example, ZT12 refers to 12 hours after light exposure.

In the intestine, circadian rhythms regulate digestion, including gastric acid production, gut motility and nutrient absorption. Circadian rhythms also affect intestinal stem cell regeneration and mucosal immunity.24, 25, 26, 27, 28 Moreover, the microbiota composition and functions depend on the time of day (Figure 3), giving rise to changes in susceptibility to disease. In this review, we discuss the changes in microbial composition and functions associated with circadian rhythms. We also discuss the mechanistic interactions between the microbiota and the immune system, and how they cross‐modulate daily oscillations. We highlight various associations that require further investigation, which will greatly enhance our knowledge and understanding of the cross‐regulation between microbiota, the immune system and circadian rhythms.

Similar to immune cells (discussed in the other review articles within this commissioned review series), the microbiota exhibit circadian rhythms, which modulate many important functions in the host (Figure 3). The microbial circadian dynamic is strongly associated with food intake and thus nutrient availability. In response to dietary glycans, Firmicutes thrive; however, once these glycans have been metabolized, Firmicutes decline in abundance, allowing Bacteroidetes and Verrucomicrobia to expand in response to accessible host glycans.29, 30, 31 These microbial rhythms promote metabolic homeostasis and can be modulated by food availability, which influences susceptibility to metabolic diseases, as discussed next.

Microbial rhythmicity is strongly influenced by nutrient availability. However, when nutrients were administered only intravenously to mice (no oral intake), microbial rhythmicity was still observed,42 suggesting that host metabolic factors/rhythms, in turn, can also impact microbial rhythmicity. Many host functions are modulated by the circadian rhythm, including the secretion of hormones such as glucocorticoids and neurotransmitters, as well as immune regulation.48, 49 Importantly, immune cells express cell‐autonomous circadian rhythms.50, 51, 52, 53 Circadian rhythms alter immune cell differentiation, such as Th17 cell differentiation, which is regulated by Rev‐erbα‐driven repression of the Rorγt promoter via Nfil3.54 Homing receptors for immune cells, including CCR7, sphingosine‐1‐phosphate receptor 1, IL7‐R and CXCR4, are also modulated by circadian rhythms driven by the host glucocorticoids.55, 56, 57, 58 Thus, the circadian rhythm influences both innate and adaptive immune cell migration at different times of day. Together, these circadian rhythm‐modulated immune influences protect the host from disease development. As mentioned earlier, microbiota oscillate and thus may impact immune functions. For example, Th17 cells can be induced by segmented filamentous bacteria (SFB),59 which are members of the daily oscillating Firmicutes phylum.30, 31, 33 Furthermore, microbiota can modulate intestinal epithelial cell (IEC)‐intrinsic Nfil3 circadian rhythms via type 3 innate lymphoid cells (ILC3).23 These observations suggest that microbial oscillations not only influence immune development, but also alter the amplitude of the type of immune responses. Thus, further understanding of circadian influences on host:microbial interactions is important.

Pattern recognition receptors (PRRs) are expressed on a wide variety of cells. These recognize conserved molecular structures that are shared by both pathogens and commensal microbes alike, and are known as pathogen‐associated molecular patterns (PAMPs).71 Through PRR signalling, the immune system is able to respond to microbial cues to regulate immunity. However, as discussed below, PRRs are also influenced by circadian rhythms (Figure 4), which can have important therapeutic effects, for example in the induction of vaccine‐induced immunity, as discussed later.

As microbiota composition and functions oscillate in a circadian manner, metabolites from gut microbiota34 in both mice and humans also exhibit circadian variations.96, 97, 98, 99, 100 Thaiss and co‐authors showed that antibiotic depletion of the microbiota led to the loss of microbial diurnal rhythmicity in mice.34 In addition, they also found that the microbiota‐induced oscillations in serum ornithine and polyamines could be induced by time‐restricted feeding, in a host that had deficient circadian rhythm (Per1 and Per2 knockout mouse). Together, these data provided pivotal evidence that microbiota modulate the host circadian metabolome. While this study did not investigate the impact of microbiota and their metabolites on host immunity, there are many potential implications for the immune system, especially mucosal immunity.

The gut microbe‐derived short‐chain fatty acids (SCFAs), butyrate, acetate and propionate, modulate immune responses, including the induction of Tregs.101, 102, 103 Interestingly butyrate, which is absent in germ‐free mice, modulates Per2 and Bmal1 rhythms, suggesting that the microbiota indirectly regulate circadian rhythms via their metabolites.42 These SCFAs typically bind to the G protein‐coupled receptors (Gpr) 41/43,104, 105 although other receptors also bind to SCFAs.106 Many types of host cells utilize SCFAs, and these promote deletion of autoreactive T cells and anti‐inflammatory responses in models of colitis, arthritis, type 1 diabetes and asthma.104, 107, 108 SCFA utilization can also restrict tumour growth and survival.109, 110, 111 Importantly, the effect of SCFAs on some cells, such as monocytes, may not always elicit the same responses from mice and humans.112 For example, activation of human monocytes with acetate induced attenuated proinflammatory responses; however, activation of mouse monocytes (on the 129/SvEv background) with acetate promoted elevated proinflammatory GM‐CSF, IL‐1α and IL‐1β cytokine secretion.112 Given that mouse and human monocytes respond differently to acetate, it is possible that additional regulators of SCFA utilization, and the rhythms that modulate them may vary between species. SCFAs such as butyrate can also regulate the integrity of the intestinal barrier by stabilizing hypoxia‐inducible factor (HIF).113, 114 Interestingly, HIFα regulates several core clock genes, allowing the cells to respond to changes in oxygen.115 This has implications, for example, for induction of tumour hypoxia in cancer therapy. Given that microbial metabolites oscillate, there may be similar oscillations in their respective SCFA receptors, or in proteins regulating the downstream responses.

Bile metabolism is also regulated in a time‐of‐day‐dependent manner due to the need to co‐ordinate metabolic responses to food intake, enabling the esterification and absorption of dietary fats and lipids.,In mice, the serum bile acids peak at the beginning and end of their active (dark) phase. The majority of bile acids are reabsorbed by the liver; however, some can enter the colon and be further metabolized, producing secondary metabolites that peak in the serum at the beginning of the dark phase.,Many species belonging to the phyla Firmicutes and Bacteroidetes encode enzymes required to metabolize bile acids, for example bile salt hydrolases. These enzymes regulate hepatic and ileal clock genes and lipid and cholesterol metabolism.,,Interestingly, the intestinal epithelium receptor, CD300lf, has been identified to be the receptor for mouse norovirus.This CD300lf receptor undergoes structural changes upon binding to the bile acid, glycochenodeoxycholic acid, which consequently enhanced the ability of norovirus to bind to the receptor.Recent studies have shown that microbiota can modulate the ability of mouse norovirus, a highly contagious viral pathogen, to infect the host.Furthermore, crosstalk between mouse norovirus and the microbiota can alter susceptibility to autoimmunity,,,, parasitic infectionand allergies.As bile acids can be modulated in a circadian manner, it is also possible that invasion of norovirus may be enhanced at different times of the day. 13278 13278 13278 13278 13278 13278 13278 13278 13278 13278 13278 13278 13278 13278 13278

In addition, microbiota synthesize a range of other molecules including xenobiotics, vitamins, polyamines and hydrogen sulphide. Little is known about how circadian rhythms modulate their production and impact on the immune system. Investment in this area would aid our understanding of their important role in health and disease.

Circadian rhythms, and their regulation, have an important influence on susceptibility to invading pathogens. Bellet and colleagues showed that the immune responses to Salmonella Typhimurium (a common foodborne pathogen) in infected mice were dependent on the time of day.129 The mice infected in the active phase (ZT16) had reduced pathogen colonization and reduced inflammation, compared with mice infected in the rest phase (ZT4).129 Host circadian rhythms are required for this response, as Clock mutant mice exhibited arrhythmic colonization and their macrophages had reduced inflammatory responses (IL‐6 and IL‐1β) to LPS (recognized by TLR4, which is known to oscillate23, 28, 50, 79, 80). Similarly, in wild‐type mice, S.pneumoniae‐induced infection at ZT12 resulted in earlier neutrophilia in the bronchoalveolar lavage fluid, with reduced local (lung) and systemic (blood) bacterial counts at 48 hours post‐infection, compared with infection at ZT0.130 In addition, intraperitoneal infection with Listeria monocytogenes confirmed that a later rest phase infection at ZT8 was important for enhanced recruitment of Ly6C^hi monocytes and stronger antimicrobial immunity, compared with infection at the start of the rest phase ZT0.53 Similar time‐of‐day‐dependent sensitivities to viral infections have also been reported.131, 132, 133 Interestingly, parasitic infection of mice by oral gavage of Trichuris muris eggs at ZT0 led to enhanced anti‐parasitic IgE titres, Th2 responses and worm expulsion, compared with the mice infected at ZT12, the end of the rest phase.134 Thus, the optimal immune responsive time to pathogens is not the same for all micro‐organisms. The different time‐dependent oscillations in pattern recognition receptors, or microbial metabolites, influence immune responsiveness. Therefore, priming the immune system at the time of greatest immune responsiveness would be vital for preventing and limiting the spread of specific infectious organisms. To date, it remains unknown as to whether the immune responses to these infectious organisms can be modulated by the commensal microbiota and whether the commensal microbial circadian rhythms are altered in pathogenic infections, which in turn subsequently change the host response. It is clear that further studies are required, especially when multiple factors are analysed in the same study.

The microbiome is an important metabolic ‘organ’, aiding in many unique host functions, which include the efficacious response to a number of therapies. It is well established that the microbiome influences susceptibility to obesity. Microbiota from obese individuals can transfer metabolic dysfunction to another host, while the microbiota from lean individuals, following transfer, protect the recipient from metabolic dysbiosis.38, 135, 136 Metformin, a widely used drug for the treatment of type 2 diabetes, improves insulin sensitivity. Metformin alters both the composition and the function of microbiota, enhancing the therapeutic effects.137 However, components of the gut microbiota can also be harmful, depending on the context. In colorectal cancer, the intestinal microbiota can influence disease progression138, 139, 140 or aid the therapeutic responses to both chemotherapeutics141, 142 and immune checkpoint inhibitors, for example CTLA‐4, PD‐1, PD‐L1.13, 143, 144 Therefore, understanding the mechanisms by which specific microbiota exert their effects (e.g. circadian modulation) could improve therapeutic success. In an animal model of colorectal cancer, induced by azoxymethane and dextran sulphate sodium, Mager and colleagues identified 3 species of tumour‐associated bacteria (Bifidobacterium pseudolongum, Lactobacillus johnsonii and Olsenella sp.) which enhanced immune checkpoint blockade therapy.145 This enhancement of therapy was orchestrated predominantly by the microbial production of inosine, which binds to the adenosine A_2A receptor, promoting Th1 differentiation in the presence of IFN‐γ and consequently anti‐tumour effects. Of note, Lactobacillus johnsonii belongs to the phylum Firmicutes, the abundance of which is known to oscillate daily.30, 31, 33Lactobacillus johnsonii is also associated with modulating immunity and preventing autoimmune diabetes.146, 147, 148 In a recent study assessing metabolism of 271 orally administered drugs by 76 different human gut bacteria, the authors reported that the microbiota can metabolize many more drugs than previously known.149 In addition, the drug‐metabolizing function of the microbiota has both local intestinal effects and important systemic effects, especially on the liver. Hepatic drug metabolism is also influenced by circadian rhythm.150, 151, 152 Thus, circadian microbial oscillations (whether through direct or indirect mechanisms) are likely to modulate the ability to metabolize drugs and therefore may have important therapeutic impacts. It should also be noted that peripheral circadian rhythms are often tissue‐specific, and therefore, microbial rhythms could be out of synchrony with the rhythms in different tissues.17, 18 This may potentially arise due to the presence of dysbiotic microbiota or stronger peripheral circadian inducers. Thus, it is critically important to identify novel pathways regulating the peripheral rhythms, which are influenced by the microbiota, and synchronize these rhythms, for maximal clinical benefits.

The types of gut microbiota that adhere to the intestinal wall may also oscillate and can play an important role in intestinal immunity. Akkermansia muciniphilia, a mucin‐degrading commensal bacteria belonging to the Verrucomicrobia phylum,153 is protective against ulcerative colitis,154, 155, 156 type 1 diabetes157, 158, 159 and obesity.160 Furthermore, Akkermansia muciniphilia improves therapeutic efficacy in cancer patients who are treated with immune checkpoint blockers.13 Given the oscillation in abundance of Verrucomicrobia,30, 31, 33 appropriately timing therapeutic administration of these bacteria may enhance efficacy. As discussed earlier, it is important to note that bacteria can be beneficial or harmful, depending on the context. Akkermansia muciniphilia, while beneficial in the aforementioned studies, can also promote T‐cell‐mediated inflammation in multiple sclerosis161 and thus microbial therapies need to be tailored appropriately.

Microbial circadian rhythms modulate long‐term immunity following immunization and vaccination. Microbiota composition can modulate vaccine efficacy to bacteria and viruses in both animal models162, 163, 164 and humans.165, 166, 167, 168 Evidence for this has been obtained from vaccine studies conducted in GF, as well as antibiotic‐treated mice, resulting in impaired antibody responses.162, 163 Similarly, in humans, administration of broad‐spectrum antibiotics also reduced the immunogenicity of the rotavirus vaccine.167 Rhythmic microbial and PRR rhythms are also likely to influence vaccine efficacy, although this remains to be studied. Host circadian genes can also alter the vaccine efficacy. Per2, one of the major circadian gene family members,169 controls TLR9‐mediated innate and adaptive immune responses to infection and sepsis.80 Moreover, Per2 also controls vaccine immune responses to TLR9 ligand‐adjuvanted immunization using CpG (a bacterial DNA, which is a TLR9 ligand and a common vaccine adjuvant72).80 These studies have provided evidence for a vital link between circadian rhythms and TLR signalling, which promotes enhanced vaccine efficacy. Furthermore, in Cry1/Cry2 double knockout mice, there are more circulating mature B cells that secrete higher titres of antibodies to T‐cell‐independent antigenic stimulation (4‐hydroxy‐3‐nitrophenyl‐acetyl (NP)‐conjugated Ficoll).170 This again highlights host rhythms influencing immunity. Oscillations in antibody responses have also been seen in other studies, including vaccination studies.56, 171, 172, 173, 174 A randomized trial investigating the efficacy of the trivalent inactivated influenza virus demonstrated that vaccine administration in the morning induced higher antibody titres than in the afternoon173; however, the oscillating antibodies post‐vaccination are likely to be viral strain‐specific171 and influenced by sex.172 As TLR9 is highly expressed on B cells175 and requires downstream MyD88 signalling to mediate functional changes,176, 177 it is likely that oscillating TLR9 levels may influence B‐cell antibody responses; however, this has yet to be fully investigated. This also applies to autoantibody production, which can be regulated by TLR expression.178, 179, 180 Murine studies, in which peptide immunization was performed at different times of day, showed that this influenced both T‐cell immunity and susceptibility to disease.56, 181, 182 Furthermore, immune‐intrinsic circadian rhythms were vital for this effect.56, 182 Thus, both Bcell and Tcell responses to antigen can be modulated by circadian rhythms. Further understanding of how the microbial rhythms may influence antigen‐specific immunity may prove even more beneficial.

While the role of microbiota‐mediated modulation of host immunity using antibiotic treatment or GF mice has been extensively studied, the impact of pro‐ or pre‐biotics or bacteriophages (bacteria‐targeting viruses) on either host or microbial circadian rhythms is not currently known. Studying bacteriophages and their role in modulating microbial circadian rhythms may therefore also prove to be an important research field, particularly as bacteriophages can adhere to the mucus layer and prevent infection from pathogenic bacteria., 13278 13278

In this review, we have discussed time‐of‐day‐dependent differences in the microbiota and how they may impact host metabolism and immunity. While it is known the microbiota are associated with the development and progression of many diseases, little is known of the influence that circadian oscillations have on the microbiota, which may also modulate disease susceptibility. We hope to have provided insight into some potential future directions in relation to microbial oscillations and how they can directly, or indirectly, regulate host immune responses in different disease settings. Given the significant influence that circadian rhythms have on host immunity, it is important that more studies consider the timing of experiments and administration of therapies. By better understanding circadian influences, we may maximize clinical success by targeting the cells/microbiota of interest, at the time they are most vulnerable. This could potentially enable reduced drug concentrations to be used, which would also limit toxic effects. Understanding these rhythms in both males and females is vital, given the importance of androgens in immunity and in shaping the host microbial communities. While the vast majority of work outlined in this review has focused on microbiota in the gut, it will also be important to study the circadian effects on microbiota in different locations that include the skin, lungs, as well as other mucosal surfaces. It is likely that most commensal microbiota, in these different locations, modulate immunity in a time‐of‐day‐dependent manner. This is a continually expanding new field and we look forward to gaining further insight, which may improve efficacy of current, as well as new therapies, for disease prevention and treatment.

This work was supported by a Medical Research Council Career Development Award (MR/T010525/1) to JAP; a NIH Research Project Grant (NIH RO1 HD097808); and a Molecular Genetic & Diabetes Mouse Core of Yale Diabetes Center grant to LW (NIH P30 DK 045735).

The authors declare no conflicts of interest.