Sustained JAK/STAT signaling is required for long-term IFNγ-induced potentiated and tolerized gene expression responses. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 6 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS-induced genes potentiated by IFNγ treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes are defined as LPS-induced genes reaching at least fourfold increase for macrophages cultured in ruxolitinib and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS in two contiguous time points. Genes are clustered by expression level 88 h after IFNγ washout: the top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given gene between PBS and IFNγ treated is quantified for each gene in F. (D) Boxplot quantifying difference in L2FC for IFNγ pre-treated and PBS-pre-treated cells at each time point. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. (E) Example of fold change for potentiated gene: CCR7. (F) Heatmap of log2 fold change in reads of LPS-induced genes tolerized by IFNγ. Tolerance is defined as twofold reduction in transcription in two contiguous time points with IFNγ before treatment and at least fourfold induction by LPS in the presence of ruxolitinib. (G) Heatmap quantifying extent of IFNγ-induced tolerance as in C. (H) Example of fold change for potentiated gene: PTX3.