Cooperative induction of a distinct subset of inflammatory genes by PGE2 and TNF.

(A) Experimental design. Primary human monocytes were stimulated with PGE2 (280 nM) and/or TNF (20 ng/ml) and harvested 3 or 24 hr after stimulation for RNAseq analysis. n = 3 independent blood donors. (B) Gene set enrichment analysis of genes induced > 2-fold by PGE2 + TNF (TP) (FDR < 0.05). (C) K-means clustering of differentially upregulated genes in any pairwise comparison relative to resting control (> 2-fold induction, FDR < 0.05). 3 hr time point. k = 5. (D) Pathway analysis of gene clusters in panel C. (E) qPCR analysis of gene expression in an additional 5 blood donors. Mean +/- SEM. Statistical significance was assessed using one-way ANOVA and Sidak’s test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (F). Western blot analysis. Representative blot out of 3 independent experiments.

PGE2 and TNF costimulation model aspects of the RA and ICI-arthritis synovial macrophage phenotype.

(A) TP costimulation recapitulates expression of 61% of the genes whose induction by TNF was augmented by coculture with synovial fibroblasts (also termed fibroblast-like synoviocytes, FLS). FLS-augmented TNF-inducible genes from data in refs. (Donlin et al., 2014; Kuo et al., 2019) were compared to the TP-induced genes at the 24 hr time point in Supplementary Fig. 2, fold-change >2, FDR < 0.05. The green area indicates extent of overlap, hypergeometric p = 8.36e-128. (B) Recapitulation of the RA synovial macrophage cluster 1 phenotype by TP-costimulated genes. The defining 128 genes of the C1 phenotype were overlapped with TP-costimulated genes and TNF/FLS-costimulated genes. TP- costimulated genes = orange + yellow = 52% of C1 defining genes (p < 10e-9 by Monte Carlo simulation). TNF/FLS-costimulated genes = orange + red = 34% of C1 defining genes. (C) Heat maps depicting expression of genes in pathogenic pathways, based on RNAseq data shown in Fig. 1. Blue font = genes expressed in C1 RA macrophages. (D) Heat map depicting regulation of representative genes that are expressed in RA C1 macrophages by P, T or TP. (E) UMAP visualization of monocyte and macrophage clusters based on scRNAseq of 14,110 macrophages and monocytes from 5 synovial fluids and 2 synovial tissues of ICI-arthritis patients. (F) Heatmap showing expression of key genes for the eight clusters identified in panel E. TNF+PGE2 signature genes are shown in red.

cAMP signaling has dichotomous suppressive and augmenting effects on the TNF-induced inflammatory response.

(A) RT-qPCR analysis of gene expression in primary human monocytes stimulated with TNF and selective agonists of PGE2 receptors EP2 and EP4 that signal predominantly via cAMP. EP2 agonist = butaprost (10 μM); EP4 agonist = CAY10598 (10 μM). n = 3 (B) RT-qPCR analysis of gene expression in primary human monocytes stimulated with TNF and increasing concentrations of cAMP analog dibutyryl cAMP (10 and 100 μM; labeled A). n = 3. Mean +/- SEM. Statistical significance was assessed using 2 way ANOVA and Sidak’s test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

PGE2 effects on TNF-induced changes in chromatin accessibility.

(A-E). Analysis of ATACseq data obtained using monocytes from 3 independent donors. (A) UPSET plot of differentially upregulated ATACseq peaks in any pairwise comparison relative to resting control (> 2-fold induction, FDR < 0.05). (B) De novo motif analysis using HOMER of ATACseq peaks induced by TNF (left panel), PGE2 (middle panel) or uniquely induced only under conditions of TP costimulation (right panel). (C) Upper. Violin plots showing normalized counts of ATACseq peaks induced by both PGE2 and TNF. ****p < 0.0001 by Wilcoxon rank sum test with Holm’s correction for multiple comparisons. Lower. HOMER de novo motif analysis of the peaks in the upper panel. (D) Heatmap of the differential TF activity scores derived from ChromVAR analysis of ATACseq data for P, T or TP treated monocytes, compared to resting control. (E) Volcano plot of differential binding analysis of ATACseq peaks between the TP and T conditions using TOBIAS.

IFN-γ opposes the effects of PGE2 on TNF-induced gene expression.

(A, C) RT- qPCR analysis of gene expression in primary human monocytes that were primed overnight with IFN-γ (100 U/ml) and then stimulated for 3 hr with P, T or TP as in Fig. 1. n = 3. Mean +/- SEM. Statistical significance was assessed using 2 way ANOVA with Sidak’s test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (B, D) Gene groups defined in Supplementary Fig. 4A based on pattern of expression in RNAseq data (n = 3) were subjected to hierarchical clustering.

Interaction analysis of IFN-γ and the TP response.

(A) Differentially expressed genes in ((IFN-γ + TP) – IFN-γ)) – (IFN-γ – Resting) contrast define statistical interactions between IFN-γ and TP treatments using the RNAseq data, n = 3, FDR < 0.05, fold change > 2. Hierarchical clustering of z-transformed gene expression values (cpm) reveals 6 interaction clusters (right). Violin plots showing relative gene expression between resting (R), TP, IFN-γ alone (I) and IFN-γ + TP (ITP) conditions (second from left). Interaction plot (left). (B) Pathway analysis of the genes in the interaction clusters defined in panel A. (C) STRING functional protein association network of transcription factors from each cluster (fold change > 4, FDR < 0.05). Lines designate functional interactions between individual TFs. The size of nodes is proportional to the number of STRINGDB interactions.

IFN-γ inhibits PGE2-induced gene expression and chromatin accessibility.

(A-B, F) Analysis of RNAseq data obtained using monocytes from 3 independent donors. (A) K-means clustering of differentially upregulated genes in any pairwise comparison relative to resting control (> 2-fold induction, FDR < 0.05). k = 5. (B) Heat maps depicting expression of genes in pathogenic pathways and expressed in C1 RA macrophages as defined in Fig. 2C and 2D. (C- E, G, H). Analysis of ATACseq data obtained using monocytes from 3 independent donors. (C) UPSET plot of differentially upregulated ATACseq peaks in any pairwise comparison relative to resting control (> 2-fold induction, FDR < 0.05). (D) De novo motif analysis using HOMER of ATACseq peaks induced uniquely by PGE2 (corresponding to G2 in panel C). (E) Heatmap of the differential TF activity scores derived from ChromVAR analysis of ATACseq data for P, IFN-γ or IFN-γ + PGE2 treated monocytes, compared to resting control. (F) Heat map depicting expression of CEBP genes in RNAseq data. (G, H) Volcano plots of differential binding analysis of ATACseq peaks of IFN-γ versus resting control (G) and IFN-γ + PGE2 versus PGE2 (H) conditions using TOBIAS. The IFN-γ versus resting results (G) reproduce results in (Mishra and Ivashkiv, 2024) that were obtained in independent experiments with different blood donors.

Crosstalk between PGE2 and TNF signaling and its regulation by IFN-γ.

(A) PGE2 costimulates TNF-induced expression of select inflammatory genes such as IL-1 and Notch pathway genes and neutrophil chemokines by inducing transcription factors including CEBP, AP-1 and NR4A1/2 that cooperate with TNF-activated NF-κB (left). IFN-γ induces IRF transcription factors that cooperate with TNF-activated NF-κB to costimulate distinct inflammatory genes such as TNF and T cell chemokines such as CXCL10, reviewed in ref. (Mishra and Ivashkiv, 2024) (right). IFN-γ inhibits induction of TP-costimulated genes by suppressing induction of AP-1 and NR4A1/2, and altering the pattern of expression of CEBP factors. PGE2 suppresses TNF-mediated induction of TNF and ISGs. As PGE2 is produced by stromal cells and IFN-γ is produced by lymphocytes, neighboring cells in inflamed tissues will help determine the macrophage response to TNF. Induction of a subset of CEBP factors by IFN-γ is not depicted. (B) IFN-γ and PGE2 oppose each other to regulate the balance between distinct TNF-induced inflammatory responses. PGE2 signaling promotes a response that activates stromal cells via IL-1, EGFR ligands and Notch pathways, and promotes recruitment of neutrophils. IFN-γ suppresses these pathways and instead promotes inflammation via TNF and recruitment of T cells.

Regulation of gene expression by PGE2 and TNF in mouse bone marrow-derived macrophages.

BMDMs were stimulated with PGE2 (280 nM) and/or mTNF (20 ng/ml) and harvested 3 or 24 hr after stimulation for qPCR analysis of gene expression, n =3. Mean +/- SEM. Statistical significance was assessed using 2-way ANOVA and Tukey’s test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Analysis of differentially regulated genes at the 24 hr time point. Monocytes were stimulated for 24 hr as described in legend to Fig. 1A. K means clustering (k = 5, genes with > 2-fold induction, FDR < 0.05) (left panel). Pathway analysis of gene clusters (right panel). n = 3.

ATACseq analysis of PGE2 and TNF stimulated monocytes.

(A- F) Additional analysis of the ATACseq data obtained using monocytes from 3 independent donors. (A) Principal component plot. (B-E) Pathway analysis of genes associated with ATACseq peaks induced by TNF (B), PGE2 (C), co-induced by TP (D) or uniquely induced by TP (E) Volcano plots of differential binding analysis of ATACseq peaks using TOBIAS.

IFN-γ opposes the effects of PGE2 on TNF-induced gene expression.

(A) RNAseq data, n = 3. K-means clustering of differentially upregulated genes in any pairwise comparison relative to resting control (> 1.25-fold induction, FDR < 0.01). 3 hr time point. k = 6; 3 gene sets (Groups 4-6) that are costimulated by T and P are apparent. (B) Gene groups 4 and 5 defined in panel A were subjected to hierarchical clustering.

(A) PCA plot of RNAseq data shown in Fig. 7. (B) Pathway analysis of gene clusters identified in Fig. 7A. (C). PCA plot of ATACseq data shown in Fig. 7.

: Clinical characteristics of ICB-arthritis patients