Characterizing functional states of macrophages via their stimulus-responses

Project Summary/Abstract Macrophages are first responder immune cells present in every tissue. Their responses are mediated by signaling pathways that activate hundreds of immune response genes. Two functional hallmarks characterize the deployment of all macrophage functions: (1) Stimulus-Response Specificity. Immune responses are powerful, and often detrimental for the host. Hence, they must be deployed on an “only-as-needed” basis. However, it remains unknown how specific macrophage responses are, and what mechanisms control Response Specificity. Quantifying the specificity of responses requires single-cell measurements of signaling or gene expression trajectories, and the development of analysis methods to compare distributions, quantify information content, precision of classification and confusion. (2) Context-Dependent Functional States. Macrophage functions adapt to the tissue microenvironment via the cytokine milieu characteristic of the tissue and the prior history of immune responses or pathogen exposure. As monocytes circulate through the body passing through tissues, they are potential biosensors of injury or infection. While prior studies have characterized these states via steady-state molecular profiling of chromatin or transcriptome, single-cell stimulus response data may be more informative of actual functional states. Considering these functional hallmarks of macrophages lead to two hypotheses that this proposal addresses: 1) Quantitative measurements of single cell stimulus responses reveal that macrophage Response Specificity is modulated by physiological and pathological context by affecting the distributions that characterize heterogeneous responses in the population. 2) Quantifying the Response Specificity of individual macrophages allows for a characterization of their Functional States, that is distinct from single-cell transcriptomic profiling. We will address these hypotheses using experimental, math modeling, and computational analysis iteratively. In Aim 1, we will determine which molecular mechanisms that drive cell-to-cell heterogeneity and why the specificity of NFκB stimulus-responses is altered by cytokine polarization states. In Aim 2, we will use a novel model-aided data integration approach to quantify for the first time the Response Specificity of individual macrophages. This will allow us to map the landscape of macrophage states based on stimulus-responses and parameters, and compare it to maps of traditional steady-state scRNA-seq data. In Aim 3, we will study how the stimulus-specificity of immune gene expression responses in single-cells are affected by polarization. With a novel model-aided approach, we are able to reconstruct dynamic trajectories and determine whether Response Specificity is better assessed by mRNA abundances or dynamical features. After finetuning these approaches on in vitro polarized macrophages, we will apply them to macrophages from mouse models of ill-health. Insights may guide future translational studies to assess Innate immune Health.