Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

(2023) 5:15 López Nadal et al. Animal Microbiome https://doi.org/10.1186/s42523-023-00230-2 Animal Microbiome Open Access RESEARCH Omics and imaging combinatorial approach reveals butyrate‑induced inflammatory effects in the zebrafish gut Adrià López Nadal1,2,3, Jos Boekhorst3, Carolien Lute1, Frank van den Berg2, Michelle A. Schorn4, Tommy Bergen Eriksen5, David Peggs5, Charles McGurk5, Detmer Sipkema4, Michiel Kleerebezem3, Geert F. Wiegertjes2 and Sylvia Brugman3* Abstract Background Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. Results We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dosedependent increase of neutrophils and macrophages in the gut area. *Correspondence: Sylvia Brugman Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. López Nadal et al. Animal Microbiome (2023) 5:15 Page 2 of 21 Conclusion The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life. Keywords Microbiome, Transcriptome, Omics, Imaging, Zebrafish, Butyrate, Soy saponin, Gut, Inflammation Background In the last decades, the implications of the microbiome in human and animal health have gained interest among and beyond the scientific community. As a consequence, food ingredients able to modulate the microbiome, such as prebiotics, became increasingly popular and accepted among the general public and have been utilized in human dietary supplements as well as in animal feed [17, 39]. A prebiotic is a substrate that is selectively utilized by host microorganisms and thereby proposed to confer a health benefit on the host (reviewed in [26]. Butyrate is a short-chain fatty acid (SCFA) derived from fiber fermentation by the gut bacteria that exhibits some prebiotic properties, playing a role in the interaction between bacterial population dynamics and host gut homeostasis [44]. Butyrate has a direct impact on the immune system via signaling G-protein coupled receptors (GPCR) on epithelial and immune cells and also induces epigenetic changes via regulation of histone acetylase and histone deacetylase enzymes (reviewed in [31]. In the last years, butyrate has been extensively used in animal feed, including its supplementation to several fish diets in the form of butyric acid or sodium butyrate due to its growth-promoting, immuno-stimulating and antioxidative properties (reviewed in [1] and to mitigate detrimental effects of sub-optimal plant-containing diets [23, 48, 70, 83]. Plant-based protein ingredients have been replacing fish meal in feed due to their more favorable price and availability. However, several anti-nutritional components derived from plant-based protein sources are reported detrimental for fish health (reviewed in [73]. For instance, soy saponin is an anti-nutritional component of soybean meal that interacts with cell membranes and promotes pore formation, vesiculation and membrane domain disruption [4]. Various studies linked the presence of soy saponin to inflammatory responses in the intestinal mucosa, enteritis as well as microbiota modulation in several fish species [13, 16, 40], including zebrafish [49]. In zebrafish larvae, the number of neutrophils increased in the gut after soybean meal feeding [30] or exposure to soy saponin in solution [49]. After assessing the inflammatory effect of soy saponin, soy-contai (...truncated)