Gut microbes and the liver circadian clock partition glucose and lipid metabolism.

Circadian rhythms govern glucose homeostasis, and their dysregulation leads to complex metabolic diseases. Gut microbes exhibit diurnal rhythms that influence host circadian networks and metabolic processes, yet underlying mechanisms remain elusive. Here, we showed hierarchical, bidirectional communication among the liver circadian clock, gut microbes, and glucose homeostasis in mice. To assess this relationship, we utilized mice with liver-specific deletion of the core circadian clock gene Bmal1 via Albumin-cre maintained in either conventional or germ-free housing conditions. The liver clock, but not the forebrain clock, required gut microbes to drive glucose clearance and gluconeogenesis. Liver clock dysfunctionality expanded proportions and abundances of oscillating microbial features by 2-fold relative to that in controls. The liver clock was the primary driver of differential and rhythmic hepatic expression of glucose and fatty acid metabolic pathways. Absent the liver clock, gut microbes provided secondary cues that dampened these rhythms, resulting in reduced lipid fuel utilization relative to carbohydrates. All together, the liver clock transduced signals from gut microbes that were necessary for regulating glucose and lipid metabolism and meeting energy demands over 24 hours.

Peptide YY: A Paneth cell antimicrobial peptide that maintains Candida gut commensalism.

The mammalian gut secretes a family of multifunctional peptides that affect appetite, intestinal secretions, and motility whereas others regulate the microbiota. We have found that peptide YY (PYY1-36), but not endocrine PYY3-36, acts as an antimicrobial peptide (AMP) expressed by gut epithelial paneth cells (PC). PC-PYY is packaged into secretory granules and is secreted into and retained by surface mucus, which optimizes PC-PYY activity. Although PC-PYY shows some antibacterial activity, it displays selective antifungal activity against virulent Candida albicans hyphae-but not the yeast form. PC-PYY is a cationic molecule that interacts with the anionic surfaces of fungal hyphae to cause membrane disruption and transcriptional reprogramming that selects for the yeast phenotype. Hence, PC-PYY is an antifungal AMP that contributes to the maintenance of gut fungal commensalism.

Phenylpropionic acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity.

The gut microbiota affects hepatic drug metabolism. However, gut microbial factors modulating hepatic drug metabolism are largely unknown. In this study, using a mouse model of acetaminophen (APAP)-induced hepatotoxicity, we identified a gut bacterial metabolite that controls the hepatic expression of CYP2E1 that catalyzes the conversion of APAP to a reactive, toxic metabolite. By comparing C57BL/6 substrain mice from two different vendors, Jackson (6J) and Taconic (6N), which are genetically similar but harbor different gut microbiotas, we established that the differences in the gut microbiotas result in differential susceptibility to APAP-induced hepatotoxicity. 6J mice exhibited lower susceptibility to APAP-induced hepatotoxicity than 6N mice, and such phenotypic difference was recapitulated in germ-free mice by microbiota transplantation. Comparative untargeted metabolomic analysis of portal vein sera and liver tissues between conventional and conventionalized 6J and 6N mice led to the identification of phenylpropionic acid (PPA), the levels of which were higher in 6J mice. PPA supplementation alleviated APAP-induced hepatotoxicity in 6N mice by lowering hepatic CYP2E1 levels. Moreover, PPA supplementation also reduced carbon tetrachloride-induced liver injury mediated by CYP2E1. Our data showed that previously known PPA biosynthetic pathway is responsible for PPA production. Surprisingly, while PPA in 6N mouse cecum contents is almost undetectable, 6N cecal microbiota produces PPA as well as 6J cecal microbiota in vitro, suggesting that PPA production in the 6N gut microbiota is suppressed in vivo. However, previously known gut bacteria harboring the PPA biosynthetic pathway were not detected in either 6J or 6N microbiota, suggesting the presence of as-yet-unidentified PPA-producing gut microbes. Collectively, our study reveals a novel biological function of the gut bacterial metabolite PPA in the gut-liver axis and presents a critical basis for investigating PPA as a modulator of CYP2E1-mediated liver injury and metabolic diseases.

Imbalanced gut microbiota predicts and drives the progression of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in a fast-food diet mouse model.

Nonalcoholic fatty liver disease (NAFLD) is multifactorial in nature, affecting over a billion people worldwide. The gut microbiome has emerged as an associative factor in NAFLD, yet mechanistic contributions are unclear. Here, we show fast food (FF) diets containing high fat, added cholesterol, and fructose/glucose drinking water differentially impact short- vs. long-term NAFLD severity and progression in conventionally-raised, but not germ-free mice. Correlation and machine learning analyses independently demonstrate FF diets induce early and specific gut microbiota changes that are predictive of NAFLD indicators, with corresponding microbial community instability relative to control-fed mice. Shotgun metagenomics showed FF diets containing high cholesterol elevate fecal pro-inflammatory effectors over time, relating to a reshaping of host hepatic metabolic and inflammatory transcriptomes. FF diet-induced gut dysbiosis precedes onset and is highly predictive of NAFLD outcomes, providing potential insights into microbially-based pathogenesis and therapeutics.

Gut microbiota modulates bleomycin-induced acute lung injury response in mice.

BACKGROUND: Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. METHODS: Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. RESULTS: Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors' response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. CONCLUSION: The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

Atypical behavioral and thermoregulatory circadian rhythms in mice lacking a microbiome.

Trillions of microbial oscillators reside throughout the mammalian body, yet their contributions toward fundamental features of host circadian rhythms (CRs) have not been characterized. Here, we demonstrate that the microbiome contributes to host CRs in activity and thermoregulation. Mice devoid of microbes (germ-free, GF) exhibited higher-amplitude CRs in a light-dark cycle and longer circadian periods in constant darkness. Circadian entrainment to food was greater in GF mice, but resetting responses to simulated jet-lag were unaffected. Microbial transplantation with cecal contents of conventionally-raised mice normalized CRs of GF mice, indicating that the concurrent activity of gut microbes modulates host circadian networks. Obesogenic effects of high-fat diet were absent in GF mice, but some circadian-disruptive effects persisted. Transkingdom (host-microbe) interactions affect circadian period and entrainment of CRs in diverse traits, and microbes alter interactions among light- and food-entrainable circadian processes in the face of environmental (light, diet) perturbations.

High-fat diet disrupts REG3γ and gut microbial rhythms promoting metabolic dysfunction.

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism ensuring optimal energy balance. However, the interplay between diet, microbes, and host factors sustaining intestinal oscillations is complex and poorly understood. Here, using a mouse model, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bidirectional manner and does not correlate with the intestinal core circadian clock. High-fat diet is the primary driver of microbial oscillators that impair host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression that secondarily drives abundance and oscillation of key gut microbes. This illustrates transkingdom coordination of biological rhythms primarily influenced by diet and reciprocal sensor-effector signals between host and microbial components, ultimately driving metabolism. Restoring the gut microbiota's capacity to sense dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.

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

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.

Mediators of Host-Microbe Circadian Rhythms in Immunity and Metabolism.

Circadian rhythms are essential for nearly all life forms, mediated by a core molecular gene network that drives downstream molecular processes involved in immune function and metabolic regulation. These biological rhythms serve as the body's metronome in response to the 24-hour light:dark cycle and other timed stimuli. Disrupted circadian rhythms due to drastic lifestyle and environmental shifts appear to contribute to the pathogenesis of metabolic diseases, although the mechanisms remain elusive. Gut microbiota membership and function are also key mediators of metabolism and are highly sensitive to environmental perturbations. Recent evidence suggests rhythmicity of gut microbes is essential for host metabolic health. The key molecular mediators that transmit rhythmic signals between microbes and host metabolic networks remain unclear, but studies suggest the host immune system may serve as a conduit between these two systems, providing homeostatic signals to maintain overall metabolic health. Despite this knowledge, the precise mechanism and communication modalities that drive these rhythms remain unclear, especially in humans. Here, we review the current literature examining circadian dynamics of gut microbes, the immune system, and metabolism in the context of metabolic dysregulation and provide insights into gaps and challenges that remain.

Microbiota Can't Keep Time in Type 2 Diabetes.

Gut microbes exhibit diurnal rhythmicity, and disruptions in this rhythmicity potentially impact host health. In this issue of Cell Host & Microbe, Reitmeier et al. (2020) employ timestamped gut microbiome sequencing data from human subjects coupled with machine learning to identify microbial rhythmicity patterns that predict Type 2 Diabetes incidence.

Microbial Colonization of Germ-Free Mice Restores Neointimal Hyperplasia Development After Arterial Injury.

Background The potential role of the gut microbiome in cardiovascular diseases is increasingly evident. Arterial restenosis attributable to neointimal hyperplasia after cardiovascular procedures such as balloon angioplasty, stenting, and bypass surgery is a common cause of treatment failure, yet whether gut microbiota participate in the development of neointimal hyperplasia remains largely unknown. Methods and Results We performed fecal microbial transplantation from conventionally raised male C57BL/6 mice to age-, sex-, and strain-matched germ-free mice. Five weeks after inoculation, all mice underwent unilateral carotid ligation. Neointimal hyperplasia development was quantified after 4 weeks. Conventionally raised and germ-free cohorts served as comparison groups. Conclusions Germ-free mice have significantly attenuated neointimal hyperplasia development compared with conventionally raised mice. The arterial remodeling response is restored by fecal transplantation. Our results describe a causative role of gut microbiota in contributing to the pathogenesis of neointimal hyperplasia.

Microbiota control acute arterial inflammation and neointimal hyperplasia development after arterial injury.

BACKGROUND: The microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice. METHODS: Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods. RESULTS: All GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson's trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-γ-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response. CONCLUSIONS: GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.

Distinct roles of intracellular heat shock protein 70 in maintaining gastrointestinal homeostasis.

The inducible heat shock protein 70 (Hsp70) is both cytoprotective and immunomodulatory, potentially accounting for its critical role in maintaining gastrointestinal homeostasis. When levels are reduced in conditions like inflammatory bowel diseases (IBD), loss of function contributes to the severity and chronicity of these diseases, although through which cell types and mechanisms remains unclear. Here, the role of Hsp70-mediated intestinal epithelial protection and immune regulation in experimental colitis was examined by using a villin promoter-driven Hsp70 transgene in the 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models and in IL-10/Hsp70 double knockout (IL10-/-/Hsp70-/-) mice. In addition, Hsp70-mediated IL-10 production and immune protection were investigated using a CD45RBhigh transfer model and measuring colonic and immune cell cytokine expression during colitis. We found that the epithelial-specific expression of Hsp70 transgene attenuated DSS-induced colitis in Hsp70-/- mice by protecting tight junctions (TJ) and their interaction with the TJ-associated protein ZO-1. In the TNBS colitis and CD45RBhigh model, Hsp70 carried out its intracellular anti-inflammatory function by maintaining IL-10 production. Impaired ERK phosphorylation, but not p38 or JNK phosphorylation pathways, was associated with decreased IL-10 production in Hsp70-deficient cells. Together, these actions can be leveraged in the context of cellular specificity to develop complementary strategies that can lead to reduction in mucosal injury and immune activation in colonic colitis development. NEW & NOTEWORTHY Using four different experimental colitis models, we filled an important gap in knowledge by defining essential roles of intracellular heat shock protein 70 in different cell types in maintaining intestinal integrity and immune regulation. These findings are relevant to human inflammatory bowel diseases and represent potential avenues for developing therapeutic strategies, not only to counter the destructive processes of inflammation but also to promote tissue healing and prevent complications frequently associated with chronic intestinal inflammation.

Mutual reinforcement of pathophysiological host-microbe interactions in intestinal stasis models.

Chronic diseases arise when there is mutual reinforcement of pathophysiological processes that cause an aberrant steady state. Such a sequence of events may underlie chronic constipation, which has been associated with dysbiosis of the gut. In this study we hypothesized that assemblage of microbial communities, directed by slow gastrointestinal transit, affects host function in a way that reinforces constipation and further maintains selection on microbial communities. In our study, we used two models - an opioid-induced constipation model in mice, and a humanized mouse model where germ-free mice were colonized with stool from a patient with constipation-predominant irritable bowel syndrome (IBS-C) in humans. We examined the impact of pharmacologically (loperamide)-induced constipation (PIC) and IBS-C on the structural and functional profile of the gut microbiota. Germ-free (GF) mice were colonized with microbiota from PIC donor mice and IBS-C patients to determine how the microbiota affects the host. PIC and IBS-C promoted changes in the gut microbiota, characterized by increased relative abundance of Bacteroides ovatus and Parabacteroides distasonis in both models. PIC mice exhibited decreased luminal concentrations of butyrate in the cecum and altered metabolic profiles of the gut microbiota. Colonization of GF mice with PIC-associated mice cecal or human IBS-C fecal microbiota significantly increased GI transit time when compared to control microbiota recipients. IBS-C-associated gut microbiota also impacted colonic contractile properties. Our findings support the concept that constipation is characterized by disease-associated steady states caused by reinforcement of pathophysiological factors in host-microbe interactions.

Insights into the pathogenesis of ulcerative colitis from a murine model of stasis-induced dysbiosis, colonic metaplasia, and genetic susceptibility.

Gut dysbiosis, host genetics, and environmental triggers are implicated as causative factors in inflammatory bowel disease (IBD), yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis (UC) patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically created blind self-filling (SFL) and self-emptying (SEL) ileal loops using wild-type (WT), IL-10 knockout (KO) (IL-10), TLR4 KO (T4), and IL-10/T4 double KO mice. After 5 wk, loop histology, host gene/protein expression, and bacterial 16s rRNA profiles were examined. SFL exhibit fecal stasis due to directional motility oriented toward the loop end, whereas SEL remain empty. In WT mice, SFL, but not SEL, develop pouchlike microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. The inflammation associated with IL-10 required TLR4, as animals lacking both pathways displayed little disease. Furthermore, germ-free IL-10 mice conventionalized with SFL, but not SEL, microbiota populations develop severe colitis. These data support essential roles of stasis-induced, colon-like microbiota, TLR4-mediated colonic metaplasia, and genetic susceptibility in the development of pouchitis and possibly UC. However, these factors by themselves are not sufficient. Similarities between this model and human UC/pouchitis provide opportunities for gaining insights into the mechanistic basis of IBD and for identification of targets for novel preventative and therapeutic interventions.

Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases.

BACKGROUND: Commensal gut microbiota play an important role in regulating metabolic and inflammatory conditions. Reshaping intestinal microbiota through pharmacologic means may be a viable treatment option. We sought to delineate the functional characteristics of glucocorticoid-mediated alterations on gut microbiota and their subsequent repercussions on host mucin regulation and colonic inflammation. METHODS: Adult male C57Bl/6 mice, germ-free, Muc2-heterozygote (±), or Muc2-knockout (-/-) were injected with dexamethasone, a synthetic glucocorticoid, for 4 weeks. Fecal samples were collected for gut microbiota analysis through 16S rRNA terminal restriction fragment length polymorphism and amplicon sequencing. Intestinal mucosa was collected for mucin gene expression studies. Germ-free mice were conventionalized with gut microbes from treated and nontreated groups to determine their functional capacities in recipient hosts. RESULTS: Exposure to dexamethasone in wild-type mice led to substantial shifts in gut microbiota over a 4-week period. Furthermore, a significant downregulation of colonic Muc2 gene expression was observed after treatment. Muc2-knockout mice harbored a proinflammatory environment of gut microbes, characterized by the increase or decrease in prevalence of specific microbiota populations such as Clostridiales and Lactobacillaceae, respectively. This colitogenic phenotype was transmissible to IL10-knockout mice, a genetically susceptible model of colonic inflammatory disorders. Microbiota from donors pretreated with dexamethasone, however, ameliorated symptoms of inflammation. CONCLUSIONS: Commensal gut bacteria may be a key mediator of the anti-inflammatory effects observed in the large intestine after glucocorticoid exposure. These findings underscore the notion that intestinal microbes comprise a "microbial organ" essential for host physiology that can be targeted by therapeutic approaches to restore intestinal homeostasis.

Diet, gut microbes, and genetics in immune function: can we leverage our current knowledge to achieve better outcomes in inflammatory bowel diseases?

Autoimmune disorders, particularly inflammatory bowel diseases (IBD), are increasing at an alarming frequency. While the exact cause remains elusive, studies have examined how the immune system is shaped in the context of genetic susceptibility, gut microbes, and environmental pressures, including dietary intake. Shifts towards a Westernized high fat, high carbohydrate diet result in changes to gut microbiota structure and function that may aid in triggering and perpetuating autoimmunity by promoting the emergence of pathobionts leading to altered immune activation. This review summarizes our current understanding of dietary-induced changes in gut microbiota on autoimmunity in the context of IBD. We provide a framework for leveraging this knowledge to develop new dietary, microbial and immune-based modulation strategies for individualized risk assessment and improving clinical outcomes.

The role of diet in triggering human inflammatory disorders in the modern age.

Previously uncommon human inflammatory disorders are emerging with alarming frequency, possibly triggered by environmental factors introduced through Westernization. This review highlights how Western diets heighten the inflammatory state promoting development of disease. Evidence that this can occur directly or indirectly through perturbations of host-microbe interactions are reviewed.

Composition of dietary fat source shapes gut microbiota architecture and alters host inflammatory mediators in mouse adipose tissue.

BACKGROUND: Growing evidence shows that dietary factors can dramatically alter the gut microbiome in ways that contribute to metabolic disturbance and progression of obesity. In this regard, mesenteric adipose tissue has been implicated in mediating these processes through the elaboration of proinflammatory adipokines. In this study, we examined the relationship of these events by determining the effects of dietary fat content and source on gut microbiota, as well as the effects on adipokine profiles of mesenteric and peripheral adipocytes. METHODS: Adult male C57Bl/6 mice were fed milk fat-based, lard-based (saturated fatty acid sources), or safflower oil (polyunsaturated fatty acid)-based high-fat diets for 4 weeks. Body mass and food consumption were measured. Stool 16S ribosomal RNA (rRNA) was isolated and analyzed via terminal restriction fragment length polymorphism as well as variable V3-4 sequence tags via next-generation sequencing. Mesenteric and gonadal adipose samples were analyzed for both lipogenic and inflammatory mediators via quantitative real-time polymerase chain reaction. RESULTS: High-fat feedings caused more weight gain with concomitant increases in caloric consumption relative to low-fat diets. In addition, each of the high-fat diets induced dramatic and specific 16S rRNA phylogenic profiles that were associated with different inflammatory and lipogenic mediator profiles of mesenteric and gonadal fat depots. CONCLUSIONS: Our findings support the notion that dietary fat composition can both reshape the gut microbiota and alter host adipose tissue inflammatory/lipogenic profiles. They also demonstrate the interdependency of dietary fat source, commensal gut microbiota, and inflammatory profile of mesenteric fat that can collectively affect the host metabolic state.