Fatty acids derived from apoptotic chondrocytes fuel macrophages FAO through MSR1 for facilitating BMSCs osteogenic differentiation

Abstract

The nonunion following a fracture is associated with severe patient morbidity and economic consequences. Currently, accumulating studies are focusing on the importance of macrophages during fracture repair. However, details regarding the process by which macrophages facilitate endochondral ossification (EO) are largely unknown. In this study, we present evidence that apoptotic chondrocytes (ACs) are not inert corpses awaiting removal, but positively modulate the osteoinductive ability of macrophages. In vivo experiments revealed that fatty acid (FA) metabolic processes up-regulated following EO. In vitro studies further uncovered that FAs derived from ACs are taken up by macrophages mainly through macrophage scavenger receptor 1 (MSR1). Then, our functional experiments confirmed that these exogenous FAs subsequently activate peroxisome proliferator-activated receptor α (PPARα), which further facilitates lipid droplets generation and fatty acid oxidation (FAO). Mechanistically, elevated FAO is involved in up-regulating the osteoinductive effect by generating BMP7 and NAD⁺/SIRT1/EZH2 axis epigenetically controls BMP7 expression in macrophages cultured with ACs culture medium. Our findings advanced the concept that ACs could promote bone regeneration by regulating metabolic and function reprogram in macrophages and identified macrophage MSR1 represents a valuable target for fracture treatments.

Keywords: Apoptotic chondrocyte; Fatty acid oxidation; MSR1; Macrophage; Osteogenic differentiation.

Fig. 1

Apoptotic chondrocytes (ACs) induce lipid droplet (LD) formation in macrophages by releasing fatty acids (FAs). (A) The distribution of ACs and macrophages in the transition zone (TZ) from the callus on day 14 (D14) post fracture was assessed. Representative images of IF staining for cleaved caspase3 (red) and F4/80 (green) are shown, and the cell nuclei were stained with DAPI (blue). Scale bars, 100 μm. (B) Lipid and FA metabolic processes were upregulated on day 14 (D14) post fracture, according to the bioinformatics analysis. Representative biological process (BP) categories obtained from GO analyses of upregulated differentially expressed genes (DEGs) and downregulated DEGs (Sup Fig. 1F) between two time points (D14 vs. D7) after bone fracture in three different GEO datasets (GSE99118, GSE99580 and GSE15267). (C) Heatmap produced from the lipidomic analysis of callus tissues in the TZ on D14 relative to D7 post fracture (n = 3). sd, standard deviation. (D) Principal component analysis (PCA) of lipidomic data from callus tissues in the TZ on D14 and D7 post fracture with three biological replicates. (E) ACs induced by staurosporine (STS) were analyzed using flow cytometry after staining with FITC-Annexin V and propidium iodide. More than 90% of chondrocytes were apoptotic in the STS-treated group. (F) Heatmap from the lipidomic analysis of culture medium (CM) representing the FAs that were statistically enriched in AC CM compared to the control group (n = 5). sd, standard deviation. (G) PCA of lipidomic data from AC CM and C CM (n = 5). (H) Venn diagrams depicting the 4 shared FAs (C22:5, C22:4, C20:4 and C20:3) in two lists (List 1: D14 vs. D7, List 2: AC CM vs. C CM). (I) Schematic of the experimental protocol for macrophages treated with AC CM or C CM. After stimulation with AC CM or C CM, the LDs in different groups were evaluated using transmission electron microscopy (TEM). Black arrows indicate LDs (green). Scale bars, 2 μm. (J and K) Flow cytometry analysis of lipid levels in BMDMs treated with AC CM or C CM and FA mix or vehicle controls. Representative histogram showing the levels of lipids in BMDMs in these groups (J). Statistical analysis of the mean fluorescence intensity (MFI) of lipids in BMDMs from the indicated groups (n = 3) (K). The results are presented as means ± s.d., **P < 0.001. (L) Gating strategy for sorting CD45⁺CD11b⁺F4/80⁺ macrophages around the area of the TZ post fracture. (M and N) Intracellular lipid levels in macrophages from the TZ were analyzed on D7 and D14 post fracture. Representative histogram displaying the lipid levels in callus-sorted macrophages on D7 and D14 post fracture (M). Measurements of the MFI of lipids were performed in triplicate. Means ± s.d., ***P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

MSR1 is essential for lipid accumulation in macrophages. (A and B) Intracellular LDs in BMDMs cultured with AC CM or C CM in the presence of DMSO, TOFA, C75 and triacsin C were visualized using BODIPY 493/503 (A). Quantification of the MFI of intracellular lipids in BMDMs from the indicated groups (n = 3) (B). The results are presented as means ± s.d., **P < 0.001. Scale bars, 10 μm. (C) Schematic representation of the Red C12 transfer assay: chondrocytes were first incubated with Red C12, apoptosis was induced by STS, and then cells were washed and cultured with CM. Finally, C12-labeled CM was collected and used to treat macrophages to determine the uptake of FAs. (D) Quantification of Red C12-positive LDs following transfer between different cell types (n = 5). The results are presented as means ± s.d., ***P < 0.0001. (E) Heatmap of DEGs in AC CM-treated BDMDs compared to C CM-treated BMDMs. Blue and red colors represent low and high expression values, respectively. (F) Representative molecular function (MF) categories identified in GO analyses based on upregulated DEGs in AC CM-treated BDMDs compared to C CM-treated BMDMs. (G) Expression patterns of the indicated scavenger receptors in BMDMs treated with AC CM, C CM, vehicle and FA mix were determined using RT–qPCR. The results of three experiments are shown. Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. (H) Representative histograms (left panel) and statistical results (right panel) showing the expression of the MSR1 protein on the surface of BMDMs treated with AC CM, C CM, vehicle and FA mix (n = 3). Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. (I) Representative histograms (left panel) and statistical analysis of the MFI (right panel) show the expression of MSR1 on the surface of callus-sorted macrophages from the TZ on D7 and D14 post fracture (n = 3). Values are presented as means ± s.d., ***P < 0.0001 (J and K) Representative histograms (J) and statistical analysis of the MFI of BODIPY 493/503 (K) showing the effect of MSR1 on FA uptake in macrophages cultured with AC CM or C CM (n = 3). Values are presented as means ± s.d., ***P < 0.0001. (L) Results of the statistical analysis showing the lipid levels in WT and MSR1 KO BMDMs in response to treatment with FA mix or vehicle controls (n = 3). Values are presented as means ± s.d., **P < 0.001. (M and N) Loss of MSR1 in AC CM-treated BMDMs significantly attenuated FA uptake. Representative confocal images of WT and MSR1 KO BMDMs following the indicated transfer assays (M). Quantification of Red C12-positive LDs in WT and MSR1 KO BMDMs treated with AC CM or C CM following transfer assays (n = 8) (N). Values are presented as means ± s.d., ***P < 0.0001. Scale bars, 10 μm. (O and P) Knockout of MSR1 reduced the LD content in callus-sorted macrophages from the TZ on D14 but not on D7 post fracture. Results from the statistical analysis of the lipid levels in WT and MSR1 KO macrophages from the TZ on D14 (O) and D7 (P) post fracture are shown (n = 3). Values are presented as means ± s.d., ***P < 0.0001.

Fig. 3

Macrophages with accumulated lipids upregulate fatty acid oxidation (FAO). (A) Compared to C CM-treated BMDMs, lipid and FA metabolic processes were upregulated in AC CM-cultured BMDMs. Representative BP categories identified in GO analyses based on upregulated DEGs in AC CM-treated BDMDs compared with C CM-treated BMDMs. (B and C) The extracellular acidification rate (ECAR) (B) and oxygen consumption rate (OCR) (C) of BMDMs treated with AC CM or C CM were detected using a Seahorse Bioscience XFp analyzer (n = 3). In the measurement of ECAR, cells were sequentially treated with glucose (Glc), oligomycin (O), and 2-deoxyglucose (2-DG) (B); when OCR was detected, cells were sequentially treated with O, FCCP (F) and antimycin A/rotenone (A&R). Etomoxir (ETO, an FAO inhibitor that blocks the transport of FAs into the mitochondrion) was further used to detect FAO. (D) Lactate production in BMDMs cultured with AC CM or C CM was determined (n = 3). Values are presented as means ± s.d., **P < 0.001. (E) Results of the statistical analysis of ΔOCR of basal respiration and respiratory capacity showing that AC CM treatment facilitates FAO in macrophages (n = 3). Values are presented as means ± s.d., *P < 0.05. ΔOCR (calculated as OCR without ETO stimulation minus OCR in the ETO-stimulated group) of basal respiration and respiratory capacity were used to determine FAO fueled by FAs present in mitochondrion. (F) Representative TEM micrographs of contacts between LDs (green) and mitochondrion (red) in BMDMs cultured with AC CM or C CM. Scale bars, 10 μm. (G) The left panel shows representative confocal images of BMDMs stained with MitoTracker (red) and BODIPY 493/503 (green) after treatment with AC CM or C CM. The right panel shows the quantification of the interaction between LDs and mitochondrion. Values are presented as means ± s.d., ***P < 0.0001. Scale bars, 10 μm. (H) The ECAR of WT and MSR1 KO BMDMs cultured with AC CM or C CM was tested (n = 3). (I) Lactate secretion from BMDMs treated with AC CM or C CM was measured (n = 3). Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. (J–L) Upon treatment with AC CM or C CM, the OCR of WT and MSR1 KO BMDMs was evaluated (n = 3) (J). Furthermore, basal respiration (K), ATP production (K), respiratory capacity (L), and respiratory reserve (L) were determined in the indicated groups of BMDMs (n = 3). Values are presented as means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. (M) The ΔOCR of basal respiration and respiratory capacity were calculated as the response of BMDMs in the indicated groups to ETO treatment (n = 3). Values are presented as means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. (N) LD-mitochondrion contact in WT and MSR1 KO BMDMs treated with AC CM or C CM (Sup Fig. 3L) was evaluated by staining cells with MitoTracker (red) and BODIPY 493/503 (green) (n = 3) (left panel). These contacts were also quantified in the indicated groups (right panel). Values are presented as means ± s.d., ***P < 0.0001. Scale bars, 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

PPARα activation in macrophages is required for AC CM-induced LD formation and FAO upregulation. (A) Representative KEGG pathway categories of upregulated DEGs affected by AC CM treatment in BMDMs. (B and C) AC CM treatment activated PPARα in macrophages. PPARα activity was measured in a series of concentrations of nuclear extracts (NEs) from WT and MSR1 KO BMDMs exposed to AC CM or C CM (n = 3) (B). Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. Furthermore, levels of the CPT1, PPARα (total), β-actin, PPARα (nuclear) and Histone H3 proteins in WT and MSR1 KO BMDMs treated with AC CM or C CM (Sup Fig. 4D) were measured using western blotting (C). (D and E) Representative histograms (D) and statistical analysis of MFI (E) showing the importance of PPARα in LD formation in BMDMs treated with AC CM or C CM based on quantifying the fluorescence intensity of BODIPY 493/503 (n = 3). Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. (F) GW6471 treatment increased lactate production in WT and MSR1 KO BMDMs cultured with AC CM (n = 3). Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. (G) The administration of GW6471 promoted glycolysis, as determined by calculating the glycolysis and glycolytic capacity of WT and MSR1 KO BMDMs treated with AC CM (n = 3). Values are presented as means ± s.d., *P < 0.05, **P < 0.001. (H and I) PPARα inhibition by GW6471 decreased OXPHOS and FAO in WT and MSR1 KO BMDMs after culture with AC CM, as evidenced by basal respiration, ATP production, respiratory capacity, respiratory reserve and ΔOCR. Values are presented as means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. (J) The left panel shows representative confocal images of GW6471-treated WT and MSR1 KO BMDMs stained with MitoTracker (red) and BODIPY 493/503 (green) after treatment with AC CM. In the right panel, the interactions between LDs and MitoTracker in BMDMs from the indicated groups were quantified. Values are presented as means ± s.d., ***P < 0.0001. Scale bars, 10 μm.

Fig. 5

Attenuating FAO in macrophages decreased the pro-osteogenic differentiation activity of macrophages. (A and B) BMSCs were cultured in osteogenesis induction medium plus CM from AC CM-treated WT or MSR1 KO BMDMs, as indicated. BMSCs cultured with osteoblast differentiation medium alone were regarded as the control group. After 7 and 14 days, matrix mineralization was tested by performing alizarin red (AR) staining (upper panel of A) and ALP staining (upper panel of B). Quantitative analyses of AR staining (lower panel of A) and ALP activities (lower panel of B) on D7 and D14 are shown. Values are presented as means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. AM = Apoptotic chondrocyte CM. (C and D) After 7 (C) and 14 (D) days, mRNA expression patterns of osteoblast-specific marker genes (Col1, Alp, Ocn, and Runx2) in the indicated groups were determined using RT–qPCR. β-Actin was used as an internal control. Values are presented as means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. AM = Apoptotic chondrocyte CM. (E and F) The importance of MSR1 during EO in vivo was evaluated using a femoral fracture model. Representative 3D images of micro-CT scans from WT and MSR1 KO mice on D7, D14 and D21 post fracture (E). Statistical analysis of mineralized callus volume/tissue volume (CV/TV, %) from micro-CT scans (F). Each group contained five animals. Values are presented as means ± s.d., *P < 0.05, ***P < 0.0001. (G) Representative X-ray images of WT and MSR1 KO mice at D14 and D21 after fracture were captured (Sup Fig. 5E), and the quantification of these images revealed significantly lower union rates in MSR1 KO mice at D14 and D21 post fracture (n = 10, Chi-square test).

Fig. 6

AC CM-treated macrophages promote BMSC osteogenic differentiation by increasing FAO-regulated BMP7 production. (A) Venn diagram depicting bmp7 as the only overlapping significantly upregulated chemokine among the three lists (List 1: DEGs of the bmp family from the RNA-seq data; List 2: significantly upregulated bmp family genes in FA mix-treated BMDMs compared to control cells; List 3: significantly upregulated bmp family genes in AC CM-stimulated BMDMs compared to C CM-treated BMDMs). (B and C) The amount of secreted BMP7 in C CM- or AC CM-cultured WT and MSR1 KO BMDMs treated with or without GW6471 (PPARα inhibitor) (B), ETO (CTP1A inhibitor) (C) or 2-DG (glycolysis inhibitor) (C) was evaluated using ELISA (n = 3). The results are presented as the means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. (D) The correlation between BMP7 production and ΔOCR of respiratory capacity in BMDMs from WT and MSR1 KO mice is shown (n = 5 mice/genotype). r indicates Pearson's correlation coefficient (r = 0.9739). (E and F) Representative images (upper panel) and statistical analysis (lower panel) of AR staining (E) and ALP staining (F) in BMSCs from the indicated groups on D7 and D14 suggest a crucial role for BMP7. The results are presented as the means ± s.d., **P < 0.001, ***P < 0.0001. (G and H) After 7 (G) and 14 (H) days, mRNA expression patterns of osteoblast-specific marker genes (Col1, Alp, Ocn, and Runx2) in the indicated groups were tested using RT–qPCR and further suggested that BMP7 might be closely associated with the osteogenic differentiation of BMSCs induced by macrophages treated with AC CM. The results are presented as the means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001.

Fig. 7

BMP7 production by macrophages in response to AC CM is regulated by the NAD⁺/SIRT1/EZH2 axis (A) The level of NAD⁺ quantified by colorimetric measurements indicated that the loss of MSR1 and inhibition of PPARα or CPT1A significantly reduced the NAD⁺ level in BMDMs cultured with AC CM (n = 3). Values are presented as means ± s.d., *P < 0.05, **P < 0.001. (B) SIRT1 protein activity was measured in AC CM-stimulated WT and MSR1 KO BMDMs treated with or without GW6471 or ETO (n = 3). Values are presented as means ± s.d., **P < 0.01, ***P < 0.0001. (C) NMN (NAD⁺ precursor) supplementation partially rescues the bmp7 mRNA level in MSR1 KO BMDMs, but not EX527-treated BMDMs (an inhibitor of SIRT1) (n = 3). Values are presented as means ± s.d., *P < 0.05, **P < 0.001. (D) Based on a public database and our literature review, 4/109 TFs (EZH2, CEBPA, FOXA2 and FOXA3) deacetylated by SIRT1 are presumed to positively modulate the expression of target genes. (E) Schematic showing the regulatory mechanism of gene transcription by the NAD⁺/SIRT1/EZH2 axis. (F) Cell lysates from WT and MSR1 KO BMDMs treated with or without GW6471 or ETO were subjected to immunoprecipitation with an EZH2 antibody followed by Western blot analysis with antibodies against acetylated lysine or EZH2. (G) Representative histograms (left panel) and statistical results (right panel) displaying the level of H3K27me3 in WT and MSR1 KO BMDMs cultured in the presence or absence of GW6471 or ETO and detected using flow cytometry (n = 3). Values are presented as means ± s.d., ***P < 0.0001. (H) The level of acetylated EZH2 in AC CM-treated WT and MSR1 KO BMDMs from the indicated groups was detected using western blotting with acetyl-lysine or EZH2 antibodies. (I) The level of H3K27me3 in AC CM-treated WT and MSR1 KO BMDMs from the indicated groups was determined using flow cytometry (n = 3). Values are presented as means ± s.d., **P < 0.001, ***P < 0.0001. (J–L) Representative histograms and statistical analysis of MFI (L) showing the levels of MSR1 (J and L) and H3K27me3 (K and L) in callus-sorted macrophages from the TZ on D7 and D14 post fracture. Values are presented as means ± s.d., ***P < 0.0001. (M and N) A ChIP-qPCR assay of BMDMs cultured with AC CM or C CM was performed to verify the potential binding site for EZH2 and H3K27me3 in the BMP7 promoter region. Integration maps of the ChIP assay are shown (M). IgG and input fractions were used as controls. A statistical analysis of qPCR was also conducted (N). Values are presented as means ± s.d., **P < 0.001.

Fig. 8

Administration of AC CM and targeting macrophage MSR1 facilitate bone regeneration in vivo. (A) The effect of AC CM on bone regeneration in vivo was assessed using a tibial monocortical defect model. The increased bone regeneration observed in the AC CM-treated group was largely diminished when CLOD (depletion of macrophages) was administered. Representative micro-CT images of the reconstruction of injured tibias (upper panel) and mineralized callus tissues (lower panel) in the defect area of the indicated groups. CLOD: clodronate liposomes. (B–E) Statistical analysis of bone volume/total volume (BV/TV) (%) (B), trabecular number (Tb.N) (C), trabecular separation (Tb.Sp) (D), and trabecular thickness (Tb.Th) (E) of the mineralized bone formed in the hole region (n = 3). The results are presented as the means ± s.d., *P < 0.05, **P < 0.001, ***P < 0.0001. (F and G) Representative micro-CT images (G) and statistical analysis of CV/TV (H) both suggested that compared to the better repair in the appropriate control group, endochondral ossification occurred during fracture repair on D14 and D21 post fracture in MSR1 KO mice transplanted with MSR1 WT bone marrow. The results are presented as the means ± s.d., *P < 0.05, ***P < 0.0001. (H) Representative X-ray images and quantification indicated that compared to control mice, irradiated MSR1 KO mice reconstituted with bone marrow from MSR1 WT mice showed higher union rates on D14 and D21 post fracture (n = 10, Chi-square test). (I) Schematic of the functional consequences and specific mechanism of AC-released FAs in macrophages during EO. As we indicated, ACs and macrophages were observed in the TZ. FAs derived from ACs are taken up by macrophages mainly through MSR1, which modifies the intracellular level of FAs and activates PPARα. Then, LD formation and FAO activity are increased by PPARα activation and subsequently increase the level of NAD⁺. Furthermore, the NAD⁺/SIRT1/EZH2 axis positively regulates bmp7 expression, which accounts for the osteoinductive effect of macrophages treated with AC CM.