Mechanisms of Epigenomic and Functional Convergence Between Glucocorticoid- and IL4-Driven Macrophage Programming

Abstract

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2_IL4). Glucocorticoids, widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2_GC), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2_IL4 and M2_GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the GC receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2_IL4:M2_GC-shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design.

Keywords: Homeostatic macrophage programming; chromatin and epigenomics; enhancer landscape; glucocorticoid receptor; kruppel-like factor 4; tissue repair and inflammation; transcriptional regulation.

Extended Data Fig. 1:. Transcriptomic analysis of the M2_IL4 and M2_GC macrophage populations.

a Expression of selected genes upregulated in M2_Cort and M2_Dex (n=3). b Volcano plot shows DEGs in M2_Cort relative to M0 (LogFC ≥ 1; FDR <0.05). Selected upregulated genes are highlighted blue, and downregulated in red. Some of the shared target genes between M2_IL4, M2_Dex, and M2_Cort are underlined. c Venn diagrams show differentially regulated pathways determined by QuSAGE in M2_IL4 and M2_Dex relative to M0 (p <0.05). d Differential pathways in M2 determined by QuSAGE as in Fig. 1d. Underlined are some of the pathways shared between M2_IL4, M2_Dex and M2_Cort (upregulated, blue; downregulated, red). e Genes from two pathways selectively repressed in M2_IL4 and M2_Dex are plotted as logFC±SD.

Extended Data Fig. 2:. M2_IL4 and M2_GC macrophage populations share epigenetic landscape.

a The distribution of ATACseq peaks relative to known genomic features in M0, M2_IL4 and M2_Dex populations across replicates (n=3). b Venn diagram shows differential M2_IL4, M2_Cort and M2_Dex H3K27ac peaks relative to M0. c H3K27ac assessed by ChIP-qPCR at enhancers of indicated genes in 2_IL4, M2_Cort and M2_Dex normalizedto that in M0. Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant. n=3, error bars are SEM. d Rank shift analysis of mean H3K27ac signals in M0 (baseline) and M2_IL4. Peaks that are also differentially regulated in M2_Dex are colored in red (hyperacetylated) and blue (hypoacetylated). The LOESS regression line (green) shows the relationships between acetylation strength in M0 and signal change in M2_IL4. Enhancer sites associated with representative IL4-specific (orange) and shared (black) genes are marked, and the expression of representative genes as determined by RNAseq (Fig. 1) is plotted on the right.

Extended Data Fig. 3:. Signal-specific and shared enhancers in M2_IL4 and M2_GC macrophages.

a-b Comparisons of TSS window-associated ATACseq (a) or H3K27ac ChIPseq (b) peaks in differential (teal) vs. non-differential (gray) genes in M2_IL4 (left panels) and M2_Dex (right panels) stratified by the number of gene-associated peaks. Only peaks with the largest positive (max) or negative (min) changes relative to M0 are shown. A subset of up- (red) and downregulated (blue) M2_IL4-unique (left) and M2_Dex-unique (right) DEGs identified by RNAseq (Fig. 1) are labeled. c-d Correlations between normalized RNAseq and ATACseq (c) or H3K27ac ChIPseq (d) signals in the TSS-proximal − 1K + 1K windows for differential (teal) and non-differential (gray) genes in M2_IL4 (left) and M2_Dex (right). A subset of 92 shared up- (red) and downregulated (blue) DEGs identified by RNAseq (Fig. 1) are labeled. e-f Correlations between normalized RNAseq and ATACseq (e) or ATACseq and H3K27ac ChIPseq (f) signals in the TSS-proximal −1K + 1K windows for differential (teal) and non-differential (gray) genes in M2_IL4 (left) and M2_Dex (right). A subset of the up- (red) and downregulated (blue) 587 M2_IL4-unique DEGs (left) or 174 M2_Dex-unique DEGs identified by RNAseq (Fig. 1) are labeled. g Differentially regulated pathways identified by Ingenuity Pathway Analysis (QIAGEN) of genes associated with change in both the ATACseq and H3K27ac ChIPseq signal in M2_IL4 and M2_Dex populations.

Extended Data Fig. 3:. Signal-specific and shared enhancers in M2_IL4 and M2_GC macrophages.

a-b Comparisons of TSS window-associated ATACseq (a) or H3K27ac ChIPseq (b) peaks in differential (teal) vs. non-differential (gray) genes in M2_IL4 (left panels) and M2_Dex (right panels) stratified by the number of gene-associated peaks. Only peaks with the largest positive (max) or negative (min) changes relative to M0 are shown. A subset of up- (red) and downregulated (blue) M2_IL4-unique (left) and M2_Dex-unique (right) DEGs identified by RNAseq (Fig. 1) are labeled. c-d Correlations between normalized RNAseq and ATACseq (c) or H3K27ac ChIPseq (d) signals in the TSS-proximal − 1K + 1K windows for differential (teal) and non-differential (gray) genes in M2_IL4 (left) and M2_Dex (right). A subset of 92 shared up- (red) and downregulated (blue) DEGs identified by RNAseq (Fig. 1) are labeled. e-f Correlations between normalized RNAseq and ATACseq (e) or ATACseq and H3K27ac ChIPseq (f) signals in the TSS-proximal −1K + 1K windows for differential (teal) and non-differential (gray) genes in M2_IL4 (left) and M2_Dex (right). A subset of the up- (red) and downregulated (blue) 587 M2_IL4-unique DEGs (left) or 174 M2_Dex-unique DEGs identified by RNAseq (Fig. 1) are labeled. g Differentially regulated pathways identified by Ingenuity Pathway Analysis (QIAGEN) of genes associated with change in both the ATACseq and H3K27ac ChIPseq signal in M2_IL4 and M2_Dex populations.

Extended Data Fig. 4:. GR, KLF4 and GRIP1 genome-wide binding in differentially polarized macrophage populations.

a Read counts in peaks Spearman’s correlation between M2_Dex and M2_Cort GR ChIPseq. b Top enriched transcription factor binding motifs in GR ChIPseq peaks in M2_Dex and M2_Cort. Peaks were analyzed by HOMER2 findMotifsGenome.pl to identify significantly enriched motifs relative to genomic background. c UpSet plot shows GC-induced peaks from GR ChIPseq and the total peak atlases from KLF4 and GRIP1 CUT&RUN as analyzed by BedSect. Set-size: number of total regions (overlaps and unique). Intersection size: number of total regions with specified overlaps. d Enrichment p-values for KLF family motifs in KLF4 CUT&RUN-derived peaks. Peaks were analyzed by HOMER2 findMotifsGenome.pl to identify significantly enriched motifs relative to a set of background regions with similar GC content. e Volcano plots of GRIP1 CUT&RUN differential peaks in M2_IL4 (left) and M2_Dex (right) relative to M0. Size of the peak point on graph is commensurate with −log10(FDR), whereas color shade is proportional to the log2FC. Peaks were scanned for the presence of transcription factor binding motifs within the HOMER database using motifmatchr and select motifs (GRE, black; STAT6, red; KLF4, blue) are indicated.

Extended Data Fig. 5:. GRIP1 is required for the M2_IL4 and M2_GC programming.

a Volcano plot shows DEGs in GRIP1 cKO vs. WT in M2_Cort relative to M0 of each genotype. (FC ≥1.5; unadjusted p-value<0.05). A subset of DEGs expressed at higher levels in the cKO relative to WT are highlighted in red, whereas those downregulated - in blue. Examples of the shared M2_IL4:M2_GC GRIP1 target genes are underlined. b Differentially regulated pathways in M2_Cort (unadjusted p < 0.01) identified by QuSAGE with MsigDB are shown for GRIP1 cKO vs. WT. Select up- (red) and downregulated (blue) pathways are labeled, and those shared between IL4 and GC are underlined. Circle size is proportional to the number of genes in the pathway and color signifies p-value. c The distribution of ATACseq peaks relative to known genomic features in M0, M2_IL4 and M2_Dex populations across replicates for WT (n=3) and GRIP1 cKO (n=4). d Venn diagram of GRIP1-dependent differential ATACseq peaks in M2_IL4 and M2_Dex (n=4, FC ≥ 2; FDR <0.05). e Heatmaps of the GRIP1-dependent ATACseq peaks in M2_IL4 and M2_Dex (FC ≥ 2; FDR <0.05) are separated into those dependent on GRIP1 for opening (blue) or closing (red).

Extended Data Fig. 6:. GRIP1 contributes to phagocytic activity of M2_IL4 and M2_GC in vitro and healing properties in vivo.

Body weight, colon length and weight changes of WT and cKO mice 2 wks after initiating DSS-induced colitis.

Fig.1 |. Transcriptomic analysis of the M2_IL4 and M2_GC populations.

a, Transcriptomic changes in M0 macrophages polarized with IL4 or GCs corticosterone (Cort) or dexamethasone (Dex) for 24 h were determined by RNAseq (n=3). Venn diagrams show DEGs in M2_IL4 and M2GC relative to M0 (FC ≥ 2; FDR <0.05); 92 of 133 genes are regulated in both M2IL4 and M2_Dex in the same direction. b, Volcano plots show DEGs in M2_IL4 (left) and M2_Dex (right) relative to M0 (LogFC ≥ 1; FDR< 0.05). Selected upregulated genes are highlighted in orange (M2_IL4) and green (M2_Dex). Downregulated genes are highlighted in red in both populations. Examples of the M2_IL4:M2_Dex shared DEGs are underlined. c, RT-qPCR validation of genes upregulated selectively in M2_IL4 (top), M2_GC (middle), or both (bottom). Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant. n=4, error bars are SEM. d, Differentially regulated pathways (unadjusted p < 0.01) identified using QuSAGE and MsigDB canonical pathways (c2.cp.v7.3, Broad Institute) are shown for M2_IL4 (top) and M2_Dex (bottom). Underlined are the shared pathways between M2_IL4 (upregulated, orange; downregulated, red) or M2_Dex (upregulated, green; downregulated, red). Circle size is proportional to the number of genes in the pathway and color signifies p-value. e, Genes from indicated pathways induced or repressed in M2_IL4, M2_Dex, or both are plotted LogFC±SD and ordered by p-value.

Fig.2 |. M2_IL4 and M2_GC macrophage populations share epigenetic landscape.

a, ATACseq was performed on M0, M2_IL4 and M2_Dex. Venn diagram shows the number of differential ATACseq peaks in M2_IL4 and M2_Dex relative to M0 (n=4, FC ≥ 2; FDR <0.05). 4,913 of 8,213 peaks were differential in both populations and regulated in the same direction. b, Heatmaps of the induced (red) and suppressed (blue) signal-specific and shared M2_IL4 and M2_Dex ATACseq peaks (FC ≥ 2; FDR <0.05). c-d, Enriched transcription factor binding motifs with associated p-values were identified by HOMER known motif analysis with JASPAR 2022 motif database in signal-specific and shared up- (c) and downregulated (d) ATACseq peaks in M2_IL4 and M2_Dex. e, Polarization-induced increases in the 5’ Tn5 cut site counts within indicated binding motifs of interest in the M2_IL4 and M2_Dex populations. f, Venn diagram shows the differential M2_IL4 and M2_Dex H3K27ac ChIPseq peaks relative to those in M0 (FDR< 0.05) with 1,304 of 1,667 peaks regulated in the same direction in both populations. g, Heatmaps of induced (red) and suppressed (blue) signal-specific and shared filtered H3K27ac peaks (FC ≥ 2; FDR <0.05) in M2_IL4 and M2_Dex. h, Volcano plots show differential H3K27ac ChIPseq peaks for M2_IL4 (left) and M2_Dex (right) relative to those in M0 (LogFC ≥ 1; FDR< 0.05); the location of selected hyperacetylated sites relative to the TSS of the closest gene are shown in orange (M2_IL4) or green (M2_Dex). Hypoacetylated sites are shown in red for each population. Sites overlapping in M2_IL4 and M2_Dex are underlined. i, Non-promoter 12,211 H3K27ac peaks after excluding 1,650 promoter-proximal peaks (−300/+200 relative to TSS) are shown as a Venn diagram with the number of differential, relative to M0, M2 population-specific and shared peaks (FC ≥ 2; FDR<0.05) indicated. j, Average H3K27ac ChIPseq signals at M2_IL4 (top), M2_Dex (middle)_, and shared (bottom) differential non-promoter peaks from (I) represented as violin plots.

Fig.3 |. M2_Dex and M2_IL4 have both distinct and shared de novo enhancers as demonstrated by H3K27ac ChIPseq.

a, Correlation between RNAseq, ATACseq and H3K27ac ChIPseq signals in selected M2_IL4-specific, M2_Dex-specific and shared genes. b, Genome-wide changes in up- or downregulated differential ATACseq (left) and H3K27ac ChIPseq (right) peaks located within −20K +20K window centered on the TSS in M2_IL4 and M2_Dex, as indicated. c-d, Comparison of TSS window-associated ATACseq (c) or H3K27ac ChIPseq (d) peaks in differential (teal) vs. non-differential (gray) genes in M2_IL4 (left panels) and M2_Dex (right panels), as indicated, stratified by the number of gene-associated peaks. Only peaks with the largest positive (max) or negative (min) changes relative to M0 are shown. A subset of 92 shared up- (red) and downregulated (blue) DEGs identified by RNAseq (Fig. 1) are labeled in each of the 4 panels. e, Correlation between normalized H3K27ac ChIPseq and ATACseq signals in the TSS-proximal −1K +1K windows for differential (teal) and non-differential (gray) genes. A subset of 92 shared upregulated (red) and downregulated (blue) DEGs identified by RNAseq (Fig. 1) are labeled.

Fig.4 |. GR, KLF4 and GRIP1 genome-wide binding in differentially polarized macrophage populations.

a, Venn diagram shows the numbers and overlap of GR ChIPseq peaks in M2_Dex and M2_Cort macrophages relative to M0 baseline (n=4; FC ≥ 2, FDR <0.05). b, Venn diagram shows the numbers and overlap of (left) GRIP1 CUT&RUN peaks in M0, M2_IL4 and M2_Dex populations (n=3; FC ≥ 1.5, FDR <0.05) or (middle and right) GRIP1 in M2_IL4 and all KLF4 peaks (n=2) or GRIP1 in M2_Dex and all KLF4 peaks, as indicated. c, GR (ChIPseq), KLF4 (CUT&RUN) and GRIP1 (CUT&RUN) read density distribution over Chil4, Hif3a and Klf9 loci in M0, M2_IL4 and M2_Dex. d, Average profiles of GR, KLF4 and GRIP1 signal from each macrophage population centered on GRIP1 CUT&RUN M2_IL4- (left), M2_Dex- (middle) specific or invariant ‘static’ (right) peaks (n=3; FC ≥ 2, FDR <0.05). e-f, Transcription factor binding motif enrichment Z-scores in KLF4 (e) and GRIP1 (f) peak subsets in each macrophage population (M2_IL4=orange, M2_Dex=green, M0=grey, as indicated) were determined in chromVAR from the HOMER list of motifs. Motifs with high variability among the samples (p_adj <= 1*10⁻⁶) were plotted and indicated motif families are highlighted in boxes.

Fig.5 |. GRIP1 is required for the establishment of M2_IL4 and M2_GC transcriptomes.

a-b, Expression of (a) GRIP1 or (b) IL4- (top) GC- (middle) or shared (bottom) M2 target genes in WT and GRIP1 cKO M0, M2_IL4, M2_Cort, and M2_Dex populations as assessed by RT-qPCR. Average relative expression of each transcript in WT M0 is arbitrarily set to 1. n=4; Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant. Error bars are SEM. c, Stacked bar plot shows the number of DEGs in GRIP1 cKO vs. WT as a fraction of WT M2_IL4, M2_Dex, or shared DEGs relative to M0 (RNAseq, WT: n=3, cKO: n=4). d, Volcano plot shows DEGs in GRIP1 cKO vs. WT in M2_IL4 (left) and M2_Dex (right) relative to M0 of each genotype. (FC≥1.5, unadjusted p-value<0.05). Selected inflammation-related genes expressed at higher levels in the cKO M2_IL4 and M2_Dex relative to WT are highlighted in red. Key M2 target genes downregulated in the cKO are highlighted in both M2_IL4 (orange) and M2_Dex (green). Examples of the shared M2_IL4:M2_Dex GRIP1 target genes are underlined. e, Differentially regulated pathways (unadjusted p < 0.01) identified using QuSAGE with MsigDB canonical pathways subset (Broad Institute) are shown for GRIP1 cKO vs. WT in M2_IL4 (left) and M2_Dex (right). Select pathways are labeled (upregulated, red; downregulated, orange for IL4 and green for Dex); M2_IL4:M2_Dex-shared pathways are underlined. Circle size is proportional to the number of genes in the pathway and color signifies p-value.

Fig.6 |. Macrophage GRIP1 contributes to phagocytic activity of M2_IL4 and M2_GC in vitro and tissue healing in vivo.

a, Relative fluorescence intensity of WT (set to 100%) and GRIP1-cKO M2_IL4, M2_Cort and M2_Dex relative to that in M0 after exposure to pHrodo Green S. aureus BioParticles (see Methods). Shown are mean values and error bars are SEM (n=3, student t-test). b, Histopathological changes assessed by H&E staining of colons from WT and cKO mice 2 wks after initiating DSS-induced colitis. Grade 1, 2 and 3 are defined in Methods. Plotted is the total area representing grade 3 for colon specimens of WT and GRIP1-cKO (n=4, student t-test). c, The expression of indicated genes was measured by RT-qPCR of total RNA from colons of WT and cKO mice. The expression of each gene was normalized to β-actin, and then fold difference to baseline, non-treated (calibrant) value calculated for WT or cKO. Shown are mean values and error bars are SEM (WT: n=10–15, cKO: n=6–8; student t-test).