Cortical bone stem cells modify cardiac inflammation after myocardial infarction by inducing a novel macrophage phenotype

Abstract

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-β, but not IL-4. CBSC therapy increased the number of galectin-3⁺ macrophages, CD4⁺ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-β, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.

Keywords: immune modulation; inflammation; macrophage; myocardial infarction; stem cell therapy.

Figure 1.

CBSCs do not have an acute cardioprotective effect. A: outline of in vivo study design. B: representative image of GFP + CBSCs in the border zone of CBSC-treated female swine myocardium. Serial transthoracic echocardiographic measurement of ejection fraction (C), left ventricle end-diastolic volume (LVEDV; D), and left ventricle end-systolic volume (LVESV; E) of swine on day 1 (D1), 3 (D3), and 7 (D7) after MI. Representative images of TTC-stained heart slices from vehicle- and CBSC-treated swine hearts (F) and quantification of infarct size (G). H: quantification of cardiac troponin I 1 h after MI by ELISA. n = 4 swine for vehicle and n = 6 swine for CBSC for all figures. Scale bar in B = 100 µm. CBSCs, cortical bone stem cells; GFP, green fluorescent protein; MI, myocardial infarction; TTC, 2,3,5-triphenyltetrazolium chloride.

Figure 2.

A: CBSC treatment alters the inflammatory response to MI in vivo. Summary of tissue harvest for analysis 7 days after MI. Representative images (B) and quantification of the number of macrophages by immunofluorescent staining for Galectin-3 in vehicle (saline) or CBSC-treated female swine hearts (C). C–H: quantification of immune cell popualtions by flow cytometry using the gating strategies outlined in Fig. S1. I: quantification of protein levels of various cytokines, chemokines, and growth factors in CBSC or vehicle (saline)-treated swine hearts. n = 3 for vehicle and n = 6 for CBSC. ***P = 0.0006 vs. vehicle. **P = 0.0014 vs. vehicle. n = 4 for vehicle, n = 6 for CBSC for all figures except I. Scale bar in B = 25 µm. CBSCs, cortical bone stem cells; IL-1RA, IL-1 receptor antagonist; MI, myocardial infarction; TGF-β, transforming growth factor-β.

Figure 3.

CBSCs increase fibroblasts and decrease apoptosis in vivo 7 days after MI. Representative confocal images (A) and quantification (B) of immunofluorescent staining for Vimentin, DAPI, and cardiac troponin I in swine myocardium 7 days after MI. Representative images of terminal deoxynucleotidyl transferase dUTP nuck end labeling (TUNEL), DAPI, and Galectin-3 staining in the infarct zone of swine myocardium 7 days after MI (C) and their quantification (D–F). n = 4 for vehicle, n = 6 for CBSC for all figures. Scale bar = 25 µm. White arrows indicate TUNEL⁺/DAPI⁺ cells. Staining for Galectin-3 and TUNEL was performed and analyzed concurrently from the same images. CBSCs, cortical bone stem cells; MI, myocardial infarction.

Figure 4.

CBSCs induce a unique macrophage phenotype in vitro. A–C: flow cytometric analysis of macrophage surface markers after stimulation with CBSC-conditioned medium (CBSC) or normal complete RPMI (control). D: representative flow cytometric analysis of inflammatory macrophage markers after treatment with 50 ng/mL LPS in CBSC or control medium for 24 h. E–F: quantification of flow cytometry analysis, ****P < 0.0001 compared with untreated, #### P < 0.0001 compared with Cont. + 50 ng/mL LPS, **P = 0.0031. G: quantification of IL-1RA in macrophage supernatant by ELISA after stimulation with CBSC-conditioned RPMI for 24 h. H: flow cytometric analysis of macrophage efferocytosis of dead cardiac myocytes after 24-h stimulation with CBSC or control medium. I and J: quantification of flow cytometry analysis; ****P < 0.0001 compared with Cont; CTV, CellTrace Violet. K and L: quantification of IL-10 and TGF-β in the supernatant of macrophages that had undergone efferocytosis of dead myocytes after stimulation with CBSC or control medium by ELISA. M and N: quantification of mitochondrial metabolism and spare respiratory capacity by Seahorse Assay, ****P < 0.0001 compared with Cont., *P = 0.042. n = 3. CBSCs, cortical bone stem cells; IL-1RA, IL-1 receptor antagonist; TGF-β, transforming growth factor-β; OCR, oxygen consumption rate.

Figure 5.

A: CBSC-treated macrophages display a unique transcriptome. Principal component analysis of CBSC, M1 (50 ng/mL LPS), M2a (40 ng/mL IL-4), M2c (40 ng/mL IL-10), and M0 (no treatment) macrophages; principal components 1 and 2 represent 64% of total variance. B: heatmap of Pearson’s correlation coefficients between all groups. C–E: volcano plots of fold change vs. −log(P value) representing differentially expressed genes (DEGs) in CBSCs vs. M2a, M2c, and M0 macrophages. F: log₂(fold change) of specific M2a- and M2c-related genes with adjusted P value in bar; values derived from CBSC vs. M2c for M2c genes, CBSC vs. M2a for M2a genes. G: Venn diagram showing DEGs and common genes between M2c, M2a, and CBSCs vs. M0 control macrophages. H: gene ontology enrichment analysis and ranking system based on DEGs of CBSC vs. M0 control. n = 4 for all groups. CBSCs, cortical bone stem cells.

Figure 6.

CBSC-treated macrophages induce proliferation and inhibit activation and contraction of human fibroblasts. A: schematic of media generation for treatment of fibroblasts. B: representative images of human fibroblasts in culture treated with CBSC or control medium outlined in A; SB525334 was used as a TGF-β inhibitor and TGF-β was used as a positive control for fibroblast activation and α-SMA. Scale bar = 50 µm. Quantification of the number of nuclei (C) and the intensity of α-SMA (D) of the 60-min treatment group. E: quantification of the percent contraction from baseline of human fibroblasts cultured on collagen gels and treated with TGF-β; vehicle group received no TGF-β. ****P < 0.0001 CBSC vs. TGF-β, ####P < 0.0001 CBSC vs. TGF-β + control media. n = 5 for all groups. F: gene ontology enrichment analysis of differentially expressed genes (DEGs) of CBSC vs. M2c of terms including search term “fibroblast,” plotted with gene ratio and −log(adjusted P value); dotted line = −log(0.05) = 1.3 indicating cutoff for significance. n = 4 for gene ontology analysis. α-SMA, α-smooth muscle actin; CBSC, cortical bone stem cell; TGF-β, transforming growth factor-β.