Multimodal analyses of immune cells during bone repair identify macrophages as a therapeutic target in musculoskeletal trauma

Abstract

Musculoskeletal traumatic injuries (MTI) involve soft tissue lesions adjacent to a bone fracture leading to fibrous nonunion. The impact of MTI on the inflammatory response to fracture and on the immunomodulation of skeletal stem/progenitor cells (SSPCs) remains unknown. Here, we used single-nucleus transcriptomic analyses to describe the immune cell dynamics after bone fracture and identified distinct macrophage subsets with successive pro-inflammatory, pro-repair and anti-inflammatory profiles. Concurrently, SSPCs transition via a pro- and anti-inflammatory fibrogenic phase of differentiation prior to osteochondrogenic differentiation. In a preclinical MTI mouse model, the injury response of immune cells and SSPCs is disrupted leading to a prolonged pro-inflammatory phase and delayed resolution of inflammation. Macrophage depletion improves bone regeneration in MTI demonstrating macrophage involvement in fibrous nonunion. Finally, pharmacological inhibition of macrophages using the CSF1R inhibitor Pexidartinib ameliorates healing. These findings reveal the coordinated immune response of macrophages and skeletal stem/progenitor cells as a driver of bone healing and as a primary target for the treatment of trauma-associated fibrosis.

Fig. 1

The immune cell atlas of bone regeneration. a Experimental design. Nuclei were extracted from the periosteum of uninjured tibia and from the periosteum and hematoma/callus at days 1, 3, 5 and 7 post-tibial fracture of wild-type mice and processed for single-nucleus RNAseq. b UMAP projection of the integration of uninjured, day 1, day 3, day 5 and day 7 datasets. Eleven populations are identified and delimited by black dashed lines. c Feature plot of the immune lineage score in the combined fracture datasets. d Percentage of cells in the immune population per time point. e UMAP projection of the subset of the immune cell clusters of the combined fracture datasets. The six populations are delimited by black dashed lines, and macrophage subpopulations are delimited by green dashed lines. f Feature plots of the expression of Arg1, Cd68, Mrc1 (CD206), and Taco1 in the macrophage subpopulations. g Percentage of cells in the immune cell populations per time point. un. uninjured, mac. macrophages, IIFCs injury-induced fibrogenic cells, Osteob. osteoblasts

Fig. 2

Dynamics of macrophage subsets during bone regeneration. a Experimental design. Cells were isolated from uninjured muscle and periosteum and from the fracture environment (hematoma/callus, activated periosteum and skeletal muscle surrounding the fracture site) at 1, 3, 5 and 7 days post-fracture from Cxc3cr1^GFP mice and analyzed by flow cytometry (n = 3–7 per group). b Percentage of CD45⁺ cells and macrophages (CD45⁺GFP⁺ F4/80⁺ cells) in the uninjured periosteum/muscle and fracture environment at 1, 3, 5 and 7 days post-fracture of Cx3cr1^GFP mice. c Percentage of Arg1⁺ macrophages (CD45⁺ GFP⁺ F4/80⁺ Arg1⁺ cells). d CD68 signal in the macrophage population (CD45⁺ GFP⁺ F4/80⁺ cells). e Percentage of CD206⁺ macrophages (CD45⁺ GFP⁺ F4/80⁺ CD206⁺ cells). f Left. Safranin’O staining and fluorescent images of GFP signal in longitudinal sections of tibial fracture at 1, 3, 5 and 7 days post-fracture. Middle and right: immunofluorescence of CD68, Arg1 and CD206 on callus hematoma/fibrosis at 1, 3, 5 and 7 days post-fracture in Cx3cr1^GFP mice showing cells labeled by Arg1 or CD68 (white arrowheads) (n = 3 sections from 3 mice). g Left: Safranin’O staining of tibial fracture section of Cx3cr1^GFP mice at 7 days post-fracture. Right: Immunofluorescence of CD206 and CD68 on adjacent sections shows that CD206⁺ macrophages are only localized in the skeletal muscle surrounding the fracture site. Scale bars: f high magnification: 1 mm, low magnification: 25 μm. g High magnification: 250 μm, low magnification: 100 μm

Fig. 3

Distinct paracrine roles of macrophage subsets in the bone fracture environment. a Number of paracrine interactions from immune cells per time point determined by Connectome package. b Incoming and outgoing interaction plot of the immune cell populations showing that early macrophages, intermediate macrophages and late macrophages 1 are the main macrophage populations with a paracrine role after fracture. c Feature plots (left) and violin plots per macrophage cluster (in all time points, middle) and per time point (in all macrophage clusters, right) of the score of the pro-inflammatory, pro-repair and anti-inflammatory secretome in the subset of macrophages. d Dot plot of the significant interactions from early, intermediate and late macrophages 1 to SSPCs, IIFCs, chondrocytes and osteoblasts determined by CellChat package. eMac early macrophages, iMac intermediate macrophages, lMac1 late macrophages 1, lMac2 late macrophages 2, un. uninjured, Ch. chondrocytes, Ob. osteoblasts

Fig. 4

Inflammatory profile of SSPCs in response to bone fracture. a UMAP projection of the subset of SSPCs, IIFCs, osteoblasts and chondrocytes from the combined fracture dataset. b Schematic representation of the differentiation trajectory of pSSPCs after bone fracture. c Percentage of cells in the different populations per time point. d UMAP projection of the cells from the day 1 and day 5 post-fracture datasets in the integrated dataset. e Heatmap of the regulon activity upregulated in pro-inflammatory IIFCs (pIIFCs, left) and upregulated in anti-inflammatory IIFCs (aIIFCs, right). f Feature plots and violin plots per time point of the score of the expression of pro-inflammatory factors (left) and anti-inflammatory factors (right). pSSPCs periosteal skeletal stem progenitor cells

Fig. 5

Musculoskeletal trauma alters macrophage dynamic during bone repair. a Left: microCT images of day 21 post-fracture (top) or post-MTI (bottom) callus showing absence of bone union in MTI (white arrowheads). b Low magnification of picrosirius staining and GFP signal on adjacent sections of 21-days post-injury callus of Cx3cr1^GFP mice and high magnification of the callus showing GFP⁺ CD206⁺ and GFP⁺ CD68⁺ cells in the fibrosis after MTI (white arrowheads). c Number of GFP⁺ and CD206⁺ cells in the callus fibrosis at day 21 post-fracture or MTI (n = 3–4 per group). d Experimental design. Cells were isolated from muscles and hematoma/callus at days 1, 3, 5 and 7 post-fracture or MTI of Cx3cr1^GFP mice and analyzed by flow cytometry (n = 3–8 per group). e Percentage of CD45⁺ cells, macrophages (CD45⁺ GFP⁺ F4/80⁺), and percentage of Arg1⁺ and CD206⁺ macrophages in uninjured tissue and at 1, 3, 5 and 7 days post-fracture or MTI of Cx3cr1^GFP mice. f CD68 signal in the macrophage population (CD45⁺ GFP⁺ F4/80⁺ cells). Statistical difference was determined between the median of fluorescent signal per sample. g Top: Safranin’O staining of day 5 post-fracture or MTI callus. Middle-bottom: high magnification of the callus and muscle sections of Cx3cr1^GFP mice showing increased amount of GFP⁺ CD206⁺ and GFP⁺ CD68⁺ cells in the fibrosis after MTI. P-value: *P < 0.05, **P < 0.01. bm bone marrow, fib fibrosis. Scale bars: a 1 mm. b low magnification: 1 mm, high magnification: 10 μm. g low magnification: 1 mm, high magnification: 100 μm

Fig. 6

Prolonged pro-inflammatory state of macrophages and SSPCs after musculoskeletal trauma. a Experimental design. Nuclei were extracted from the fracture callus at day 5 post-fracture or post-MTI and processed for snRNAseq. UMAP projection of the integration of the day 5 post-fracture and post-MTI datasets. b UMAP projection of the integrated dataset separated by dataset of origin. c Percentage of cells in the IIFC, immune cell, chondrocyte and osteoblast populations per dataset. d UMAP projection of the subset of immune cells from the day 5 post-fracture and MTI datasets. e Percentage of early, intermediate and late macrophages in the total dataset from day 5 post-fracture and MTI. f Gene ontology (GO) of the upregulated genes in macrophages (clusters 1–5) from MTI dataset compared to fracture dataset. g Violin plots of the score of pro-inflammatory, pro-repair and anti-inflammatory secretome in the subset of immune cells. h Top: UMAP projection of the subset of IIFCs, chondrocytes, and osteoblasts from the day 5 post-fracture and post-MTI datasets. Bottom: UMAP projection per dataset of origin. i Dot plot of genes markers in IIFCs, chondrocytes, osteoblasts and fibrosis genes specifically upregulated in MTI. j Percentage of pro-inflammatory IIFCs (pIIFCs) and anti-inflammatory IIFCs (aIIFCs) in the fracture and MTI datasets. k Violin plots of the expression of pro-inflammatory and anti-inflammatory factors in the subset dataset. l Dot plot of genes encoding for pro-inflammatory factors upregulated in MTI and genes encoding for anti-inflammatory factors upregulated in fracture. pIIFC pro-inflammatory injury induced fibrogenic cells, aIIFC anti-inflammatory injury induced fibrogenic cells, eMac early macrophages, iMac intermediate macrophages, lMac1 late macrophages 1, Chond. chondrocyte. Osteob. Osteoblast

Fig. 7

Macrophage depletion prevents fibrotic accumulation in musculoskeletal trauma. a Experimental design. MTI was induced in Ccr2^+/+ and Ccr2^−/− mice, in which macrophages fail to infiltrate tissues during inflammation. Representative microCT scan of 35 days post-MTI callus from Ccr2^+/+ and Ccr2^−/− mice, showing nonunion in Ccr2^+/+ mice (white arrowheads) and bone bridging in Ccr2^−/− mice. b Percentage of healed calluses from Ccr2^+/+ and Ccr2^−/− mice showing bone union (white), semi-union (gray) or nonunion (black) on microCT scan at day 35 post-MTI (n = 4-5 mice per group). c Volume of fibrosis in the callus of Ccr2^+/+ and Ccr2^−/− mice at 21 days post-MTI. d Immunofluorescence on callus sections of Ccr2^+/+ and Ccr2^−/− mice at 21 days post-MTI showing the presence of CD206⁺ and CD68⁺ macrophages only in the fibrosis of Ccr2^+/+ mice. e Experimental design. MTI was induced to LysM^Cre; R26^tdTom and LysM^Cre; R26^tdTom/IDTR mice, and diphteria toxin (DT) was injected at days 2, 3 and 4 post-injury to induce macrophage depletion. Representative microCT scan of 35 days post-MTI callus from LysM^Cre; R26^tdTom and LysM^Cre; R26^tdTom/IDTR mice, showing nonunion in LysM^Cre; R26^tdTom mice (white arrowheads) and bone bridging in LysM^Cre; R26^tdTom/IDTR mice. f Percentage of healed calluses from LysM^Cre; R26^tdTom and LysM^Cre; R26^tdTom/IDTR mice showing bone union (white), semi-union (gray) or nonunion (black) on microCT scan at day 35 post-MTI (n = 7 mice per group). g Volume of fibrosis in the callus of LysM^Cre; R26^tdTom and LysM^Cre; R26^tdTom/IDTR mice at 21 days post-MTI. h Immunofluorescence on callus sections of LysM^Cre; R26^tdTom and LysM^Cre; R26^tdTom/IDTR mice at 21 days post-MTI showing the presence of CD206⁺ and CD68⁺ macrophages only in LysM^Cre; R26^tdTom mice. Scale bars: a–e: 1 mm, d–h: 100 μm. P-value: *P < 0.05

Fig. 8

CFS1R inhibition prevents fibrous nonunion after musculoskeletal trauma. a Experimental design. Wild-type mice were treated by oral gavage of Pexidartinib or vehicle from days 3 to 14 post-MTI. b Representative microCT scan of 21 days post-MTI callus from mice treated with Pexidartinib or vehicle, showing nonunion in vehicle-treated mice (white arrowheads) and bone bridging in Pexidartinib-treated mice (n = 6–9 mice per group). c Number of bridged sides of day 21 post-MTI callus of mice treated with Pexidartinib or vehicle. d Volume of mineralized bone in 21 days post-MTI callus of mice treated with Pexidartinib or vehicle (n = 6–9 mice per group). e Percentage of cartilage and fibrosis in day 21 post-MTI callus of mice treated with Pexidartinib or vehicle. f Immunofluorescence on callus sections of vehicle- and Pexidartinib-treated mice at 21 days post-MTI showing fewer CD206⁺ and CD68⁺ macrophages in Pexidartinib-treated mice. Scale bars: b: 1 mm, f: 100 μm. P-value: *P < 0.05, **P < 0.01