向幼年斑马鱼(Danio rerio)体内显微注射β-葡聚糖以评估训练有素的免疫表型

Hannah Darroch, Jonathan Astin, Christopher J. Hall
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摘要

先天免疫系统可以记住以前的炎症,在一个被称为“训练免疫”的过程中,长期增强对二次免疫挑战的反应。经过训练的先天免疫细胞经过代谢和表观遗传重塑,并在二次挑战后,提供增强的保护与治疗潜力。训练免疫已经在体外或体外再刺激先天免疫细胞中进行了大量研究,其中主要的损伤通常注射到小鼠,成年斑马鱼或人体内。虽然信息量很大,但有机会在一个未受干扰的完整生物体中完全在体内研究训练过的免疫。斑马鱼幼虫的专属先天免疫反应提供了一个有吸引力的系统来模拟训练免疫。斑马鱼幼虫在受精后2天具有功能性先天免疫系统(dpf),可以进行高分辨率,高通量分析。这一点,再加上它们的光学透明性、保守的抗菌反应和转基因报告系的可用性,使它们成为研究体内训练免疫的一个有吸引力的替代模型。我们设计了一种方案,其中β-葡聚糖(最广泛使用的训练免疫的实验触发器之一)通过显微注射系统地传递到幼体斑马鱼中,以刺激训练样表型。刺激后,评估幼虫基因表达的变化,这表明β-葡聚糖的刺激作用。该方案描述了一种稳健的递送方法,将训练免疫的金标准刺激物之一送入模型生物,该模型生物高度适合于几种非侵入性下游分析。•本协议概述了一种最常见的训练免疫的实验刺激物进入斑马鱼幼虫。•该方案能够在体内评估训练样表型。•该方案可应用于转基因或突变斑马鱼系,以研究细胞或基因对β-葡聚糖刺激的反应。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Microinjection of β-glucan Into Larval Zebrafish (Danio rerio) for the Assessment of a Trained-Like Immunity Phenotype
The innate immune system can remember previous inflammatory insults, enabling long-term heightened responsiveness to secondary immune challenges in a process termed “trained immunity.” Trained innate immune cells undergo metabolic and epigenetic remodelling and, upon a secondary challenge, provide enhanced protection with therapeutic potential. Trained immunity has largely been studied in innate immune cells in vitro or following ex vivo re-stimulation where the primary insult is typically injected into a mouse, adult zebrafish, or human. While highly informative, there is an opportunity to investigate trained immunity entirely in vivo within an unperturbed, intact whole organism. The exclusively innate immune response of larval zebrafish offers an attractive system to model trained immunity. Larval zebrafish have a functional innate immune system by 2 days post fertilisation (dpf) and are amenable to high-resolution, high-throughput analysis. This, combined with their optical transparency, conserved antibacterial responses, and availability of transgenic reporter lines, makes them an attractive alternative model to study trained immunity in vivo. We have devised a protocol where β-glucan (one of the most widely used experimental triggers of trained immunity) is systemically delivered into larval zebrafish using microinjection to stimulate a trained-like phenotype. Following stimulation, larvae are assessed for changes in gene expression, which indicate the stimulatory effect of β-glucan. This protocol describes a robust delivery method of one of the gold standard stimulators of trained immunity into a model organism that is highly amenable to several non-invasive downstream analyses. Key features • This protocol outlines the delivery of one of the most common experimental stimulators of trained immunity into larval zebrafish. • The protocol enables the assessment of a trained-like phenotype in vivo. • This protocol can be applied to transgenic or mutant zebrafish lines to investigate cells or genes of interest in response to β-glucan stimulation.
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