Loss of synovial tissue macrophage homeostasis precedes rheumatoid arthritis clinical onset

doi:10.1126/sciadv.adj1252

. 2024 Sep 27;10(39):eadj1252.

doi: 10.1126/sciadv.adj1252. Epub 2024 Sep 25.

Loss of synovial tissue macrophage homeostasis precedes rheumatoid arthritis clinical onset

Megan M Hanlon^{1

2}, Conor M Smith³, Mary Canavan^{1

2

4}, Nuno G B Neto⁵, Qingxuan Song⁶, Myles J Lewis⁷, Aoife M O'Rourke^{1

2

4}, Orla Tynan^{1

2}, Brianne E Barker^{1

2}, Phil Gallagher², Ronan Mullan⁸, Conor Hurson⁹, Barry Moran³, Michael G Monaghan⁵, Costantino Pitzalis^{7

10}, Jean M Fletcher^{3

11}, Sunil Nagpal⁶, Douglas J Veale², Ursula Fearon^{1

2}

Affiliations

¹ Molecular Rheumatology, School of Medicine, Trinity College Dublin, Dublin, Ireland.
² Centre for Arthritis and Rheumatic Diseases, St. Vincent's University Hospital, University College Dublin, Dublin, Ireland.
³ School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
⁴ Translational Immunopathology, School of Biochemistry and Immunology and School of Medicine, Trinity College Dublin, Dublin, Ireland.
⁵ Department of Mechanical and Manufacturing Engineering, Trinity Biomedical Sciences Institute, Dublin, Ireland.
⁶ Immunology and Discovery Sciences, Janssen Research and Development, Spring House, PA, USA.
⁷ Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Queen Mary University of London and Barts NIHR BRC and Barts Health NHS Trust, London, UK.
⁸ Department of Rheumatology, Adelaide and Meath Hospital, Dublin, Ireland.
⁹ Department of Orthopaedics, St. Vincent's University Hospital, Dublin, Ireland.
¹⁰ Department of Biomedical Sciences, Humanitas University and Humanitas Research Hospital, Milan, Italy.
¹¹ School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.

PMID: 39321281
PMCID: PMC11423874
DOI: 10.1126/sciadv.adj1252

Loss of synovial tissue macrophage homeostasis precedes rheumatoid arthritis clinical onset

Megan M Hanlon et al. Sci Adv. 2024.

. 2024 Sep 27;10(39):eadj1252.

doi: 10.1126/sciadv.adj1252. Epub 2024 Sep 25.

Authors

Affiliations

¹ Molecular Rheumatology, School of Medicine, Trinity College Dublin, Dublin, Ireland.
² Centre for Arthritis and Rheumatic Diseases, St. Vincent's University Hospital, University College Dublin, Dublin, Ireland.
³ School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
⁴ Translational Immunopathology, School of Biochemistry and Immunology and School of Medicine, Trinity College Dublin, Dublin, Ireland.
⁵ Department of Mechanical and Manufacturing Engineering, Trinity Biomedical Sciences Institute, Dublin, Ireland.
⁶ Immunology and Discovery Sciences, Janssen Research and Development, Spring House, PA, USA.
⁷ Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Queen Mary University of London and Barts NIHR BRC and Barts Health NHS Trust, London, UK.
⁸ Department of Rheumatology, Adelaide and Meath Hospital, Dublin, Ireland.
⁹ Department of Orthopaedics, St. Vincent's University Hospital, Dublin, Ireland.
¹⁰ Department of Biomedical Sciences, Humanitas University and Humanitas Research Hospital, Milan, Italy.
¹¹ School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.

PMID: 39321281
PMCID: PMC11423874
DOI: 10.1126/sciadv.adj1252

Abstract

This study performed an in-depth investigation into the myeloid cellular landscape in the synovium of patients with rheumatoid arthritis (RA), "individuals at risk" of RA, and healthy controls (HC). Flow cytometric analysis demonstrated the presence of a CD40-expressing CD206⁺CD163⁺ macrophage population dominating the inflamed RA synovium, associated with disease activity and treatment response. In-depth RNA sequencing and metabolic analysis demonstrated that this macrophage population is transcriptionally distinct, displaying unique inflammatory and tissue-resident gene signatures, has a stable bioenergetic profile, and regulates stromal cell responses. Single-cell RNA sequencing profiling of 67,908 RA and HC synovial tissue cells identified nine transcriptionally distinct macrophage clusters. IL-1B⁺CCL20⁺ and SPP1⁺MT2A⁺ are the principal macrophage clusters in RA synovium, displaying heightened CD40 gene expression, capable of shaping stromal cell responses, and are importantly enriched before disease onset. Combined, these findings identify the presence of an early pathogenic myeloid signature that shapes the RA joint microenvironment and represents a unique opportunity for early diagnosis and therapeutic intervention.

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Figures

**Fig. 1.. Synovial tissue macrophage phenotypic characterization.**
(A) Representative flow cytometric dot plots of four independent RA synovial tissue samples demonstrating the frequency of CD206⁺CD163⁺ macrophages and coexpression of CD40. (B) Dot plots indicating percentage frequency of CD206⁺CD163⁺, CD206⁻CD163⁻, and CD206⁺CD163⁺CD40⁺ macrophages and median fluorescence intensity (MFI) of CD40 on CD206⁺CD163⁺ macrophages in RA synovial tissue (n = 9) in comparison to synovial fluid (n = 9). Data are presented as mean ± SEM with each symbol representing a different sample. Statistical analysis using Mann-Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.005, significantly different from synovial tissue. (C) Visual representation of multidimensional flow cytometric data. SPICE analysis was performed for the identification of distinct macrophage subsets in an average of RA synovial tissue (n = 9) and fluid (n = 6). Each pie segment indicates the different combinations of marker expression as denoted by the legend below. The surrounding pie arcs indicate the specific macrophage markers produced by each pie segment. High-quality RNA was isolated from sorted CD206⁺CD163⁺ and CD206⁻CD163⁻ synovial tissue macrophages and bulk RNA-seq was performed. (D) PCA was performed on the total dataset of RA synovial tissue sorted CD206⁺CD163⁺ and CD206⁻CD163⁻ macrophage subsets (n = 9). (E) Hierarchical clustered heatmap displaying DEGs involved in adhesion/cell growth, cytoskeletal rearrangement, cytokine/chemokines, macrophage markers/phagocytosis, and metabolism in RA synovial tissue CD206⁺CD163⁺ macrophages compared to CD206⁻CD163⁻ macrophages (n = 9). (F) Representative multiphoton microscopy FLIM analysis of flow sorted RA CD206⁺CD163⁺ and CD206⁻CD163⁻ synovial tissue macrophages. Representative FLIM images whereby a red/green cell is predominantly using OXPHOS, while a blue cell that indicates glycolysis is being used as the main energy source. (G) Summary of macrophage emission lifetime (τ_avg) (n = 7) and optical redox ratio (ORR) (n = 3). Data expressed as mean ± SEM using Wilcoxon signed rank or paired t test, *P < 0.05, ***P < 0.005 significantly different from each other.

**Fig. 2.. CD206⁺CD163⁺ RA macrophages correlate with RA disease activity and treatment response.**
Analysis of CD206⁺CD163⁺CD40⁺ based on RA disease activity scores (DAS28). Low DAS28 indicates less than 2.6 with high DAS28 greater than 2.6. (A) Dot plot, representative flow cytometry plots, and relative histogram of CD40 expression on double-positive CD206⁺CD163⁺ macrophage population in high versus low disease activity. (B) Correlation and relative proportion bar chart representing the relationship between baseline CD206⁺CD163⁺CD40⁺ expression and DAS28. (C) Correlation and bar charts representing the relationship between baseline CD206⁺CD163⁺CD40⁺ expression and change in DAS28 upon 1-year follow-up. Data are presented as mean ± SEM, using Mann-Whitney U test, *P < 0.05.

**Fig. 3.. Pathogenic role of transitional CD40-expressing CD206⁺CD163⁺ macrophages.**
(A) Clustered heatmap displaying unsupervised hierarchical clustering of genes that are distinct in RA synovial tissue CD206⁺CD163⁺ macrophages (n = 9) and patient-matched polarized RA monocyte-derived M1 and M2 macrophages (n = 9). Heatmaps representing log-normalized expression values of (B) the top M2-like and (C) M1-like genes in RA M1 and M2 macrophages and CD206⁺CD163⁺ RA synovial tissue macrophages (n = 9). (D) Violin plots representing log-normalized expression values of macrophage tissue-resident–associated genes in the CD206⁺CD163⁺ macrophages (n = 9) compared to CD206⁻CD163⁻ macrophages (n = 9), synovial fluid CD206⁺CD163⁺ (n = 4), and polarized RA M1 macrophages (n = 9) with medians marked by black dots. Data expressed as mean ± SEM using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 significantly different from each other. (E) Bar graphs represent cytokines secreted from sorted RA synovial tissue CD206⁺CD163⁺ macrophages measured by MSD assay. (F) Schematic of experimental workflow of CD206⁺CD163⁺ macrophage conditioned media on healthy synovial fibroblasts. (G) Dot plots of cytokine expression following the addition of CD206⁺CD163⁺ macrophage conditioned media on healthy synovial fibroblasts (n = 4). (H) Line plots indicating sorted CD206⁺CD163⁺ synovial tissue macrophage cytokine expression with or without preincubation with CD40 inhibitor (n = 3). Data are presented as mean ± SEM with each symbol representing a different sample. Statistical analysis using Mann–Whitney U test.

**Fig. 4.. Synovial tissue macrophage expression in healthy versus RA synovial tissue.**
(A) SPICE analysis of macrophage subsets in an average of healthy synovial tissue (n = 3) and RA synovial tissue (n = 9). (B) Representative flow cytometry plots indicating the expression of double-positive CD206⁺CD163⁺ and coexpression of CD40 macrophages in healthy and RA synovial tissue. (C) Dot plots indicating the percentage frequency of CD206⁺CD163⁺ and CD206⁺CD163⁺CD40⁺ macrophage subsets in RA synovial tissue (n = 17) compared to healthy control tissue (n = 6 to 11) along with (D) representative histogram and relative proportion graphs of CD40 expression on the double-positive CD206⁺CD163⁺ macrophage subset in healthy versus RA synovial tissue. (E) Representative flow cytometry dot plots of CX₃CR1 expression with accompanying (F) quantification of percentage frequency and MFI and (G) representative histogram of CX₃CR1 expression in healthy (n = 8) and RA (n = 7) synovial tissue macrophages. Data are presented as mean ± SEM with each symbol representing a different sample. Statistical analysis using Mann-Whitney U test, **P < 0.01, ***P < 0.001, ****P < 0.0001 significantly different from each other.

**Fig. 5.. scRNA-seq defines synovial tissue macrophage heterogeneity from health to disease.**
(A) Analysis of pathways enriched in CD206⁺CD163⁺ macrophages in healthy (n = 4) and RA (n = 4) synovial tissue, color intensity represents significance, and dot size denotes the number of genes within each pathway that are differentially expressed. (B) Term plot of the indicated pathways with significant enrichment in RA compared with healthy control macrophages. Color indicates up- or down-regulation of specific genes within the pathway and dot size represents statistical significance of change. (C) UMAP representation of 12 synovial tissue myeloid clusters based on 67,908 synovial tissue cells identified by high-dimensionality scRNA-seq analysis. (D) Heatmap of the top 20 DEGs per cluster. (E) Proposed classification and (F) hierarchical clustering of human synovial tissue macrophages. (G) Proportional cluster abundance between patients with established RA and healthy control individuals. (H) UMAP depicting synovial tissue macrophage cluster distribution between RA and healthy synovium. Two-tailed Spearman’s correlation between synovial expression of (I) IL-1B⁺CCL20⁺ and (J) SPP1⁺MT2A⁺ synovial tissue macrophage clusters with DAS28 CRP and changes in DAS28 CRP in the PEAC cohort (n = 90).

**Fig. 6.. Synovial tissue macrophage CD40 expression and cellular interactions.**
(A) UMAP illustrating CD206⁺CD163⁺ mRNA expression in synovial tissue macrophages. Violin plot representing the expression level of CD206⁺CD163⁺ in healthy and RA synovium. (B) UMAP illustrating CD206⁺CD163⁺CD40⁺ mRNA expression in synovial tissue macrophages. Violin plot representing the expression level of CD206⁺CD163⁺CD40⁺ in healthy and RA synovium. (C) Violin plot demonstrating CD40 signaling module expression between healthy and RA synovium. (D) Violin plot representing CD40 gene expression levels across all synovial tissue macrophage clusters. (E and F) Circos plots depicting the top predicted receptor and ligand interactions between specific indicated macrophage clusters and synovial tissue fibroblasts. (G) DoRothEA analysis of transcription factor usage by RA compared with healthy control synovial tissue macrophage clusters, based on expression of known downstream ligands.

**Fig. 7.. Synovial tissue macrophage expression in individuals at risk of developing rheumatoid arthritis.**
(A) Multiparametric flow cytometric data of total CD64⁺ macrophages subjected to tSNE algorithm following data concatenation of one healthy individual at risk and RA synovial tissue. Data concatenation and relative expression for the indicated parameters are shown. (B) Identification of three populations of CD206, distinguished by CD40 and CX₃CR1 expression, and (C) expression of three clusters in healthy, IAR, and RA synovial tissue. Representative flow cytometry dot plots and accompanying percentage frequency quantification of (D) CD206⁺CD163⁺CD40⁺ in healthy (n = 11), arthralgia (n = 4), and RA (n = 17) and (E) CX₃CR1 in healthy (n = 8), arthralgia (n = 5), and RA (n = 6) synovial tissue macrophages. (F) Dot plots of expression of CD206⁺CD163⁺CD40⁺ and (G) IL-1B⁺CCL20⁺ and SPP1⁺ MT2A⁺ gene signatures in IAR (n = 10) and established RA (RA) (n = 85) compared to normal healthy synovium (n = 44) synovial tissue. Data expressed as mean ± SEM using one-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ****P < 0.0001 significantly different from each other.

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References

1. McInnes I. B., Schett G., The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205–2219 (2011). - PubMed
1. Aletaha D., Neogi T., Silman A. J., Funovits J., Felson D. T., Bingham C. O., Birnbaum N. S., Burmester G. R., Bykerk V. P., Cohen M. D., Combe B., Costenbader K. H., Dougados M., Emery P., Ferraccioli G., Hazes J. M. W., Hobbs K., Huizinga T. W. J., Kavanaugh A., Kay J., Kvien T. K., Laing T., Mease P., Ménard H. A., Moreland L. W., Naden R. L., Pincus T., Smolen J. S., Stanislawska-Biernat E., Symmons D., Tak P. P., Upchurch K. S., Vencovský J., Wolfe F., Hawker G., 2010 Rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 62, 2569–2581 (2010). - PubMed
1. Armstrong D. J., McCausland E. M., Quinn A. D., Wright G. D., Obesity and cardiovascular risk factors in rheumatoid arthritis. Rheumatology 45, 782–783 (2006). - PubMed
1. Naranjo A., Sokka T., Descalzo M. A., Calvo-Alen J., Horslev-Petersen K., Luukkainen R. K., Combe B., Burmester G. R., Devlin J., Ferraccioli G., Morelli A., Hoekstra M., Majdan M., Sadkiewicz S., Belmonte M., Holmqvist A.-C., Choy E., Tunc R., Dimic A., Bergman M., Toloza S., Pincus T., QUEST-RA Group , Cardiovascular disease in patients with rheumatoid arthritis: Results from the QUEST-RA study. Arthritis Res. Ther. 10, R30 (2008). - PMC - PubMed
1. Kennedy A., Ng C. T., Chang T. C., Biniecka M., O’Sullivan J. N., Heffernan E., Fearon U., Veale D. J., Tumor necrosis factor blocking therapy alters joint inflammation and hypoxia. Arthritis Rheum. 63, 923–932 (2011). - PubMed

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[1] McInnes I. B., Schett G., The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205–2219 (2011). - PubMed

[2] McInnes I. B., Schett G., The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365, 2205–2219 (2011). - PubMed

[3] Aletaha D., Neogi T., Silman A. J., Funovits J., Felson D. T., Bingham C. O., Birnbaum N. S., Burmester G. R., Bykerk V. P., Cohen M. D., Combe B., Costenbader K. H., Dougados M., Emery P., Ferraccioli G., Hazes J. M. W., Hobbs K., Huizinga T. W. J., Kavanaugh A., Kay J., Kvien T. K., Laing T., Mease P., Ménard H. A., Moreland L. W., Naden R. L., Pincus T., Smolen J. S., Stanislawska-Biernat E., Symmons D., Tak P. P., Upchurch K. S., Vencovský J., Wolfe F., Hawker G., 2010 Rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 62, 2569–2581 (2010). - PubMed

[4] Aletaha D., Neogi T., Silman A. J., Funovits J., Felson D. T., Bingham C. O., Birnbaum N. S., Burmester G. R., Bykerk V. P., Cohen M. D., Combe B., Costenbader K. H., Dougados M., Emery P., Ferraccioli G., Hazes J. M. W., Hobbs K., Huizinga T. W. J., Kavanaugh A., Kay J., Kvien T. K., Laing T., Mease P., Ménard H. A., Moreland L. W., Naden R. L., Pincus T., Smolen J. S., Stanislawska-Biernat E., Symmons D., Tak P. P., Upchurch K. S., Vencovský J., Wolfe F., Hawker G., 2010 Rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 62, 2569–2581 (2010). - PubMed

[5] Armstrong D. J., McCausland E. M., Quinn A. D., Wright G. D., Obesity and cardiovascular risk factors in rheumatoid arthritis. Rheumatology 45, 782–783 (2006). - PubMed

[6] Armstrong D. J., McCausland E. M., Quinn A. D., Wright G. D., Obesity and cardiovascular risk factors in rheumatoid arthritis. Rheumatology 45, 782–783 (2006). - PubMed

[7] Naranjo A., Sokka T., Descalzo M. A., Calvo-Alen J., Horslev-Petersen K., Luukkainen R. K., Combe B., Burmester G. R., Devlin J., Ferraccioli G., Morelli A., Hoekstra M., Majdan M., Sadkiewicz S., Belmonte M., Holmqvist A.-C., Choy E., Tunc R., Dimic A., Bergman M., Toloza S., Pincus T., QUEST-RA Group , Cardiovascular disease in patients with rheumatoid arthritis: Results from the QUEST-RA study. Arthritis Res. Ther. 10, R30 (2008). - PMC - PubMed

[8] Naranjo A., Sokka T., Descalzo M. A., Calvo-Alen J., Horslev-Petersen K., Luukkainen R. K., Combe B., Burmester G. R., Devlin J., Ferraccioli G., Morelli A., Hoekstra M., Majdan M., Sadkiewicz S., Belmonte M., Holmqvist A.-C., Choy E., Tunc R., Dimic A., Bergman M., Toloza S., Pincus T., QUEST-RA Group , Cardiovascular disease in patients with rheumatoid arthritis: Results from the QUEST-RA study. Arthritis Res. Ther. 10, R30 (2008). - PMC - PubMed

[9] Kennedy A., Ng C. T., Chang T. C., Biniecka M., O’Sullivan J. N., Heffernan E., Fearon U., Veale D. J., Tumor necrosis factor blocking therapy alters joint inflammation and hypoxia. Arthritis Rheum. 63, 923–932 (2011). - PubMed

[10] Kennedy A., Ng C. T., Chang T. C., Biniecka M., O’Sullivan J. N., Heffernan E., Fearon U., Veale D. J., Tumor necrosis factor blocking therapy alters joint inflammation and hypoxia. Arthritis Rheum. 63, 923–932 (2011). - PubMed

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Loss of synovial tissue macrophage homeostasis precedes rheumatoid arthritis clinical onset

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Loss of synovial tissue macrophage homeostasis precedes rheumatoid arthritis clinical onset

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