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.

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Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C et al. (2016) Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection. J Immunol 196:846-56
Sun, Jing; Li, Ning; Oh, Kyu-Seon et al. (2016) Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use. Sci Signal 9:ra3
Gottschalk, Rachel A; Martins, Andrew J; Angermann, Bastian R et al. (2016) Distinct NF-κB and MAPK Activation Thresholds Uncouple Steady-State Microbe Sensing from Anti-pathogen Inflammatory Responses. Cell Syst 2:378-90
Dutta, Bhaskar; Azhir, Alaleh; Merino, Louis-Henri et al. (2016) An interactive web-based application for Comprehensive Analysis of RNAi-screen Data. Nat Commun 7:10578
Miller, Alexandra H; Vayttaden, Sharat J; Al-Khodor, Souhaila et al. (2015) Assay Development for Image-Based Quantification of Intracellular Bacterial Replication and Analysis of the Innate Immune Response to Infection. Assay Drug Dev Technol 13:515-28
Li, Ning; Sun, Jing; Benet, Zachary L et al. (2015) Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages. Sci Rep 5:9559
Al-Khodor, Souhaila; Marshall-Batty, Kimberly; Nair, Vinod et al. (2014) Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages. Cell Microbiol 16:378-95
Sung, Myong-Hee; Li, Ning; Lao, Qizong et al. (2014) Switching of the relative dominance between feedback mechanisms in lipopolysaccharide-induced NF-*B signaling. Sci Signal 7:ra6
Grainger, John R; Wohlfert, Elizabeth A; Fuss, Ivan J et al. (2013) Inflammatory monocytes regulate pathologic responses to commensals during acute gastrointestinal infection. Nat Med 19:713-21
Rodriguez-Martin, Sara; Kropp, Kai Alexander; Wilhelmi, Vanessa et al. (2012) Ablation of the regulatory IE1 protein of murine cytomegalovirus alters in vivo pro-inflammatory TNF-alpha production during acute infection. PLoS Pathog 8:e1002901

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