Research ArticleRheumatoid Arthritis

HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness

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Science Translational Medicine  08 May 2019:
Vol. 11, Issue 491, eaau8587
DOI: 10.1126/scitranslmed.aau8587
  • Fig. 1 HBEGF+ inflammatory macrophages in RA joints identified by scRNA-seq.

    (A) Human synovial CD14+ single-cell clusters (940 cells). CD45+CD14+ cells were flow-sorted from synovial tissue of patients with RA (n = 10) or OA (n = 2) and processed by plate-based scRNA-seq. (B) CD14+ single-cell cluster marker genes. Median expression of a gene across all cells in cluster is indicated by color and the percentage of cells expressing the gene in each cluster is indicated by size. (C) Differential gene expression of bulk CD14+ synovial cell populations from patients with RA (n = 16) versus OA (n = 13) plotted as log2 fold change with −log10 FDR adjusted P value; dark gray <0.1. Right: Positively enriched pathways in RA bulk CD14+ cells. (D) CD14+ single-cell cluster marker genes highlighted on the bulk RA versus OA plot from (C) (up to 100 genes shown per cluster, Bonferroni-corrected P < 0.1, expressed in ≥30% of cells). Hypergeometric test: ***P < 10−6, *P < 10−3. Right: Number of cluster genes higher in RA or OA bulk comparison or not significant (ns) (FDR adjusted P < 0.1). (E) GSEA using the CD14+ single-cell markers as gene sets and ranked gene lists from human blood–derived macrophages exposed to various stimuli (colored bars) or the bulk CD14+ RA versus OA analysis (background: gray bars). |NES| (normalized enrichment scores) > 2.5 were significant at FDR adjusted P < 0.001. (F) Gene expression for each CD14+ single cell plotted as log2 counts per million (CPM) + 1. (G) HBEGF expression in CD14+ bulk cell populations from individual patients, plotted as log2 transcripts per million (TPM) + 1. n = 16 RA and n = 13 OA samples. *Bonferroni-corrected P < 10−3.

  • Fig. 2 HBEGF+ inflammatory macrophage polarization by tissue fibroblasts.

    (A) Human blood–derived macrophage genes regulated by synovial fibroblasts and TNF (3709 genes, FDR adjusted P < 0.1; n = 4 donors). Expression changes plotted as log2 fold. The x-axis plots fibroblasts + TNF versus TNF; the y axis plots TNF versus untreated. HBEGF+ cluster 1 single-cell markers labeled in red (expressed in >55% of cells, Bonferroni-corrected P < 10−6). (B) Expression of select synovial CD14+ cluster 1 genes in the blood-derived macrophages exposed to TNF (T) or fibroblasts + TNF (F + T). n = 3 donors. (C) qPCR of blood-derived macrophage gene expression over time, plotted as percentage (%) of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Representative of n = 4 donors. (D) The PLAUR gene cell surface protein product (CD87) detected by flow cytometry in blood-derived macrophage cultures at 24 hours. (E) Western blot of STAT4 in blood-derived macrophage cultures at 24 hours. Representative of n = 4 donors. Asterisk (*) denotes nonspecific band. Hsp90, loading control. (F) PGE2 enzyme-linked immunosorbent assay (ELISA) using supernatants from blood-derived macrophage (Mϕ) cultures at 24 hours. n = 4 donors. Data are means ± SEM. (G) qPCR of blood-derived macrophage gene expression in cultures at 24 hours. n = 8 donors. (H) qPCR of blood-derived macrophage gene expression in culture at 24 hours. Nap, COX inhibitor naproxen (150 nM). (I) ATAC-seq tracks from PLAUR gene promoter regions in blood-derived macrophage cultures at 3 hours. The region with light orange highlight is further examined in (J). The arrow indicates transcription start site. (J) FAIRE-qPCR analysis of open chromatin for the region highlighted in (I), % total input reported as mean ± SEM. n = 4 donors. *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 3 HBEGF+ inflammatory macrophages promote EGFR-dependent synovial fibroblast pathologic activity.

    (A) Human synovial fibroblast genes altered by blood-derived macrophages during a TNF response (885 genes differentially expressed), RNA-seq, 48 hours. n = 2 donors. x axis: log2 fold change by macrophages. y axis: significance as the −log10 FDR adjusted P value. (B) Fibroblast pathways regulated by macrophages under TNF conditions (GSEA and IPA). Macrophage-induced, brown. Macrophage–down-regulated, maroon. (C) ELISA assay using supernatants of synovial fibroblast (F) cultures with or without macrophages (M) and TNF (T) for 48 hours. n = 4 donors for both, reported as mean with SEM. (D) qPCR on fibroblast cultures at 32 hours. EGF receptor inhibitor, AG 1478 (AG, 4 μM). Mean ± SEM. n = 6 donors. (E) Cell counts and photomicrographs of synovial fibroblast Matrigel invasion assay with crystal violet stain, 18 hours after a 24-hour preincubation with macrophages (M), TNF (T), and the EGFR inhibitor (AG). n = 3 donors. *P < 0.05, **P < 0.01, and ***P < 0.001 by paired Student’s t test, respectively.

  • Fig. 4 Clinically effective RA medications and a therapeutic EGFR inhibitor target HBEGF+ inflammatory macrophage-fibroblast cross-talk in RA tissue.

    (A) Number of blood-derived macrophage genes affected by RA medications in the presence of TNF and synovial fibroblasts from cultures at 24 hours. Black, fibroblast-regulated genes opposed by drug. Gray, all other genes regulated by drug. FDR adjusted P < 0.1. n = 2 to 4 donors. (B) RA patient synovial tissue ex vivo drug response assay using highly inflamed synovium (scored by histology) from patients with positive blood titers for anti–cyclic citrullinated peptide (CCP+) antibodies. Dissociated cells placed into culture were exposed to a panel of medications for 24 hours. αTNF, anti-TNF antibodies. (C) ELISA using supernatants from RA tissue ex vivo assay. n = 8 donors. *P < 0.05 and **P < 0.01 by Wilcoxon signed-rank test. Bottom: IFN-α response upon tofacitinib exposure, bulk RNA-seq, and GSEA. n = 2 donors. (D) Direction and intensity of change for CD14+ single-cell cluster markers in RA synovial cells exposed to various medications. Normalized enrichment scores (GSEA). (E, H, and I) ELISAs as described in (C). (F) PGE2 ELISA using supernatants. Data are means ± SEM. n = 7 donors. *P < 0.05, paired Student’s t test. (G) qPCR using ex vivo synovial cells treated with naproxen, plotted as percentage (%) of TBP. n = 7 donors. Data are means ± SEM. *P < 0.05, paired Student’s t test. (J) Gene expression changes induced by the EGFR inhibitor AG-1478 in RA patient ex vivo synovial cells (y axis) compared to changes in synovial fibroblasts induced by macrophages and TNF (x axis, data from Fig. 3A); n = 2 donors. FDR adjusted P < 0.1, plotted as a log2 fold change. Highlighted genes are from Fig. 3A.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/491/eaau8587/DC1

    Materials and Methods

    Fig. S1. Genes defining the CD14+ single-cell clusters and variance across patient samples.

    Fig. S2. Identification of synovial HBEGF+ inflammatory macrophages in an independent RA patient study.

    Fig. S3. Synovial fibroblasts down-regulate several pathways in TNF-induced macrophages and impose a transcriptome consistent with TNF and prostaglandin exposure.

    Fig. S4. Synovial fibroblasts express EGF receptors whereas HBEGF+ inflammatory macrophages express two EGF ligands.

    Fig. S5. Synovial fibroblast exposure modifies how RA medications affect TNF-induced macrophages.

    Table S1. Patient characteristics for CD14+ synovial single-cell samples.

    Table S2. Patient characteristics for CD14+ synovial bulk-sorted samples.

    Table S3. RA patient characteristics for the ex vivo synovial tissue assay.

    Table S4. Individual patient disease diagnosis, serology, and histology for the ex vivo synovial tissue assay.

    Data file S1. Individual subject-level data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Genes defining the CD14+ single-cell clusters and variance across patient samples.
    • Fig. S2. Identification of synovial HBEGF+ inflammatory macrophages in an independent RA patient study.
    • Fig. S3. Synovial fibroblasts down-regulate several pathways in TNF-induced macrophages and impose a transcriptome consistent with TNF and prostaglandin exposure.
    • Fig. S4. Synovial fibroblasts express EGF receptors whereas HBEGF+ inflammatory macrophages express two EGF ligands.
    • Fig. S5. Synovial fibroblast exposure modifies how RA medications affect TNF-induced macrophages.
    • Table S1. Patient characteristics for CD14+ synovial single-cell samples.
    • Table S2. Patient characteristics for CD14+ synovial bulk-sorted samples.
    • Table S3. RA patient characteristics for the ex vivo synovial tissue assay.
    • Table S4. Individual patient disease diagnosis, serology, and histology for the ex vivo synovial tissue assay.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data.

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