Project description: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.
Project description:Macrophages have distinct characteristics depending on their microenvironment. We performed proteomic analysis between M1 and M2 macrophages and found that cellular metabolism is the key regulator of macrophage function. We used microarray to support proteomic data between M1 and M2 macrophages. M1 macrophages are obtained using cell sorting of CD45+MHCII+CD8a-F4/80+ population from C57BL/6J bone marrow cell derived heterogenous cells under GM-CSF conditioning for 7 days. M2 macrophages are differentiated with 20% L929 cell supernatant for 7 days and sorted from CD45+F4/80+CD11b+ population.
Project description:Bone-marrow macrophages polarized to M2 phenotype are immunosuppressive. Interestingly, treatment with whole-glucan particles converts M2 macrophages to M1 phenotype with an anti-tumor phenotype. In this study, the effect of WGP treatment for 6 hours on the gene expression of M2 macrophages was assessed.
Project description:We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.
Project description:Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPβ, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Since the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas.
Project description:Classically (M1) and alternatively activated (M2) macrophages play distinct roles in various physiological and disease processes. Understanding the gene transcription programs that contribute to macrophage polarization along the M1/M2 spectrum may lead to new tools to detect and therapeutically manipulate macrophage phenotype. Here, we define the M1 and M2 macrophage signature through mRNA microarray. The M1 macrophage signature was defined by 629 up-regulated and 732 down-regulated genes while the M2 macrophage signature was formed by 388 up-regulated and 425 down-regulated genes. While a subset of probes was common to both M1 and M2 cells, others were exclusive to each macrophage subset. The common M1/M2 pathways were characterized by changes in various transcriptional regulators and signaling partners, including increases in Kruppel-like Factor (Klf) 4, but decreases in Klf2. To identify M1 and M2 biomarkers that help discriminate these populations, we selected genes that were increased during M1 or M2 differentiation but decreased in the opposite population. Among top novel M1-distinct genes, we identified CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2). Among top M2 genes, we found early growth response protein 2 (Egr2) and Myc. We validated these genes by Real-Time PCR and developed a CD38/Egr2-based flow cytometry assay that discriminates between M1 and M2 macrophages. Overall, this work defines the M1 and M2 signature and identifies several novel M1 and M2 genes that may be used to distinguish and manipulate M1 and M2 macrophages. Total RNA was prepared from bone marrow-derived macrophages of wild-type mice (n=2-3 independent mice) treated in M0, M1 or M2 conditions (n=2-3 replicates per condition originating from different mice)
Project description:In this study, we used mass spectrometry and label-free quantification (LFQ) to characterize the global proteomics of polarized (M1, M2a, M2b, M2c, and M2d) and unpolarized (M0) phenotypes of macrophages from human THP-1 monocytes. The results described the biological functions of the four M2 macrophages subtypes and provided available references for identifying M2 macrophages or subtypes of M2 macrophages.
Project description:PMMA bone cement is biologically inert and exhibits poor biological activity. Following implantation, it generates bone cement particles, which can easily induce tissue inflammation around the implant . Compared with PMMA bone cement, ES-PMMA bone cement had anti-inflammatory effect by inducing M2 polarization of macrophages. RNA sequencing was used to explore the underlying molecular mechanisms.
Project description:Bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 mice and differentiated under standard culture conditions. Upon completion of differentiation, cells were subjected to cytokine stimulation to induce distinct M2 macrophage phenotypes. Specifically, M0 macrophages were polarized into M2a macrophages with 20 ng/mL interleukin-4 (IL-4), into M2b macrophages with 100 ng/mL lipopolysaccharide (LPS) in combination with 50 μg/mL mouse immunoglobulin G (IgG), and into M2c macrophages with 40 ng/mL interleukin-10 (IL-10). Following induction, the functional heterogeneity of M0, M2a, M2b and M2c macrophages was systematically assessed through proteomic profiling. The resulting data revealed distinct protein expression patterns reflective of the specialized functional roles of each macrophage subtype.