The effects of chronic immune stimulation on muscle growth in rainbow trout

Successful production of aquaculture species depends on efficient growth with low susceptibility to disease. Therefore, selection programs have focused on rapid growth combined with disease resistance. However, chronic immune stimulation diminishes muscle growth (a syndrome referred to as cachexia), and decreases growth efficiency in production animals, including rainbow trout. In mammals, recent results show that increased levels of pro-inflammatory cytokines, such as those seen during an immune assault, specifically target myosin and MyoD and inhibit muscle growth. This suggests that increased disease resistance in fish, a desired trait for production, may actually decrease the growth of muscle, the main aquacultural commodity. To test this possibility, a rainbow trout model of cachexia was developed and characterized. A six-week study was conducted in which rainbow trout were chronically immune stimulated by repeated injections of LPS. Growth indices were monitored, and whole body and muscle proximate analyses, real-time PCR, and immunohistochemistry were conducted to examine the resulting cachectic phenotype. Muscle ratio was decreased in fish chronically immunostimulated, however expression levels of MyoD2 and myosin were not decreased compared to fish that were not immunostimulated, indicating that while muscle accretion was altered, the mechanism by which it occurred was somewhat different than that characterized in mammals. Microarray analysis was used to compare gene expression in fish that had been chronically immunostimulated versus those that had not to identify possible alternative mechanisms of cachexia in fish. Keywords: muscle, cachexia, rainbow trout, chronic immune stimulation Overall design: Fish of approximately 60g were separated into either an experimental group receiving an LPS injection or a control group receiving a sterile PBS injection. The LPS group was injected with 10 mg/kg bw (body weight) LPS in a volume of 0.1ml sterile PBS, and the PBS group was injected with an equal volume of sterile PBS. Liver, and muscle tissue were harvested from 10 fish from (saline and LPS injected) at day 43 of the experiments. During the experiment, fish were fed to satiation once daily with a commercial feed (Silver Cup, Murray, UT), and feed consumption was recorded daily. In the first week following injection, fish injected with LPS consumed 45-70% of the amount of feed consumed by the saline-injected fish. The following week, LPS-injected fish consumed 80-90% of the amount of feed consumed by the saline-injected fish.Total RNA from either muscle or liver was pooled by treatment (saline- or LPS-injected, 9 fish per treatment). cDNA was prepared and either Cy3- or Cy5-labeled using Superscript II Reverse Transcriptase (Invitrogen) and Genisphere’s Array 50 Kit.Scanning was performed using a Perkin Elmer ScanArray 5000 and images and data were processed using ScanArray Express software (Perkin Elmer, Wellesley, MA). The adaptive circle method was used for quantitation, and mean background of spots was subtracted and LOWESS normalization was performed. Data was filtered by eliminating sequences that did not have a minimum of 2-fold difference in expression on at least one of the dye-flip slides and that did not have a spot mean minus background pixel intensity of at least 1000. For the microarray work presented in the paper, only the samples from day 43 were analyzed, because day 43 is the relevant time point for testing the hypothesis that chronic immune stimulation suppresses muscle growth.水产养殖物种的成功养殖，依赖于高效生长与低疾病易感性。因此，遗传选育项目一直聚焦于兼顾快速生长与抗病性的性状。然而，慢性免疫刺激会抑制肌肉生长（该综合征被称为恶病质（cachexia）），并降低包括虹鳟（rainbow trout）在内的养殖动物的生长效率。 在哺乳动物中，近期研究表明，免疫攻击过程中升高的促炎细胞因子（pro-inflammatory cytokines）可特异性靶向肌球蛋白与MyoD，进而抑制肌肉生长。这提示，作为养殖目标性状的鱼类抗病性提升，反而可能降低作为水产养殖主要商品的肌肉产量。 为验证这一可能性，我们构建并表征了虹鳟的慢性免疫刺激诱导恶病质模型。本研究为期六周，通过反复注射脂多糖（Lipopolysaccharide, LPS）对虹鳟进行慢性免疫刺激。期间监测生长相关指标，并开展全身体常规营养成分分析、肌肉常规营养成分分析、实时定量PCR（real-time PCR）与免疫组织化学（immunohistochemistry）实验，以探究诱导产生的恶病质表型。 慢性免疫刺激组的鱼体肌肉占比出现下降，但与未免疫刺激的对照组相比，MyoD2与肌球蛋白的表达水平并未降低，这表明尽管肌肉沉积过程发生改变，其背后的分子机制与哺乳动物中已表征的机制存在一定差异。 我们采用基因芯片（microarray）分析，对比了慢性免疫刺激组与对照组鱼体的基因表达谱，以筛选鱼类恶病质的潜在替代调控机制。 关键词：肌肉、恶病质（cachexia）、虹鳟（rainbow trout）、慢性免疫刺激 实验设计概况：将体重约60g的虹鳟随机分为两组，实验组接受脂多糖注射，对照组接受无菌磷酸盐缓冲液（PBS）注射。实验组按10 mg/kg体重的剂量注射脂多糖，注射体积为0.1ml无菌PBS；对照组注射等体积的无菌PBS。实验第43天时，分别从两组（生理盐水注射组与脂多糖注射组）各采集10尾鱼的肝脏与肌肉组织。 实验期间，每日以商用饲料（Silver Cup, Murray, UT）投喂至鱼体饱食，并每日记录摄食量。注射后的第一周，脂多糖注射组的摄食量为生理盐水注射组的45%~70%；第二周，脂多糖注射组的摄食量为生理盐水注射组的80%~90%。 分别按处理组（生理盐水注射组或脂多糖注射组，每组9尾鱼）混合肌肉或肝脏组织的总RNA。采用Superscript II逆转录酶（Invitrogen）与Genisphere的Array 50试剂盒，制备互补DNA（cDNA）并进行Cy3或Cy5荧光标记。 使用Perkin Elmer ScanArray 5000扫描仪进行信号扫描，并通过ScanArray Express软件（Perkin Elmer, Wellesley, MA）处理图像与数据。采用自适应圆法进行点信号定量，扣除斑点的平均背景信号，并进行LOWESS归一化处理。通过筛选去除至少一张染料互换芯片中表达差异未达2倍以上，且斑点平均信号减去背景像素强度未达1000以上的序列。 本研究中的基因芯片实验仅分析了第43天的样本，因为第43天是验证"慢性免疫刺激抑制肌肉生长"这一假说的关键时间节点。