No abstract

14Macrophages are innate immune cells that contribute to fighting infections, tissue repair, and 15 maintaining tissue homeostasis. To enable such functional diversity, macrophages resolve 16 potentially conflicting cues in the microenvironment via mechanisms that remain unclear. Here, 17we used single-cell RNA sequencing to explore how individual macrophages respond when co-18 stimulated with the inflammatory stimuli, LPS+IFN-γ, and the resolving cytokine, IL-4. We found 19 that co-stimulated macrophages displayed a distinct global transcriptional program. However, 20 variable negative cross-regulation between some LPS+IFN-γ-and IL-4-specific genes resulted in 21 significant cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation led 22to mutually exclusive expression of the T-cell-polarizing cytokines Il6 and Il12b versus the IL-4-23 associated factors Arg1 and Chil3 in single co-stimulated macrophages, and single-cell secretion 24 measurements showed that these specialized functions were maintained for at least 48 hours. 25Overall, our study suggests that increasing functional diversity in the population is one strategy 26 macrophages use to respond to conflicting environmental cues. 27 LPS+IFN-γ or the IL-4 transcriptional program in response to co-stimulation. 131 132 Co-stimulation with LPS+IFN-γ and IL-4 induces transcriptional cross-regulation that 133 varies substantially across individual cells 134We next focused on how co-stimulation with LPS+IFN-γ and IL-4 affected the expression 135 of genes uniquely upregulated by either LPS+IFN-γ or IL-4 alone ( Fig. 2a), which we refer to as 136 the core gene programs. For both LPS+IFN-γ-and IL-4-induced genes, co-stimulation with the 137 other cue caused both transcriptional upregulation and inhibition in a subset of core genes 138 belonging to each program, consistent with our own and previously reported observations 14 . 139Among the LPS+IFN-γ-induced core genes, 87 were inhibited by co-stimulation and 97 were 140 augmented by co-stimulation, while among the IL-4-induced core genes, 196 were inhibited and 141 16 were augmented by co-stimulation (Fig. 2b). 142We compared observations from our single-cell dataset to population-level RT-qPCR data 514