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.