Characterization of the inflammatory niche at injury site
To understand the role of inflammation and inflammatory mediators in a post-traumatic response leading to aberrant wound healing, we analyzed both systemic and local cytokine and chemokine production by multiplex bead-based assays after a HO inducing injury. Burn tenotomy (burn/tenotomy) was performed on wild type mice, and tissue homogenates from the extremity injury site, and plasma were collected at days 0 (no burn/tenotomy), 3, and 7 post burn/tenotomy. Multiplex protein analysis revealed local increases in monocyte and neutrophil associated cyto/chemokines. In particular, chemokines responsible for recruitment and activation of these cells were increased at the tenotomy site at 3 days post injury, including CXCL1, CXCL2, and CCL2 (MCP-1) (Fig. 1a). Additionally, monocyte produced chemokines CCL3 and CCL4 were increased (Fig. 1a). CCL3 and CCL4 have been shown to induce the expression of other pro-inflammatory molecules including IL-1, IL-6, and TNF-α19, which were also increased at the site of extremity injury in our model (Fig. 1b). Cytokines G-CSF and GM-CSF, important in neutrophil and monocyte/macrophage maturation respectively, were also increased (Fig. 1c). These data point towards neutrophils and monocytes being important players of the immune system during the initial response to musculoskeletal trauma. In addition to neutrophil and monocyte associated factors being increased locally at the site of injury, transforming growth factor beta (TGF-β) 1, 2, and 3 were also increased (Fig. 1d). Interestingly, LIF and CXCL5 were increased, which, in addition to immune modulatory functions, are believed to be important in stem cell recruitment and maintenance (Fig. 1e). Systemically, there were less changes in these inflammatory mediators in the plasma from days 0, 3, and 7 post burn/tenotomy with no significant fluctuations identified over the time course of the experiment (Supplemental Fig. 1). This suggests that changes in monocyte and granulocyte associated factors may be important in the local microenvironment leading to aberrant tissue regeneration as seen in HO formation.
a–c Injury site homogenates harvested from burn/tenotomy mice on day 0, 3, and 7 post burn/tenotomy. a Monocyte/Macrophage associated factors. b Monocyte/Macrophage and neutrophil maturation factors. c Cytokines stimulated by monocyte factors. d TGF family members. e Stem cell maintaining factors. Levels of cytokines and chemokines in pg/ug of total protein, data represented as the median with interquartile range. Changes in cytokines and chemokines across day 3 and day 7 vs. day 0 were analyzed by an analysis of variance (ANOVA) with post-hoc Dunnett test (n = 3 mice/time point) significance. Non-heteroscedastic data identified by Levene’s test for homogeneity of variances were alternatively analyzed by Welch statistic and post-hoc Dunnett T3. Degrees of freedom (df or df1) across samples = 2. F statistic and significant post-hoc p-values respectively: CXCL1: 30.359, p(D0 vs. D7) = 0.036, CXCL2: 8.504, CCL2: 268.773, p(D0 vs. D3) = 0.000, p(D0 vs. D7) = 0.000, CCL3: 16.430, CCL4: 22.441, p(D0 vs. D3) = 0.014, G-CSF: 12.579, GM-CSF: 4.988, IL-1b: 3.486, IL-6: 13.019, TNF-α: 38.435, p(D0 vs. D7) = 0.019, TGF-β1: 9.156, TGF-β2: 11.376, TGF-β3: 7.362, CCL5: 0.825, CXCL5: 0.825, LIF: 25.368, p(D0 vs. D3) = 0.001, p(D0 vs. D7) = 0.002 *p < .05 **p < .01. f t-SNE dimensionality reduction analysis of single cell sequencing from day 3 cells harvested at the extremity injury site revealed 14 distinct cell clusters (representative, performed in triplicate). g, h Feature plots displaying the single cell gene expression of g monocyte/macrophage cytokines and chemokines increased in the homogenates and h their receptors. Source data are provided as a Source Data file.
To obtain an unbiased characterization of cells infiltrating the area of injury and producing these cyto/chemokines, we harvested the tissue at the tenotomy site at day 3, isolated, and performed single cell RNA sequencing using the 10× genomics platform. Cluster analysis using the t-distributed stochastic neighbor embedding (tSNE) dimensionality reduction identified that there were 14 different cell clusters present at the day 3 injury site. Using the Cten and Immgen databases, we identified 5 monocyte/macrophage clusters and two granulocyte clusters based on their top ~50 gene expressing signatures (Fig. 1f). Using feature plots to identify the expression of the above immune mediators; we found that the monocyte and macrophage clusters were responsible for much of this expression. The stromal cells also appeared to contribute to recruitment, as these cells expressed Cxcl1, Cxcl2, and Ccl2 (Fig. 1g). Tgfb1 was the only TGF beta family member expressed in the monocyte and macrophage clusters (Fig. 1g). Clusters 0, 1, and 3 represent the clusters with most of the Tgfb1 gene expression. Analyzing the top genes specific in these clusters and doing a literature search of other single cell analysis, we find that populations similar to those seen in our clusters 1 and 3 have been described (Supplemental Table 1). Gene signature similar to our cluster 1 has been seen in atherosclerosis20 and acute lung injury21 while signatures similar to cluster 3 were seen in myocardial infarction22 and atherosclerosis20. Cluster 0 does appear unique, as this cluster appears similar to a signature seen when analyzing monocyte-derived clusters from the central nervous system under homeostasis23. Further, to understand the cells responding to these mediators, analysis of respective receptors was performed. As would be expected, we found that granulocyte colony stimulating factor receptor, Csf3r, and the neutrophil chemokine Cxcl2 receptor (Cxcr2) were expressed in the granulocyte clusters. The macrophage colony stimulating factor receptor, Csf1r, responsible for binding M-CSF leading to survival, proliferation, and differentiation of macrophages24, was expressed in the macrophage clusters (Fig. 1h). Unexpectedly, Csf1r expression was also seen in our lymphocyte and granulocyte clusters. Previous studies have shown that while the protein is not produced, transcripts for Csf1r can be seen in neutrophils, dendritic cells, and splenic T cells25,26,27. Together these data highlight a key role for monocytes, macrophages, and granulocytes in the initial phases of the pathogenesis of HO.
Critical initial functions of monocyte-derived macrophages
Since monocyte and macrophage clusters compose the majority of the immune cells present at day 3 in our model and appear to be the cells producing mediators increased at this time point, we performed in depth in vivo analysis of the cellular composition and function of infiltrating immune cells over the course of HO formation. To do this, we first monitored the recruitment of myeloid cells to the site of injury using a reporter mouse model driven by myeloid lineage specific protein LysM (LysM-Cre/mTmGfl/fl). Using these mice, we found that green fluorescent myeloid cells were present in high numbers at the injury site 1 week after injury and remained present as late as 3 weeks after injury (Fig. 2a). Next, we monitored myeloperoxidase (MPO) activity, an enzyme commonly expressed in activated neutrophils, monocytes, and macrophages, at the injured site using bioluminescent in vivo imaging over a course of 3 weeks. MPO activity was detected specifically at the ankle and reached a peak on day 1 after injury (Fig. 2b) consistent with an influx of neutrophils observed by flow cytometry at the same time point (Fig. 2c, d). MPO activity decreased thereafter but remained detectable even 3 weeks post-trauma at the tendon injury site, after neutrophils were no longer present, indicating that MPO is derived from recruited monocytes/macrophages at later time points (Fig. 2b, d). In contrast, MPO activity almost entirely disappeared at the burn site on the back where no HO formed (Fig. 2b).
a Immunofluorescence of LysmCre+/−/mTmGfl/fl mice at 1 and 3 weeks after injury localizing EGFP+ myeloid cells. Scale bars correspond to 100 μm. b In vivo measurement of inflammation with imaging of myeloperoxidase (MPO) activity. n = 3 mice per indicated time point. Top: representative images of each time point (scale shown: blue = low, red = high). Bottom: quantification of the total bioluminescent signal at the injury site using standardized region of interest (ROIs). c Gating strategy for flow cytometry analysis to identify inflammatory cell populations. d Left: Quantification of recruitment of inflammatory cell populations over time was analyzed using flow cytometry (n = 4 mice per time point). Right: Representative flow cytometry plots demonstrating dynamic changes in Ly6C, F4/80 and CD206 monocyte populations over 3 weeks (n = 4 mice per time point). Source data are provided as a Source Data file.
Using flow cytometry, we observed an early infiltration of CD11b+Ly6G+ neutrophils that peaked at day 1 comprising 19% of all cells at the injury site (Fig. 2d, negative gates Supplemental Fig. 2). Similarly, CD11b+Ly6G− monocytes were recruited in large numbers comprising over 40% of all cells at day 2. While neutrophils mostly disappeared after 3 weeks, monocytes/macrophages remained the prevalent inflammatory cell population comprising nearly 15% of all cells by 3 weeks (Fig. 2d). Further, we noted that Ly6Chi monocytes, commonly described as the classical pro-inflammatory monocytes28, were recruited during the initial stage of the early inflammatory phase and rapidly dissipated by day 5, while Ly6Clo macrophages were present later in the inflammatory response and were the prevalent population that persisted out to 3 weeks after injury (Fig. 2d). Ly6Clo cells also co-expressed the markers F4/80 and CD206 suggesting a more M2 like phenotype (Fig. 2d).
Taken together, this data suggests that monocyte-derived macrophages are important in the initial immune response to musculoskeletal injury, and responses by these cells after injury in this model might contribute to aberrant tissue regeneration as seen in HO.
Macrophage depletion attenuates aberrant wound healing
Having shown that monocytes are recruited to the site of injury in large numbers we aimed to understand their role in promoting HO formation by using intravenous liposomal clodronate depletion of circulatory monocytes prior to and immediately following burn/tenotomy (Fig. 3a). Clodronate liposomes injected intravenously cannot cross capillary barriers29 and therefore do not affect resident macrophages30 allowing us to validate the role of primarily circulatory monocytes. Depletion of monocytes was confirmed using flow cytometry, demonstrating significant reduction of the circulatory monocyte population (6.5% vs. 1.1%, p < 0.001) at one week after injury without depletion of neutrophils (Fig. 3b). Administration of clodronate for the first three weeks after injury resulted in a significantly dampened inflammatory response at the injury site, reinforced by decreased MPO activity (Fig. 3c) and reduced ankle edema size (Fig. 3d), confirming a critical role of infiltrating monocytes in mounting inflammation. Correspondingly, depletion of circulatory monocytes resulted in decreased recruitment of total monocytes to the site of injury while neutrophil recruitment remained unaffected (Fig. 3b). Interestingly, the decrease in monocytes was attributable to a significant reduction of Ly6Clo monocytes; however, the percentage of Ly6Chi monocytes remained unchanged. Consistent with these findings, the presence of differentiated mature macrophages (CD11b+F4/80+) were significantly reduced (18.0% vs. 10.7%, p < 0.001) (Fig. 3b). To determine changes in the inflammatory milieu both locally and systemically after total monocyte depletion, we used LysMcre-iDTR mice where monocytes were depleted by pre-injection of diptheria toxin (DT) two days before the burn/tenotomy, the day of burn/tenotomy and 2 days after the burn/tenotomy. demonstrated changes to the pro-inflammatory cyto/chemokine levels in the plasma at day 3 (Supplemental Fig. 3). We found changes with regards to pro-inflammatory cyto/chemokines are in the serum at day 3. This suggests that the depletion of macrophages affects the systemic response to the dorsal burn but does not change the early response occurring at the HO site. We found a trend for decreases in plasma CCL2, G-CSF, GM-CSF, IL-1b, IL-6, and TNF-α levels when macrophages were depleted. There were also decreased levels of TGF-β1 at the tenotomy site, albeit not significant (Supplemental Fig. 3). This suggests that depletion of monocytes alters the peripheral response after injury, subsequently affecting the downstream immune cell response which occurs at the injury site.
a Schematic of experimental set-up. b Flow cytometry analysis of injury site 1 week after burn/tenotomy in mice treated with clodronate or PBS for monocytes (CD11b+ Ly6G−); neutrophils (CD11b+Ly6G+); classical monocytes (CD11b+ Ly6G− Ly6Chi), alternatively activated monocytes (CD11b+ Ly6G− Ly6Clo) and macrophages (F4/80+). Circulating monocytes: n = 4/group, df = 8, t = −0.213, p = 0.000. Injury site: n = 4/group, df = 8. Neutrophils: t = −0.213,p = 0.837; Monocytes: t = 3.490, p = 0.008; Ly6C−: t = 3.193, p = 0.013; Ly6Clow: t = 4.139, p = 0.003; Ly6Chi: t = −0.979, p = 0.356; F4/80 macrophages: t = 3.552, p = 0.007. c IVIS imaging of MPO activity 1 week after injury in mice treated with clodronate or control (n = 4 mice per treatment). Total bioluminescent signal at the injury site using standardized region of interest (ROIs) was calculated and presented as total flux in photons per second per ROI. df = 3.182, t = 1.995, p = 0.135. d Left: representative images of ankle edema present in each treatment group. Right: quantification of ankle size. n(PBS) = 5, n(Clod) = 4, df = 7, t = 11.350, p = 0.000. e Representative Safranin O staining of tendon injury site 3 weeks after burn/tenotomy in clodronate and PBS treated mice. n = 3/group. f MicroCT analysis of tenotomy site 9 weeks after burn/tenotomy in clodronate and PBS control treated mice. Left: representative 3D reconstruction. Right: quantification of unthresholded total HO, floating HO (HO not associated with tibia or calcaneus) and proximal HO (HO proximal to the calcaneus). n(PBS) = 6, n(Clod) = 5. Total HO: t = 3.302, df = 5.312, p = 0.020; Floating HO: t = 1.867, df = 5.002, p = 0.121; Proximal HO: t = 1.313, df = 9, p = 0.222. All analyses assess for homoscedasticity and difference in means via Levene’s F-test and two-tailed Student’s t-test, respectively. *p < 0.05. Source data are provided as a Source Data file.
To determine whether the suppression of the early inflammatory response through monocyte depletion affects pathologic wound healing, we examined the effect on early formation of HO. Mice treated with liposomal clodronate demonstrated decreased formation of the cartilage precursor as evidenced in Safranin O staining of the injury site at 3 weeks (Fig. 3e). As expected, early depletion of circulatory monocytes further resulted in reduced volume of mature HO by microCT quantification in the clodronate group as compared to control (Fig. 3f). These findings reveal a critical role of monocytes/macrophages in initiating the acute inflammatory phase after tissue injury confirming a contribution to aberrant mesenchymal cell differentiation.
Monocyte/macrophage heterogeneity during trauma induced HO
To appreciate the changes of monocytes/macrophages during the initial phases leading to HO formation in an unbiased approach, single cell RNA (scRNA) sequencing was performed from tissue at the extremity injury site collected at days 0, 3, 7, and 21-post injury. Canonical correlation analysis of the 4 datasets yielded 12 transcriptionally unique cell clusters identifiable at the injury site, several with characteristic profiles attributable to known cell types including those classified as stromal cells by expression of Pdgfrα (clusters 0 and 6) and a large number of cells expressing markers being related to granulocytes and monocytes/macrophages (clusters 1, 3, 4, 7; Fig. 4a). Distinct clusters expressed genes commonly linked to M2 monocytes including Mrc1 (CD206) and Arg1 (cluster 1, 3, and 4; Fig. 4b). These findings confirm cellular heterogeneity among monocytes and macrophages at the site of injury during trauma induced HO. Analysis of cells from day 0 (prior to injury) in the canonical analysis (Fig. 4c) demonstrated for the first time in this model, macrophages that were present at steady state (resident macrophages) that may play a role in the pathogenesis and the formation of HO (Fig. 4c; cluster 1). To determine markers for this population, we analyzed feature plots from day 0 tSNE plots. Resident macrophages appear to be defined as Siglec1+, Timd4+, Lyve1+, Mrc1+, Cd163+, Arg1−, Fcgr1+ cells (Fig. 4d).
a Day 0, 3, 7, and 21 combined canonical correlation analysis and T-distributed stochastic neighbor embedding (t-SNE) plot identified 12 distinct cell clusters based on gene expression differences. b Violin plots of monocyte/macrophage markers (Siglec1, Arg1, Mrc1, Csfr1), Tgfb1, and Tgfbr1. c t-SNE plot displaying only those cells in the canonical correlation analysis from day 0. d Feature plots of monocyte/macrophage genes expressed to identify possible resident macrophages at the extremity injury site from day 0 cells.
To understand the hypothetical developmental relationships that might exist within the monocyte and macrophage clusters, we performed trajectory analysis on clusters 1, 3, 4, and 7 using the Monocle algorithm. Three branch points were determined based on changes in monocytes and macrophages gene expression, and this was plotted in pseudotime (Fig. 5a). Clusters were superimposed on the monocle pseudotime plot and revealed that cluster 1 fell towards the beginning of pseudotime while clusters 4, 3, and 7 in that order, followed along the remaining trajectory (Fig. 5b). Gene expression plotted by cluster across pseudotime, revealed that increased expression of Arg1 was accompanied by a decreased expression of Siglec1, Mrc1, and Csfr1 (Fig. 5c). Analysis of gene expression changes over pseudotime at each branch point (Fig. 5d) allowed us to determine cell markers associated with each branch of the trajectory. Cells at the beginning of the trajectory are similar to our resident population having markers Siglec1, Mrc1, Folr2, and are Arg1 negative (Fig. 5d; left at midline). The most up-regulated markers for each trajectory are listed in Fig. 5a. Tracking gene expression changes across macrophage states revealed coordinated patterns defining macrophage subset identification and functions. In short, changes after the first branch resulted in two populations: one that was IL-1b+Ccl4+, Erg1+, Mrc1−, Siglec1−, and Csfr1−, while the other was Mfge8+, Mgp+, Siglec1+, Arg1+, and Cd68+. After the second branch there was a terminal Siglec1+, Arg1+, Cd68+, Tgfb1+, Clec4d+, Tnf+, Cxcl3+, Cd14+, and Chil3+ signature and an intermediate Mmp2+, Cd81+, Sparc+, and Igfbp7+ subset. At the final branch, there were two subsets characterized by Trem2+, Apoe+, Spp1+, Arg1+, and Tgfb1+ expression in the first, and Plac8+, Ifitm6+, Igfbp4+, and interestingly, Col1a1+Col1a2+and Col3a1+ expression in the other (Fig. 5a). Taken together this highlights the heterogeneity that is present in the macrophages at the site of extremity injury during trauma-induced HO.
a Focused Monocle pseudotime trajectory analysis including only the monocyte/macrophage defined clusters 1, 3, 4, and 7 from Fig. 4a. b Monocyte/ macrophage clusters superimposed on pseudotime branches. c Cluster-defined gene expression plotted as a function of pseudotime. d Heatmaps of differentially expressed genes ordered based on their common kinetics through pseudotime displayed at each trajectory branch point as defined in our Monocle trajectory analysis of the monocyte/macrophage clusters (5a).
Tgfb1 expression in macrophages drives HO formation
Having established that monocytes and monocyte-derived macrophages are important for HO formation in our burn/tenotomy model, we further explored the pathways underlying this process. Microarray gene set enrichment analysis of peripheral blood mononuclear cells from patients who suffered burn injuries, predisposing them to a higher risk of HO formation, demonstrated elevated TGF-β1 signaling (Fig. 6a; left). Subsequently, HO anlagen was harvested from injured wild type mice and subjected to RNAseq analysis. Interrogation of the data using a publicly accessible gene set enrichment database revealed clustering of highly expressed Tgfb1 signature genes indicating an activation of the TGF-β1 pathway at the injury site (Fig. 6a; right). Furthermore, we observed increased pSMAD2/3 in tissue homogenates of injured sites by 5 days after injury corresponding to the peak of monocyte recruitment (Fig. 6b). Further, TGF-β1 co-localized with F4/80+ and CD68+ macrophages at the early HO site in both mouse (Fig. 6c) and human (Fig. 6d; left) samples, respectively. In early human HO, pSMAD2 co-localized with PDGFRα, an MSC marker, suggesting TGF-β1 signaling present in these cells at the HO site (Fig. 6d; right). Homogenates from the day 3 extremity after burn/tenotomy displayed high levels of TGF-β1 (Fig. 1d) and single cell analysis from day 3 post injury demonstrated that Tgfb1, but not Tgfb2 or Tgfb3, was increased within the monocyte and macrophage clusters (Fig. 1g). Finally, in our single cell and trajectory analysis Tgfb1 is up-regulated in many of the macrophage cell clusters at the HO site (Fig. 5a). Together, these data suggest that Tgfb1 expressing monocytes/macrophages during the early stages of inflammation, may be important in the aberrant formation of HO in human and mouse.
a Left: GSEA analysis of microarray data collected from buffy coat of human burn injury patients at increased risk of HO compared to post-surgical control patients. Right: GSEA analysis of RNAseq performed of tendon injury site 3 weeks after burn/tenotomy in mice. b Western blot of whole tissue protein collected from the injury site of C57BL/6 J mice after burn/tenotomy at indicated time points. A western blot for p-SMAD2 and H3 was performed on the nuclear fraction and SMAD2 and alpha-Tubulin were performed on the cytosolic fraction. n = 5 were pooled for each time point. c Co-localization of F4/80+ and TGF-β1 at tendon injury site 1 week after burn/tenotomy. Scale bars correspond to 100 μm. d Left: Co-localization of CD68+ and TGF-β1 in early human HO. Right: Co-localization of p-SMAD2 and PDGFRα in human HO. Source data are provided as a Source Data file.
To confirm this finding, we hypothesized that monocyte Tgfb1 deletion would result in attenuation of HO formation. To test this, we crossed LysMCre mice with Tgfb1fl/fl mice, creating a myeloid cell specific deletion of Tgfb1. These mice did not have differences in body weight or defects in cortical bone thickness (Supplemental Fig. 4) and had demonstrated decreased TGF-β1 expression in bone marrow derived macrophages (Supplemental Fig. 5). LysMCre-Tgfb1fl/fl (mTgfb1KO) mice demonstrated a significantly decreased inflammatory response after burn/tenotomy as evidenced in lower MPO activation at the ankle and on the back indicating that Tgfb1 critically regulates inflammatory function of the infiltrating monocytes in the acute injury (Fig. 7a). Given that Tgfb1 can alter monocyte chemotaxis31, we next interrogated whether inflammatory cell recruitment was altered in mTgfb1KO mice after an HO inducing injury. Seven days after injury, deletion of Tgfb1 in myeloid lineage cells did not alter the recruitment of cells to the site of injury (Fig. 7b). Most interesting, mTgfb1KO mice demonstrated significantly reduced HO formation at 9 weeks after injury by microCT (Fig. 7c). Analyzing TGF-β1 levels in homogenates and serum from mTgfb1KO mice after burn/tenotomy, we observed no decrease in TGF-β1 at the injury site, but a trend towards a decrease in the serum of mTgfb1KO mice compared to wild type (Fig. 7d), albeit not significant. These data point to Tgfb1 being deleted from myeloid lineage cells in these mice (lower levels in serum); however we still identified high levels at the site of injury, suggesting that it is not the change in the microenvironment, but the loss of TGF-β1 resulting in an altered macrophage phenotype and functional capacity to promote HO at the site of injury. Analysis of the plasma and tenotomy site in LysMCre-Tgfb1fl/fl mice was also performed. There were no significant changes in the levels of pro-inflammatory cyto/chemokines at the tenotomy site. There was a trend for decreases in plasma GM-CSF, IL-1b, IL-6, CCL3, CCL4, CCL5, but no differences in the levels of TGF-β1 (Supplemental Fig. 6). This suggests depletion of TGF-β1 producing monocytes alter the peripheral response and subsequent response occurring at the injury site. This data points to the TGF-β1 production in monocytes as a marker of the cell phenotype important in potentiating ectopic bone formation.
a Representative images of IVIS analysis for MPO of C57BL/6 J and LysMCre-Tgfb1fl/fl mice bred on a C57BL/6 J background at 1 week after burn/tenotomy. Right: quantification of total bioluminescence/region of interest. n = 5/group. Injury site: t = 1.898, df = 8, p = 0.094; dorsum: t = 6.385, df = 4.007, p = 0.003. b Quantification of neutrophils and monocyte subpopulations based on Ly6C using flow cytometry of injury site 1 week after burn/tenotomy in C57BL/6 J and LysMCre-Tgfb1fl/fl mice bred on a C57BL/6 J background. n = 4 mice/group. n = 3/group. Neutrophils: t = −1.125, fd = 4, p = 0.324; Ly6C-: t = 1.685, df = 4, p = 0.167; Ly6Clo: t = 0.315, df = 4, p = 0.768; Ly6Chi: t = −1.272, df = 4, p = 0.272. c MicroCT analysis of tenotomy site 9 weeks after burn/tenotomy in C57BL/6 J and LysMCre-Tgfb1fl/fl mice. Left: Representative 3D reconstruction. Right: Quantification of total HO, floating HO (not associated with tibia or calcaneus) and proximal HO (all HO proximal to calcaneus). n = 5/group. Total HO: t = 2.290, df = 0, p = 0.051; Floating HO: t = 4.591, df = 4, p = 0.008; Proximal HO: t = 2.578, df = 8, p = 0.033. d Levels of TGFβ1 in pg/ug total protein from homogenates from the extremity injury and plasma from LysMCre-Tgfb1fl/fl, LysMCre-Tgfb1fl/wt, or wild type mice 3 days after burn/tenotomy n = 3 mice in each genotype. n = 3/group, df across groups = 2. F statistics: homogenate TGFB1: 0.588, plasma TGFB1: 1.008. All pairwise comparisons were analyzed for homoscedasticity and difference in means via Levene’s F-test and two-tailed Student’s t-test, respectively. Homoscedastic and heteroscedastic multi-group analyses were performed via ANOVA + post-hoc Dunnett’s test and Welch’s comparison of means + post-hoc Dunnett’s T3 test, respectively. *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.
CD47-activating peptide alters macrophage phenotype
Given that complete macrophage ablation would abrogate phagocytosis and oxidative burst capacity of macrophages necessary for normal wound healing, and because TGF-β1 signaling plays a critical role in the homeostasis of several essential physiologic processes, global systemic depletion of macrophages could have considerable side effects. Because of this, we sought to find a more clinically applicable approach to altering macrophage phenotype. Previous studies have suggested that CD47 activation modulates Tgfb1 expression32,33, therefore, we treated mice that underwent burn/tenotomy with daily systemic injections of CD47-activating peptide (p7N3) for 3 weeks. Treatment with CD47-activating peptide lead to significantly decreased cartilage formation indicated by Safranin O staining (Fig. 8a) as well as decreased mature HO formation seen by microCT (Fig. 8b). CD47 treatment did not impact the cortical thickness of the tibia (Supplemental Fig. 7). As was seen with mTgfb1KO mice, local TGF-β1 protein levels were not different with CD47 treatment, however there was a decrease in the systemic levels of circulating TGF-β1 (Fig. 8c). Further, QPCR analysis of markers of M1 (iNos) or M2 (Arginase, Mrc1, and Tgfb1) from sorted macrophages from the injury site of day 0, and 3 days post burn/tenotomy, and day 3 post burn/tenotomy with PBS or p7N3 treatment revealed that with p7N3 treatment, the phenotypic characteristics of the macrophages changed at the injury site (Fig. 8d). As expected, p7N3 treatment resulted in decreased levels of Tgfb1 expression in macrophages; however, while the cells still expressed Arg1 and Mrc1, the levels were decreased and this was accompanied by increased iNos expression, suggesting a change in phenotype of these cells (Fig. 8d). There was no difference in phagocytosis capacity of macrophages sorted from p7N3 treated compared to PBS treated animals (Fig. 8e); however, there was a difference in circularity of these cells (Fig. 8f). Protein analysis of the homogenates revealed significant increases in Eotaxin and CCL2 in p7N3 treated animals. Interestingly, there was also an increase in LIF, an IL-6 class cytokine that prevents stem cell differentiation, however this did not reach statistical significance (Fig. 9a).
a Representative Safranin O stain of tendon injury site 3 weeks after burn/tenotomy in p7N3 (CD47 agonist) treated and PBS control mice. n = 3/group. b MicroCT analysis of tenotomy site 9 weeks after burn/tenotomy in PBS and p7N3 (CD47 agonist) treated mice. Left: Representative 3D reconstruction. Right: Quantification of total HO, floating HO and proximal HO. n = 7/group. Total HO: t = 3.415, df = 7.840, p = 0.009; Floating HO: t = 2.201, df = 12, p = 0.048; Proximal HO: t = 2.686, df = 8.549, p = 0.026. c Levels of TGF-β1, TGFβ2, and TGFβ3 in pg/ug total protein and represented as median with interquartile range from Top: homogenates from the extremity injury (TGF-β1: t = −0.635, df = 4, p = 0.560; TGF-β2: t = −0.643, df = 4, p = 0.555; TGF-β3: t = −1.272, df = 2.186, p = 0.322) and Bottom: plasma from PBS and p7N3 (CD47) peptide treated mice 3 days after burn/tenotomy (TGF-β1: t = 1.544, df = 2.037, p = 0.260; TGF-β2: t = 2.747, df = 4, p = 0.052; TGF-β3: t = −1.492, df = 4, p = 0.210). n = 3 mice per treatment group. d qPCR analysis of M1 (iNos) and M2 (Arg1 and Mrc1) macrophage markers and Tgfb1 in macrophages isolated from the extremity injury site of naive (day 0), burn/tenotomy day 3, burn/tenotomy day 3 treated with PBS, and burn/tenotomy day 3 treated with p7N3 (CD47) peptide. Day 0 vs. Day 3—iNOS: t = 2.020, df = 2, p = 0.181; Arg1: t = −6.084, df = 3, p = 0.009; Mrc1: t = 0.703, df = 4, p = 0.521; Tgfb1: t = 0.253, df = 4, p = 0.812. PBS vs. CD47 – iNOS: t = −0.834, df = 2.043, p = 0.491; Arg1: t = 0.895, df = 4, p = 0.421; Mrc1: t = 1.176, df = 4, p = 0.305; Tgfb1: t = 1.186, df = 4, p = 0.301. e Representative images of phagocytosis assay using macrophages isolated from the extremity injury at day 3 after burn/tenotomy in mice treated with PBS or p7N3 (CD47). PBS n = 3, CD47 n = 4 approximately 25 cells/mouse. f Measurement of cellular circularity Circularity: t = 6.119, df = 55.537, p = 0.000 and quantification of mean fluorescent intensity phagocytosed by each macrophage. MFI: t = −0.357, df = 111, p = 0.722. Source data are provided as a Source Data file.
a Changes in cytokine and chemokine levels from homogenates in mice treated with PBS or p7N3 (CD47-activating peptide) 3 days afte