TPL-2 inhibits interferon-beta expression via an ERK1/2-TCF-FOS axis in TLR4-stimulated macrophages

Studies in mice have indicated that TPL2 kinase plays important roles in immune responses to pathogens and in various models of inflammatory disease. We set out to determine how TPL2 (Map3k8) regulates transcription using an in vitro system of LPS-stimulated primary macrophages. TPL2 regulated genes are involved in a number of different processes including inflammation as well as reproduction, cell cycle and signal transduction. Differentially expressed genes were used in a K means analysis, grouping based on expression profile, which identified 12 different expression profiles. There was a strong correlation between rapidly-induced genes regulated by TPL2 kinase activity and their expression in cells treated with the MKK1/2 inhibitor (PD032590)1 or from cells where phosphorylation of P105 by IKK2 is blocked. Cluster 1 genes were found to be enriched for TCF family members. TPL2 was shown to regulate ELK1 phosphorylation and known TCF targets genes such as Egr1. TPL2 is known to regulate transcription of multiple cytokines and chemokines including IFNb. Like Map3k8[D270A] macrophages, expression of Ifnb1 was increased in Elk1-/-Elk4-/- macrophages compared to WT macrophages following LPS stimulation. In fact, this increase was also seen in MKK1/2 inhibitor treated macrophages. Expression of Fos was decreased in both Map3k8[D270A] and Elk1-/-Elk4-/- macrophages. Macrophages generated from mice with myeloid-specific deletion of Fos also had elevated levels of Ifnb1 compared to controls. Similar results were obtained following activation of TLR1/2, TLR9 and TNF-R in macrophages. Furthermore, LPS or TNF stimulation of MEFs lacking Elk1, Elk3 and Elk4 had reduced FOS protein levels and increased Ifnb1 mRNA suggesting a general role for ERK1/2 > TCF pathway regulating Ifnb1 levels through Fos expression. Overall design: Bone marrow derived macrophages (BMDMs) from LysM-Cre (LysM, control) and LysM-Cre Fos-fl/fl (FosKO) mice were generated and stimulated with LPS to determine the genes and processes controlled by FOS. BMDMs were stimulated with LPS (100ng/ml) for various times up to 2 hours or left unstimulated.

小鼠研究表明，TPL2激酶（TPL2 kinase）在宿主对抗病原体的免疫应答以及多种炎症疾病模型中发挥关键作用。本研究旨在借助脂多糖（LPS）刺激的原代巨噬细胞体外模型，阐明TPL2激酶（TPL2 kinase，Map3k8）调控基因转录的具体机制。受TPL2调控的基因参与多种生物学过程，涵盖炎症、生殖、细胞周期及信号转导等领域。研究人员对差异表达基因开展K均值（K-means）聚类分析，依据表达谱进行分组，最终鉴定出12种不同的表达模式。受TPL2激酶活性调控的快速诱导型基因，与经MKK1/2抑制剂（PD032590）处理的细胞、或IKK2介导的P105磷酸化被阻断的细胞中的基因表达模式存在显著相关性。聚类簇1的基因显著富集于TCF家族（TCF family）成员相关的功能模块。实验证实TPL2可调控ELK1的磷酸化水平，以及已知TCF靶基因如Egr1的转录活性。已有研究表明，TPL2可调控多种细胞因子与趋化因子的转录，其中包括IFNb。与Map3k8[D270A]巨噬细胞的表型一致，经LPS刺激后，Elk1-/-Elk4-/-巨噬细胞内Ifnb1的表达水平较野生型（WT）巨噬细胞显著升高。事实上，经MKK1/2抑制剂处理的巨噬细胞中也观测到了这一表达上调现象。在Map3k8[D270A]与Elk1-/-Elk4-/-巨噬细胞中，Fos的表达水平均出现下调。从髓系特异性Fos敲除小鼠体内分离得到的巨噬细胞，其Ifnb1的表达水平同样较对照组显著升高。在巨噬细胞中激活TLR1/2、TLR9及肿瘤坏死因子受体（TNF-R）后，均可得到一致的实验结果。此外，在缺失Elk1、Elk3及Elk4的小鼠胚胎成纤维细胞（MEFs）中，经LPS或肿瘤坏死因子（TNF）刺激后，FOS蛋白水平降低而Ifnb1 mRNA水平升高，这表明ERK1/2-TCF通路可通过调控Fos的表达，普遍调节Ifnb1的表达水平。实验整体设计：分别提取LysM-Cre（LysM，对照组）与LysM-Cre Fos-fl/fl（FosKO）小鼠的骨髓来源巨噬细胞（BMDMs），经LPS刺激后探究FOS所调控的基因及生物学过程。将BMDMs以100ng/ml的LPS刺激不同时长（最长2小时），同时设置未刺激的空白对照组。