Approaches to discern if microbiome associations reflect causation in metabolic and immune disorders.

Our understanding of microorganisms residing within our gut and their roles in the host metabolism and immunity advanced greatly over the past 20 years. Currently, microbiome studies are shifting from association and correlation studies to studies demonstrating causality of identified microbiome signatures and identification of molecular mechanisms underlying these interactions. This transformation is crucial for the efficient translation into clinical application and development of targeted strategies to beneficially modulate the intestinal microbiota. As mechanistic studies are still quite challenging to perform in humans, the causal role of microbiota is frequently evaluated in animal models that need to be appropriately selected. Here, we provide a comprehensive overview on approaches that can be applied in addressing causality of host-microbe interactions in five major animal model organisms (Caenorhabditis elegans, Drosophila melanogaster, zebrafish, rodents, and pigs). We particularly focused on discussing methods available for studying the causality ranging from the usage of gut microbiota transfer, diverse models of metabolic and immune perturbations involving nutritional and chemical factors, gene modifications and surgically induced models, metabolite profiling up to culture-based approached. Furthermore, we addressed the impact of the gut morphology, physiology as well as diet on the microbiota composition in various models and resulting species specificities. Finally, we conclude this review with the discussion on models that can be applied to study the causal role of the gut microbiota in the context of metabolic syndrome and host immunity. We hope this review will facilitate important considerations for appropriate animal model selection.

A Critical Evaluation of In Vitro Hesperidin 2S Bioavailability in a Model Combining Luminal (Microbial) Digestion and Caco-2 Cell Absorption in Comparison to a Randomized Controlled Human Trial.

SCOPE: Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. METHOD AND RESULTS: We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. CONCLUSIONS: In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability.

Ferulic acid-4-O-sulfate rather than ferulic acid relaxes arteries and lowers blood pressure in mice.

Consumption of foods rich in ferulic acid (FA) such as wholegrain cereals, or FA precursors such as chlorogenic acids in coffee, is inversely correlated with risk of cardiovascular disease and type 2 diabetes. As a result of digestion and phase II metabolism in the gut and liver, FA is converted predominantly into ferulic acid-4-O-sulfate (FA-sul), an abundant plasma metabolite. Although FA-sul is the main metabolite, very little has been reported regarding its bioactivities. We have compared the ex vivo vasorelaxing effect of FA and FA-sul (10-7-3.10-5M) on isolated mouse arteries mounted in tissue myographs. FA-sul, but not FA, elicited a concentration-dependent vasorelaxation of saphenous and femoral arteries and aortae. The FA-sul-mediated vasorelaxation was blunted by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a soluble guanylate cyclase (sGC) inhibitor. The role of sGC was confirmed in femoral arteries isolated from sGCα1(-/-) knockout mice. Furthermore, 4-aminopyridine, a specific inhibitor of voltage-dependent potassium channels, significantly decreased FA-sul-mediated effects. In anesthetized mice, intravenous injection of FA-sul decreased mean arterial pressure, whereas FA had no effect, confirming the results obtained ex vivo. FA-sul is probably one of the major metabolites accounting for the blood pressure-lowering effects associated with FA consumption.

Chronic exposure to short-chain fatty acids modulates transport and metabolism of microbiome-derived phenolics in human intestinal cells.

Dietary fiber-derived short-chain fatty acids (SCFA) and phenolics produced by the gut microbiome have multiple effects on health. We have tested the hypothesis that long-term exposure to physiological concentrations of SCFA can affect the transport and metabolism of (poly)phenols by the intestinal epithelium using the Caco-2 cell model. Metabolites and conjugates of hesperetin (HT) and ferulic acid (FA), gut-derived from dietary hesperidin and chlorogenic acid, respectively, were quantified by LC-MS with authentic standards following transport across differentiated cell monolayers. Changes in metabolite levels were correlated with effects on mRNA and protein expression of key enzymes and transporters. Propionate and butyrate increased both FA transport and rate of appearance of FA glucuronide apically and basolaterally, linked to an induction of MCT1. Propionate was the only SCFA that augmented the rate of formation of basolateral FA sulfate conjugates, possibly via basolateral transporter up-regulation. In addition, propionate enhanced the formation of HT glucuronide conjugates and increased HT sulfate efflux toward the basolateral compartment. Acetate treatment amplified transepithelial transport of FA in the apical to basolateral direction, associated with lower levels of MCT1 protein expression. Metabolism and transport of both HT and FA were curtailed by the organic acid lactate owing to a reduction of UGT1A1 protein levels. Our data indicate a direct interaction between microbiota-derived metabolites of (poly)phenols and SCFA through modulation of transporters and conjugating enzymes and increase our understanding of how dietary fiber, via the microbiome, may affect and enhance uptake of bioactive molecules.