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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Lin, Bin; Dutta, Bhaskar; Fraser, Iain D C (2017) Systematic Investigation of Multi-TLR Sensing Identifies Regulators of Sustained Gene Activation in Macrophages. Cell Syst 5:25-37.e3
Koppenol-Raab, Marijke; Sjoelund, Virginie; Manes, Nathan P et al. (2017) Proteome and Secretome Analysis Reveals Differential Post-transcriptional Regulation of Toll-like Receptor Responses. Mol Cell Proteomics 16:S172-S186
Sun, Jing; Katz, Samuel; Dutta, Bhaskar et al. (2017) Genome-wide siRNA screen of genes regulating the LPS-induced TNF-? response in human macrophages. Sci Data 4:170007
Sakai, Jiro; Cammarota, Eugenia; Wright, John A et al. (2017) Lipopolysaccharide-induced NF-?B nuclear translocation is primarily dependent on MyD88, but TNF? expression requires TRIF and MyD88. Sci Rep 7:1428
Li, Ning; Katz, Samuel; Dutta, Bhaskar et al. (2017) Genome-wide siRNA screen of genes regulating the LPS-induced NF-?B and TNF-? responses in mouse macrophages. Sci Data 4:170008
Welsbie, Derek S; Mitchell, Katherine L; Jaskula-Ranga, Vinod et al. (2017) Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons. Neuron 94:1142-1154.e6
Klionsky, Daniel J (see original citation for additional authors) (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1-222
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
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
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

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