Analysis of Innate Immune Signaling Networks

该项目旨在定义控制原理，以确定哺乳动物细胞中信号输入和输出函数之间的复杂关系，并最终生成描述与传染病相关的环境中细胞行为的定量计算模型。使用巨噬细胞作为模型系统，我们正在通过各种读数来测量细胞对模式识别受体(PRR)配体和完整病原体的反应，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。 我们研究了RAW264.7小鼠巨噬细胞对4种Toll样受体(TLR)配体(脂多糖、Pam2CSK4、Pam3CSK4和Resiquimod 848)的反应。尽管这些受体具有共同的信号成分，但我们发现NFkB和MAPK信号模块的反应谱在动力学、反应幅度和途径选择性方面有所不同。分析对复合刺激的反应(模仿完整病原体的反应)导致下游信号通路的非相加水平的激活。我们已经扩展了我们对原始细胞中信号输出的非加性的初步观察，以确认最初的信号效应反映在分泌的细胞因子中。我们还证实，这种效应在其他巨噬细胞系和原代骨髓来源的巨噬细胞中也有复制。我们已经克隆了由上述配体激活的TLR通路中很大一部分信号成分的cDNA，并正在使用荧光标记的表达构建物来评估近端信号成分亚细胞定位的变化是否是配体激活诱导的不同信号反应的基础。今年，我们还分析了对相同TLR配体组合的转录反应，并确定了非相加转录输出的某些配体特定模式。 在我们对巨噬细胞对上述双TLR配体的反应的描述中，以及在之前发表的研究中，协同反应特别发生在MyD88和TRIF信号通路都被激活的情况下。这可能被宿主用作一种检测机制，用于联合暴露于病毒和细菌病原体，或显著感染细胞内病原体，并导致细胞因子如IL-6和IL-12的产生增加，以推动对感染的强大适应性免疫反应。今年，我们启动了一项研究，以确定巨噬细胞对诱导TRIF依赖(Poly I：C/TLR3)和MyD88依赖通路(R848/TLR7)的配体的反应，强烈高于相加释放的IL-6和IL-12 p40亚单位的基础。确定这种非线性反应背后的细胞机制将对巨噬细胞PRR途径串扰的建模和确定炎性疾病的治疗靶点具有重要意义。 去年，我们还启动了一项针对盲肠伯克霍尔德氏菌(BCC)的巨噬细胞反应的专门研究，BCC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其存在问题，并且与A类选择药物马利伯克霍尔德氏菌和假鼻疽密切相关。巨噬细胞可能在BCC引起的肺部感染中发挥关键作用，但对BCC感染和复制这些细胞的机制知之甚少。我们研究了BCC强毒力株(J2315)和弱毒力株(K56-2)感染人单核细胞的生长动力学、细胞毒性、细胞内转运以及自噬和凋亡等细胞反应的诱导。为了确定TLR信号反应对感染的贡献，我们比较了活细菌和福尔马林灭活细菌启动早期信号反应和后期分泌一系列细胞因子的能力。我们发现，J2315毒株的毒力部分是通过延迟巨噬细胞的内体成熟来调节其毒力的，这使得它能够避免溶酶体融合，并从内吞途径逃脱，在感染的细胞质中复制。对巨噬细胞对感染的信号反应的分析表明，NFkB和MAPK信号模块的特定成分的异常激活是对野生型细菌的反应，而不是福尔马林灭活的细菌，这表明除了识别细菌细胞表面的TLR配体外，还涉及一种机制。今年，我们在确定感染期间诱导的宿主途径和细菌用来避免宿主清除的颠覆机制方面取得了重大进展。我们还开发了一种384孔格式的感染检测方法，以允许筛选调节感染过程的宿主细胞因子。

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. We have extended our initial observations of non-additivity in signaling outputs in RAW cells to confirm that the initial signaling effects are reflected in 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. This year, we have also analyzed transcriptional responses to the same TLR ligand combinations and have identified certain ligand-specific patterns of non-additive transcriptional output. In our profiling of the response of macrophage cells to dual TLR ligands described above, and in previously published studies, synergistic responses occur specifically in cases where both the MyD88 and TRIF signaling pathways are activated. This is likely used by the host as a detection mechanism for either combined exposure to viral and bacterial pathogens, or to a significant infection with intracellular pathogen, and it leads to the increased production of cytokines such as IL-6 and IL-12 that serve to drive a robust adaptive immune response to infection. This year, we have initiated a study to identify the basis of a strongly greater than additive release of IL-6 and IL-12 p40 subunit from macrophages in response to ligands which induce the TRIF-dependent (Poly I:C/TLR3) and MyD88- dependent pathways (R848/TLR7). Identifying the cellular mechanism underlying this non-linear response will have important implications both for modeling of PRR pathway crosstalk in macrophages and also for identifying therapeutic targets for inflammatory disorders. Last year, we 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. 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. This year we have made significant progress identifying the host pathways induced during infection and subversion mechanisms used by the bacteria to avoid host clearance. We have also developed an infection assay in 384-well format to permit screening for the host cell factors that regulate the course of infection.