Dr Silke Kiessling

Targeted sequencing of 16S rRNA genes enables the analysis of microbiomes. Here, we describe a protocol for the collection, storage, and preparation of fecal samples. We describe how we cluster similar sequences and assign bacterial taxonomies. Using diversity analysis and machine learning, we can extract disease-associated features. We also describe a circadian analysis to identify the presence or absence of rhythms in taxonomies. Differences in rhythmicity between cohorts can contribute to determining disease-associated bacterial signatures. For complete details on the use and execution of this protocol, please refer to Reitmeier et al. (2020).

Diurnal (i.e., 24-hour) oscillations of the gut microbiome have been described in various species including mice and humans. However, the driving force behind these rhythms remains less clear. In this study, we differentiate between endogenous and exogenous time cues driving microbial rhythms. Our results demonstrate that fecal microbial oscillations are maintained in mice kept in the absence of light, supporting a role of the host’s circadian system rather than representing a diurnal response to environmental changes. Intestinal epithelial cell-specific ablation of the core clock gene Bmal1 disrupts rhythmicity of microbiota. Targeted metabolomics functionally link intestinal clock-controlled bacteria to microbial-derived products, in particular branched-chain fatty acids and secondary bile acids. Microbiota transfer from intestinal clock-deficient mice into germ-free mice altered intestinal gene expression, enhanced lymphoid organ weights and suppressed immune cell recruitment. These results highlight the importance of functional intestinal clocks for microbiota composition and function, which is required to balance the host’s gastrointestinal homeostasis.

Abstract Objective Impaired clock genes expression has been observed in biopsy samples from patients with inflammatory bowel disease (IBD). Disruption of circadian rhythms, which occurs in shift workers, has been linked to an increased risk of gastrointestinal diseases, including IBD. The intestinal clock balances gastrointestinal homeostasis by regulating the microbiome. Here we characterize intestinal immune functions in mice lacking the intestinal clock and IBD-relevant mouse model under different feeding conditions to describe the functional impact of the intestinal clock in the development of gastrointestinal inflammation. Design Tissues and fecal samples from intestinal clock-deficient mice (Bmal1IEC-/-) and mouse models for colitis (IL-10-/-, Bmal1IEC-/-xIL-10-/-, dextran sulfate sodium (DSS) administration) under ad libitum and restricted feeding (RF) conditions were used to determine the causal role of the intestinal clock for colitis. Results In IL-10-/- mice, inflammation correlated with disrupted colon clock genes expression. Genetic loss of intestinal clock functions promoted DSS and IBD inflammatory phenotypes and dramatically reduces survival, and colonization with disease-associated microbiota in germ- free Bmal1IEC-/- hosts increased their inflammatory responses, demonstrating the causal role of colonic clock disruption and the severity of IBD. RF in IL-10-/- mice restored the colon clock and related immune functions, improved the inflammatory responses and rescued the histopathological phenotype. In contrast, RF failed to improve IBD symptoms in Bmal1IEC-/- xIL-10-/- demonstrating the significance of the colonic clock to gate the effect of RF. Conclusion We provide evidence that inflammation-associated intestinal clock dysfunction triggers host immune imbalance and promotes the development and progression of IBD-like colitis. Enhancing intestinal clock function by RF modulates the pathogenesis of IBD and thus could become a novel strategy to ameliorate the symptoms in IBD patients. Competing Interest Statement The authors have declared no competing interest.

Circadian disruption, e.g. through shift work, causes microbial dysbiosis and increases the risk of metabolic diseases. Microbial rhythmicity in mice depends on a functional intestinal clock and frequent jetlag as well as high-caloric energy intake induces loss of these oscillations. Similarly, arrhythmic microbiota was found in obese and T2D populations. However, the interplay between the intestinal circadian clock, the microbiome, diet and host metabolism is poorly understood. In intestinal-specific Bmal1 knockout mice (Bmal1IEC-/- ) we demonstrate the relevance of the intestinal clock in microbiome oscillations and host and microbial nutrient metabolism. Microbiota transfer from Bmal1IEC-/- mice into germ-free recipients led to obesity, reflected by increased bodyweight and fat mass. Western diet-fed Bmal1IEC-/- mice increased bodyweight likely through mechanisms involving the intestinal clock-control of lipid and hexose transporters. Additionally, we identified dietary fiber as novel link between circadian microbial rhythmicity, intestinal clock functioning and host physiology. Thus, revealing the potential of fiber-rich diet intervention as a non-invasive strategy targeting microbial oscillations in metabolic disease prevention.

Lifestyle, obesity, and the gut microbiome are important risk factors for metabolic disorders. We demonstrate in 1,976 subjects of a German population cohort (KORA) that specific microbiota members show 24-h oscillations in their relative abundance and identified 13 taxa with disrupted rhythmicity in type 2 diabetes (T2D). Cross-validated prediction models based on this signature similarly classified T2D. In an independent cohort (FoCus), disruption of microbial oscillation and the model for T2D classification was confirmed in 1,363 subjects. This arrhythmic risk signature was able to predict T2D in 699 KORA subjects 5 years after initial sampling, being most effective in combination with BMI. Shotgun metagenomic analysis functionally linked 26 metabolic pathways to the diurnal oscillation of gut bacteria. Thus, a cohort-specific risk pattern of arrhythmic taxa enables classification and prediction of T2D, suggesting a functional link between circadian rhythms and the microbiome in metabolic diseases. [Display omitted] •Human gut microbiome exhibits diurnal rhythmicity across populations and individuals•Obese and T2D individuals show disrupted circadian rhythms in the gut microbiome•Arrhytmic bacterial signatures contribute to risk classification and prediction of T2D•These risk signatures show regional differences in applicability across three cohorts Reitmeier et al. show that specific gut microbes exhibit rhythmic oscillations in relative abundance and identified taxa with disrupted rhythmicity in individuals with type 2 diabetes (T2D). This arrhythmic signature contributed to the classification and prediction of T2D, suggesting functional links between circadian rhythmicity and the microbiome in metabolic diseases.

Objective Internal clocks time behavior and physiology, including the gut microbiome, in a circadian (∼24 h) manner. Mismatch between internal and external time, e.g. during shift work, disrupts circadian system coordination promoting the development of obesity and type 2 diabetes (T2D). Conversely, body weight changes induce microbiota dysbiosis. The relationship between circadian disruption and microbiota dysbiosis in metabolic diseases, however, remains largely unknown. Methods Core and accessory clock gene expression in different gastrointestinal (GI) tissues were determined by qPCR in two different models of circadian disruption - mice with Bmal1 deficiency in the circadian pacemaker, the suprachiasmatic nucleus (Bmal1SCNfl/-), and wild-type mice exposed to simulated shift work (SSW). Body composition and energy balance were evaluated by nuclear magnetic resonance (NMR), bomb calorimetry, food intake and running-wheel activity. Intestinal permeability was measured in an Ussing chamber. Microbiota composition and functionality were evaluated by 16S rRNA gene amplicon sequencing, PICRUST2.0 analysis and targeted metabolomics. Finally, microbiota transfer was conducted to evaluate the functional impact of SSW-associated microbiota on the host's physiology. Results Both chronodisruption models show desynchronization within and between peripheral clocks in GI tissues and reduced microbial rhythmicity, in particular in taxa involved in short-chain fatty acid (SCFA) fermentation and lipid metabolism. In Bmal1SCNfl/- mice, loss of rhythmicity in microbial functioning associates with previously shown increased body weight, dysfunctional glucose homeostasis and adiposity. Similarly, we observe an increase in body weight in SSW mice. Germ-free colonization experiments with SSW-associated microbiota mechanistically link body weight gain to microbial changes. Moreover, alterations in expression of peripheral clock genes as well as clock-controlled genes (CCGs) relevant for metabolic functioning of the host were observed in recipients, indicating a bidirectional relationship between microbiota rhythmicity and peripheral clock regulation. Conclusions Collectively, our data suggest that loss of rhythmicity in bacteria taxa and their products, which likely originates in desynchronization of intestinal clocks, promotes metabolic abnormalities during shift work.