Diet in the driving seat: natural diet-immunity-microbiome interactions in wild fish

Natural interactions between the diet, microbiome and immunity are largely unstudied. Here we employ wild three-spined sticklebacks as a model, combining field observations with complementary experimental manipulations of diet designed to mimic seasonal variation in the wild. We clearly demonstrate that season-specific diets are a powerful causal driver of major systemic immunophenotypic variation. This effect occurred largely independently of the bulk composition of the bacterial microbiome (which was also driven by season and diet) and of host condition, demonstrating neither of these, per se, constrain immune allocation in healthy individuals. Nonetheless, through observations in multiple anatomical compartments, differentially exposed to the direct effects of food and immunity, we found evidence of immune-driven control of bacterial community composition in mucus layers. This points to the interactive nature of the host-microbiome relationship, and is the first time, to our knowledge, that this causal chain (diet → immunity → microbiome) has been demonstrated in wild vertebrates. Microbiome effects on immunity were not excluded and, importantly, we identified outgrowth of potentially pathogenic bacteria (especially mycolic-acid producing corynebacteria) as a consequence of the more animal-protein-rich summertime diet. This may provide part of the ultimate explanation (and possibly a proximal cue) for the dramatic immune re-adjustments that we saw in response to diet change

Robotic injection of zebrafish embryos for high-throughput screening in disease models

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines

Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet β-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription

Using fish models to investigate the links between microbiome and social behaviour: the next step for translational microbiome research?

Recent research has revealed surprisingly important connections between animals’ microbiome and social behaviour. Social interactions can affect the composition and function of the microbiome; conversely, the microbiome affects social communication by influencing the hosts’ central nervous system and peripheral chemical communication. These discoveries set the stage for novel research focusing on the evolution and physiology of animal social behaviour in relation to microbial transmission strategies. Here, we discuss the emerging roles of teleost fish models and their potential for advancing research fields, linked to sociality and microbial regulation. We argue that fish models, such as the zebrafish (Danio rerio, Cyprinidae), sticklebacks (‎Gasterosteidae), guppies (Poeciliidae) and cleaner–client dyads (e.g., obligate cleaner fish from the Labridae and Gobiidae families and their visiting clientele), will provide valuable insights into the roles of microbiome in shaping social behaviour and vice versa, while also being of direct relevance to the food and ornamental fish trades. The diversity of fish behaviour warrants more interdisciplinary research, including microbiome studies, which should have a strong ecological (field‐derived) approach, together with laboratory‐based cognitive and neurobiological experimentation. The implications of such integrated approaches may be of translational relevance, opening new avenues for future investigation using fish models

Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations

Animal hosts must co-exist with beneficial microbes while simultaneously being able to mount rapid, non-specific, innate immune responses to pathogenic microbes. How this balance is achieved is not fully understood, and disruption of this relationship can lead to disease. Excessive inflammatory responses to resident microbes are characteristic of certain gastrointestinal pathologies such as inflammatory bowel disease (IBD). The immune dysregulation of IBD has complex genetic underpinnings that cannot be fully recapitulated with single-gene-knockout models. A deeper understanding of the genetic regulation of innate immune responses to resident microbes requires the ability to measure immune responses in the presence and absence of the microbiota using vertebrate models with complex genetic variation. Here, we describe a new gnotobiotic vertebrate model to explore the natural genetic variation that contributes to differences in innate immune responses to microbiota. Threespine stickleback, Gasterosteus aculeatus, has been used to study the developmental genetics of complex traits during the repeated evolution from ancestral oceanic to derived freshwater forms. We established methods to rear germ-free stickleback larvae and gnotobiotic animals monoassociated with single bacterial isolates. We characterized the innate immune response of these fish to resident gut microbes by quantifying the neutrophil cells in conventionally reared monoassociated or germ-free stickleback from both oceanic and freshwater populations grown in a common intermediate salinity environment. We found that oceanic and freshwater fish in the wild and in the laboratory share many intestinal microbial community members. However, oceanic fish mount a strong immune response to residential microbiota, whereas freshwater fish frequently do not. A strong innate immune response was uniformly observed across oceanic families, but this response varied among families of freshwater fish. The gnotobiotic stickleback model that we have developed therefore provides a platform for future studies mapping the natural genetic basis of the variation in immune response to microbes

Surfactants, Aromatic and Isoprenoid Compounds, and Fatty Acid Biosynthesis Inhibitors Suppress Staphylococcus aureus Production of Toxic Shock Syndrome Toxin 1▿

Menstrual toxic shock syndrome is a rare but potentially life-threatening illness manifest through the actions of Staphylococcus aureus toxic shock syndrome toxin 1 (TSST-1). Previous studies have shown that tampon additives can influence staphylococcal TSST-1 production. We report here on the TSST-1-suppressing activity of 34 compounds that are commonly used additives in the pharmaceutical, food, and perfume industries. Many of the tested chemicals had a minimal impact on the growth of S. aureus and yet were potent inhibitors of TSST-1 production. The TSST-1-reducing compounds included surfactants with an ether, amide, or amine linkage to their fatty acid moiety (e.g., myreth-3-myristate, Laureth-3, disodium lauroamphodiacetate, disodium lauramido monoethanolamido, sodium lauriminodipropionic acid, and triethanolamine laureth sulfate); aromatic compounds (e.g. phenylethyl and benzyl alcohols); and several isoprenoids and related compounds (e.g., terpineol and menthol). The membrane-targeting and -altering effects of the TSST-1-suppressing compounds led us to assess the activity of molecules that are known to inhibit fatty acid biosynthesis (e.g., cerulenin, triclosan, and hexachlorophene). These compounds also reduced S. aureus TSST-1 production. This study suggests that more additives than previously recognized inhibit the production of TSST-1

Supporting Biomedical Research Training for Historically Underrepresented Undergraduates Using Interprofessional, Nonformal Education Structures

Research experience provides critical training for new biomedical research scientists. Students from underrepresented populations studying science, technology, engineering, and mathematics (STEM) are increasingly recruited into research pathways to diversify STEM fields. However, support structures outside of research settings designed to help these students navigate biomedical research pathways are not always available; nor are program support components outside the context of laboratory technical skills training and formal mentorship well understood. This study leveraged a multi-institutional research training program, Enhancing Cross-Disciplinary Infrastructure and Training at Oregon (EXITO), to explore how nine institutions designed a new curricular structure (Enrichment) to meet a common goal of enhancing undergraduate research training and student success. EXITO undergraduates participated in a comprehensive, 3-year research training program with the Enrichment component offered across nine sites: three universities and six community colleges, highly diverse in size, demographics, and location. Sites’ approaches to supporting students in the training program were studied over a 30-month period. All sites independently created their own nonformal curricular structures, implemented interprofessionally via facilitated peer groups. Site data describing design and implementation were thematically coded to identify essential programmatic components across sites, with student feedback used to triangulate findings. Enrichment offered students time to critically reflect on their interests, experiences, and identities in research; network with peers and professionals; and support negotiation of hidden and implicit curricula. Students reported the low-pressure setting and student-centered curriculum balanced the high demands associated with academics and research. Core curricular themes described Enrichment as fostering a sense of community among students, exposing students to career paths and skills, and supporting development of students’ professional identities. The non-formal, interprofessional curricula enabled students to model diverse biomedical identities and pathways for each other while informing institutional structures to improve diverse undergraduate students’ success in academia and research