Green Benjamin B, McKay Stephanie D, Kerr David E
Department of Animal Science, University of Vermont, Terrill Hall, 570 Main Street, Burlington, VT, 05405, USA.
BMC Genomics. 2015 Jan 27;16(1):30. doi: 10.1186/s12864-015-1223-z.
By comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness.
Age-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2'deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals.
The fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.
通过比较从5月龄或16月龄动物收集的成纤维细胞，我们之前发现来自老年动物的培养物在脂多糖（LPS）刺激下产生更多的IL-8。我们现在通过检查来自同一动物不同年龄的培养物在LPS反应中的全转录组差异来扩展这一发现，并研究DNA甲基化对反应性年龄依赖性增加的表观遗传基础的贡献。
用DNA去甲基化剂5-氮杂-2'-脱氧胞苷（AZA）预处理培养物可消除成纤维细胞在暴露于LPS 24小时后IL-8产生的年龄依赖性差异。对从5个月或16个月大的同一个体收集的成纤维细胞进行RNA-Seq分析，并将其平行暴露于LPS 0、2和8小时，结果显示对LPS有强烈反应，且在来自老年动物的培养物中反应更大。包括IL-8、IL-6、TNF-α和CCL20（以及许多其他免疫相关基因）在内的促炎基因在LPS暴露后的16月龄培养物中表达更高（FDR<0.05）。甲基化CpG岛回收分析测序（MIRA-Seq）揭示了遍布基因组的大量甲基化峰，同时基因启动子区域整体低甲基化，并且除了基因组上的20个区域外，来自同一动物两个年龄的成纤维细胞之间具有显著相似性。
在个体动物中，成纤维细胞对LPS的促炎反应从5个月到16个月龄急剧增加。更好地理解这一过程的潜在机制可以阐明先天免疫反应发展的生理过程以及先天免疫反应中可能出现的个体差异。此外，尽管我们的数据显示年龄对甲基化的影响相对较小，但它提供了牛成纤维细胞甲基化组的总体概况，为未来利用这种细胞类型进行牛表观遗传学研究提供了指导。
