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Type I interferons and the cytokine TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation

Nature Immunology volume 18, pages 1104–1116 (2017)Cite this article

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Abstract

Cross-regulation of Toll-like receptor (TLR) responses by cytokines is essential for effective host defense, avoidance of toxicity and homeostasis, but the underlying mechanisms are not well understood. Our comprehensive epigenomics approach to the analysis of human macrophages showed that the proinflammatory cytokines TNF and type I interferons induced transcriptional cascades that altered chromatin states to broadly reprogram responses induced by TLR4. TNF tolerized genes encoding inflammatory molecules to prevent toxicity while preserving the induction of genes encoding antiviral and metabolic molecules. Type I interferons potentiated the inflammatory function of TNF by priming chromatin to prevent the silencing of target genes of the transcription factor NF-κB that encode inflammatory molecules. The priming of chromatin enabled robust transcriptional responses to weak upstream signals. Similar chromatin regulation occurred in human diseases. Our findings reveal that signaling crosstalk between interferons and TNF is integrated at the level of chromatin to reprogram inflammatory responses, and identify previously unknown functions and mechanisms of action of these cytokines.

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Figure 1: Pretreatment with TNF reprograms the subsequent response to TLR4 stimulation in human macrophages.
Figure 2: Distinct epigenetic landscape at different TLR4-induced gene classes.
Figure 3: Distinct TF binding at different gene classes and gene expression patterns in human disease.
Figure 4: Type I interferons block TNF-mediated tolerization of genes encoding inflammatory molecules, without affecting LPS signaling.
Figure 5: Integration of signaling crosstalk between interferons and TNF at the chromatin level.
Figure 6: Interferons and TNF prime chromatin by cooperatively recruiting TFs to class 1 promoters.
Figure 7: Colocalization of IRF1 and p65 and altered transcriptional requirements in IFN-α- and TNF-treated macrophages.
Figure 8: Chromatin accessibility in monocytes from people with SLE.

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Acknowledgements

We thank A. Pernis and I. Rogatsky for discussions and review of the manuscript, and F. Barrat (Hospital for Special Surgery) for monocytes from donors with SLE. Supported by the US National Institutes of Health and The Tow Foundation (for the David Z. Rosensweig Genomics Center).

Author information

Authors and Affiliations

  1. Arthritis and Tissue Degeneration Program and the David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, New York, USA

    Sung Ho Park, Kyuho Kang, Eugenia Giannopoulou, Yu Qiao, Geonho Kim, Kyung-Hyun Park-Min & Lionel B Ivashkiv

  2. Biological Science Department, New York City College of Technology, City University of New York, Brooklyn, New York, USA

    Eugenia Giannopoulou

  3. Department of Microbiology, Dankook University, Cheonan, Chungnam, Republic of Korea

    Keunsoo Kang

  4. Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, New York, USA

    Lionel B Ivashkiv

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Contributions

S.H.P. conceived of, designed and performed most of the experiments, performed bioinformatic analysis and wrote the manuscript; Kyuho K., Y.Q., G.K., and K.-H.P.-M. contributed experiments and expertise; E.G. and Keunsoo K. performed bioinformatic analysis; L.B.I. conceived of and oversaw the study and edited the manuscript; and all authors reviewed and provided input on the manuscript.

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Correspondence to Lionel B Ivashkiv.

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Integrated supplementary information

Supplementary Figure 1 Transcriptional profiles of TNF-treated macrophages, stratified by K-means clustering.

(a) This figure corresponds to Fig. 1b and shows more detail of gene expression in the different clusters within each class (right bar graphs). The left panel depicts K-means clustering (K = 12) of 1,574 LPS-induced genes (>3-fold) in the indicated conditions, showing relative gene expression for each gene. The adjacent bar graphs represent FPKM values for a given cluster in RNA-seq analysis. Error bars indicate SEM. The right two panels are bar graphs and box plots depicting FPKM values for the gene classes shown in Fig. 1b. (b) RT-qPCR analysis of representative genes in human primary macrophages stimulated with TNF (10 ng/ml) for 24 h and challenged with LPS (10 ng/ml) for 3 h; results are presented relative to baseline expression in naive macrophages (N), set as 1. Data are from one representative out of four-seven independent experiments with different blood donors with similar results. Error bars indicate SEM. (c) Induction of Class 3 genes by LPS under tolerized conditions is dependent on IFNAR signaling. RT-qPCR analysis of representative Class 3 and 4 genes in human primary macrophages stimulated with TNF (10 ng/ml) for 24 h in the presence of anti-IFNAR (5 μg/ml) or isotype CTL, and challenged with LPS (10 ng/ml) for 3 h; results are presented relative to baseline expression in unstimulated (N) cells, set as 1. Data are from one representative of three independent experiments with different blood donors with similar results. Error bars indicate SEM.

Supplementary Figure 2 Kinetic analysis of TNF-induced mRNA transcripts and TNF-induced memory.

(a) Kinetic analysis of TNF-induced mRNA transcripts. RNA-seq analysis of a time course of TNF stimulation (1, 3, 6, 24 h) was performed and the genes in the 12 clusters and 6 gene classes from Fig. 1b were visualized on a heat map (left). The gene order in each column is the same as the heat maps in Fig. 1b. Bar graphs (right) represent FPKM values for each cluster in the various gene classes. Error bars indicate SEM. (b) Venn diagram showing the overlap between LPS (10 ng/ml for 3 h) or TNF (10 ng/ml for 1 or 3 h) inducible genes, and how the genes in each region segregate into the above-defined gene classes. Genes that are not LPS-inducible are not included in the analysis in this study. (c) TNF-induced tolerization of TNF and IL6 genes persists after TNF washout. RT-qPCR analysis of TNF or IL6 genes in macrophages cultured with TNF (20 ng/ml) for 24 h, followed by washout of TNF and incubation in fresh medium for 24 or 48 h. Then, mRNA levels were measured after LPS (10 ng/ml for 3 h) stimulation. Data are from one representative of three independent experiments with different blood donors with similar results. Error bars indicate SEM.

Supplementary Figure 3 Class-specific chromatin regulation in TNF-treated macrophages.

(a) Quantitation of the data shown as a heat map in Fig. 2a. Box graphs represent H4ac, H3K4me3, H2Bub ChIP-seq and ATAC-seq normalized tag densities (log2) at the promoters (-2kb < TSS < +2kb) of a given class under the indicated conditions. **** p < 0.0001, ** p < 0.01, * p < 0.05, Kolmogorov-Smirnov test. (b) Representative UCSC Genome Browser tracks displaying normalized profiles for H4ac, H3K4me3, H2Bub ChIP-seq and ATAC-seq signals at Class 1(TNF, IL27, TNFSF9 and TNFAIP2), Class 3 (OASL, IFIT1, IRF7 and MX1) and Class 4 (FPR1/2, PTGES, CH25H, CLU) genes under indicated conditions (N, L, T, T-L). Boxes enclose genomic regions regulated across conditions. (c) Box graphs represent quantitation of the normalized tag densities (log2) for 6 histone modifications and chromatin accessibility at the promoters (-2kb < TSS < +2kb) in naïve macrophages. Each class we tested is indicated in x-axis. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, Kolmogorov-Smirnov test. (d) Heatmaps of H3K36me3, H3K56ac, and H3K79me2 normalized tag densities at gene bodies (TSS to TES) of a given class under indicated conditions. (e) Tolerization with TNF does not increase negative H3K27me3 or H3K9me3 histone marks at Class 1 and 2 tolerized gene promoters (-2kb < TSS < +2kb). Relative ChIp-seq normalized tag counts (upper) and ChIP-qPCR confirmation at IL6 promoter (lower).

Supplementary Figure 4 Class-specific chromatin regulation.

(a) Box graphs represent quantification of the normalized tag densities for H2Bub, H3K4me3 ChIP-seq and ATAC-seq at the promoters (-2kb < TSS < +2kb) in L and T-L conditions. ATAC-seq data represent pooled samples from three (T-L) or five (L) replicates from independent donors, and H2Bub, H3K4me3 ChIP-seq data are from an additional independent donor. p-value, Kolmogorov-Smirnov test. (b) Formaldehyde-assisted isolation of regulatory elements (FAIRE) assay at representative TNF, IL6 and CCL5 genes under indicated conditions. Data from two experiments out of four to six independent experiments with different donors are shown. The decrease in chromatin accessibility in T-L versus L conditions (condition 4 versus 2) at TNF and IL6 promoters was consistently observed. A decrease in basal chromatin accessibility at TNF and IL6 promoters after TNF treatment (condition 3 versus 1) was not observed in all donors, and data representative of the two patterns observed are shown separately as donor 1 (left) and donor 2 (right). (c) Distribution of ATAC-seq footprints and occupancy of transcription factor binding sites. Heatmaps showing per nucleotide ATAC-seq cleavage sites for NRF, NFY and SP1 motifs in LPS-stimulated human primary macrophages ranked by tag density. The number of ATAC-seq footprints for each TF is shown in y-axis. 200 bp windows are shown centered at the midpoints of the ATAC-seq footprint. Footprinting was performed with two independent ATAC-seq replicates. (d-f) Reversal of tolerization of IL6 by GSK3 inhibition is mediated by increased type I IFN. (d) Inhibition of GSK3 results in increased IFNB expression after TNF stimulation. RT-qPCR analysis of IFNB mRNA in human primary macrophages stimulated with TNF (10 ng/ml) for indicated times in the presence of SB216763 (10 μM) or control DMSO. (e) Increased Ifnb expression in GSK3-deficient macrophages. RT-qPCR analysis of Ifnb mRNA in BMDMs obtained from mice lacking myeloid GSK3 (Gsk3bfl/fl Cre) or genetically matched control mice (Gsk3b+/+ Cre), then stimulated with TNF (40 ng/ml) for indicated times (left) or for 24 h and then challenged for 3 h with LPS (10 ng/ml, right). (f) IFNAR blockade prevents reversal of tolerance by GSK3 inhibitor SB216763. RT-qPCR analysis of IL6 mRNA in human primary macrophages stimulated with TNF (10 ng/ml) for 24 h and then challenged with LPS (10 ng/ml) for 3 h in the presence of SB216763 (10 μM), anti-IFNAR (5 μg/ml) and isotype CTL as indicated. (g) RT-qPCR analysis of TNF and IL-6 mRNA in human primary macrophages under indicated conditions. IFN-α reversed TNF-induced tolerance (bar 12) but did not reverse LPS-induced endotoxin tolerance (bar 10). mRNA results are presented relative to baseline expression in unstimulated cells, set as 1 (d-g). Data are representative of three independent experiments (d, f, g) or two independent experiments (e).

Supplementary Figure 5 Enhancer profiles of TNF-treated macrophages.

(a) Heatmaps of H3K27ac and ATAC-seq normalized tag densities at the enhancers (see Methods) of genes in different classes based on Fig. 1b (upper). Box graphs represent quantitation of the normalized tag densities (log2) for a given class (lower). **** p < 0.0001, ** p < 0.01, * p < 0.05, Kolmogorov-Smirnov test. (b) The number of genes (%) that are associated with super enhancers and typical enhancers in a given gene class in the indicated conditions. (c-d) Bar graphs represent the number of genes and % of genes in each class that are associated with latent enhancers (c) or CpG islands (d). (e) Significantly enriched motifs (p < 10-5) in ATAC-seq footprints at enhancers in each gene class (see Methods) in indicated conditions. Motifs are grouped according to transcription factor families. (f) De novo motif analysis of enhancers (entire ATAC-seq peak in all experimental conditions) associated with the 6 gene classes using HOMER.

Supplementary Figure 6 Regulation of chromatin accessibility by TNF and IFN-α.

(a) Correlation matrix heatmap based on Pearson correlation coefficients comparing normalized RNA-seq tag counts across all indicated conditions and replicates. The concordance between replicates was very high (the number in each box indicates Pearson correlation coefficient, r). (b) Heatmap of ATAC-seq, and H2Hub and H3K4me3 ChIP-seq normalized tag densities of Class 1 genes (upper) under all eight experimental conditions performed together in one experiment. Gene order is the same as in Fig. 1b. Box graphs represent quantitation of the normalized tag densities (log2) for Class 1 gene promoters (lower). p-value, Kolmogorov-Smirnov test. (c) Heatmap of ATAC-seq normalized tag densities at enhancers (see Methods) of Class 1 genes in indicated conditions (upper). Box graphs represent quantitation of the normalized tag densities (log2) for Class 1 gene enhancers (lower). **** p < 0.0001, Kolmogorov-Smirnov test. (d) Representative UCSC Genome Browser tracks displaying normalized profiles for ATAC-seq and H2Bub, H3K4me3 ChIP-seq signals at IL27, TNFSF9, SPHK1, IL6, CCL20 and CXCL2 Class 1 genes under indicated conditions. Boxes enclose genomic regions regulated across conditions.

Supplementary Figure 7 Co-occupancy of IRF1 and p65 at genomic regions associated with class 1 genes encoding inflammatory molecules.

(a) De novo motif enrichment analysis of Class 1 gene enhancers using ATAC-seq footprints under same conditions as the promoter analysis in Fig. 6a. (b) Bar graphs represent cumulative values for IRF family members in RNA-seq analysis from three replicates. (c) Representative UCSC Genome Browser tracks displaying normalized profiles for p65 and IRF1 ChIP-seq signals at CXCL10, CXCL11, IL8, TNF, TNIP2, CCL20 and IL6 (Class 1) genes under indicated conditions. Boxes enclose co-localization of p65 and IRF1 binding peaks to the same genomic regions. (d) ChIP-qPCR analysis of recruitment of p65 and IRF1 to representative genes under indicated conditions. Data are representative of three independent experiments. (e-f) Venn diagram showing the overlap between p65 ChIP-seq peaks in human macrophages (stimulated with TNF (10 ng/ml) for 24 h) and IRF1 Chip-seq peaks (stimulated with IFN-γ (100 U/ml) for 24 h and challenged with LPS (10 ng/ml) for 3 h) within 10 kb ± TSS of Class 1 genes. p-value, Fisher’s exact test relative to a randomly selected control gene set. Representative UCSC Genome Browser tracks displaying normalized profiles for p65 and IRF1 ChIP-seq signals at IL27 and IL1B genes. Boxes enclose co-localization of p65 and IRF1 binding peaks to the same genomic regions. IRF1 ChIP-seq data are from GSE43036.

Supplementary Figure 8 ‘Sequential rheostat’ model of integrated regulation of the TLR-induced transcriptional response by TNF and IFN-α.

(a) Sequential rheostat model of integrated regulation of the TLR-induced transcriptional response by TNF and IFN-α via upstream signaling and downstream chromatin-based epigenetic mechanisms. Regulation of TLR-induced signaling pathways and chromatin states of TLR-inducible genes by TNF and IFN-α determines transcriptional output. TNF treatment ‘tolerizes’ macrophages to diminish signaling responses to TLR4 agonist LPS. Left panel. In TNF-stimulated cells, IFN-α promotes opening of chromatin at tolerized (T) genes, thereby allowing gene induction in response to weak upstream signals. Right panel. At nontolerized (NT) genes, TNF increases chromatin accessibility concomitantly with suppression of upstream signaling. This enables NT gene induction in response to weak TLR4 signaling upon secondary LPS challenge. (b) Left panel. In naïve macrophages LPS activates a strong signal that is sufficient to broadly remodel chromatin at target genes promoters, resulting in expression of the full set of TLR-inducible genes. Right panel. TNF induces chromatin remodeling at subsets of TLR-inducible genes in a class-specific manner. In parallel, TNF induces expression of multiple signaling inhibitors such that LPS activates only a weak signal. This weak signal is insufficient to induce chromatin remodeling required for transcriptional activation of Class 1 and 2 T genes, but is sufficient to activate transcription at Class 3 and 4 NT genes where chromatin has been opened and primed by TNF. Exogenous type I IFN cooperates with TNF to open and prime chromatin at Class 1 and 2 genes, enabling transcriptional activation by weak LPS-induced signals. Thus, environmental signals are integrated at the epigenomic level to regulate the TLR response in a gene class-specific manner.

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Park, S., Kang, K., Giannopoulou, E. et al. Type I interferons and the cytokine TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation. Nat Immunol 18, 1104–1116 (2017). https://doi.org/10.1038/ni.3818

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