Unique Genetic Responses Revealed in RNA-seq of the Spleen of Chickens Stimulated with Lipopolysaccharide and Heat

Climate change and disease have large negative impacts on poultry production, but little is known about the interactions of responses to these stressors in chickens. Fayoumi (heat and disease resistant) and broiler (heat and disease susceptible) chicken lines were stimulated at 22 days of age, using a 2x2x2 factorial design including: breed (Fayoumi or broiler), inflammatory stimulus [lipopolysaccharide (LPS) or saline], and temperature (35°C or 25°C). Transcriptional changes in spleens were analyzed using RNA-sequencing on the Illumina HiSeq 2500. Thirty-two individual cDNA libraries were sequenced (four per treatment) and an average of 22 million reads were generated per library. Stimulation with LPS induced more differentially expressed genes (DEG, log2 fold change ≥ 2 and FDR ≤ 0.05) in the broiler (N=283) than the Fayoumi (N=85), whereas heat treatment resulted in fewer DEG in broiler (N=22) compared to Fayoumi (N=107). The double stimulus of LPS+heat induced the largest numbers of changes in gene expression, for which broiler had 567 DEG and Fayoumi had 1471 DEG of which 399 were shared between breeds. Further analysis of DEG revealed pathways impacted by these stressors such as Remodelling of Epithelial Adherens Junctions due to heat stress, Granulocyte Adhesion and Diapedesis due to LPS, and Hepatic Fibrosis/Hepatic Stellate Cell Activation due to LPS+heat. The genes and pathways identified provide deeper understanding of the response to the applied stressors and may serve as biomarkers for genetic selection for heat and disease tolerant chickens. Overall design: At 22 days of age, divergent chicken breeds (Fayoumi and broiler) were treated with a thermal treatment (heat stress at 35C, or thermoneutral at 25C as a control) for 3.5 hours, then stimulated subcutaneously with an inflammatory stimulus (LPS, or saline as a control) for another 3.5 hours. Chickens were euthanized and spleens were harvested. A total of 32 indivudally coded cDNA libraries were prepared using TruSeq v2 library preparation kit which selects for polyA mRNA. In this 2x2x2 full factorial design with the factors of breed, thermal treatment, and inflammatory stimulus, there were a total of 8 treatment groups. Each treatment group had a total of 4 animal biological replicates. Therefore, a total of 32 individual barcoded samples were sequenced. A total of 8 individually barcoded cDNA libraries were sequenced per lane using the HiSeq Illumina 2500, and we used 4 lanes total. Reads were mapped to Galgal 2.0.

气候变化与疾病对家禽生产产生了巨大的负面影响，然而关于鸡对这些压力源的响应相互作用的研究知之甚少。本研究选取了抗热抗病的法尤米鸡和易感热的肉鸡品种，在22日龄时采用2x2x2的析因设计，包括品种（法尤米或肉鸡）、炎症刺激[脂多糖（LPS）或盐水]和温度（35°C或25°C）三个因素。利用Illumina HiSeq 2500进行RNA测序，分析了脾脏中的转录组变化。共测序了32个单独的cDNA文库（每个处理4个），每个文库平均生成2200万个测序读数。LPS刺激在肉鸡（N=283）中诱导了更多差异表达基因（DEG，log2倍数变化≥2且FDR≤0.05），而热处理在肉鸡（N=22）中与法尤米鸡（N=107）相比，差异表达基因较少。LPS+热的双重刺激导致了基因表达的最大变化，其中肉鸡有567个DEG，法尤米鸡有1471个DEG，其中399个在品种间共有。进一步分析DEG揭示了受这些压力源影响的通路，如热应激引起的上皮粘附斑重塑、脂多糖引起的粒细胞粘附和渗出，以及LPS+热引起的肝纤维化/肝星状细胞活化。所确定的基因和通路为我们对施加的压力源的响应提供了更深入的理解，并可能作为耐热耐病鸡遗传选择的生物标志物。总体设计：在22日龄时，不同的鸡品种（法尤米和肉鸡）被给予热处理（35°C的热应激或25°C的恒温作为对照）3.5小时，然后皮下注射炎症刺激（LPS，或盐水作为对照）另外3.5小时。随后对鸡进行安乐死并采集脾脏。使用TruSeq v2文库制备试剂盒制备了32个单独编码的cDNA文库（每个处理4个），该试剂盒选择polyA mRNA。在这个包含品种、热处理和炎症刺激三个因素的2x2x2全析因设计中，共有8个处理组。每个处理组有4个动物生物重复。因此，共测序了32个单独条码化的样品。每个条码化的cDNA文库在每个Illumina HiSeq 2500通道中测序8个，总共使用了4个通道。测序读数被映射到Galgal 2.0。