Analysis of Innate Immune Signaling Networks

先天免疫信号网络分析

• 批准号: 8157089
• 负责人: Iain Fraser
• 金额: $ 31.39万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/8157089
• 关键词: Immune; Signal Transduction

This project seeks to define the control principles that determine the complex relationship between signal input and output function in mammalian cells, and ultimately to generate quantitative computational models to describe cellular behavior in circumstances relevant to infectious disease. Using macrophages as a model system, we are characterizing the cellular response both to pattern recognition receptor (PRR) ligands and also to intact pathogens by measurement of the cellular response through a variety of readouts such as; signaling protein phosphorylation, intracellular trafficking, pathogen replication, transcription and production of immune mediators. We have profiled the response of RAW264.7 murine macrophage cells to a group of 4 toll-like receptor (TLR) ligands (LPS, Pam2CSK4, Pam3CSK4 and Resiquimod 848). Despite these receptors sharing common signaling components, we find that the response profiles of the NFkB and MAPK signaling modules vary with respect to kinetics, response magnitude and pathway selectivity. Analysis of the response to combined stimuli (mimicking what would occur with an intact pathogen) leads to non-additive levels of activation of downstream signaling pathways. In collaboration with the PSIIM Computational Biology group, we will use these observations to develop quantitative simulations of single and multi-receptor activation in an effort to understand the non-additive outputs observed in our experiments. These models will be used as a basis for understanding the interplay between PRR-activated pathways during an infection and the subsequent host response. We have also initiated a specific study of the macrophage response to Burkholderia cenocepacia (Bcc), an opportunistic bacteria particularly problematic in cystic fibrosis and chronic granulomatous disease patients, and closely related to the category A select agents Burkholderia mallei and pseudomallei. Macrophages are likely to play a key role in Bcc-induced pulmonary infections, but very little is known about the mechanism of Bcc infection and replication in these cells. We have studied the infection of human monocytic cells with virulent (J2315) and less virulent (K56-2) strains of Bcc to characterize growth kinetics, cytotoxicity, intracellular trafficking and induction of cellular responses such as autophagy and apoptosis. To determine the contribution of TLR signaling responses to infection, we have compared the ability of live and formalin killed bacteria to initiate early signaling responses and later secretion of a range of cytokines. In addition to providing important insight to the macrophage response to this pathogen in clinically relevant circumstances, the model described earlier for combined TLR activation will be used in an effort to determine the relative contribution of different TLR pathways to the host response to Bcc, and possibly gain insight to the mechanism of virulence adopted by this pathogen. In a continuation of a collaborative project with colleagues from the Alliance for Cellular Signaling, we published a study on the subversion of Ca2+ signaling pathways in macrophages by Clostridium difficile, a major cause of acute colitis. We determined that the bacterial toxin, ToxB, specifically attenuated Ca2+ signaling through one class of G-protein coupled receptor while enhancing signaling through another, which may have a key role in altering the inflammatory state of macrophages resident in the gut mucosa.

该项目旨在定义哺乳动物细胞中信号输入和输出功能之间复杂关系的控制原理，并最终生成定量计算模型，以描述与传染病相关的环境中的细胞行为。 使用巨噬细胞作为模型系统，我们正在表征对模式识别受体（PRR）配体的细胞反应，也表征了通过通过多种读数（例如；信号蛋白磷酸化，细胞内运输，病原体复制，转录和免疫介质的产生。 我们已经介绍了RAW264.7鼠巨噬细胞对4个Toll样受体（TLR）配体（LPS，PAM2CSK4，PAM3CSK4和Resiquimod 848）的反应。 尽管这些受体共享共同的信号传导成分，但我们发现NFKB和MAPK信号模块的响应曲线在动力学，响应幅度和途径选择性方面有所不同。 分析对组合刺激的反应（模仿完整病原体会发生的事情）导致下游信号通路的激活水平非添加水平。 与PSIIM计算生物学组合作，我们将使用这些观察结果来开发单个和多受体激活的定量模拟，以了解我们实验中观察到的非addive效率。 这些模型将被用作理解感染过程中PRR激活途径与随后的宿主反应之间的相互作用的基础。 我们还开始了对巨噬细胞对伯克霍尔德·西帕西亚（BCC）的巨噬细胞反应的具体研究，这是一种机会性细菌，在囊性纤维化和慢性肉芽肿性疾病患者中尤为问题，并且与选择的药物Burkholderia Mallei和Pseudomallei紧密相关。 巨噬细胞可能在BCC诱导的肺部感染中起关键作用，但是对于这些细胞中BCC感染和复制的机制知之甚少。 我们已经研究了人类单核细胞的感染（J2315）（J2315）和BCC的毒性较小（K56-2）菌株，以表征生长动力学，细胞毒性，细胞内运输和诱导细胞反应，例如自噬和凋亡。 为了确定TLR信号反应对感染的贡献，我们比较了福尔马林和福尔马林杀死细菌启动早期信号反应的能力，并随后分泌一系列细胞因子。 除了在临床相关的情况下对巨噬细胞对这种病原体的巨噬细胞反应提供重要的见解外，还将使用前面描述的TLR激活的模型，以确定不同TLR途径对宿主对BCC的相对贡献，并可能获得对这种病原体采用的VAIRENIS的洞察力。 在与细胞信号联盟同事的协作项目继续进行的一项协作项目中，我们发表了一项研究，该研究是关于巨噬细胞中Ca2+信号传导途径的颠覆，这是艰难梭菌的主要原因，这是急性结肠炎的主要原因。 我们确定细菌毒素，毒素，特异性地通过一类G蛋白偶联受体减弱了Ca2+信号传导，同时通过另一种受体增强信号传导，这可能在改变肠道粘膜中驻留的巨噬细胞炎症状态中具有关键作用。