Analysis of Innate Immune Signaling Networks

该项目旨在定义确定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则，并最终生成定量计算模型来描述与传染病相关的情况下的细胞行为。 使用巨噬细胞作为模型系统，我们通过测量细胞反应来表征对模式识别受体（PRR）配体和对完整病原体的细胞反应，所述细胞反应通过各种读数，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。 我们分析了RAW264.7鼠巨噬细胞对一组4种toll样受体（TLR）配体（LPS、Pam 2CSK 4、Pam 3CSK 4和Resiquimod 848）的应答。 尽管这些受体共享共同的信号传导组件，我们发现，NF κ B和MAPK信号传导模块的响应曲线不同的动力学，响应幅度和途径的选择性。 对组合刺激的反应的分析（模拟完整病原体将发生的反应）导致下游信号传导途径的非加和水平的激活。 与PSIIM计算生物学小组合作，我们将利用这些观察结果来开发单受体和多受体激活的定量模拟，以了解我们实验中观察到的非加性输出。 这些模型将被用作理解感染期间PRR激活途径与随后的宿主反应之间相互作用的基础。 我们还启动了巨噬细胞对新洋葱伯克霍尔德氏菌（Bcc）反应的特定研究，新洋葱伯克霍尔德氏菌是一种机会性细菌，在囊性纤维化和慢性肉芽肿病患者中特别成问题，与A类选择剂鼻疽伯克霍尔德氏菌和类鼻疽伯克霍尔德氏菌密切相关。 巨噬细胞可能在Bcc诱导的肺部感染中起关键作用，但对Bcc感染和这些细胞中复制的机制知之甚少。 我们已经研究了人单核细胞与Bcc的毒性（J2315）和毒性较小（K56-2）菌株的感染，以表征生长动力学，细胞毒性，细胞内运输和诱导细胞反应，如自噬和凋亡。 为了确定TLR信号应答对感染的贡献，我们比较了活的和福尔马林杀死的细菌启动早期信号应答和随后分泌一系列细胞因子的能力。 除了在临床相关情况下提供对巨噬细胞对该病原体的应答的重要见解之外，将使用先前描述的用于组合TLR活化的模型来努力确定不同TLR途径对宿主对Bcc的应答的相对贡献，并可能获得对该病原体所采用的毒力机制的见解。 在与细胞信号传导联盟的同事合作项目的继续中，我们发表了一项关于艰难梭菌（急性结肠炎的主要原因）对巨噬细胞中Ca 2+信号通路的颠覆的研究。 我们确定细菌毒素ToxB通过一类G蛋白偶联受体特异性地减弱Ca 2+信号传导，同时通过另一类G蛋白偶联受体增强信号传导，这可能在改变肠道粘膜中巨噬细胞的炎症状态中起关键作用。

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.