Synovial macrophage diversity and activation of M-CSF signaling in post-traumatic osteoarthritis
- Department of Orthopaedic Surgery, University of Michigan, United States
- Department of Biomedical Engineering, University of Michigan, United States
- Department of Physiology and Pharmacology, Western University, Canada
- Bone and Joint Institute, Western University, Canada
- Department of Medicine, Schulich School of Medicine and Dentistry, Western University, Canada
- Department of Internal Medicine – Division of Rheumatology, University of Michigan, United States
Figures
Figure 1 with 1 supplement
Diverse immune cell types are present in healthy and injured synovium.
(A) Uniform manifold approximation and projection (UMAP) plot of all immune cells by single-cell RNA sequencing (scRNA-seq) of synovium from mice subjected to Sham, 7 days anterior cruciate ligament …
Figure 1—figure supplement 1
Computational subsetting of immune cells from whole synovium single-cell RNA sequencing (scRNA-seq).
(A) Immune cell clusters (2, 5, 8) were computationally subset out of all synovial cells from previously published dataset available at NCBI GEO Accession number GSE211584 (Knights et al., 2023b). (B…
Figure 2 with 5 supplements
Integration of joint immune cells from mouse arthritis single-cell RNA sequencing (scRNA-seq) datasets.
(A) Integrated uniform manifold approximation and projection (UMAP) plot of all immune cells from GSE134420 (Culemann), GSE200843 (Sebastian), GSE211584 (Knights), and GSE184609 (Muench). (B) Violin …
Figure 2—figure supplement 1
Recapitulation of published single-cell RNA sequencing (scRNA-seq) datasets: Culemann.
(A) Uniform manifold approximation and projection (UMAP) plot of all cell clusters from GSE134420 (Culemann et al., 2019). Cells were sorted CD45+ CD11b+ Ly6G- hindpaw synovial cells from mice …
Figure 2—figure supplement 2
Recapitulation of published single-cell RNA sequencing (scRNA-seq) datasets: Sebastian.
(A) Uniform manifold approximation and projection (UMAP) plot of all cell clusters from GSE200843 (Sebastian et al., 2022). Cells were sorted CD45+ cells obtained from a whole knee joint digestion …
Figure 2—figure supplement 3
Recapitulation of published single-cell RNA sequencing (scRNA-seq) datasets: Muench.
(A) Uniform manifold approximation and projection (UMAP) plot of all cell clusters from GSE184609 (Muench et al., 2022). Cells were sorted live cells obtained from hindpaw synovia of mice with …
Figure 2—figure supplement 4
Integration of immune single-cell RNA sequencing (scRNA-seq) datasets.
(A) Heatmap of top 5 gene markers for each cell cluster in the integrated immune object of all scRNA-seq datasets. (B) Total abundance of each major immune cell grouping for each dataset. (C) …
Figure 2—figure supplement 5
Assessment of neutrophils in the whole joint.
(A) Healthy mouse knee joints were subjected to two-step digestion described by Leale et al., 2022, resulting in a soft and hard tissue fraction. Joints were taken from naïve mice (unperfused) or …
Figure 3 with 2 supplements
Comparison of macrophages between arthritis disease states.
(A) Monocytes and MΦ from all post-traumatic osteoarthritis (PTOA) and rheumatoid arthritis (RA) immune cell datasets were computationally isolated, resulting in three clusters: MΦ, monocytes and …
Figure 3—figure supplement 1
Macrophages in osteoarthritis (OA) and rheumatoid arthritis (RA).
(A) Uniform manifold approximation and projection (UMAP) plots showing clusters of MΦ, monocytes, and osteoclast-like cells across datasets.
Figure 3—figure supplement 2
Outgoing signaling patterns from macrophages in post-traumatic osteoarthritis (PTOA) and rheumatoid arthritis (RA).
(A–B) River plots of outgoing communication patterns of MΦ, monocytes (Mono), and osteoclasts (Osteo) in PTOA (A) or RA (B), generated in CellChat. *Same signals from the same cell type in PTOA and …
Figure 4 with 1 supplement
Synovial macrophage subsets and trajectories in post-traumatic osteoarthritis (PTOA).
(A) Uniform manifold approximation and projection (UMAP) plot of monocytes and MΦ from synovium of Sham, 7 days anterior cruciate ligament rupture (ACLR) and 28 days ACLR mice, or split by condition …
Figure 4—figure supplement 1
Synovial macrophage subsets and trajectories.
(A) To generate an object containing only monocytes and MΦ from our dataset (GSE211584), we first subset all myeloid cell clusters from Seurat into Monocle3, then removed dendritic cell (DC), mast …
Figure 5 with 2 supplements
Stromal-immune crosstalk via M-CSF signaling.
(A) Cartoon depicting multi-directional and uni-directional crosstalk between stromal and immune cells in synovium, used for calculation of crosstalk communication probability scores. (B) Induction …
Figure 5—figure supplement 1
Stromal-immune crosstalk communication probability ratios in Sham and 7 days anterior cruciate ligament rupture (ACLR).
(A–C) Crosstalk communication probability ratios for multi-directional (stromal ←→ immune) or uni-directional (stromal → immune or immune → stromal) signaling in Sham synovium. A higher ratio …
Figure 5—figure supplement 2
M-CSF signaling hierarchy to stromal cells.
(A–C) CellChat hierarchy plots for the M-CSF signaling pathway in Sham, 7 days anterior cruciate ligament rupture (ACLR) and 28 days ACLR, with stromal cells as the receiving cell type in each case. …
Figure 6 with 1 supplement
Transcriptional control of monocyte differentiation in synovium.
(A–B) Pseudotime trajectory from monocytes to infiltrating MΦ showing directionality (arrowheads), starting point (root, star), branching points (nodes, black circles), and endpoints (termini, gray …
Figure 6—figure supplement 1
Expression of key transcription factors across pseudotime from monocytes to macrophages.
(A) RcisTarget analysis was performed to screen promoters of genes enriched in modules 1, 4, and 6 of the monocyte-to-infiltrating MΦ trajectory. Pseudotime regression plots for the top four …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Mus musculus) | C57Bl/6 mice | Jackson Laboratory | Strain #000664 | Male and female |
| Antibody | TruStain FcX PLUS | BioLegend | RRID:AB_2783137 | Clone S17011E (1:1000) |
| Antibody | Anti-mouse CD3-APC/Fire750 (rat monoclonal) | BioLegend | RRID:AB_2572117 | Clone 17A2 (1:100) |
| Antibody | Anti-mouse CD11b-BV605 (rat monoclonal) | BioLegend | RRID:AB_11126744 | Clone M170 (1:400) |
| Antibody | Anti-mouse CD11c-PE/Dazzle594 (Armenian hamster monoclonal) | BioLegend | RRID:AB_2563654 | Clone N418 (1:200) |
| Antibody | Anti-mouse CD19-FITC (rat monoclonal) | BioLegend | RRID:AB_2629813 | Clone 1D3/CD19 (1:100) |
| Antibody | Anti-mouse CD45-BV650 (rat monoclonal) | BioLegend | RRID:AB_2565884 | Clone 30-F11 (1:400) |
| Antibody | Anti-mouse F4/80-APC/R700 (rat monoclonal) | BD Horizon | RRID:AB_2869711 | Clone T45-2342 (1:400) |
| Antibody | Anti-mouse FceRIa-PE/Cy7 (Armenian hamster monoclonal) | BioLegend | RRID:AB_10640122 | Clone MAR-1 (1:100) |
| Antibody | Anti-mouse Ly6G-BV421 (rat monoclonal) | BioLegend | RRID:AB_10897944 | Clone 1A8 (1:200) |
| Antibody | Anti-mouse MHC Class II-BV421 (rat monoclonal) | BioLegend | RRID:AB_2650896 | Clone M5/114.15.2 (1:200) |
| Antibody | Anti-mouse NK1.1-BV510 (mouse monoclonal) | BioLegend | RRID:AB_2562216 | Clone PK136 (1:200) |
| Peptide, recombinant protein | Type IV Collagenase | Sigma | CAS 9001-12-1 | |
| Peptide, recombinant protein | Liberase | Sigma | 5401119001 | |
| Peptide, recombinant protein | DNaseI | Sigma | CAS 9003-98-9 | |
| Chemical compound, drug | TO-PRO-3 iodide | Invitrogen | T3605 | |
| Software, algorithm | FlowJo v10 | BD/Treestar | NA | |
| Software, algorithm | Seurat v4.1 | Hao et al., 2021 | https://doi.org/10.1016/j.cell.2021.04.048 | |
| Software, algorithm | Cluster Identity PRedictor (CIPR) | Ekiz et al., 2020 | https://doi.org/10.1186/s12859-020-3538-2 | |
| Software, algorithm | Monocle 3 | Cao et al., 2019 | https://doi.org/10.1038/s41586-019-0969-x | |
| Software, algorithm | RCisTarget (SCENIC) | Aibar et al., 2017 | https://doi.org/10.1038/nmeth.4463 | |
| Software, algorithm | CellChat | Jin et al., 2021 | https://doi.org/10.1038/nmeth.4463 | |
| Other | scRNA-seq data of synovial cells from ACLR | NCBI GEO | GSE211584 | Knights et al., 2023a |
| Other | scRNA-seq data of synovial macrophages from RA | NCBI GEO | GSE134420 | Culemann et al., 2019 |
| Other | scRNA-seq data of joint immune cells from ACLR | NCBI GEO | GSE200843 | Sebastian et al., 2022 |
| Other | scRNA-seq data of hindpaw joint cells from arthritis | NCBI GEO | GSE184609 | Muench et al., 2022 |
| Other | Bulk RNA-seq data of synovium | NCBI GEO | GSE271903 | Bergman et al., 2024 |
Additional files
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Supplementary file 1
Flow cytometry antibodies.
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp1-v1.docx
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Supplementary file 2
Top 10 Cluster Identity PRedictor (CIPR) hits for each neutrophil cluster from the integrated object of all immune cells, all datasets.
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp2-v1.docx
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Supplementary file 3
Ctrl vs post-traumatic osteoarthritis (PTOA) mac differentially expressed genes (DEGs).
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp3-v1.xlsx
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Supplementary file 4
Ctrl vs rheumatoid arthritis (RA) mac differentially expressed genes (DEGs).
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp4-v1.xlsx
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Supplementary file 5
Gene ontology outputs for post-traumatic osteoarthritis (PTOA) and rheumatoid arthritis (RA) macrophages.
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp5-v1.xlsx
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Supplementary file 6
Gene ontology outputs for resident-like macrophages.
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp6-v1.xlsx
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Supplementary file 7
Monocyte-to-infiltrating macrophage trajectory gene modules.
- https://cdn.elifesciences.org/articles/93283/elife-93283-supp7-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/93283/elife-93283-mdarchecklist1-v1.docx
