Analysis of Innate Immune Signaling Networks

我们于2008年8月成立了PSIIM分子和细胞生物学小组，最近才为该项目聘请了实验室人员，但在评估RAW和THP1细胞对各种TLR配体的反应方面取得了进展。 使用为筛选应用而开发的RAW和THP1报告细胞系，我们测定了八种TLR配体(脂多糖、Pam2CSK4、Pam3CSK4、FSL-1、肽聚糖、Resiquimod 848、CpG DNA和PolyI：C)激活NFkB和TNFα转录的动力学时间曲线和剂量反应数据。我们发现，在未分化的单核细胞状态下，THP1细胞对内毒素的反应最弱，但在5 ng/ml佛波酯(PMA)处理72小时后，1 ng/ml的内毒素对THP1细胞的反应变得高度敏感。这表明PMA的分化正在诱导该细胞系中更多的巨噬细胞样状态。 我们目前正试图将每个配体激活NFkB的动力学与随后诱导的肿瘤坏死因子α启动子活性联系起来。以前的报道已经描述了基于负反馈调节因子的阶段表达和降解的NFkB活性振荡模型。我们在我们的细胞系中观察到了p65/rela和肿瘤坏死因子α启动子激活的振荡动力学，目前我们正在评估这些数据是否可以为识别不同刺激之间的异同提供一个初步框架。 我们还开始努力收集TLR激活的巨噬细胞中磷蛋白图谱的数据。我们将把定量Western blotting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多个目标(最多100个)，这减少了因样本之间的可变性而引入的数据中的噪声。我们还将对这项技术进行评估，以测量特定的转录本和分泌的细胞因子，以努力生成更全面地描述TLR激活诱导的细胞状态的广泛数据集。

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.