Quantifying stimulus-response specificity to probe the functional state of macrophages.

Immune sentinel macrophages initiate responses to pathogens via hundreds of immune response genes. Each immune threat demands a tailored response, suggesting that the capacity for stimulus-specific gene expression is a key functional hallmark of healthy macrophages. To quantify this property, termed "stimulus-response specificity" (SRS), we developed a single-cell experimental workflow and analytical approaches based on information theory and machine learning. We found that the response specificity of macrophages is driven by combinations of specific immune genes that show low cell-to-cell heterogeneity and are targets of separate signaling pathways. The "response specificity profile," a systematic comparison of multiple stimulus-response distributions, was distinctly altered by polarizing cytokines, and it enabled an assessment of the functional state of macrophages. Indeed, the response specificity profile of peritoneal macrophages from old and obese mice showed characteristic differences, suggesting that SRS may be a basis for measuring the functional state of innate immune cells. A record of this paper's transparent peer review process is included in the supplemental information.