Transcriptional responses of human monocyte-derived macrophages (HMDM) to lipopolysaccharide (LPS). Homo sapiens

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing inter-species differences in the transcriptional responses of primary human and mouse macrophages to the TLR4 agonist, LPS. Using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5’ ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologs, http://www.macgate.qfab.org). Divergently regulated (DR) orthologs were enriched for genes encoding cellular “inputs” such as cell surface receptors (e.g. TLR6, IL-7Rα), and functional “outputs” such as inflammatory cytokines/chemokines (e.g. CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. DR genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced inter-species promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change. Overall design: Human monocyte-derived macrophages (HMDM) were stimulated with the TLR4 agonist, lipopolysaccharide, over a time course (0, 2, 6, 24h) and analysed in biological quadruplicate (each of which represents a pool of two independent blood donors), in total representing 8 macrophage preparations from independent blood donors, on a custom-designed, focused microarray.

基因表达的进化改变通常被认为是物种间表型差异的主要驱动因素。本研究通过分析原代人源与小鼠巨噬细胞（macrophage）对Toll样受体4（TLR4）激动剂脂多糖（Lipopolysaccharide, LPS）的转录应答差异，探究先天免疫的多样化进程。本研究采用可支持跨物种分析的定制化平台，结合mRNA 5'端深度测序技术，鉴定出人与小鼠间受LPS调控的同源基因（orthologous gene）表达存在广泛分化（占同源基因的24%，相关数据见http://www.macgate.qfab.org）。差异调控（Divergently Regulated, DR）的同源基因富集于编码细胞“输入信号”的基因（如细胞表面受体TLR6、IL-7Rα）以及功能“输出信号”的基因（如炎性细胞因子/趋化因子（inflammatory cytokine/chemokine）CCL20、CXCL13）。反之，连接输入与输出信号的细胞内信号传导组分通常呈现协同调控模式。差异调控基因与宽泛的基因表达动态范围以及特定的启动子结构特征（TATA盒（TATA box）富集、CpG岛（CpG island）缺失）相关。令人意外的是，调控分化还与物种间启动子保守性增强存在关联。因此，受可实现动态调控的复杂且高度保守的启动子调控的基因，同时也是最易发生进化改变的基因。实验整体设计：采用定制化聚焦型微阵列，对经TLR4激动剂脂多糖刺激的人源单核细胞衍生巨噬细胞（human monocyte-derived macrophages, HMDM）进行时间梯度（0、2、6、24小时）处理，并开展四组生物学重复实验（每组样本均来自2名独立献血者的混合细胞），总计使用8份来自独立献血者的巨噬细胞制备物进行分析。