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. 2015 Oct;14(10):2722-32.
doi: 10.1074/mcp.M115.048744. Epub 2015 Jul 30.

Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells

Yi Rang Na  1 , Ji Hye Hong  2 , Min Yong Lee  2 , Jae Hun Jung  2 , Daun Jung  1 , Young Won Kim  1 , Dain Son  1 , Murim Choi  3 , Kwang Pyo Kim  4 , Seung Hyeok Seok 2nd  1
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Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells

Yi Rang Na et al. Mol Cell Proteomics. 2015 Oct.

Abstract

Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs.

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Figures

Fig. 1.
Fig. 1.
Quantitative proteomic/phosphoproteomic analysis of GM-BMMs/M-BMMs. A, GM-BMMs and M-BMMs were differentiated over 7 days from C57BL/6J male mice bone marrow cells with GM-CSF or M-CSF supplementation, respectively. Cells were sorted by CD45+F4/80+MHCII+ population for GM-BMMs, or CD45+F4/80+CD11b+ population for M-BMMs. B, Surface marker examination of GM-BMMs and M-BMMs. Attached cells were stained with the appropriate antibodies and analyzed for surface MHCII, CD11c, CD80, CD64, and MerTK expressions per gated populations in A. Three independent experiments were performed. **, p < 0.01, ***, p < 0.001 by Student t test. C, Experimental strategy. Macrophage proteins were pooled to generate three biological replicates for each of the GM-BMM/M-BMM/GM-BMM+LPS/M-BMM+LPS experimental groups. D, Results of protein expression experiments. E, Results of phosphoproteome experiments. F, Scatter plot of gene symbols detected both in proteome and microarray analyses. Data represents the fold change of log2(GM-BMM/M-BMM) of 2491 genes. Regression p value = 2.1987e-242, R-squre = 0.31352. G, GO analysis of differentially expressed proteins and phosphopeptides. Heat-map shows significant GO biological process terms (p < 0.05) for differentially expressed proteins/phosphoproteins between GM-BMM/M-BMM and GM-BMM+LPS/M-BMM+LPS. Red color indicates increased protein/phosphoprotein abundances in GM-BMM or GM-BMM/LPS, and blue color indicates increased abundances in M-BMM or M-BMM/LPS. Star markings show the most enriched GO category in a heatmap.
Fig. 2.
Fig. 2.
GM-BMMs have higher glycolytic capacities than M-BMMs. A, Expression profiles of glycolytic genes and proteins in GM-BMMs compared with M-BMMs. ***, p < 0.001 by two-way ANOVA. B, Western blot results for RPIA, PFKP, and actin. C, Basal OCR and ECAR of GM-BMMs and M-BMMs. Data obtained using XF-24 extracellular flux analyzer. Three independent experiments were performed. **, p < 0.01 by Student t test. D, Maximum glycolytic capacities of GM-BMMs and M-BMMs. Perturbation profiling of lactate production (ECAR) was achieved by the addition of glucose (10 mm), oligomycin (5 μm), and 2-deoxyglucose (2-DG, 100 mm). Three independent experiments were performed. ***, p < 0.001 by two-way ANOVA. E, Quantitative graph representing mean maximum glycolytic capacities of GM-BMMs and M-BMMs. Three independent experiments were performed. *, p < 0.05 by Student t test. F, LPS induced acute glycolysis. GM-BMMs and M-BMMs were stimulated with 100 ng/ml E. coli LPS and recorded for the ECAR changes. Data showing relative ECAR (%) changes from baseline. Three independent experiments were performed. ***, p < 0.001 by two-way ANOVA. G, Glucose effect on acute glycolysis upon LPS stimulation. GM-BMMs were stimulated with LPS with or without media glucose and ECAR changes were recorded. H–K, GM-BMMs produced more inflammatory cytokines than M-BMMs in a glycolysis-dependent manner. GM-BMMs and M-BMMs were sorted and replated in 96-well plates at 5 × 105 density. Cells were treated with LPS (100 ng/ml) with or without 2-DG (100 mm) and cytokines were measured at 4 h (TNFα) and 24 h (IL-6, IL-1β, IL-10). ELISA was performed to quantify TNFα (H), IL-6 (I), IL-10 (K) production in culture supernatants, or IL-1β synthesis (J) in cell lysates. Three independent experiments were performed. **, p < 0.01, ***, p < 0.001 by two-way ANOVA.
Fig. 3.
Fig. 3.
M-BMMs phagocytosed more beads than GM-BMMs. A, Relative mRNA and protein expression levels of endocytosis participants in M-BMMs compared with GM-BMMs. **, p < 0.01, ***, p < 0.001 by two-way ANOVA. B, Western blot results for TFRC and actin. C, Histograms showing the number of beads per cell. Sorted macrophages were replated and incubated with Alexa350 tagged latex beads for 2 h. Bead phagocytosis was analyzed per macrophage population (GM-BMMs: F4/80+MHCII+, M-BMMs: F4/80+CD11b+) using FACS. Cell percentages containing more than four beads are indicated in both histograms. D, Quantitative graph showing cell percentages with more than four beads per cells. Three independent experiments were performed. *, p < 0.05 by Student t test.
Fig. 4.
Fig. 4.
Regulated protein networks in GM-BMMs versus M-BMMs. Protein abundance and phosphorylation data were analyzed with the Ingenuity Pathway Analysis (IPA) using 145 differentially expressed proteins and phosphoproteins involved in differentially regulated biological process between GM-BMMs and M-BMMs. Glucose metabolism, lipid metabolism, amino acid metabolism, DNA replication, endocytosis, and Rho/GTPase signaling were the subnetworks most significantly affected. Proteins/nodes are grouped according to their function. Red color indicates GM-BMMs up-regulation and green is M-BMMs up-regulation. Circle means DEP, diamond means DPP. White circles are predicted interacting signaling molecules as well as transcription factors. All indicated by gene symbol.
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