Table_2_Early Life Glucocorticoid Exposure Modulates Immune Function in Zebrafish (Danio rerio) Larvae.DOCX

In this study we have assessed the effects of increased cortisol levels during early embryonic development on immune function in zebrafish (Danio rerio) larvae. Fertilized eggs were exposed to either a cortisol-containing, a dexamethasone-containing (to stimulate the glucocorticoid receptor selectively) or a control medium for 6 h post-fertilization (0–6 hpf). First, we measured baseline expression of a number of immune-related genes (socs3a, mpeg1.1, mpeg1.2, and irg1l) 5 days post-fertilization (dpf) in larvae of the AB and TL strain to assess the effectiveness of our exposure procedure and potential strain differences. Cortisol and dexamethasone strongly up-regulated baseline expression of these genes independent of strain. The next series of experiments were therefore carried out in larvae of the AB strain only. We measured neutrophil/macrophage recruitment following tail fin amputation (performed at 3 dpf) and phenotypical changes as well as survival following LPS-induced sepsis (150 μg/ml; 4–5 dpf). Dexamethasone, but not cortisol, exposure at 0–6 hpf enhanced neutrophil recruitment 4 h post tail fin amputation. Cortisol and dexamethasone exposure at 0–6 hpf led to a milder phenotype (e.g., less tail fin damage) and enhanced survival following LPS challenge compared to control exposure. Gene-expression analysis showed accompanying differences in transcript abundance of tlr4bb, cxcr4a, myd88, il1β, and il10. These data show that early-life exposure to cortisol, which may be considered to be a model or proxy of maternal stress, induces an adaptive response to immune challenges, which seems mediated via the glucocorticoid receptor.

本研究评估了胚胎发育早期皮质醇（cortisol）水平升高对斑马鱼（Danio rerio）幼鱼免疫功能的影响。本研究将受精后的斑马鱼卵在受精后0~6小时（hours post-fertilization, hpf）期间，分别暴露于含皮质醇、含地塞米松（dexamethasone，可选择性激活糖皮质激素受体（glucocorticoid receptor））的培养基，或对照培养基中，总暴露时长为6小时。首先，为评估本暴露处理的有效性及品系间潜在差异，我们分别检测了AB品系与TL品系斑马鱼幼鱼在受精后5天（days post-fertilization, dpf）时，多种免疫相关基因（socs3a、mpeg1.1、mpeg1.2及irg1l）的基础表达水平。结果显示，皮质醇与地塞米松均可显著上调上述基因的基础表达水平，且该效应不受品系影响。因此后续实验仅使用AB品系斑马鱼幼鱼开展。我们检测了幼鱼在3 dpf时进行尾鳍截肢后的中性粒细胞/巨噬细胞招募情况，以及在4~5 dpf时以150 μg/ml脂多糖（lipopolysaccharide, LPS）诱导脓毒症后的表型变化与存活率。实验结果表明，在0~6 hpf期间经地塞米松（而非皮质醇）暴露处理的幼鱼，其尾鳍截肢后4小时的中性粒细胞招募水平显著升高。与对照组相比，0~6 hpf期间经皮质醇或地塞米松暴露的幼鱼，在接受LPS攻毒处理后表型更温和（如尾鳍损伤程度更轻）且存活率更高。基因表达分析显示，tlr4bb、cxcr4a、myd88、il1β及il10的转录本丰度也随之出现差异。上述结果表明，生命早期阶段暴露于皮质醇（可作为母体应激的模型或替代指标）可诱导机体对免疫挑战产生适应性应答，且该应答似乎由糖皮质激素受体介导。