A zebrafish immunodeficiency model induced by the combination of Tacrolimus and Everolimus for rapid assessment of immune-enhancing agents

The integrity of the immunity system serves as the linchpin of an individual’s overall well-being and quality of life. In recent years, there has been a surge in the development of immune-enhancing drugs, nutraceuticals, and functional foods. However, an easy-to-use, rapid and intuitive immunity assessment framework is still missing. Zebrafish, an ideal model for high-throughput drug screening and assessing therapeutic effectiveness, provides us with new ideas. Here we report a methodology for the assessment of the immune-enhancing effects of various substances within a period of 5 days. Experimentally, it was determined that the continuous treatment of Tacrollimus (0.5 ng/mL) and Everolimus (5 μg/mL) on developing zebrafish from 48 hpf to 120 hpf was sufficient to establish an immunodeficient model. The results showed that the model exhibited a significant reduction in the number of immune cells (thymocytes, myeloid cells, and macrophages) and a greater than 50% decrease in the expression of immune-related genes ( itk, hexb, cpa5, isg15, rsad2 , and il10 ). Astragalus powder can effectively increase the content of thymic T cells and macrophages, but its effect on myeloid cells is limited. Ganoderma lucidum spore powder had the most obvious influence on macrophages, with limited impact on thymus cells and myeloid cells. Royal jelly showed a significant effect on thymus T cells and macrophages, but its effect on myeloid cells was limited. Bailing capsule can significantly increase the content of myeloid cells and thymus T cells, while its effect on macrophages was limited. The study demonstrated that the immunity-related indexes recovered differently in response to various immunomodulators relative to the model group, suggesting that these immunomodulators may exert their effects through distinct mechanisms.