Analysis of Innate Immune Signaling Networks

先天免疫信号网络分析

基本信息

批准号：
7964768
负责人：
Iain Fraser
金额：
$ 5.71万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7964768
关键词：
Aftercare Asbestos Attenuated Behavior Cell Line Cell model Cells Cellular biology Collaborations Communicable Diseases Complex Computational Biology Computer Simulation DNA Data Data Set Development Dose Feedback Genetic Transcription Human Human Resources Immune Immune response Individual Inflammatory Kinetics Life Ligands Literature Macrophage Activation Mammalian Cell Measurement Modeling Molecular Mus Noise Output Peptidoglycan Phorbol Esters Phosphoproteins Poly I-C Post-Translational Protein Processing Proteins Proteomics RNA Interference Receptor Activation Reporter Reporting Research Sampling Screening procedure Signal Transduction Signaling Protein Silicon Dioxide Statistical Models Stimulus Technology Time Toll-like receptors Transcript Tumor Necrosis Factor-alpha Undifferentiated Western Blotting base cytokine experience human TNF protein insight macrophage models and simulation monocyte network models p65 pathogen promoter resiquimod response transcription factor

项目摘要

We established the PSIIM Molecular and Cell Biology group in August 2008, and lab personnel have been hired for this project only recently, however progress has been made in evaluating RAW and THP1 cell responses to a variety of TLR ligands. Using RAW and THP1 reporter cell lines developed for screening applications, we have determined kinetic time courses and dose response data for the activation of NFkB and TNF alpha transcription for eight TLR ligands (LPS, Pam2CSK4, Pam3CSK4, FSL-1, Peptidoglycan, Resiquimod 848, CpG DNA, and poly I:C). We find the THP1 cell line is minimally responsive to LPS in its undifferentiated monocyte state, but it becomes highly responsive to LPS at 1ng/ml 72 hr after treatment with 5ng/ml phorbol ester (PMA). This suggests that PMA differentiation is inducing a more macrophage-like state in this cell line. We are currently attempting to correlate the kinetics of NFkB activation by each ligand with the subsequent induction of TNF alpha promoter activity. Previous reports have described models for oscillation of NFkB activity based on phasic expression and degradation of negative feedback regulators. We have observed oscillatory kinetics for both p65/RelA and TNF alpha promoter activation in our cell lines, and we are currently assessing whether these data might provide an initial framework for identifying similarities and differences between varied stimuli. We have also begun our efforts to collect data on the phosphoprotein profile in TLR-activated macrophages. We will compare data from quantitative western blotting with the xMAP (multi-analyte profiling) technology developed by Luminex. A key feature of the xMAP approach is the ability to assess numerous targets (up to 100) in a single sample, which reduces the noise in the data introduced by sample-to-sample variability. We will also evaluate this technology for the measurement of specific transcripts and secreted cytokines in an effort to generate a broad dataset that more comprehensively describes the cellular state induced by TLR activation.

我们于2008年8月建立了PSIIM分子和细胞生物学组，直到最近才雇用了实验室人员，但是在评估对各种TLR配体的RAW和THP1细胞反应方面才取得了进展。 Using RAW and THP1 reporter cell lines developed for screening applications, we have determined kinetic time courses and dose response data for the activation of NFkB and TNF alpha transcription for eight TLR ligands (LPS, Pam2CSK4, Pam3CSK4, FSL-1, Peptidoglycan, Resiquimod 848, CpG DNA, and poly I:C). 我们发现THP1细胞系在其未分化的单核细胞状态下对LP的反应很小，但是用5ng/ml phorbol酯（PMA）处理后，它以1NG/ml 72小时的速度对LPS高度响应。这表明PMA分化正在诱导该细胞系中更类似巨噬细胞的状态。我们目前正在尝试将每种配体的NFKB激活动力学与随后的TNFα启动子活性相关联。先前的报告描述了基于阶段表达和负反馈调节剂的降解的NFKB活性振荡模型。我们已经观察到在细胞系中p65/rela和TNFα启动子激活的振荡动力学，并且我们目前正在评估这些数据是否可以为识别各种刺激之间的相似性和差异提供初始框架。我们还开始努力收集有关TLR激活巨噬细胞中磷酸蛋白谱的数据。我们将将来自定量蛋白质印迹的数据与Luminex开发的XMAP（多分析物分析）技术进行比较。 XMAP方法的一个关键特征是能够在单个样本中评估众多目标（最多100个），从而降低了样本对样本变异性引入的数据中的噪声。我们还将评估这项技术，以测量特定的转录本和分泌的细胞因子，以生成一个广泛的数据集，该数据集更全面地描述了TLR激活引起的细胞状态。