Investigating the signaling codes that specify immune responses by macrophages

• Applicant: Dr. Stefanie Luecke
• Subject Area: Immunology
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 419234150

Macrophages and other cells of the innate immune system use more than a dozen pattern recognition receptors (PRRs) to detect pathogen-derived molecules (PAMPs). These distinct receptors activate just three key signaling kinases, IKK, TBK1, and MAPK. Their downstream transcription factors specify the diverse, highly stimulus-specific expression of primary immune response genes. Two hypotheses have been proposed to explain how just three signaling pathways can produce stimulus-specific gene expression. The first is a “combinatorial code”, i.e. that the pathways may be activated in stimulus-specific combinations and that immune response genes are differentially responsive to different pathways. The second hypothesis is a “dynamic code”, i.e. that information about the stimulus is encoded in the dynamic control of signaling activity and that target genes may be able to distinguish between different dynamic profiles. Recent work has confirmed for some of the pathways that activation dynamics are stimulus-specific. However, it is unknown how the dynamic and combinatorial control are integrated to provide information on stimulus identity to the nucleus. The encoding of information can be studied using information theory, a mathematical approach developed for telecommunications. We propose to study the stimulus-specific integration of combinatorial and dynamic activation of three key PRR-activated signaling pathways, the IKK/NFκB, TBK1/IRF3, and p38 MAPK pathway. This requires analysis of single cell responses with repeated measures, since the detailed, highly heterogeneous response dynamics of individual cells are lost when using bulk population read-outs. Thus, we will develop a systems biology approach allowing for quantitative assessment of signaling activity in live single cells using a novel triple fluorescent reporter model system closely resembling primary macrophages. For this, we will introduce fluorescent reporters for NFκB, IRF3 and p38 into murine hematopoietic stem cell-derived macrophages using an enabling, newly developed technology. Then, we will optimize 24 h live cell image acquisition and automated high-throughput image analysis. We will then apply recently developed information-theoretic algorithms to identify the dynamic features and combinatorial coding that specify stimulus identity. Using this workflow, we will study how dose-specificity for individual PAMPs and PAMP-specificity are defined by the interface of the dynamic and combinatorial codes, how pathogen-identity is encoded in the integration of multiple PAMP-specific activation patterns of the converging pathways, and how macrophage polarization by IFNγ changes the stimulus-specificity of signaling. This research will provide novel insights into the balance of tight control and high specificity of responses by macrophages, which is highly relevant to the study and treatment of diseases involving dysregulated macrophage activity, such as autoimmune and inflammatory diseases.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Alexander Hoffmann