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A Population of M2 Macrophages Associated with Bone Formation

ABSTRACT: Purpose: We had previously published that very early during bone formation we observed a very transient cell that had the phenotype of brown fat and was important in creating a hypoxic microenvironment. This is critical for cartilage formation, the first stage in bone formation. The purpose of the experiments in this manuscript were to determine the phenotype of these "brown fat" cells in more detail. Methods: To determine the phenotype of these cells they were isolated by FACS for the beta 3 adrenergic receptor that we had previously found was a good marker for these cells during bone formation. After isolation, the cells both from HO and from the callus of a fracture were subjected to single cell RNA seq. Results: The results of the single cell RNA seq showed that the cells in HO had many of the phenotypic markers of type 2 macrophages. However, they also retained the markers of brown fat including Ucp1 and Ucp2 as well as many markers of adipogenesis and extreme elevation of mitochondrial proteins. This mixture of proteins is required to burn oxygen in the microenvionment to create hypoxia. The cells made during fracture repair were further along in bone formation because they had other blood cells (B and NK cells) besides these macrophages. However, a small fraction was the same as the macrophages found during HO. Conclusions: The type 2 macrophages made during bone formation had many of the properties of type 2 macrophages in the blood. However, they were also different from macrophages found in blood in that they lacked many of the key markers of blood macrophages. These unique macrophages are made rapidly at the start of bone formation and then disappear four days later probably differentiating to osteoclasts. However, they are critical in determining where the new bone forms as well as its size and shape.

ORGANISM(S): Mus musculus

PROVIDER: GSE185500 | GEO | 2021/10/09

REPOSITORIES: GEO

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Project description:Runx2 and Axin2 regulate skeletal development. We recently determined that Axin2 and Runx2 molecularly interact in differentiating osteoblasts to regulate intramembranous bone formation, but the relationship between these factors in endochondral bone formation was unresolved. To address this, we examined the effects of Axin2 deficiency on the cleidocranial dysplasia (CCD) phenotype of Runx2+/- mice, focusing on skeletal defects attributed to improper endochondral bone formation. Axin2 deficiency unexpectedly exacerbated calvarial components of the CCD phenotype in the Runx2+/- mice; the endocranial layer of the frontal suture, which develops by endochondral bone formation, failed to mineralize in the Axin2-/-:Runx2+/-mice, resulting in a cartilaginous, fibrotic and larger fontanel than observed in Runx2+/- mice. Transcripts associated with cartilage development (e.g., Acan, miR140) were expressed at higher levels, whereas blood vessel morphogenesis transcripts (e.g., Slit2) were suppressed in Axin2-/-:Runx2+/-calvaria. Cartilage maturation was impaired, as primary chondrocytes from double mutant mice demonstrated delayed differentiation and produced less calcified matrix in vitro. The genetic dominance of Runx2 was also reflected during endochondral fracture repair, as both Runx2+/- and double mutant Axin2-/-:Runx2+/- mice had enlarged fracture calluses at early stages of healing. However, by the end stages of fracture healing, double mutant animals diverged from the Runx2+/- mice, showing smaller calluses and increased torsional strength indicative of more rapid end stage bone formation as seen in the Axin2-/- mice. Taken together, our data demonstrate a dominant role for Runx2 in chondrocyte maturation, but implicate Axin2 as an important modulator of the terminal stages of endochondral bone formation. 4 mice per genotype X 4 genotypes: wildtype (WT), Runx2+/- (R-Het), Axin2-/- (A-KO), Axin2-/-:Runx2+/- (A-KO:R-Het). Total = 16 samples

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A Population of M2 Macrophages Associated with Bone Formation

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