Although evidence has shown that vitamin D (VD) influences gut homeostasis, limited knowledge is available how VD regulates intestinal immunity against bacterial infection. In the present study, cyp2r1 mutant zebrafish, lacking the capacity to metabolize VD, and zebrafish fed a diet devoid of VD, were utilized as VD-deficient animal models. Our results confirmed that the expression of antimicrobial peptides (AMPs) and IL-22 was restrained and the susceptibility to bacterial infection was increased in VD-deficient zebrafish. Furthermore, VD induced AMP expression in zebrafish intestine by activating IL-22 signaling, which was dependent on the microbiota. Further analysis uncovered that the abundance of the acetate-producer Cetobacterium in VD-deficient zebrafish was reduced compared to WT fish. Unexpectedly, VD promoted the growth and acetate production of Cetobacterium somerae under culture in vitro. Importantly, acetate treatment rescued the suppressed expression of β-defensins in VD-deficient zebrafish. Finally, neutrophils contributed to VD-induced AMP expression in zebrafish. In conclusion, our study elucidated that VD modulated gut microbiota composition and production of short-chain fatty acids (SCFAs) in zebrafish intestine, leading to enhanced immunity.

Short-chain fatty acids (SCFAs) are mainly produced by microbiota through the fermentation of carbohydrates in the intestine. Acetate, propionate, and butyrate are the most abundant SCFA metabolites and have been shown to be important in the maintenance of host health. In this study, head kidney macrophages (HKMs) were isolated and cultured from turbots. We found that the antibacterial activity of HKMs was increased after these cells were incubated with sodium butyrate, sodium propionate or sodium acetate. Interestingly, our results showed that all three SCFAs enhanced the expression of hypoxia inducible factor-1 α (HIF-1α) in HKMs, and further study confirmed that butyrate augmented the oxygen consumption of these cells. Moreover, HIF-1α inhibition diminished the butyrate-promoted intracellular bacterial killing activity of macrophages, and SCFAs also raised the gene expression and activity of lysozymes in HKMs via HIF-1α signaling. In addition, our results suggested that butyrate induced HIF-1α expression and the bactericidal activity of HKMs through histone deacetylase inhibition, while G protein-coupled receptors did not contribute to this effect. Finally, we demonstrated that butyrate induced a similar response in the murine macrophage cell line RAW264.7. In conclusion, our results demonstrated that SCFAs promoted HIF-1α expression via histone deacetylase inhibition, leading to the enhanced production of antibacterial effectors and increased bacterial killing of macrophages.

Vitamin D (VD) plays a vital role in various physiological processes in addition to its classic functions on maintaining the balance of Ca and P metabolism. However, there still are gaps to understand in depth the issues on the precise requirement, metabolic processes and physiological functions of VD in fish. In this study, we investigated the effects of VD on the growth, intestinal health, host immunity and metabolism in turbot (Scophthalmus maximus L.), one important commercial carnivorous fish in aquaculture, through the supplementation of different doses of dietary VD₃ (0, 200, 400, 800 and 1600 μg VD₃/kg diet). According to our results, the optimal VD₃ level in the feed for turbot growth was estimated to be around 400 μg/kg, whereas VD₃ deficiency or overdose in diets induced the intestinal inflammation, lowered the diversity of gut microbiota and impaired the host resistance to bacterial infection in turbot. Moreover, the level of 1α,25(OH)₂D₃, the active metabolite of VD₃, reached a peak value in the turbot serum in the 400 μg group, although the concentrations of Ca and phosphate in the turbot were stable in all groups. Finally, the deficiency of dietary VD₃ disturbed the nutritional metabolism in turbot, especially the metabolism of lipids and glucose. In conclusion, this study evaluated the optimal dose of dietary VD₃ for turbot and provided the evidence that VD has a significant impact on intestinal health, host immunity and nutritional metabolism in fish, which deepened our understanding on the physiological functions and metabolism of VD₃ in fish.

Vitamin D (VD) is a major regulator of calcium metabolism in many living organisms. In addition, VD plays a key role in regulating innate and adaptive immunity in vertebrates. Neutrophils constitute an important part of the first line of defense against invading microbes; however, the potential effect of VD on neutrophils remains elusive. Thus, in this study zebrafish in different developmental stages were utilized to identify the potential role of VD in the basal homeostasis and functions of neutrophils. Our results showed that addition of exogenous VD₃ promoted granulopoiesis in zebrafish larvae. Reciprocally, neutrophil abundance in the intestine of adult zebrafish with a cyp2r1 mutant, lacking the capacity to 25-hydroxylate VD, was reduced. Moreover, VD-mediated granulopoiesis was still observed in gnotobiotic zebrafish larvae, indicating that VD regulates neutrophil generation independent of the microbiota during early development. In contrast, VD was incapable to influence granulopoiesis in adult zebrafish when the commensal bacteria were depleted by antibiotic treatment, suggesting that VD might modulate neutrophil activity via different mechanisms depending on the developmental stage. In addition, we found that VD₃ augmented the expression of il-8 and neutrophil recruitment to the site of caudal fin amputation. Finally, VD₃ treatment significantly decreased bacterial counts and mortality in zebrafish infected with Edwardsiella tarda (E. tarda) in a neutrophil-dependent manner. Combined, these findings demonstrate that VD regulates granulopoiesis and neutrophil function in zebrafish immunity.