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. 2018 Jan 16;48(1):75-90.e6.
doi: 10.1016/j.immuni.2017.12.010.

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

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Free PMC article

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

Zsolt Czimmerer et al. Immunity. .
Free PMC article

Abstract

The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

Keywords: IL-1β; IL-4; STAT6; alternative macrophage polarization; inflammasome activation; inflammation; macrophage epigenomics; pyroptosis; repression; transcription.

Figures

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Figure 1
Figure 1
The IL-4-STAT6 Signaling Pathway Induced Gene Expression during Mouse Alternative Macrophage Polarization (A) Heatmap of IL-4-regulated (p value ≤ 0.05, FC ≥ 2) gene expression clusters in WT murine BMDMs. Data represent the average fold changes of four individual animals. (B) The average fold change from the IL-4-repressed gene cluster at the indicated time points following IL-4 stimulation in WT (n = 4) and Stat6−/− (n = 2) BMDMs. Error bars represent means ± SD. (C) RT-qPCR analysis of gene expression on a set of IL-4-repressed genes in WT and Stat6−/− BMDMs. BMDMs were treated with IL-4 for 6 hr. Data are representative of five individual animals per genotype from two independent experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant change. Error bars represent means ± SD. (D) GSEA analysis of IL-4-repressed genes (in vitro) against a ranked list of genes regulated in the Brugia malayi-implanted mice-derived macrophages (Ne-Mac) compared to the intraperitoneal thioglycollate-administrated mice-derived peritoneal macrophages (Thio-Mac). (E) Expression of IL-4-repressed genes in the Brugia malayi-implanted mice-derived macrophages (Ne-Mac) and the intraperitoneal thioglycollate-administrated mice-derived peritoneal macrophages (Thio-Mac). Reads per kilobase per million values (RPKM) are presented as the mean and SD of three individual animals per group quantified by RNA-seq. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 2
Figure 2
Changes in RNAPII and H3K27Ac Enrichments as well as Nascent RNA Transcription Are Immediate Early Markers of IL-4-STAT6-Regulated Transcription (A) Metagene plot of RNAPII-pS5-, RNAPII-pS2-, and H3K27Ac-specific ChIP-seq enrichments and GRO-seq signals on the gene bodies of regulated gene clusters (Figure 1A) in the presence of IL-4 in WT BMDMs (TSS, transcription start site; TTS, transcription termination site). Coverage is defined as read count per million mapped reads. Data (H3K27Ac, RNAPII-pS2, and RNAPII-pS5) are combined from two independent biological replicates. (B) H3K27Ac, RNAPII-pS5, and RNAPII-pS2 ChIP-seq signals at the selected IL-4-repressed gene bodies. ChIP-seq signals are visualized by the Integrative Genomics Viewer. Data are representative of two independent biological replicates. BMDMs were treated with IL-4 for 1 hr.
Figure 3
Figure 3
RNAPII-pS5-Based Characterization of IL-4-Activated STAT6 Cistrome in Mouse Macrophages (A) Histograms of the average coverage of STAT6 peaks at the indicated period of time following IL-4 treatment in WT BMDMs. (B) Pie chart of the RNAPII-pS5-positive and -negative STAT6-bound regulatory regions, 1 hr of IL-4 stimulation. (C) Read distribution plot of ChIP-seq intensities for STAT6 and RNAPII-pS5 around the summit of the detected STAT6 peaks in a 4 kb window (left). Clustering of STAT6-RNAPII-pS5 co-bound genomic regions was based on the usage of DiffBind analysis (p ≤ 0.05). Boxplots of the average RPKM values for RNAPII-pS5 in each cluster (right). Boxes encompass the 25th to 75th percentile RPKMs. Whiskers extend to the 10th and 90th percentiles. (D) Boxplots of RNAPII-pS2 and H3K27Ac read enrichments (RPKM) around the identified STAT6 peak clusters in WT BMDMs. Boxes encompass the 25th to 75th percentile RPKMs. Whiskers extend to the 10th and 90th percentiles. (E) Heatmap of correlations between STAT6 peak (C) and IL-4-regulated gene clusters (Figure 1A) based on genomic proximity and functional chromatin domain prediction. (F) Integrative Genomics Viewer snapshots of STAT6, H3K27Ac, RNAPII-pS5, and RNAPII-pS2 ChIP-seq signals on a set of IL-4-repressed and activated genomic loci. (G) RT-qPCR measurements of eRNA expression at IL-4-repressed enhancers in WT and Stat6−/− macrophages. (H) Bar plots showing the percentage of the STAT6 motif-positive STAT6 peaks in the clusters on (C). The STAT6 binding motif was used for targeted search. Dashed line indicates background (Bg: 9.51%). (I) Boxplot of STAT6 motif scores at the functionally distinct STAT6 peak clusters. Boxes encompass the 25th to 75th percentile motif scores. Whiskers extend to the 10th and 90th percentiles. BMDMs (B–D, F, and G) were treated with IL-4 for 1 hr. Data in (A)–(D) are combined from two independent biological replicates. Changes in (D) were considered significant at p < 0.00001 using paired t test and an average fold change cut off value of ≥ 1.15 was used between control and IL-4-treated samples. # means significant difference, n.s. indicates not significant change. Data in (F) are representative of two independent biological replicates. Data in (G) are representative of five individual animals per genotype from two independent experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant change. Error bars represent mean ± SD.
Figure 4
Figure 4
IL-4-Induced Changes at Repressor and Activator STAT6 Sites and the Role of HDAC3 in IL-4-STAT6-Mediated Repression (A–D) Boxplots of ATAC-seq (A) and ChIP-seq (B–D) signals for PU.1 (B), p300 (C), HDAC1 (D), HDAC2 (D), and HDAC3 (D) on the repressor and activator STAT6 sites in WT BMDMs. Boxes encompass the 25th to 75th percentile RPKMs. Whiskers extend to the 10th and 90th percentiles. (E) Boxplots of the expression of IL-4-HDAC3-dependent repressed genes in WT (n = 3) and Hdac3fl/fl Lyz2 Cre (n = 3) BMDMs using publicly available microarray results. Boxes encompass the 25th to 75th percentile changes. Whiskers extend to the 10th and 90th percentiles. (F) Metagene plots of RNAPII-pS5 and RNAPII-pS2 signals at the gene bodies of IL-4-HDAC3-dependent repressed genes. Coverage is defined as read count per million mapped reads. (G) Read distribution plot of ChIP-seq intensities for RNAPII-pS5, STAT6, HDAC3, NCoR, and SMRT around the summit of the detected STAT6 peaks at the IL-4-repressed enhancers (n = 325) in the subTADs of HDAC3-dependent repressed genes, 1 hr of IL-4 stimulation. (H) Boxplot of the average HDAC3 binding intensity on the genomic regions (G). Boxes encompass the 25th to 75th percentile RPKMs. Whiskers extend to the 10th and 90th percentiles. (I) Normalized microarray signal intensity of Fos, Lyz1, Lyz2, and Smad3 in control or IL-4-stimulated WT and Hdac3fl/fl Lyz2 Cre BMDMs. (J) RT-qPCR measurements of Fos, Lyz1, Lyz2, and Smad3 expression in control or IL-4-stimulated WT and Ncor1fl/fl Lyz2 Cre iBMDMs. BMDMs were treated with IL-4 for 1 hr (A–D, F–H) or 24 hr (E, I, and J). Data (A–D, F, and H) are combined from two independent biological replicates. Changes (A–D and H) were considered significant at p < 0.00001 using paired t test and an average fold change cut off value of ≥ 1.15 was used between control and IL-4-treated samples. # means significant difference, ns indicates not significant change. Data (I) represent the mean and SD of three independent biological replicates. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant difference. Error bars represent means ± SD. Data (J) represent the mean and SD of three independent biological replicates. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant difference. Error bars represent means ± SD.
Figure 5
Figure 5
Selective Repression of LPS-Activated Inflammatory Program by IL-4-Activated STAT6 (A) Schematic representation of the experimental system. (B) Boxplot of the fold changes of LPS-activated genes (RNA-seq). Clustering was based on the different LPS-induced gene expression effects on IL-4-pretreated and untreated BMDMs (p < 0.05). Data represent the average fold changes of three individual animals from two independent experiments. Boxes encompass the 25th to 75th percentile changes. Whiskers extend to the 10th and 90th percentiles. (C) Metagene plot of RNAPII-pS2 signals over the gene bodies of the genes in the clusters (B). Coverage is defined as read count per million mapped reads. Data are combined from two independent biological replicates. (D) RT-qPCR measurements of basal and LPS-induced expression of the inflammation-associated genes in IL-4-pretreated and unstimulated WT and Stat6−/− BMDMs. Data are cumulative of four individual animals per genotype from two independent experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant difference. Error bars represent means ± SD. BMDMs were pretreated with IL-4 for 24 hr followed by LPS exposure for 3 hr (B and D) or 1 hr (C).
Figure 6
Figure 6
Attenuated Inflammatory Response Is Conferred by the Repressive Action of IL-4-STAT6 Signaling on a Subset of Enhancers (A) Venn diagram of the overlap between the STAT6-bound regulatory regions associated to IL-4-inhibited LPS-responsive genes and the LPS-activated p65 cistrome. (B) Flowchart of the identification of IL-4-repressed, LPS-inducible inflammatory enhancers. Significant changes in RNAPII binding were identified by DiffBind analysis (p ≤ 0.05). (C) Read distribution plot of ChIP-seq intensities for RNAPII-pS5 and RNAPII-pS2 around the summit of STAT6 peaks on the identified 448 overlapping STAT6 and p65-bound regulatory elements exhibiting IL-4-dependent attenuation of LPS response. (D) Boxplot of the average coverage (RPKM) for RNAPII-pS5 and RNAPII-pS2 binding at the regulatory regions presented on the read distribution plot on (C), exhibiting attenuated LPS response in IL-4-pretreated BMDMs. Boxes encompass the 25th to 75th percentile RPKMs. Whiskers extend to the 10th and 90th percentiles. (E) Boxplot of the average coverage (RPKM) for p65 binding at the regulatory regions presented on the read distribution plot on (C), exhibiting attenuated LPS response in IL-4-pretreated BMDMs. Regulatory regions showing repressed (left) and not influenced (right) p65 binding are shown. Boxes encompass the 25th to 75th percentile RPKMs. Whiskers extend to the 10th and 90th percentiles. (F) Genome browser views of the IL-4 repressed regulatory regions showing attenuated LPS response in IL-4-pretreated BMDMs. ChIP-seq signals for RNAPII-pS5, RNAPII-pS2, and p65 are shown. BMDMs were pretreated with IL-4 for 24 hr followed by 1 hr LPS exposure (A–E). Data (A–E) are combined from two independent biological replicates. Changes (D, E) were considered significant at p < 0.00001 using paired t test and an average fold change cut off value of ≥ 1.15 was used between control and IL-4-treated samples. # means significant difference, ns indicates not significant difference. Data (F) are representative of two independent biological replicates.
Figure 7
Figure 7
IL-4 Pretreatment Dampens the Inflammatory Response of Macrophages, Including Inflammasome Activation, IL-1β Production, and Pyroptosis (A) Genome browser view of STAT6-, RNAPII-pS5-, RNAPII-pS2-, and p65-specific ChIP-seq signals on the Il1b locus. Data are representative of two independent biological replicates. (B) RT-qPCR-based measurement of basal and LPS-induced Il1b expression in IL-4-pretreated and unstimulated WT and Stat6−/− BMDMs. Data are cumulative of four individual animals per genotype from two independent experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant change. Error bars represent means ± SD. (C) ChIP-qPCR measurement of p65 binding at Il1b_−9.7Kb enhancer from WT and Stat6−/− BMDMs. Data represent the mean and SD of two biological replicates. +p < 0.1, ns, not significant change. (D) RT-qPCR measurement of basal and LPS-induced Il1b_−9.7Kb eRNA expression in IL-4-pretreated and unstimulated WT and Stat6−/− BMDMs. Data are cumulative of four individual animals per genotype from two independent experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant change. Error bars represent means ± SD. (E) Western blot determination of basal and LPS-regulated Nlrp3, pro-IL-1β, pro-Caspase1, ASC, and β-actin expression in IL-4-pretreated and unstimulated WT and Stat6−/− BMDMs. Data are representative of five individual animals per genotype from two independent experiments. (F) Contour map representation of laser-scanning imaging cytometry of Caspase-1 activity in WT and Stat6−/− BMDMs. Data are representative of two independent experiments. (G) ELISA measurement of IL-1β secretion in IL-4-pretreated and unstimulated WT and Stat6−/− mouse BMDMs. Data represent the mean and SD of three individual animals. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant change. (H) Lactate dehydrogenase activity assay measurement of LPS and ATP co-stimulation-induced LDH release in IL-4-pretreated and unstimulated WT and Stat6−/− BMDM supernatants. LDH release expressed as the percentage of Triton X-100-liberated total LDH release. Data represent the mean and SD of three individual animals. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant change. (I) Contour map representation of laser-scanning imaging cytometry analysis of PI-labeled WT and Stat6−/− BMDMs. Data are representative of two independent experiments. (J) Basal, LPS, and Salmonella Typhimurium (SL3261)-induced expression of Nlrp3 and Il1b expression in naive and Heligmosomoides polygyrus (H. polygyrus)-infected mice-derived peritoneal macrophages. Each data point represents the mean and SD of five to six individual animals per group. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant change. BMDMs were pretreated with IL-4 for 24 hr followed by LPS exposure for 1 hr (A and C), 3 hr (B, D, E, F, H, and I), or the indicated period (G).

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