Circadian rhythms refer to 24-h oscillations of biological or physiological activity, which are found in almost all life on earth (Loudon, 2012; Westwood et al., 2019). In mammals, the timing of these rhythms is determined by a central core located in the suprachiasmatic nucleus (SCN) of the mammalian brain, with additional inputs from peripheral oscillators (circadian modulators located in cells or tissues outside of the SCN). These rhythms are strongly induced by light signals via the retinohypothalamic tract (Meijer et al., 1986, 1992) within the SCN, stimulating neuronal firing and inducing rhythmic neurotransmitter secretion, e.g., glucocorticoids such as vasoactive intestinal polypeptide (Shinohara et al., 1994), which coordinate and regulate peripheral rhythms located in other tissues of the body. This coordination by the SCN enables synchronization of these light-controlled rhythms for maximal host benefit, that includes induction of metabolic pathways in anticipation of dietary intake. Peripheral rhythms themselves are also influenced by other factors including hormones and food intake/nutrient availability (Balsalobre et al., 2000; Damiola et al., 2000). Together, the regulation of these rhythms is vital to maintain good health, as disruptions to these rhythms alter susceptibility to infections, autoimmunity, metabolic diseases, and cancer (Turek et al., 2005; Lee et al., 2010; Gibbs et al., 2014; Edgar et al., 2016; Voigt et al., 2016; Ehlers et al., 2018; Hopwood et al., 2018; Krakowiak and Durrington, 2018).

At the transcriptional level, circadian rhythms are controlled by integrated autoregulatory transcription-translation feedback loops, which direct proteins to bind to sequence-specific elements, resulting in the initiation or repression of circadian gene expression. The interlocking of the three transcriptional feedback loops, shown in Figure 1, generates ∼24-h cycles of transcription with varied phases of expression depending on the proteins available to bind to the promotors or enhancers of target clock-controlled genes (CCGs) (Takahashi, 2017). Together these loops enable a more responsive feedback mechanism that modulates the circadian rhythm in response to environmental changes or stimulations, and these include temperature, hormones, food intake, and microbes.

It has been estimated that ∼1,000 microbial species reside in the human intestines, encoding a vast metagenome with millions of unique genes, modulating host metabolism and regulation of immune responses (Whitman et al., 1998; Qin et al., 2010; Li et al., 2014). Microbial composition can be strongly influenced by many factors including genetics, age, lifestyle and diet (Ley et al., 2006; Turnbaugh et al., 2009; Wu et al., 2011; Goodrich et al., 2014; Bokulich et al., 2016; Odamaki et al., 2016; Sonnenburg and Bäckhed, 2016; Sonnenburg et al., 2016). Crosstalk between bacteria and the immune system is important for the education and development of a mature immune system, including the development of adaptive T-helper 17 cells (Th17) and regulatory T cells (Treg) (Ivanov et al., 2009; Atarashi et al., 2011, 2013), as well as the development of innate lymphoid cells (Gury-BenAri et al., 2016). Importantly, host bacterial composition influences disease susceptibility to obesity (Bäckhed et al., 2004; Turnbaugh et al., 2006; Vrieze et al., 2012), autoimmunity (Frank et al., 2007; Wen et al., 2008; Feng et al., 2010; Lee et al., 2011; de Goffau et al., 2013; Cekanaviciute et al., 2017; Pearson et al., 2019a), cancer (Arthur et al., 2012), and infectious diseases (Dubourg et al., 2017; Kaul et al., 2020; Yeoh et al., 2021). Furthermore, host bacterial composition can modulate responses to immunotherapy (Gopalakrishnan et al., 2018; Routy et al., 2018; Mager et al., 2020) and drugs (Wu et al., 2017; Zimmermann et al., 2019). Taken together, these reciprocal interactions highlight the important role bacteria play in shaping both our immune responses and therapeutic success against a variety of microbial infections, but also other conditions.

Similarly to bacteria, viruses, which are obligate parasites requiring infection of host cells in order to multiply, have co-adapted to their hosts and endogenous viral elements have been incorporated into host genomes millions of years ago (Simmonds et al., 2019). Alongside bacteria, viruses are often associated with many public health outbreaks, e.g., norovirus and rotavirus (Atmar and Estes, 2006; Harris et al., 2008; Lysén et al., 2009; Wikswo and Hall, 2012). Viruses have also been associated with global pandemics including the H1N1 influenza virus (Smith et al., 2009a, b) and SARS-CoV-2 (Wu Y. et al., 2020; Zhu et al., 2020), which have been responsible for the deaths of millions of people worldwide. Thus, there is great need to better understand how antiviral responses are stimulated and how they can be modulated to achieve protective antiviral responses, without promoting significant inflammation and cell damage. Therefore, better understanding of the mechanisms by which microbes can be modulated, including by circadian rhythms, are essential for identifying ways to improve the harnessing of microbes for promoting disease protection and therapeutic success.

It is known that viral infections can alter the gut microbiota composition, including HIV, influenza virus, norovirus, rotavirus, HBV and HCV in humans, which can in turn influence disease severity (reviewed in Li et al., 2019; Yuan et al., 2020). Individuals with HIV infection had reduced microbial richness and depleted Bacteroidia members but their stool samples were enriched in species belonging to the Proteobacteria phyla (Vujkovic-Cvijin et al., 2013). Interestingly, the reduced alpha diversity (number of different microbial species present) (Noguera-Julian et al., 2016), was associated with the severity of immunodeficiency (Noguera-Julian et al., 2016). However, this could be restored following anti-retroviral therapy (Ji et al., 2018). Importantly, the alterations in microbial composition were also associated with altered microbial functions, whereby HIV infection repressed the generation of proline, phenylalanine, and lysine (all amino acids) (Serrano-Villar et al., 2016), while promoting tryptophan catabolism (Vujkovic-Cvijin et al., 2013) by the microbiota. Given Proteobacteria and Bacteroidetes (which includes the Bacteroidia members) oscillate, it is conceivable that circadian rhythms influence the microbiota composition in those individuals. Administration of antibiotics/probiotics/prebiotics in mice, as well as studies in germ-free (GF; which do not have any bacteria) or gnotobiotic mice (which have a defined microbial community), have confirmed that viral-bacterial crosstalk can modulate susceptibility to viral infection and disease, e.g., norovirus infection (Schaffer et al., 1963; Isaak et al., 1988; Cadwell et al., 2010; Ichinohe et al., 2011; Basic et al., 2014; Osborne et al., 2014; Robinson K. M. et al., 2014; Pearson et al., 2019b).

Commensal bacteria exhibit both stimulatory and suppressive functions in controlling viral infection, through direct and indirect mechanisms, which can lead to harmful or beneficial outcomes for the host (Figure 4). Commensal microbiota can promote viral infections directly through the production of microbial ligands (e.g., LPS) or metabolites (e.g., Bile acids or SCFAs), which can promote: (1) target cell proliferation and upregulation of virus receptors, e.g., CD300lf (a receptor for murine norovirus) on Tuft cells (von Moltke et al., 2016; Nelson et al., 2018; Wilen et al., 2018), (2) reactivation of viruses (Gorres et al., 2014), (3) increased virion stability (Robinson C. M. et al., 2014; Berger et al., 2017), and (4) increased viral replication (Kuss et al., 2011). Importantly, norovirus infection of B cells can be promoted or prevented depending on the presence of bacteria expressing H-type histo-blood group antigens (Jones et al., 2014; Karst, 2015). In addition, enteric bacteria have been reported to enhance enteric viral (poliovirus) co-infection, which consequently can facilitate genetic recombination and enhance viral fitness (Erickson et al., 2018). Importantly, commensal bacteria can also modulate the immune response, promoting viral infection (Jude et al., 2003; Kane et al., 2011; Young et al., 2012; Uchiyama et al., 2014; Baldridge et al., 2015; Wilks et al., 2015). Two examples with different mechanisms of virus-mediated immune suppression include mouse mammary tumor virus (MMTV), which primarily infects intestinal dendritic cells, B cells and T cells (Held et al., 1993; Beutner et al., 1994) and Norovirus, which primarily infects intestinal Tuft cells (Wilen et al., 2018). In MMTV infection, MMTV in conjunction with bacterial LPS (signaling through TLR4), strongly induces proinflammatory IL-6, which in turn promotes Tregs to produce IL-10, a potent immune-suppressing cytokine, promoting viral escape from the immune system (Jude et al., 2003; Kane et al., 2011; Wilks et al., 2015). However, in norovirus infection, the presence of gut bacteria downregulates antiviral IFNλ receptor expression in the intestine and subsequently reduces intracellular downstream signaling via Stat1 and Irf3 (Baldridge et al., 2015). This should limit antiviral immunity and the survival of the virus.

On the other hand, commensal microbiota can also suppress viral infections by multiple mechanisms including: (1) directly binding to the virus, whereby the virus may be destabilized, prevented from being internalized and/or viral replication is suppressed (Botić et al., 2007; Conti et al., 2009; Mastromarino et al., 2011; Chen et al., 2016; Bandoro and Runstadler, 2017), and (2) boosting antiviral immunity and mucosal barrier integrity. This antiviral immunity includes enhancing viral antigen presentation to antigen-specific T and B cells, leading to the activation of cytotoxic CD8⁺ T cells, generation of IFN-γ-producing Th1 cells and IL-17a-secreting Th17 cells (Hensley-McBain et al., 2016), as well as synthesis of virus-specific antibodies (Ichinohe et al., 2011; Oh et al., 2014) to control and limit the infection. Furthermore, bacterial and viral ligands can activate toll-like receptors and inflammasomes, both of which promote the secretion of IFNs, IL-1β and IL-18 (Ichinohe et al., 2011; Wang et al., 2012; Oh et al., 2014) from a range of cells, while reducing IL-33 secretion (Oh et al., 2016) and enhancing IL-22 (Hensley-McBain et al., 2016) and IFN responses (Steed et al., 2017). Together, they boost antiviral immune responses and mucosal barrier integrity.

Interestingly, microbial products, such as LPS, can also both promote and suppress viral infections, which may be due to different LPS structures that have different immunostimulatory effects (Vatanen et al., 2016). This has been reported in MMTV studies, where MMTV virions bound to E. coli LPS had a greater ability to stimulate IL-6 production from splenocytes than B. theta LPS (Wilks et al., 2015). However, further studies are required to determine whether the differences in LPS from the various bacterial sources, and/or their structures, differentially modulate virus stability. Interestingly, different serotypes of E. coli LPS can differentially alter body temperature in rats (Dogan et al., 2000). As the peripheral circadian oscillators sense temperature changes, it is also possible that the origin of the LPS may differentially induce temperature changes in vivo and alter circadian rhythmicity. Furthermore, as both bacteria and TLRs oscillate, including TLR4, it is highly likely this will have an effect on viral promotion or suppression.

Bacteriophages, viruses which infect and subsequently destroy bacteria, are highly abundant and can modulate bacterial composition of the host (Hsu et al., 2019; Khan Mirzaei et al., 2020) and can prevent infection of the host by pathogenic bacteria by adhering to the mucus layer (Dubos et al., 1943; Matsuzaki et al., 2003). Importantly, bacteriophages have also been shown to be influenced by circadian rhythms (Kao et al., 2005; Liu et al., 2019). Cyanophages, which infect cyanobacteria, have been shown to exhibit diurnal rhythms in their ability to infect bacteria and in gene expression, which relates to the ability of the cyanobacteria to undergo photosynthesis (Liu et al., 2019). While this provides the first evidence of circadian modulation of phages, future studies are needed to determine whether phages in humans or mice exhibit similar changes. It will also be important to develop new technologies to fully understand the potential of these as microbial modulators and as modulators of circadian rhythms (Khan Mirzaei and Deng, 2021).

Further studies evaluating crosstalk between viruses and bacteria in circadian rhythms are urgently needed. Studies involving GF and gnotobiotic mice would be greatly insightful, so would co-infection models. Better understanding of these interactions would undoubtedly help not only to identify novel pathways for therapeutic targeting but also to better understand how therapy may inadvertently alter susceptibility to other pathogens, including opportunistic pathogens.

Therapies targeting the circadian clock have shown promise in controlling bacterial and viral infection. SR9009, a REV-ERB agonist, which suppresses Bmal1, has successfully inhibited viral entry and replication of HCV (Zhuang et al., 2019), HBV (Zhuang et al., 2021), and HIV-1 (Borrmann et al., 2020). Likewise, SR8278, a REV-ERB antagonist, which represses Rev-erbα and promotes Bmal1 expression, has rendered mice more susceptible to VSV infection when infected at ZT12, compared to the more protected mice infected at ZT12 without REV-ERB antagonist treatment (Gagnidze et al., 2016). It is unknown, thus far, if Rev-erb agonists change the gut bacterial composition and function. Thus, these studies need to be conducted, and combined with studies of viral infection. While the therapies targeting circadian proteins have shown promise with respect to infection, it is important to consider broader effects. For example, Rev-erbα modulates Th17 cell development (Yu et al., 2013), and thus Rev-erbα agonists may promote cell development, while antagonists may prevent Th17 expansion. It is possible that Th17-promoting Rev-erbα agonists may subsequently result in intestinal dysbiosis in the host, due to dysregulated Th17 immune responses to gut bacteria. In addition, given the plasticity of Th17, Treg, and Th1 responses (Lee et al., 2009; Wei et al., 2009; Mukasa et al., 2010), any Rev-erbα modulation needs to be considered in all these aspects for additional immune impacts that may alter both the efficacy of treatment and susceptibility to other health problems.

The specific timing of therapeutic intervention, referred to as chronotherapy, may also play a role in modulating bacterial and viral crosstalk. In murine HSV-2 infection, time-of-day influences the survival of the infected mice, with infections at ZT6 repressing viral replication and promoting survival of the host, compared to infections at ZT18 (Matsuzawa et al., 2018). Interestingly, when these mice were infected at ZT18, a fourfold higher dose of acyclovir (administered 30 min prior to infection) was required for mice infected at ZT18, compared to those infected at ZT6 to obtain similar survival and clinical scores (100 mg/kg vs. 25 mg/kg). Therefore, the time of infection can make a substantial difference to the drug dosage needed to control the infection, which for some therapies may not be possible due to the enhanced toxicity or side effects of the drug when higher doses are used. In addition, the use of higher doses may also promote host resistance to the effective therapy. It is unclear whether antibiotics may have a similar effect on bacterial pathogens at different times-of-day. If so, administering the antibiotics at a time synchronized with the timing of optimal anti-microbial host immune responses may prove beneficial and prevent suboptimal doses of antibiotics being used, and thus reduce antibiotic resistance.

Given that the microbiota have the ability to metabolize many drugs (Zimmermann et al., 2019), and that the abundance of bacteria can oscillate, it is likely that this change in the microbiota at different times-of-day may alter the therapeutic responses that the drugs elicit. For example, certain bacterial species can enhance the success of therapy with immune checkpoint blockade in mice and cancer patients (Sivan et al., 2015; Vétizou et al., 2015; Mager et al., 2020) and gut bacteria could also enhance the responses to chemotherapy via modulating the tumor microenvironment and immune cell function (Iida et al., 2013; Viaud et al., 2013). Importantly, not only does drug-metabolism by microbiota have a local effect, but systemic consequences can occur, particularly in the liver, where hepatic drug metabolism can also be influenced by circadian oscillations (Kim and Lee, 1998; Lin et al., 2019; Wang et al., 2019). Interestingly, in the liver, Bmal1 regulates oscillations in drug metabolizing genes, as Bmal1-deficient mice showed higher levels of toxicity from metabolizing xenobiotics (Lin et al., 2019). Given that Bmal1 promotes oscillations in drug-metabolizing genes and toxicity sensitivity, and in addition, Bmal1 promotes both HBV and HCV infection in hepatocytes, some potential adverse effects of circadian therapies (i.e., Rev-erb agonists) on drug metabolism, which may result in increasing toxicity, should also be considered.

Circadian oscillations also modulate vaccine responses to bacterial and viral pathogens. The magnitude of antibody response to the trivalent inactivated influenza vaccine in humans correlated with early TLR5 expression (Oh et al., 2014). Microbial flagellin is a ligand for TLR5 (Hayashi et al., 2001) and stimulation of TLR5 boosted plasma cell numbers and antibody titers in response to the influenza vaccine (Oh et al., 2014). TLR5 was also involved in boosting antibody responses to the inactivated polio vaccine but not to adjuvanted (Tetanus-Diphtheria-pertussis) vaccine or the live-attenuated yellow fever vaccine (YF-17D). Interestingly, antibody responses to the trivalent influenza vaccine are also altered at different times-of-day, with higher titers observed in elderly individuals vaccinated in the morning compared with the afternoon (Long et al., 2016). It is unclear whether circadian oscillations in gut bacteria contribute to the alteration seen in anti-influenza antibody response; however, it is certainly possible, as TLRs oscillate and are likely to be involved in bacterial and viral ligand sensing. Further studies need to be conducted for other vaccinations and in different age groups. Given that elderly people have reduced immune responses compared to younger individuals (Gustafson et al., 2020), timing of the vaccination to boost the protective response is clinically relevant.

As discussed, it is clear animal models have a circadian rhythm and that both bacterial and viral organisms are able to modulate these rhythms to either promote better survival of the host or better advantage to the pathogen. While these studies have been enlightening and have some similarities with human circadian rhythm studies, it is important to consider the true implication of animal circadian studies and how these rhythms may be different to humans.

Both mice and humans have an evolutionarily preserved molecular clock and SCN, which controls the circadian rhythms in response to light; however, as mice are nocturnal and humans are diurnal their responses to light can be different. Mice can entrain to light exposure of 1 lux for a few minutes, while humans require both a high light intensity (>100 lux’s) and a longer duration (>30 min) to entrain (Daan and Pittendrigh, 1976; Khalsa et al., 2003). Thus, differences in study design related to light intensity and duration can have different effects between the species and thus the induction of circadian rhythms. Similarly, laboratory animals are generally housed in abrupt 12-h light/dark cycles, whereas humans can experience seasonal variations in light exposure (Jasser et al., 2006; Higuchi et al., 2007). Interestingly, nocturnal vs. diurnal animal species can also have a different time window and magnitude of response when serotonin is able to modulate circadian rhythms (Horikawa and Shibata, 2004; Novak and Albers, 2004; Cuesta et al., 2008). Thus, changes in response and timing need to be considered when considering translation and applicability of animal studies into human studies.

There are many other factors that may also be involved in modulating circadian differences between mice and humans. For example, changes in microbial composition between the species, as well as food type and time eaten, will also be important in determining microbial influences on circadian rhythms. Furthermore, the laboratory animals studied are often in-bred and thus, in comparison to humans, exhibit much less genetic diversity, which also may influence the applicability of murine studies to humans. However, as for other types of research that use animal models, murine studies provide a signpost for areas of investigation when considering circadian rhythms in humans. Thus, it is important for studies conducted in animal models to be confirmed in humans.

Circadian rhythms modulate the composition and function of commensal bacteria, and host susceptibility to bacterial and viral infection. While the interactions between circadian rhythms and viral or bacterial infection have been separately studied, there is a significant knowledge gap regarding the three-way crosstalk among viruses, bacteria and circadian rhythms. Thus, the studies with co-infection (bacteria and virus) may provide important insight into the crosstalk. It is clear that circadian clock manipulation and vaccination at particular times-of-day may have important implications for boosting host protection from infection and inflammation. Thus, there is a need to better understand these interactions. As discussed earlier, not all viruses have the same time-of-day virulence to infect the host; thus, we need to consider how to target individual viruses that may alter susceptibility to infection by other viruses, opportunistic bacteria or immune-mediated diseases. By better understanding these interactions we may be able to reduce antiviral/antibiotic resistance by using lower doses of drugs, which may also result in fewer toxic side effects. Although it is difficult to pinpoint the time when an infection occurs in humans, it is possible to boost the protective immune responses at the optimal time to enhance therapeutic success, whether by vaccination or immune-modulating therapies. Finally, given the impact that circadian rhythms have on microbes and immunity, it would be important to consider the role of circadian rhythms in the design and implementation of experiments in both animal and human studies.

JAP wrote the manuscript. ACV and KB-B conducted research for the manuscript. FSW and LW edited the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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