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.
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