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。 今年，我们扩展了RAW细胞中信号输出的非加和性观察，以确认初始信号效应反映在转录反应和分泌的细胞因子中。 我们还证实，在其他巨噬细胞系和原代骨髓源性巨噬细胞中也能重现这种效应。 我们已经克隆了由所述配体激活的TLR途径中的大部分信号传导组分的cDNA，并且正在使用荧光标记的表达构建体来评估近端信号传导组分的亚细胞定位的变化是否是由配体激活诱导的不同信号传导应答的基础。 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。 今年，我们发现J2315菌株部分通过延迟巨噬细胞中的内体成熟来介导其毒力，这使得它能够避免溶酶体融合，并且它从内吞途径中逃脱以在感染的细胞质中复制。 对感染的巨噬细胞信号传导反应的分析表明，NFkB和MAPK信号传导模块的特定组分的异常活化响应于野生型而不是福尔马林杀死的细菌而发生，表明涉及除细菌细胞表面TLR配体的识别之外的机制。 我们目前正在研究这些影响的基础。 在与来自细胞信号传导联盟的同事的合作项目的延续中，我们发表了描述使用微流体设备来分析巨噬细胞单细胞对G蛋白偶联受体（GPCR）配体的反应的研究，以及如何通过磷脂酶C β家族的蛋白质从多个Ca 2+诱导GPCR配体整合信号的分析。

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. This year we have extended our observations of non-additivity in signaling outputs in RAW cells to confirm that the initial signaling effects are reflected in transcriptional responses and secreted cytokines. We have also confirmed that the effects are reproduced in other macrophage cell lines and in primary bone marrow derived macrophages. We have cloned cDNAs for a large proportion of the signaling components in the TLR pathways activated by the described ligands, and are using fluorescently tagged expression constructs to assess whether changes in subcellular localization of the proximal signaling components underlies the different signaling responses induced by ligand activation. 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. This year we have found that the J2315 strain mediates its virulence in part through delay of endosomal maturation in macrophages, which allows it to avoid lysosomal fusion and it escapes from the endocytic pathway to replicate in the infected cell cytoplasm. Analysis of the macrophage signaling response to infection suggests that aberrant activation of specific components of the NFkB and MAPK signaling modules occurs in response to wild type but not formalin killed bacteria, suggesting a mechanism other than recognition of bacterial cell surface TLR ligands is involved. We are currently investigating the basis for these effects. In a continuation of collaborative projects with colleagues from the Alliance for Cellular Signaling, we published studies describing the use of a microfluidics device to analyze the macrophage single cell response to G Protein-coupled receptor (GPCR) ligands and also an analysis of how signals are integrated from multiple Ca2+ inducing GPCR ligands by proteins from the phospholipase C beta family.