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.
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Data availability
Sequencing data from this study have been deposited at the GEO and will be publicly available from the date of publication. the accession numbers are GSE261243, GSE261244 and GSE261245. Source data are provided with this paper.
Code availability
Any additional information required to reanalyze the data (including original code) reported in this paper is available from the first or last authors on request.
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Acknowledgements
We thank the Weill Cornell Medicine Genomics Core for sequencing and the David Z. Rosensweig Genomics Center at HSS for data analysis. This work was supported by grants from the National Institutes of Health AI046712, AI044938, DE019420, AR084248 (to L.B.I.) and T32AR071302-01 (to B.M.). The David Z. Rosensweig Genomics Center is supported by The Tow Foundation. Figures were created using Biorender.com.
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B.M. conceptualized, designed and performed most of the experiments, performed bioinformatic analysis, prepared figures and wrote the manuscript. M.B. performed ATAC-seq experiments. R.Y., C.W., V.C., R.B. and C.B., contributed experiments or experimental expertise. L.B.I. conceptualized and oversaw the study and edited the manuscript. All authors reviewed and provided input on the manuscript.
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Nature Immunology thanks Peter Murray, John O’Shea and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Ioana Staicu was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.
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Extended data
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.
Supplementary information
Supplementary Information
Supplementary Tables 1–3.
Supplementary Data 1
List of packages used for analyzing and visualizing genomic data in this study.
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Source Data Figs. 1–7
Statistical source data in clearly labeled excel sheet; unprocessed western blots are attached inside excel sheet.
Source Data Extended Data Fig. 1–7
Statistical source data in clearly labeled excel sheet; unprocessed western blots are attached inside excel sheet.
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Mishra, B., Bachu, M., Yuan, R. et al. IL-10 targets IRF transcription factors to suppress IFN and inflammatory response genes by epigenetic mechanisms. Nat Immunol 26, 748–759 (2025). https://doi.org/10.1038/s41590-025-02137-3
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DOI: https://doi.org/10.1038/s41590-025-02137-3
