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

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 dose-dependent increase of neutrophils and macrophages in the gut area. 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.

Assessing Larval Zebrafish Survival and Gene Expression Following Sodium Butyrate Exposure and Subsequent Lethal Bacterial Lipopolysaccharide (LPS) Endotoxin Challenge

“…Contrarily, in sea bass, supplementation of butyrate in the diet is reported to increase the expression of TNF-alpha in the intestines and promote the turnover of cells within the intestinal crypts [25]. This variation in the expression of TNF-alpha in response to butyrate could be due to the fact that butyrate is not known to be naturally occurring within the zebrafish gut [26]. Moreover, LPS was not used to elicit an inflammatory response within the sea bass but rather a soybean meal diet [25].…”

Importance of Probiotics in Fish Aquaculture: Towards the Identification and Design of Novel Probiotics

“…Butyrate also inhibits histone deacetylase 3 (HDAC3) involved in chromatin remodeling and produces epigenetic changes [ 53 ] that modify the cell fate of immune cells. In fish, butyrate has been identified in the gut of carnivorous and herbivorous specimens [ 54 , 55 ], promotes the expression of heat shock protein HSP70, pro-inflammatory factors (IL-1β and TNF-α), and anti-inflammatory cytokines (TGF-β) in Cyprinus carpio [ 56 ], and improves the inflammatory response in juvenile zebra fish [ 57 ]. Butyrate has been detected in the intestinal feces of Atlantic salmon at a concentration of around 1 mM [ 58 , 59 ].…”

Small fish, big discoveries: zebrafish shed light on microbial biomarkers for neuro-immune-cardiovascular health

“…This was consistent with another study that found that pre-treatment with butyrate increased survival and decreased production of TNF-α following exposure to LPS in 3 dpf larvae ( Wang, 2023 ). Noteworthy, a recent high throughput quantitative histological and transcriptomic analyses of the zebrafish gut showed that sodium butyrate supplemented in feed increased the expression of pro-inflammatory genes and activity of oxidoreductase, decreased expression of genes involved in histone modification, mitotic process, GPCR activity, and increased relative abundance of Rhodobacter , Flavobacterium and Bacteroides, which are associated with inflammation in juvenile zebrafish gut ( López Nadal et al, 2023 ). Histological analysis also showed that butyrate supplementation increased eosinophils, rodlet cells and reduced mucus producing cells in the gut ( López Nadal et al, 2023 ), suggesting the responsiveness of the zebrafish gut immune system to the changes in the gut microbiota.…”

“…Noteworthy, a recent high throughput quantitative histological and transcriptomic analyses of the zebrafish gut showed that sodium butyrate supplemented in feed increased the expression of pro-inflammatory genes and activity of oxidoreductase, decreased expression of genes involved in histone modification, mitotic process, GPCR activity, and increased relative abundance of Rhodobacter , Flavobacterium and Bacteroides, which are associated with inflammation in juvenile zebrafish gut ( López Nadal et al, 2023 ). Histological analysis also showed that butyrate supplementation increased eosinophils, rodlet cells and reduced mucus producing cells in the gut ( López Nadal et al, 2023 ), suggesting the responsiveness of the zebrafish gut immune system to the changes in the gut microbiota. Moreover, Morales Fenero et al showed that SCFA can increase survival rate, reduce expression of inflammatory cytokines, and reduce the recruitment of neutrophils in a colitis model induced by 2,4,6-trinitrobenzene sulfonic acid in larval zebrafish ( Morales Fénero et al, 2021 ).…”