IL-10 targets IRF transcription factors to suppress IFN and inflammatory response genes by epigenetic mechanisms

Abstract

Interleukin-10 (IL-10) is pivotal in suppressing innate immune activation, in large part by suppressing induction of inflammatory genes. Despite decades of research, the molecular mechanisms underlying this inhibition have not been resolved. Here we utilized an integrated epigenomic analysis to investigate IL-10-mediated suppression of LPS and TNF responses in primary human monocytes. Instead of inhibiting core TLR4-activated pathways such as NF-κB, MAPK-AP-1 and TBK1-IRF3 signaling, IL-10 targeted IRF transcription factor activity and DNA binding, particularly IRF5 and an IRF1-mediated amplification loop. This resulted in suppression of inflammatory NF-κB target genes and near-complete suppression of interferon-stimulated genes. Mechanisms of gene inhibition included downregulation of chromatin accessibility, de novo enhancer formation and IRF1-associated H3K27ac activating histone marks. These results provide a mechanism by which IL-10 suppresses inflammatory NF-κB target genes, highlight the role of IRF1 in inflammatory gene expression and describe the suppression of IFN responses by epigenetic mechanisms.

Extended Data Fig. 1 |. IL-10 preferentially suppresses IFN relative to inflammatory responses in LPS- and TNF-stimulated monocytes.

a–f, Additional analysis of the RNAseq data shown in Fig. 1 obtained using monocytes from 3 independent donors. a, Experimental design. Human monocytes were cultured −/+ IL-10 (100 ng/mL) for 18 h and then stimulated with LPS (10 ng/ml) for 3 h. b Principal component analysis (PCA) of RNA-seq data for Resting, IL-10, LPS, and IL-10 + LPS treatments. c, Volcano plot of DEGs regulated by LPS (up=1745 and down=1738) relative to resting control. d, Monocytes or in vitro differentiated macrophages were pretreated with IL-10 and stimulated with LPS, and IL-6 mRNA was measured by RT-qPCR and normalized relative to GAPDH mRNA (dots correspond to independent donors; n = 3). ***p < 0.0005; ****p < 0.0001, 2-way ANOVA with Tukey’s multiple comparisons test. e, Venn diagram depicting LPS-induced genes that are suppressed vs those not suppressed by IL-10. f, Scatterplot of LPS-induced genes showing log2 fold change values for LPS and IL-10 + LPS relative to resting control. g, UpSet plot showing overlap between suppressed genes identified in e and genes in individual k-means clusters from Fig. 1c. h, FACS to analyze phosphorylation of NF-κB in −/+ IL-10 treated human monocytes challenged with LPS for the times (mins) indicted in the representative histogram plot. Left panels, representative histogram plots; right panels, box plot of MFI from FACS experiments (n = 4 independent donors). Dots correspond to independent donors, box plot showing the distribution of the data with markers for the minimum, 25th percentile, mean, 75th percentile, and maximum values. ***p < 0.0005; ****p < 0.0001, 2-way ANOVA with Tukey’s multiple comparisons test. i-m, analysis of RNAseq data obtained using monocytes from 3 independent donors and treated with −/+ IL-10 (100 ng/mL) for 18 h and then stimulated with TNF (20 ng/ml) for 6 h. i, PCA of RNA-seq data for Resting, IL10, TNF, and IL-10 + TNF treatments. j, GSEA plot of RNA-seq data performed on DEGs ranked by log2CPMs in TNF and IL-10 + TNF treated human monocytes. k, k-means (k = 6) clustering of differentially upregulated genes in primary human monocytes in any pairwise comparison to resting control. l, Hallmark pathway enrichment analyses of the gene clusters identified in k. m, Venn diagram depicting TNF-induced genes that are suppressed vs not suppressed by IL-10. Created in https://BioRender.com.

Extended Data Fig. 2 |. IL-10 suppresses H3K27ac at genomic regions enriched in IRF1 binding motifs.

a–e, Additional analysis of H3K27ac CUT&RUN data shown in Fig. 2 obtained using monocytes from 2 independent donors and treated as depicted in Extended Data Fig. 1a. a, PCA plot of H3K27ac data for Resting, IL-10, LPS and IL-10 + LPS treatments of primary human monocytes. b, Bar plot depicting total number and the direction of regulation of the differentially regulated H3K27ac peaks relative to resting control. c, Hallmark pathway enrichment analyses of the genes associated with peaks in groups identified in Fig. 2a. d, UpSet plot showing interaction between suppressed genes identified in Extended Data Fig. 1e and individual groups of H3K27ac peaks from Fig. 2a. e, De novo motif analysis results using HOMER on Group 3 H3K27ac peaks. Created in https://BioRender.com.

Extended Data Fig. 3 |. IL-10 decreases chromatin accessibility and preferentially suppresses IRF activity.

a–e, Additional analysis of ATACseq data shown in Fig. 3 obtained using monocytes from 4 independent donors and treated as depicted in Extended Data Fig. 1a. a, Hallmark pathway enrichment analyses of genes associated with peaks identified as suppressed or non-suppressed in Fig. 3b. b, Heatmap of the H3K27ac normalized signal density surrounding LPS-induced ATAC peaks identified as suppressed or non-suppressed and plotted under the indicated conditions. Results are presented in RPKM values within a range of ± 3.0 kb around peak centers. c–e, Volcano plot of differential binding analysis of JASPAR motifs by TOBIAS using BINDetect algorithm c, for peaks associated with IL-10, LPS, and IL-10 + LPS compared to resting control, d, for TNF vs. IL-10 + TNF, e, for TNF, and IL-10 + TNF treated human monocytes compared to resting control. Created in https://BioRender.com.

Extended Data Fig. 4 |. IL-10 broadly suppresses LPS-induced IRF1 binding and associated histone acetylation.

a–d, Additional analysis of IRF1 CUT&RUN data shown in Fig. 4 obtained using monocytes from 3 independent donors and treated as depicted in Extended Data Fig. 1a. a, Bar plot depicting total number of IRF1 binding sites from CUT&RUN experiments. b, PCA plot of IRF1 CUT&RUN experiments for Resting, IL-10, LPS, and IL-10 + LPS treated primary human monocytes. c, Hallmark pathway enrichment analyses of genes associated with LPS-induced IRF1 binding peaks. d, Representative IGV gene tracks are shown, illustrating LPS-induced IRF1 binding, H3K4me3 and H3K27ac peaks, and ATACseq peaks at the OAS3 locus. Created in https://BioRender.com.

Extended Data Fig. 5 |. IL-10 suppresses expression of IRF1 and IRF5 which mediate IFNB induction in human monocytes.

a, Immunoblot of IRF1 (top panel) and IRF5 (bottom panel) using whole cell lysates of monocytes pretreated with IL-10 followed by the indicated time course of LPS stimulation. Representative blots from 3 independent donors are depicted, HPRT is used as loading control. b, mRNA of indicated genes was measured by RT-qPCR and normalized relative to GAPDH mRNA in cells stimulated with IL-10 for 18 h and then challenged with LPS for 3 h under two conditions. No washout of IL-10 (labeled 0): IL-10 remained in the culture during the LPS challenge; washout of IL-10 (labeled 24): IL-10 was removed and the cells were cultured for an additional 24 h before being challenged with LPS for 3 h. (n = 3 independent donors). Data are depicted as mean ± SEM. **p < 0.001; ***p ≤ 0.0005; ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. c, IGV gene tracks of IL-10-induced STAT3 binding at IRF1 regulatory elements. d, IFNB mRNA was measured by qPCR and normalized relative to GAPDH mRNA in cells stimulated IL-10 for 18 h and challenged with LPS at various time points (n = 3 independent donors). Data are depicted as mean ± SEM. ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. e, mRNA of indicated genes was measured by RT-qPCR and normalized relative to GAPDH. Cells were treated with IL-10 for 18 h and stimulated with exogenous IFN-β to assess whether IL-10 blocks induction of ISGs (n = 6 independent donors). Data are depicted as mean ± SEM. *p < 0.05; ***p ≤ 0.0005; ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. f, h Heatmaps of z-score normalized expression of f, TNF RNA-seq showing regulation of IRF and ISG genes by IL-10, h, of LPS RNA-seq showing regulation of IRFs and ISGs by IL-10. g, Immunoblot of IRF1 and p38 using whole cell lysates from IRF1 or HPRT-edited cells (CRIPSR-Cas9 mediated edits) and stimulated with LPS for 3 h (representative blot from one out of 3 independent donors). Created in https://BioRender.com.

Extended Data Fig. 6 |. Inflammatory and interferon-stimulated genes suppressed by IL-10 are IRF1/5 targets.

a, b, mRNA of indicated IFN-response genes (CXCL10 and ISG15) (a) and b, inflammatory genes (TNF and IL6) was measured by qPCR and normalized relative to GAPDH mRNA. This analysis was conducted after CRISPR-Cas9 gRNA targeting of IRF1 + IRF5 or HPRT-control for 48 h, followed by stimulation with LPS for 3 h (n = 13 independent donors, some donors are same as Fig. 7a, b). Data are depicted as mean ± SEM. ****p < 0.0001 by Mann-Whitney two-sided test. c–h, Additional analysis of RNA-seq data shown in Fig. 7 obtained using monocytes from 3 independent donors and treated as depicted in Extended Data Fig. 1a. c, PCA plot depicting the RNA-seq data from primary human monocytes subjected to CRISPR-Cas9-mediated deletion of IRF1/5 or HPRT, followed by stimulation with LPS for 3 h. d, k-means clustering analysis (k = 6) conducted on z-score normalized data of differentially upregulated genes in any pairwise comparison relative to resting control. e, Hallmark pathway enrichment analyses performed on clusters identified in d. f, Scatterplot of LPS-induced genes showing log2 fold change values for HPRT-edited and IRF1/5-edited monocytes relative to resting control. g, Venn diagram of LPS-induced genes in HPRT-edited controls compared to IRF1/5-edited monocytes. h, UpSet plot showing interaction between suppressed genes identified in g to individual k-means clusters from d. Created in https://BioRender.com.

Extended Data Fig. 7 |. IL-10 downregulates an IRF1-mediated rheostat to suppress TLR4-induced expression of inflammatory NF-κB target genes and ISGs.

TLR4 signaling induces expression of IRF1, which amplifies induction of inflammatory NF-κB target genes, IFNB, and ISGs in partnership with IRF5 and ISGF3. IFN-β further augments expression of IRF1 (lower right), which in turn will increase IFN-β production; this identifies an amplification loop for increasing expression of ISGs and inflammatory genes. IRF1 also auto-amplifies its own expression. IL-10 turns down this IRF1-mediated rheostat, interrupts the IFN-β-IRF1 amplification loop, and suppresses IRF transcription factor activity to broadly downregulate induction of inflammatory and interferon response genes. Created in BioRenders.com.

Fig. 1 |. IL-10 preferentially suppresses IFN relative to inflammatory responses in LPS- and TNF-stimulated monocytes.

a, qPCR assay showing the expression levels of IL6, TNF, CXCL10 and ISG15 mRNA, normalized to GAPDH mRNA, in CD14⁺ human monocytes cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with LPS (10 ng ml^–1) for 3 h. Dots correspond to independent donors (n = 7). b, GSEA of RNA-seq data performed on differentially expressed genes (DEGs), ranked by log₂(counts per million), in human CD14⁺ monocytes treated with IL-10 and LPS as in a. Hallmark gene sets from the Broad Institute and LPS-regulated IFN and inflammatory pathways are displayed. NES, normalized expression score; ES, expression score; adj. P, adjusted P value. c, k-means clustering analysis (k = 6) of differentially upregulated genes in any pairwise comparison of CD14⁺ monocytes treated with IL-10 and LPS as in a, relative to resting unstimulated monocytes. d,e, Hallmark pathway enrichment analyses on clusters as in c (d) or on LPS-induced genes suppressed by IL-10 (e). f,g, Heatmaps of LPS-induced genes suppressed by IL-10 (n = 436), clustered by inflammatory and IFN response genes (f) and representative inflammatory and IFN response genes suppressed by IL-10 (g). h, Hallmark pathway enrichment analysis performed on TNF-induced genes suppressed by IL-10 in CD14⁺ monocytes cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with TNF (20 ng ml^–1) for 6 h. i, Heatmap of TNF-induced genes suppressed by IL-10 as in h (n = 268), clustered by inflammatory and IFN response genes. j, qPCR of CXCL10 and TNF mRNA normalized relative to GAPDH mRNA in CD14⁺ monocytes stimulated with IL-10 and TNF as in h (n = 4 independent donors). In b–i, data are from three independent donors. In c, f, g, and i, z-score-normalized data were used to make heatmaps. In a and j, data are depicted as mean ± s.e.m. ***P < 0.0005; ****P < 0.0001 by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test. Created with BioRender.com.

Fig. 2 |. IL-10 suppresses H3K27ac at genomic regions enriched in IRF1-binding motifs.

a, Venn diagram of CUT&RUN data, showing the numbers of differentially upregulated H3K27ac peaks in CD14⁺ human monocytes from two independent donors. Cells were cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with LPS (10 ng ml^–1) for 3 h (group 1, LPS unique, n = 3,450; group 2, common, n = 2,299; group 3, IL-10 + LPS unique, n = 515). b, Heatmap of CUT&RUN data showing the normalized signal density of H3K27ac binding in CD14⁺ monocytes treated with IL-10 and LPS as in a. The results show reads per kilobase per million mapped reads (RPKM) values within a range of ±3.0 kb around peak centers. c, Violin plots showing normalized average signal density of H3K27ac binding in CD14⁺ monocytes treated with IL-10 and LPS as in a. Data are plotted as log₂(RPKM + 1) counts of H3K27ac reads (n = 3,450 for group 1; 2,299 for group 2; and 515 for group 3). In the embedded box plots, the midline represents the median, the box spans the interquartile range (IQR) from the 25th (Q1) to the 75th (Q3) percentile, the whiskers extend to the minimum and maximum values within 1.5 × IQR, and any points beyond this range are plotted as outliers (black dots). *P < 0.05; ****P < 0.0001 by two-way ANOVA with Tukey’s multiple-comparisons test. d was calculated using Cohen’s d method. d, Feature distribution plot showing localization of H3K27ac peak genomic coordinates in CD14⁺ monocytes treated with IL-10 and LPS as in a. UTR, untranslatable region. e,f, Representative Interactive Genome Viewer (IGV) gene tracks of CUT&RUN data showing H3K27ac (e) or H3K4me3 (f) binding at IFIT and IL6 loci in CD14⁺ monocytes treated with IL-10 and LPS as in a. H3K4Me3 CUT&RUN data were obtained using monocytes from two independent donors. g, De novo motif analysis results using HOMER showing enriched motifs under H3K27ac peaks in CD14⁺ monocytes treated with IL-10 and LPS as in a. Created with BioRender.com.

Fig. 3 |. IL-10 decreases chromatin accessibility and preferentially suppresses IRF activity.

a, Principal component (PC) analysis plot of ATAC-seq data in CD14⁺ human monocytes from four independent donors. Cells were cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with LPS (10 ng ml^–1) for 3 h. b, Venn diagram showing the numbers of ATAC-seq peaks in CD14⁺ monocytes treated with IL-10 and LPS as in a. c, Heatmap showing normalized signal density of ATACseq peaks in CD14⁺ monocytes treated with IL-10 and LPS as in a. The results are presented as RPKM values within a range of ± 3.0 kb around peak centers. d, Violin plots showing normalized average signal density of the ATAC-seq peaks in c. Data are plotted as log₂(RPKM + 1) counts of ATAC-seq reads (n = 7,701 for non-suppressed ATAC peaks, and 1,112 for suppressed ATAC-seq peaks). In the embedded box plots, the midline represents the median, the box spans the interquartile range (IQR) from the 25th (Q1) to the 75th (Q3) percentile, the whiskers extend to the minimum and maximum values within 1.5 × IQR, and any points beyond this range are plotted as outliers (black dots). ****P < 0.0001 by two-way ANOVA with Tukey’s multiple-comparisons test. n.s., not significant. e, Representative IGV gene tracks showing ATAC-seq peaks at the IL1B and IFIT loci in CD14⁺ monocytes treated with IL-10 and LPS as in a. f, De novo motif analysis using HOMER of LPS-induced ATAC-seq peaks that were suppressed (left) or were not suppressed (right) by IL-10 in CD14⁺ monocytes treated with IL-10 and LPS as in a. g, Heatmap depicting expression of LPS-induced ISGs associated with ATAC-seq peaks that were suppressed by IL-10 in CD14⁺ monocytes treated with IL-10 and LPS as in a. h, Volcano plot of differential transcription-factor-binding analysis of ATAC-seq peaks using TOBIAS in CD14⁺ monocytes treated with IL-10 and LPS as in a. i, Heatmap of the differential transcription factor activity scores derived from ChromVAR analysis of ATACseq peaks in CD14⁺ monocytes treated with IL-10 and LPS as in a. ATAC-seq data (a–i) were obtained using CD14⁺ monocytes from four independent donors. Created with BioRender.com.

Fig. 4 |. IL-10 broadly suppresses LPS-induced IRF1 binding and associated histone acetylation.

a, UpSet plot of IRF1 CUT&RUN data showing the numbers of IRF1 binding peaks (log₂(FC) ≥ 1 and FDR ≤ 0.05) in CD14⁺ human monocytes from three independent donors. Cells were cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with LPS (10 ng ml^–1) for 3 h. b, Heatmap showing the normalized signal density of LPS-induced IRF1 peaks (top) and violin plot showing normalized average signal density of IRF1 peaks (bottom, n = 4,395) in CD14⁺ monocytes treated with IL-10 and LPS as in a. c, De novo motif analysis using HOMER, showing motifs enriched under IRF1-binding peaks in CD14⁺ monocytes treated with IL-10 and LPS as in a. d, Heatmap showing ATAC-seq normalized signal density surrounding LPS-induced IRF1 peaks (top), and violin plot showing ATAC-seq normalized average signal density (bottom, n = 4,395) under IRF1 peaks in CD14⁺ monocytes treated with IL-10 and LPS as in a. e, Heatmap showing H3K27ac normalized signal density surrounding LPS-induced IRF1 peaks (top), and violin plot showing H3K27ac normalized average signal density (bottom, n = 4,395) in CD14⁺ monocytes treated with IL-10 and LPS as in a. f, Representative IGV gene tracks showing IRF1 binding, H3K4me3 and H3K27ac peaks and ATAC-seq peaks at the CXCL10 locus in CD14⁺ monocytes treated with IL-10 and LPS as in a. In b, d and e, heatmaps were generated using RPKM values within a range of ±3.0 kb around peak centers, and violin plot data are plotted as log₂(RPKM + 1) counts. In the embedded box plots, the midline represents the median, the box spans the interquartile range (IQR) from the 25th (Q1) to the 75th (Q3) percentile, the whiskers extend to the minimum and maximum values within 1.5 × IQR, and any points beyond this range are plotted as outliers (black dots). ****P < 0.0001 by two-way ANOVA with Tukey’s multiple-comparisons test. d was calculated using Cohen’s d method. IRF1 CUT&RUN analysis (a–f) was done using monocytes from three independent donors. Created with BioRender.com.

Fig. 5 |. IL-10 alone induces limited epigenomic reprogramming.

a, Volcano plot of RNA-seq data showing DEGs in CD14⁺ human monocytes treated with IL-10 (100 ng ml^–1) for 18 h. Rest, resting unstimulated control CD14⁺ monocytes (n = 3 independent donors). b, Hallmark pathway enrichment analysis of genes suppressed by IL-10 in CD14⁺ monocytes treated with IL-10 as in a. c, Volcano plot of H3K27ac CUT&RUN peaks in CD14⁺ monocytes (n = 2 independent donors). d, Volcano plot of ATAC-seq peaks in CD14⁺ monocytes treated with IL-10 (100 ng ml^–1) for 18 h (n = 4 independent donors). e,f, HOMER de novo motif analysis showing motifs enriched under ATAC-seq peaks suppressed (top) or induced (bottom) by IL-10 (e), or under H327ac-binding peaks induced by IL-10 (f) in CD14⁺ monocytes treated with IL-10 for 18 h. e, n = 4 donors; f, n = 2 donors. g, UpSet plot showing numbers of ATAC-seq peaks in CD14⁺ monocytes from four independent donors cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with LPS (10 ng ml^–1) for 3 h. Created with BioRender.com.

Fig. 6 |. IL-10 suppresses expression of IRF1 and IRF5 which mediate IFNB induction in human monocytes.

a, qPCR assay showing expression of IRF1 mRNA normalized relative to GAPDH mRNA in CD14⁺ human monocytes cultured with IL-10 (100 ng ml^–1) for 18 h and then stimulated with LPS (10 ng ml^–1) for 3 h (n = 7 independent donors; same donors as in Fig. 1a). Data are depicted as mean ± s.e.m. ****P < 0.0001 by one-way ANOVA with Tukey’s multiple-comparisons test. b, Flow cytometry assay showing IRF1 protein expression in CD14⁺ monocytes treated with IL-10 and LPS as in a. Representative histogram plot (left) and cumulative MFI data (right) from seven independent donors. c, Immunoblot of IRF1, p38, pSTAT1 and STAT1 in whole cell lysates of monocytes treated with IL-10 and LPS as in a (n = 3 independent donors, p38 used as loading control). d, qPCR assay showing expression of IRF1 mRNA normalized relative to GAPDH mRNA in CD14⁺ monocytes pre-treated with baricitinib (1 μM) for 30 min before stimulation with IFN-β (5 ng ml^–1) or LPS (10 ng ml^–1) for 3 h (n = 3 independent donors). e, IGV gene tracks of H3K27ac (top) and H3K4me3 (bottom) peaks at IRF1 locus in CD14⁺ monocytes stimulated with IL-10 and LPS as in a. f, qPCR assay showing expression of IFNB mRNA normalized relative to GAPDH mRNA in CD14⁺ monocytes stimulated with IL-10 and LPS as in a (n = 7 independent donors; same donors as in Fig. 1b). g, Immunoblot of phosphorylated TBK1 and TBK1 using whole-cell lysates of CD14⁺ monocytes stimulated with IL-10 and LPS as in a (representative blot from one out of three independent donors). h, IGV gene tracks of LPS-induced IRF1 binding at IFNB enhancer (n = 3). i, qPCR assay showing expression of IFNB mRNA normalized relative to GAPDH mRNA in CD14⁺ monocytes in which IRF1, IRF5, IRF1 + IRF5 or HPRT (control) genes had been edited using CRISPR–Cas9. Monocytes were stimulated with LPS for 3 h (n = 9 independent donors and data are depicted as mean ± s.e.m.). Gene expression for each donor is plotted relative to the resting condition, which is set at 100. ****P < 0.0001 by Mann–Whitney two-sided test. For a, b, d, and f, each dot represents an independent donor and data are depicted as mean ± s.e.m. *P < 0.05; **P < 0.001; ***P ≤ 0.0005; ****P < 0.0001 by one-way ANOVA with Tukey’s multiple-comparisons test. Created with BioRender.com.

Fig. 7 |. Inflammatory and IFN-stimulated genes suppressed by IL-10 are targets of IRF1 and IRF5.

a,b, qPCR assay showing expression of ISG15 and CXCL10 (a) or TNF and IL6 (b) mRNA normalized relative to GAPDH mRNA in CD14⁺ monocytes in which IRF1 and IRF5 (IRF1/5-KO) or HPRT (HPRT-KO) genes had been edited using CRISPR–Cas9, followed by treatment with IL-10 (100 ng ml^–1) for 18 h and stimulation with LPS (10 ng ml^–1) for 3 h (dots represent independent donors). Data are depicted as mean ± s.e.m. Gene expression for each donor is plotted relative to the resting condition, which is set at 100. *P < 0.05 by by Mann–Whitney two-sided test. c,d, Hallmark IFN-α response enrichment plot (c) and inflammatory response enrichment plot (d) of RNA-seq data DEGs ranked by log₂(counts per million) in IRF1/5 KO versus HPRT KO CD14⁺ monocytes stimulated with LPS for 3 h (n = 3 independent donors). e, UpSet plot showing overlap between LPS-induced genes suppressed by IL-10, as identified by RNA-seq in Fig. 1 (n = 3), and genes dependent upon IRF1 and IRF5, identified by RNA-seq as in c and d, in CD14⁺ monocytes as described in a (n = 3). f, Hallmark pathway enrichment analysis of the genes in e. g, Hallmark pathway enrichment analysis of genes showing decreased expression in IRF1/5 KO relative to control HPRT KO CD14⁺ monocytes stimulated with LPS (10 ng ml^–1) for 3 h. h,i, Heatmaps showing genes downregulated in IRF1/5-KO monocytes relative to HPRT-KO CD14⁺ monocytes stimulated with LPS (10 ng ml^–1, n = 530) for 3 h, clustered by inflammatory and IFN response genes (h) or showing representative ISGs (i). Rest, unstimulated CD14⁺ monocytes. Created with BioRender.com.