Host-microbe circadian dynamics: Finding a rhythm and hitting a groove in scientific inquiry

Abstract

Gut microbes are mediators of organismal-level circadian rhythms, responding to and transducing environmental cues. Gut microbes also exhibit rhythms, yet their contribution to a healthy microbiome remains unclear. We present our path to identifying host-microbe circadian dynamics related to health and outline a series of forward-thinking questions requiring further exploration.

What defines a “healthy” microbiome? This question has eluded both physician scientists and basic scientists alike for decades and remains a key roadblock to our ability to functionally manipulate the gut microbiome across species to promote wellness. While the gut microbiome is clearly associated with the incidence of several diseases, ranging from inflammatory bowel diseases to metabolic diseases to allergic airway diseases, we have just recently begun to gain insights into how gut microbes mechanistically contribute to the transition from a state of health to disease (Pierre and Leone, 2020). The progress in microbiome sciences to date related to the gut and other environments, including our oceans and soils, has been aided by the advent of high-throughput next-generation sequencing and the development of bioinformatics tools to analyze these large datasets. Thus far, these data have revealed that innumerable factors contribute to the heterogeneity observed in the gut microbiota between individuals, including host genetics, nutritional status, dietary composition, meal timing, exercise, and sleep patterns, to name a few (Frazier et al., 2020). This person-to-person variability makes it particularly difficult to define a “healthy” microbiome in any given population. Additionally, multiple aspects of a microbiome can be utilized to “define” its status, including microbial community membership, genetic potential, functional outputs, and network stability, as well as additional measures that have yet to be identified (Pierre and Leone, 2020). Further knowledge of how this dynamic, non-stochastic pseudo-organ behaves and engages with host physiology will enable functional modulation of the gut microbiota to target aspects of health and disease.

For over half a century, the use of gnotobiotic animals in biomedical and agricultural research, particularly germ-free rodents, has allowed for insightful interrogations to understand host-microbe interactions as they relate to microbial ecology, host physiology, growth, and development, as well as disease pathogenesis (Pierre and Leone, 2020). Resurgence of gnotobiotic work began in the late 1990s with pioneering work performed by Dr. Jeffrey Gordon’s laboratory, where the mechanistic underpinnings of how gut microbiota contribute to global metabolic homeostasis became evident. Using gnotobiotic mice as an essential tool, several intriguing findings were presented by the Gordon group revealing that (1) germ-free mice eat more calories than their conventionally raised counterparts to maintain the same amount of weight; (2) germ-free mice are resistant to high-fat, high-simple-sugar diet-induced weight gain; and (3) gut microbes contribute to enhanced energy extraction from certain diets that could tip energy balance and promote fat deposition (reviewed in Ecklu-Mensah et al., 2022). While several molecular metabolic mechanisms were identified by the Gordon group and others, it seemed that the global physiological outputs of the germ-free mouse were so unique that additional mechanisms must be at play.

As trainees in the laboratory of Dr. Eugene B. Chang, we were challenged to identify novel molecular pathways involved in the resistance of germ-free mice to diet-induced obesity. To do so, we first examined the transcriptome of the liver between germ-free, ex-germ-free (mice that had received cecal microbiota transplant), and conventionally raised counterparts. Serendipitously, we observed that the most significantly and differentially expressed pathways between germ-free and colonized animals were those containing genes associated with driving circadian rhythmicity (Leone et al., 2015). Circadian rhythms are essential to nearly all life forms, governing molecular metabolic and immunological outputs as well as behavioral responses, i.e., fasting/feeding, sleeping/waking, in response to light:dark cues over a 24 h period (Frazier and Chang, 2020). Disruptions in circadian rhythms are often associated with metabolic diseases, including obesity and type 2 diabetes. In lower organisms, such as Euprymna scolopes (Hawaiian bobtail squid), work by the groups of Drs. Margaret McFall-Ngai and Edward Ruby had already identified and eloquently described mechanisms outlining the contribution of gut microbes to diel rhythms in the host (Nyholm and McFall-Ngai, 2021). Here, the “squid-Vibrio fischeri” model system revealed that host-microbe diurnal interactions are well-conserved ecological principles that drive both molecular and behavioral outputs on both sides of this two-way street of communication. But what could this mean, and is it important to mammalian host physiology? How could this phenomenon be studied meaningfully in more complex model systems, such as the mouse? And so began the journey to define the contribution of gut microbes to circadian rhythms in the context of metabolic homeostasis.

Here, we had to quickly gain proficiency in how to study the circadian system, leaning on collaborators with expertise in the field and coming together as a laboratory to perform sometimes grueling, around-the-clock studies. The study of circadian rhythms requires a dedicated and determined team, and it doesn’t hurt to have several people classified as “owls” versus “larks” to keep the machine running over 24 and 48 h periods of sample collections and behavior assessments. Additionally, germ-free work poses its own challenges. The use of germ-free isolators, coupled with working under dim red-light conditions to perform timed tissue collections, gavages, and injections, significantly upped the ante on already difficult tasks, oftentimes requiring creative solutions on the fly. Nevertheless, we persisted, and our work revealed that gut microbes serve as a key element for the establishment of the circadian network in metabolic organs, particularly the liver, transducing how much, when, and what is acquired from the diet (Leone et al., 2015). In the absence of microbial cues, germ-free mice exhibited dampened rhythms not only in core circadian genes but also in circadian clock-controlled genes associated with metabolic outputs. High-fat diets elicited little to no change to transcriptional programming in germ-free mice. However, high-fat diet in conventionally raised animals dampened rhythms of microbial diurnal oscillations in specific members of the community (determined via 16S rRNA gene amplicon sequencing), as well as functional outputs including the short-chain fatty acid, butyrate, which is a microbial product that contributes to health maintenance.

But are food intake and diet composition the only drivers of oscillations in this dynamic host-microbe interaction? In a key collaboration with the laboratory of surgeon Dr. Kenneth Kudsk (one of the only groups worldwide to perform parenteral feeding studies in mice), we examined both host and microbe diurnal dynamics in animals receiving constant parenteral infusion of nutrients as compared to their enterally fed counterparts. Surprisingly, gut microbiota rhythms persisted in animals receiving parenteral feeding, despite a complete reshaping of the microbial community relative to enterally fed mice (Leone et al., 2015). This finding remains intriguing—what are the host factors that contribute to rhythmicity of certain members of the microbial community, and which component, host or microbe, governs local diurnal dynamics in the intestine? Are these interactions necessary for host health? These and other questions are the basis for an independent NIH NIDDK K01 career development award (PI:Leone), and the studies to address them are currently ongoing in the Leone laboratory. This work (Frazier et al., 2022), along with recently published studies by several other groups (Brooks et al., 2021; Thaiss et al., 2016), has revealed that host antimicrobial peptides, such as Regenerating Islet-derived 3 gamma, which targets Gram-positive bacteria predominantly in the small intestine, are engaged in maintaining host-microbe circadian dynamics. Host-derived signals, such as antimicrobial peptides, appear to be essential for the prevention of enteric inflammation and infection (Brooks et al., 2021) and can become disrupted by high-fat diet (Frazier et al., 2022; Thaiss et al., 2016).

While our initial paper published in Cell Host & Microbe (Leone et al., 2015) serves as a seminal manuscript in the field of examining host-microbe circadian dynamics, it was not the first nor the second publication to hit the press. Within 6 months of each other, all within Cell or Cell family journals, both Thaiss et al. and Zarrinpar et al. presented unique yet complementary findings to ours, underscoring the repeatability and importance of these dynamics to host physiology (Thaiss et al., 2014; Zarrinpar et al., 2014). Following these papers, additional studies emerged examining the contribution of other gene components of the host circadian gene network to microbial oscillations, a central role for the immune system as a transducer of circadian dynamics of gut microbial cues, and an initiation of studies providing proof-of-concept for gut microbiota oscillations in human subjects (reviewed in Frazier et al., 2022). Indeed, the crosstalk between gut microbes and peripheral tissue clocks serves as the basis for a NIH NIDDK F31 predoctoral fellowship (PI, Frazier; comentors, Chang and Leone) examining the mechanistic contribution of gut microbes to the circadian dynamics of the gut-liver axis in metabolic health. Importantly, all three first authors on the initial seminal papers are now junior investigators at different institutions across the United States. Yet, while these three labs could be seen as competitors, competition oftentimes bores collaboration, which can allow for creative explorations that ultimately advance the overall goals of the field. In some instances, supportive collaborations have developed, serving as the basis for the next generation of scientists to dig deeper into the mechanistic contributions of gut microbes to circadian networks.

In the context of understanding what constitutes a healthy gut microbial community and bridging off the discoveries of these initial investigations that gut microbial oscillations are both impacted by and associate with diet-induced obesity, we now must wrestle with additional questions and unknowns (as outlined in Figure 1), including what defines a “healthy” oscillation, especially for a microbiome? Oscillations and diurnal patterns contribute to the overwhelming heterogeneity of gut microbiota, which has become a novel and expanding area of focus in microbiome research (Frazier and Chang, 2020). At the very least, these emerging studies provide evidence that time of microbiome sample collection, i.e., saliva or stool, in both the preclinical and clinical arena must be considered and recorded (Frazier et al., 2020). For instance, time-stamped stool samples collected from a large-scale cohort of individual human subjects were used to recreate gut microbiota rhythms, where loss of rhythmicity of certain microbiome-derived features both in relative abundance and functional outputs correlates with and is predictive of metabolic diseases, such as type 2 diabetes (Reitmeier et al., 2020).

Figure 1. Environmental factors (i.e., diet) coupled with circadian disturbances (i.e., sleep loss or jet lag) significantly impact diurnal rhythms of gut microbes driving metabolic disruption.

Circadian alignment (left panel) coupled with a balanced, low-fat, high-fiber diet and proper sleep results in normal oscillations of host-derived antimicrobial peptides (AMPs) in the gut with corresponding rhythms in relative abundance of ~20% of the bacterial community and microbial outputs, including secondary bile acids and short-chain fatty acids (SCFAs). Circadian disruptions (right panel) induced by diets high in saturated fat and sugar or disrupted sleep (i.e., jet lag) result in a loss of gut AMP rhythmicity, gut microbiota imbalances with overall decreased oscillations in relative abundance of specific microbial community members. However, under these conditions, “gain-of-function” microbial oscillations of certain community members and their outputs, such as H₂S and trimethylamine (TMA), are also observed and correspond with metabolic disruption. Several knowledge gaps remain that require further investigation to gain novel insights into host-microbe circadian interactions and define what constitutes a healthy microbiome.

Importantly, the general observation across nearly all preclinical and clinical studies, regardless of the manipulation, is that perturbations, including genetic ablation of circadian genes, high-fat diet, and jet lag, result in a significant reduction in total gut microbial oscillations both in community membership and in functional outputs (Frazier et al., 2020). This “loss of function” in the gut microbiota appears to be extremely important and to contribute to health disturbances. However, nearly all circadian microbiome studies, whether highlighted or not, have observed a concomitant gain in oscillation in other microbial community members and functional outputs. This is particularly evident in studies examining diet-induced obesity and other diseases associated with metabolic syndrome. For instance, in our initial study, we noted a gain in oscillation of potential H₂S-producing bacteria via 16S rRNA gene amplicon sequencing as well as via culture-based, indirect H₂S measurements (Leone et al., 2015). Further, Reitmeier et al. noted that while microbiota oscillations were diminished in a subset of disease-predicting bacteria in human subjects with type 2 diabetes, gains in oscillations were also evident (Reitmeier et al., 2020). Yet, we still do not understand the implications, positive or negative, of a gain in oscillation as it relates to the microbiome. There is a need to develop tools that allow for more precise manipulations to interrogate the relevance of both a loss and a gain in oscillation as it relates to host-microbe circadian dynamics to fully understand their roles in health versus disease.

Perhaps one key to understanding the physiological meaning of a gain or loss in microbial oscillation is the identification and further definition of key microbial mediators responsible for this dynamic host-microbe communication. While there are likely many candidates that have yet to be discovered, several microbially derived metabolites exhibiting circadian dynamics that are involved in host signaling have been explored. In addition to short-chain fatty acids and H₂S outlined in our studies (Leone et al., 2015), secondary bile acids, which are produced via microbial modifications of host-derived primary bile acids in the gut, are known modulators of host energy balance. Not only do key bile acid synthesis enzymes exhibit diurnal expression patterns, but both primary and secondary bile acids exhibit oscillations in host circulation (Frazier and Chang, 2020). Zarrinpar et al. revealed that, in mice, timed feeding of an obesogenic diet can partially restore oscillations in both gut microbiota relative abundances and secondary bile acids relative to ad libitum low-fat-and high-fat-diet-fed counterparts (Zarrinpar et al., 2014). Most recently, microbially derived trimethylamine N-oxide (TMAO), which has been implicated in the incidence of cardiovascular disease, is yet another metabolite that exhibits host-microbe circadian dynamics. Schugar et al. demonstrated that inhibition of gut microbial TMAO production significantly improves host metabolic parameters including insulin sensitivity, weight gain, and energy expenditure, in part via reorganization of both the gut microbiota community and the host circadian clock (Schugar et al., 2022). These are just a few examples of known microbially mediated metabolites, likely just a drop in the bucket for understanding the full scope of diurnal and complex dynamics of the microbially derived metabolome.

While much of the focus on host-microbe circadian dynamics has been on the bacterial component of the gut microbiota via 16S rRNA gene amplicon sequencing, along with interrogation via shotgun metagenomics and metabolomics (Brooks et al., 2021; Leone et al., 2015; Schugar et al., 2022; Thaiss et al., 2014, 2016; Zarrinpar et al., 2014), several additional microbial kingdoms have been left unexplored. For instance, little to no research has examined whether fungi or archaea, both important, albeit less abundant, components of the gut microbiome, exhibit oscillations in abundance and function in a similar manner to bacteria. Due to this, it remains unclear whether oscillations in these community members could be functionally relevant to the host circadian coordination or misalignment present in health versus disease. Other microbial kingdoms that are less relevant to gut microbiomes of rodent or humans have also yet to be explored. For example, protozoa, a key element of the gut microbiome in ruminant animals, such as cows and sheep, exhibit variability over 24 h in both abundance and outputs involved in the production of methane by their symbiotic archaea partners (Brask et al., 2015). Much remains to be explored regarding the mechanistic contribution of these additional microbial rhythms in the context of host health in both animals and humans. Examining key aspects of diurnal oscillations in these additional microbial community members, including fungi, archaea, and protozoa, is necessary to gain insights into their functional contributions to host-microbe ecological circadian dynamics and, in general, what constitutes a healthy microbiome.

The necessity to gain insights into circadian dynamics of additional microbial community members also extends to examining their interactions in other mammalian and avian hosts. While several publications have provided evidence for microbial oscillations in small-scale human subject research, the difficulty of 24 h sampling in humans, particularly beyond stool and saliva collection, limits their utility in moving beyond description. More frequent sampling can be performed in other animal species, such as the pig, to gain translational insights. Further, both the small and large intestine can be cannulated in many agricultural animals used in both biomedical and non-biomedical research, aiding in round-the-clock, time-matched collections of both host and microbial samples from different gut regions. Importantly, pigs, cattle, and poultry are all diurnal animals, exhibiting far more similarities to humans in their circadian behaviors in comparison to the highly popular and readily available rodent model systems. For instance, night-restricted feeding in the pig, a diurnal animal that eats primarily during the daytime, similarly to humans, resulted in loss of oscillations in relative abundances of specific microbial community members with concomitant changes in the circulating proteome (Wang et al., 2021). Importantly, animals such as the pig and chicken can be rederived under germ-free conditions, allowing for the interrogation of circadian dynamics in a growing animal coupled with assembly and oscillations of a microbial community upon colonization.

The implications for host-microbe circadian work in alternative animal models extends far beyond identifying translational applications for human health. Species such as pigs, cattle, and poultry are essential components to global food production systems, and circadian rhythms are crucial in maintaining animal health and welfare. With consumer pressures to move away from the use of antibiotics in animal agriculture due to concerns surrounding antibiotic resistance, defining and understanding what constitutes a “healthy microbiome” has become a central focus, transcending human health alone. Indeed, agricultural animal production systems have pushed outside the circadian norms across species using extended photoperiods to maximize yields, resulting in near around-the-clock milk, egg, and meat production. The consequences of these extreme husbandry conditions and circadian disruptions to gut health in the production setting remain largely unexplored, particularly from a gut microbiome perspective. However, recent work has just begun to explore these issues. Work in poultry model systems has revealed that photoperiod length during early life impacts host circadian gene expression as well as the acquisition and oscillations in relative abundances of gut microbiota in the developing chick (Hieke et al., 2019). Whether and how these early-life phenomena induced by extended photoperiods impact microbial metabolite outputs as well as host immune function and gut health later in life remain to be determined. However, additional inquiry could provide meaningful insights into host-microbe circadian dynamics for both animal agriculture and human health that create sustainable, healthy ecosystems.

The next frontier in host-microbe circadian dynamics is to ask, can we manipulate this system to determine not only how it can be broken but also how it can be repaired? Since gut microbes exhibit some degree of plasticity, could their outputs be a way to reset central and peripheral clocks more rapidly? Regardless of the outcomes of these questions, the discovery that gut microbes are important mediators of circadian networks across species has opened a new direction in microbiome sciences. This focus area offers exciting opportunities to ask a series of questions that will take a lifetime and multiple generations of investigators to address; however, the ability to manipulate this conserved mediator of health could have enormous impact.