Quantitative cytokine profiling of primary human macrophages reveals distinct single-cell modes of trained immunity

Macrophages can remember prior activation and subsequently augment their response to restimulation through trained immunity. However, it remains uncertain how trained immunity phenotypes manifest in individual cells. Here, we leverage highly quantitative single-molecule RNA imaging across 90,857 individual macrophages from 26 human donors to reveal inflammatory response dynamics in trained vs. untrained populations at single-cell resolution. Different inflammatory response genes showed distinct single-cell behavior in trained populations upon restimulation. Although training increased transcription of these response genes early after restimulation, untrained populations eventually "caught up" to the transcriptional output of trained populations, highlighting the importance of sampling timescale when interpreting transcriptional assays of training. Training did not significantly alter the relationship between the transcriptional activation of different genes within the same single cell, and any single cell appeared to be capable of training. Overall, these results revealed gene-specific single-cell transcriptional changes that generate population-wide training phenotypes in macrophages.