Investigating the signaling codes that specify immune responses by macrophages

巨噬细胞和其他先天免疫系统细胞使用十几个模式识别受体(PRR)来检测病原体衍生分子(PAMP)。这些不同的受体只激活三个关键的信号通路：IKK、TBK1和MAPK。它们的下游转录因子指定了初级免疫反应基因的多样化、高度刺激特异性表达。已经提出了两个假设来解释只有三条信号通路如何产生刺激特异的基因表达。第一种是“组合密码”，也就是说，通路可以在刺激特定的组合中被激活，免疫反应基因对不同的通路有不同的反应。第二个假设是“动态密码”，即有关刺激的信息在信号活动的动态控制中被编码，目标基因可能能够区分不同的动态特征。最近的研究已经证实，对于某些通路，激活动力学是刺激特有的。然而，目前尚不清楚如何将动态控制和组合控制相结合，以向核团提供有关刺激身份的信息。可以使用信息论来研究信息的编码，信息论是为电信开发的一种数学方法。我们建议研究刺激特异性整合PRR激活的三个关键信号通路，即IKK/NFκB、Tbk1/IRF3和p38MAPK通路。这需要通过重复测量来分析单个细胞的响应，因为当使用批量总体读出时，单个细胞的详细的、高度异质的响应动力学就会丢失。因此，我们将开发一种系统生物学方法，允许使用与原始巨噬细胞非常相似的新的三重荧光报告模型系统来定量评估活的单细胞中的信号活性。为此，我们将使用一项新开发的使能技术将NFκB、IRF3和p38的荧光报告引入小鼠造血干细胞来源的巨噬细胞。然后，我们将优化24小时活细胞图像采集和自动化高通量图像分析。然后，我们将应用最新开发的信息论算法来识别指定刺激身份的动态特征和组合编码。利用这一工作流程，我们将研究如何通过动态和组合代码的界面定义单个PAMP的剂量特异性和PAMP特异性，如何在聚合途径的多个PAMP特异性激活模式的整合中编码病原体身份，以及干扰素γ引起的巨噬细胞极化如何改变信号的刺激特异性。这项研究将为巨噬细胞反应的严密控制和高度特异性之间的平衡提供新的见解，这对于研究和治疗涉及巨噬细胞活性失调的疾病，如自身免疫性疾病和炎症性疾病具有重要意义。

Macrophages and other cells of the innate immune system use more than a dozen pattern recognition receptors (PRRs) to detect pathogen-derived molecules (PAMPs). These distinct receptors activate just three key signaling kinases, IKK, TBK1, and MAPK. Their downstream transcription factors specify the diverse, highly stimulus-specific expression of primary immune response genes. Two hypotheses have been proposed to explain how just three signaling pathways can produce stimulus-specific gene expression. The first is a “combinatorial code”, i.e. that the pathways may be activated in stimulus-specific combinations and that immune response genes are differentially responsive to different pathways. The second hypothesis is a “dynamic code”, i.e. that information about the stimulus is encoded in the dynamic control of signaling activity and that target genes may be able to distinguish between different dynamic profiles. Recent work has confirmed for some of the pathways that activation dynamics are stimulus-specific. However, it is unknown how the dynamic and combinatorial control are integrated to provide information on stimulus identity to the nucleus. The encoding of information can be studied using information theory, a mathematical approach developed for telecommunications. We propose to study the stimulus-specific integration of combinatorial and dynamic activation of three key PRR-activated signaling pathways, the IKK/NFκB, TBK1/IRF3, and p38 MAPK pathway. This requires analysis of single cell responses with repeated measures, since the detailed, highly heterogeneous response dynamics of individual cells are lost when using bulk population read-outs. Thus, we will develop a systems biology approach allowing for quantitative assessment of signaling activity in live single cells using a novel triple fluorescent reporter model system closely resembling primary macrophages. For this, we will introduce fluorescent reporters for NFκB, IRF3 and p38 into murine hematopoietic stem cell-derived macrophages using an enabling, newly developed technology. Then, we will optimize 24 h live cell image acquisition and automated high-throughput image analysis. We will then apply recently developed information-theoretic algorithms to identify the dynamic features and combinatorial coding that specify stimulus identity. Using this workflow, we will study how dose-specificity for individual PAMPs and PAMP-specificity are defined by the interface of the dynamic and combinatorial codes, how pathogen-identity is encoded in the integration of multiple PAMP-specific activation patterns of the converging pathways, and how macrophage polarization by IFNγ changes the stimulus-specificity of signaling. This research will provide novel insights into the balance of tight control and high specificity of responses by macrophages, which is highly relevant to the study and treatment of diseases involving dysregulated macrophage activity, such as autoimmune and inflammatory diseases.