Transcription profiling of spleens from dual specificity phosphatase-1 (DUSP1) deficient and wild type mice to demonstrate a specific regulatory role of DUSP1 on a subset of LPS-induced genes that control the outcome of endotoxin shock

Activation of the Mitogen activated protein kinase (MAPK) cascade following Toll-like receptor (TLR) stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase Dual specificity phosphatase-1 (DUSP1) as an essential endogenous regulator of the inflammatory response to LPS. DUSP1-deficient (DUSP1-/-) bone marrow derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1-/- mice with LPS caused increased lethality and overshooting production of IL-6 and TNF. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, that includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4 and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, IFNgamma and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock. Experiment Overall Design: Mice were injected intraperitoneally with E. coli LPS (10µg/g bodyweight) and sacrificed 6h later. Total spleen RNA (5 µg) was prepared, labeled and hybridized to Affymetrix MOE430A 2.0 GeneChips according to the manufacturer's instructions. Three biological replicates per condition were analysed. CEL Files were processed for global normalization and generation of expression values using the rma algorithm in the R affy package (www.bioconductor.org).丝裂原活化蛋白激酶（Mitogen activated protein kinase, MAPK）级联通路在Toll样受体（Toll-like receptor, TLR）受到刺激激活后，可介导先天免疫细胞快速激活细胞因子基因的表达。针对感染危险信号的平衡应答要求细胞活化过程具有一过性特征。本研究鉴定出丝裂原活化蛋白激酶磷酸酶双特异性磷酸酶1（Dual specificity phosphatase-1, DUSP1）是脂多糖（LPS）诱导炎症应答的关键内源性调控因子。 DUSP1缺陷型（DUSP1-/-）小鼠骨髓来源巨噬细胞中，p38 MAPK的活化会出现选择性持续延长，且细胞因子的产生量显著升高。向DUSP1-/-小鼠腹腔内注射脂多糖可导致其死亡率升高，IL-6与TNF的生成过量。转录组分析显示，DUSP1可调控脂多糖诱导表达的大量基因，其中包括IL-6、IL-10以及趋化因子CCL3、CCL4与CXCL2。与之相反，内毒素致死的重要介导因子IFNγ与IL-12的表达，在DUSP1缺失后并未出现显著变化。上述实验结果共同证明，DUSP1可特异性调控脂多糖诱导表达的部分基因亚群，而这些基因决定了内毒素休克的转归。 实验整体设计：向小鼠腹腔内注射大肠杆菌脂多糖（剂量为10μg/g体重），6小时后处死小鼠。提取脾脏总RNA（5μg），依照厂商说明书进行标记后，与Affymetrix MOE430A 2.0基因芯片进行杂交。每个实验条件设置3次生物学重复。使用R语言affy包中的rma算法（www.bioconductor.org）对CEL文件进行全局归一化处理并生成表达量数值。