Summary of Study ST003323

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002067. The data can be accessed directly via it's Project DOI: 10.21228/M8F82F This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003323
Study TitleGlobal Metabolomics and U-13C6-Glucose Tracing in Bone Marrow Derived Macrophages (BMDM)
Study SummaryWe evaluated alterations in cellular metabolism via comprehensive metabolic profiling of BMDMs derived from control and trained HSCLT. Thus, we performed ultra-high-performance mass spectrometry (UHPLC-MS) analysis of control and trained donor HSCLT-derived BMDMs following 6hr IC stimulation. We identified 206 metabolites, of which 88 were differentially abundant in control vs. trained BMDMs. Notably, we observed reductions in global metabolite abundance across multiple pathways in trained HSCLT-derived BMDMs including multiple amino acid, nucleotide, glutathione, and fatty acid synthesis pathways among others. In agreement with our Seahorse data, BMDMs from trained HSCLT exhibited a reduction in the abundance of metabolites in and downstream of the glycolysis pathway following IC stimulation, compared to BMDMs from control donor HSCLT. This phenotype was particularly evident in pathways constituting central carbon metabolism, including glycolysis itself (green), the pentose phosphate pathway (red), the tricarboxylic acid cycle (TCA, blue), as well as alternative carbon metabolites (grey). Furthermore, we observed significant reductions in products of central carbon metabolism, including levels of ATP, AMP (gold), and amino acids (grey) as well as fatty acids, pentose phosphates and lactate. Along these lines, even in the absence of IC stimulation there were significant reductions in metabolites associated with several pathways, primarily amino acid metabolism, nucleotide metabolism, and fatty acid metabolism (42 metabolites total). Given the significant reduction in glycolytic metabolites and their downstream products in IC-stimulated BMDMs derived from trained HSCLT, we also measured changes in glucose fate by culturing the BMDMs for 24h in media containing U-13C-glucose. 6hr prior to harvest, we stimulated them with IC. This approach facilitates analysis of IC-induced glycolytic activity levels and details changes in glucose fate based on isotopic labeling of downstream metabolites with 13C, while also providing information on global (13C labeled + unlabeled) metabolite levels. Consistent with our global metabolic analysis and Seahorse assays above, we noted reduced global metabolite levels as well as broad reductions in overall 13C peak areas in multiple metabolic pathways downstream of glucose, including lower glycolysis (lactate production), the pentose phosphate pathway, TCA cycle and TCA cycle-derived amino acids, based on measurement of the peak area of 13C labeled isotope species (M+#). To further evaluate potential shifts in glucose utilization we also expressed the data as a proportion of M+x# 13C labeled plus M+0 unlabeled metabolite isotopologues. Of note, in trained HSCLT donor-derived BMDMs we identified a slight though significant increase in the proportion of M+6 13C labeling in upper glycolysis (hexose phosphate), which culminated in preferential entry into the pentose phosphate pathway (based on increased though non-significant proportions of M+6 13C labeled phospho-gluconate, M+7 13C labeled sedoheptulose 1/7-p) and M+5 labeled pentose phosphates, likely indicative of 13C label cycling through the non-oxidative PPP. On the other hand, BMDMs from trained donor HSCLT exhibited a trending reduction in the proportion of 13C labeling of lactate and a significant reduction in the proportion of 13C labeling in the TCA cycle indicative of reduced glucose oxidation through central carbon metabolic pathways downstream of pyruvate. Furthermore, we observed reduced 13C labeling in key amino acids derived from central carbon metabolism including alanine and glutamate (purple), reflecting reduced contribution of glucose to amino acid synthesis and consistent with reduced global levels of these metabolites (Fig. 6D). As these data could be consistent with increased glucose entry into fatty acid synthesis pathways, we measured 13C label accumulation into palmitate but did not identify significant changes in 13C labeling. Taken together, these data are consistent with broad metabolic remodeling of IC-stimulated BMDMs derived from trained HSCLT, characterized by smaller metabolite pools and preferential routing of glucose into the PPP, with concomitant reduction of glucose entry into the TCA cycle.
Institute
University of Colorado Anschutz Medical Campus
Last NameBevers
First NameShaun
Address12801 E 17th Ave Bldg. RC-1S Rm L2400 Aurora CO 80045
Emailshaun.bevers@cuanschutz.edu
Phone970-217-3413
Submit Date2024-06-14
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2024-08-05
Release Version1
Shaun Bevers Shaun Bevers
https://dx.doi.org/10.21228/M8F82F
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002067
Project DOI:doi: 10.21228/M8F82F
Project Title:Trained immunity in hematopoietic stem cell-derived macrophages is defined by a distinct metabolic and epigenetic state
Project Summary:In the present study, we investigate the contribution of long-term hematopoietic stem cells (HSCLT) to trained immunity (TI) in the setting of chronic autoimmune disease. Using a mouse model of systemic lupus erythematosus (SLE), we show that bone marrow derived macrophages (BMDMs) from autoimmune mice exhibit hallmark features of TI, including increased Mycobacterium avium killing and inflammatory cytokine production. Furthermore, these functional properties are mechanistically linked to increased glycolytic metabolism in BMDMs from primary autoimmune mice. While we find that HSC from autoimmune mice constitute a transplantable, long-term reservoir for macrophages that exhibit the functional properties of TI, these BMDMs exhibit a distinctive metabolic state typified by attenuated glycolytic activity. Furthermore, and in contrast to BMDMs from primary autoimmune mice, BMDMs and myeloid progenitors derived from autoimmune donor HSC exhibit a unique pattern of molecular remodeling characterized by reduced chromatin accessibility at a broad array of metabolic genes, while retaining elevated expression of TI-associated transcriptional regulators such as Jun and Fos. Taken together, our data show that HSC exposed to autoimmune inflammation gives rise to macrophages in which the hallmark functional properties of TI are decoupled from glycolytic metabolism. Altogether, our data support a model in which TI is characterized by a spectrum of distinct molecular and metabolic states capable of driving augmented immune function.
Institute:University of Colorado Anschutz Medical Campus
Last Name:Bevers
First Name:Shaun
Address:12801 E 17th Ave Bldg. RC-1S Rm L2400 Aurora CO 80045
Email:shaun.bevers@cuanschutz.edu
Phone:970-217-3413

Subject:

Subject ID:SU003444
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA3604455_prim-Ctrl-5Bone Marrow Primary Cell Control
SA3604462_prim-Ctrl-2Bone Marrow Primary Cell Control
SA3604471_prim-Ctrl-1Bone Marrow Primary Cell Control
SA3604484_prim-Ctrl-4Bone Marrow Primary Cell Control
SA3604493_prim-Ctrl-3Bone Marrow Primary Cell Control
SA36045012_prim-Lup-2Bone Marrow Primary Cell Lup
SA36045114_prim-Lup-4Bone Marrow Primary Cell Lup
SA36045213_prim-Lup-3Bone Marrow Primary Cell Lup
SA36045315_prim-Lup-5Bone Marrow Primary Cell Lup
SA36045411_prim-Lup-1Bone Marrow Primary Cell Lup
SA36045510_prim-pIC-5Bone Marrow Primary Cell Pristane
SA3604569_prim-pIC-4Bone Marrow Primary Cell Pristane
SA3604578_prim-pIC-3Bone Marrow Primary Cell Pristane
SA3604587_prim-pIC-2Bone Marrow Primary Cell Pristane
SA3604596_prim-pIC-1Bone Marrow Primary Cell Pristane
SA36046050_sec-C13-24H-Ctrl-5Bone Marrow Secondary Cell Control
SA36046149_sec-C13-24H-Ctrl-4Bone Marrow Secondary Cell Control
SA36046248_sec-C13-24H-Ctrl-3Bone Marrow Secondary Cell Control
SA36046347_sec-C13-24H-Ctrl-2Bone Marrow Secondary Cell Control
SA36046446_sec-C13-24H-Ctrl-1Bone Marrow Secondary Cell Control
SA36046535_sec-C13-6H-Ctrl-5Bone Marrow Secondary Cell Control
SA36046634_sec-C13-6H-Ctrl-4Bone Marrow Secondary Cell Control
SA36046732_sec-C13-6H-Ctrl-2Bone Marrow Secondary Cell Control
SA36046833_sec-C13-6H-Ctrl-3Bone Marrow Secondary Cell Control
SA36046931_sec-C13-6H-Ctrl-1Bone Marrow Secondary Cell Control
SA36047016_sec-Ctrl-1Bone Marrow Secondary Cell Control
SA36047117_sec-Ctrl-2Bone Marrow Secondary Cell Control
SA36047218_sec-Ctrl-3Bone Marrow Secondary Cell Control
SA36047319_sec-Ctrl-4Bone Marrow Secondary Cell Control
SA36047420_sec-Ctrl-5Bone Marrow Secondary Cell Control
SA36047542_sec-C13-6H-Lup-2Bone Marrow Secondary Cell Lup
SA36047660_sec-C13-24H-Lup-5Bone Marrow Secondary Cell Lup
SA36047759_sec-C13-24H-Lup-4Bone Marrow Secondary Cell Lup
SA36047858_sec-C13-24H-Lup-3Bone Marrow Secondary Cell Lup
SA36047957_sec-C13-24H-Lup-2Bone Marrow Secondary Cell Lup
SA36048056_sec-C13-24H-Lup-1Bone Marrow Secondary Cell Lup
SA36048145_sec-C13-6H-Lup-5Bone Marrow Secondary Cell Lup
SA36048227_sec-Lup-2Bone Marrow Secondary Cell Lup
SA36048343_sec-C13-6H-Lup-3Bone Marrow Secondary Cell Lup
SA36048426_sec-Lup-1Bone Marrow Secondary Cell Lup
SA36048541_sec-C13-6H-Lup-1Bone Marrow Secondary Cell Lup
SA36048630_sec-Lup-5Bone Marrow Secondary Cell Lup
SA36048728_sec-Lup-3Bone Marrow Secondary Cell Lup
SA36048829_sec-Lup-4Bone Marrow Secondary Cell Lup
SA36048944_sec-C13-6H-Lup-4Bone Marrow Secondary Cell Lup
SA36049036_sec-C13-6H-pIC-1Bone Marrow Secondary Cell Pristane
SA36049121_sec-pIC-1Bone Marrow Secondary Cell Pristane
SA36049222_sec-pIC-2Bone Marrow Secondary Cell Pristane
SA36049323_sec-pIC-3Bone Marrow Secondary Cell Pristane
SA36049451_sec-C13-24H-pIC-1Bone Marrow Secondary Cell Pristane
SA36049540_sec-C13-6H-pIC-5Bone Marrow Secondary Cell Pristane
SA36049653_sec-C13-24H-pIC-3Bone Marrow Secondary Cell Pristane
SA36049754_sec-C13-24H-pIC-4Bone Marrow Secondary Cell Pristane
SA36049855_sec-C13-24H-pIC-5Bone Marrow Secondary Cell Pristane
SA36049924_sec-pIC-4Bone Marrow Secondary Cell Pristane
SA36050025_sec-pIC-5Bone Marrow Secondary Cell Pristane
SA36050137_sec-C13-6H-pIC-2Bone Marrow Secondary Cell Pristane
SA36050238_sec-C13-6H-pIC-3Bone Marrow Secondary Cell Pristane
SA36050339_sec-C13-6H-pIC-4Bone Marrow Secondary Cell Pristane
SA36050452_sec-C13-24H-pIC-2Bone Marrow Secondary Cell Pristane
Showing results 1 to 60 of 60

Collection:

Collection ID:CO003437
Collection Summary:Bone marrow derived macrophages (BMDMs) were generated by flushing one femur with 3mL of SM. BM cells were then placed into culture in T-75 flasks with BMDM media (DMEM base, 10% FBS, 1x Anti Anti (Gibco, 15240-062), 1x MEM-NEAA (Gibco, 11140-050), 1x Sodium Pyruvate (Gibco, 11360-070), 1x L-glutamine (Gibco, 25030-081) containing 50ng/mL of M-CSF (Peprotech). On day 5 of culture, adherent cells were washed three times with sterile 1x DPBS (Gibco, 14190-136) and isolated via treatment with 0.25% Trypsin (Gibco, 25200-056) for 3 minutes at 37oC before mechanical scraping. Cells were counted via hemocytometer before being seeded overnight for functional assessment. The positive generation of BMDMs in culture was determined by examination of Gr1+/CD11b+/F4/80+ cells via flow cytometry using staining cocktails described above. For BMDM differentiation in the presence of rapamycin (1µg/mL, Sigma R0395), rapamycin was added to the BMDM media at the time of initial plating. After three days of culture, rapamycin was replenished by adding it directly to the culture flask for a final concentration of 1µg/mL.
Sample Type:Bone marrow

Treatment:

Treatment ID:TR003453
Treatment Summary:BMDMs (3x105 cells) were plated and cultured for a total of 24 hours. After 18 hours of culture, the cells were stimulated with IC for the remaining 6 hours. For stable isotope-labeled U-13C6-glucose tracing experiments, the cells were cultured in BMDM media using DMEM base media without glucose that was supplemented with U-13C-glucose (Sigma Aldrich 389374) at a concentration of 4.5g/L for 24 hours, with IC stimulation starting at hour 18 for the remaining 6 hours. Subsequently, the BMDMs were washed with DPBS, all liquid was removed, and the cells were flash frozen using liquid nitrogen

Sample Preparation:

Sampleprep ID:SP003451
Sampleprep Summary:The BMDMs were then prepared for metabolomics analyses via ultra-high pressure-liquid chromatography-mass spectrometry (UHPLC-MS – Vanquish and Q Exactive, Thermo Fisher) as previously reported (Nemkov, D’Alessandro and Hansen, 2015). Briefly, cells were extracted in ice cold methanol:acetonitrile:water (5:3:2 v/v/v) at a concentration of 2x106 cells/mL of buffer. After vortexing for 30 min at 4°C, samples were centrifuged at 12,000  g for 10 min at 4°C and supernatants processed for metabolomics analyses.

Combined analysis:

Analysis ID AN005438 AN005439
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE POSITIVE
Units Intensity Counts Intensity Counts

Chromatography:

Chromatography ID:CH004126
Chromatography Summary:After sample randomization, 10 μL of extracts were injected into a Thermo Vanquish UHPLC system (San Jose, CA, USA) and resolved on a Kinetex C18 column (150 × 2.1 mm, 1.7 μm, Phenomenex, Torrance, CA, USA) at 450 μL/min through a 5 min gradient from 0 to 100% organic solvent B (mobile phases: A = 95% water, 5% acetonitrile, 1 mM ammonium acetate; B = 95% acetonitrile, 5% water, 1 mM ammonium acetate) in negative ion mode. Solvent gradient: 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B
Flow Rate:0.45 mL/min
Solvent A:95% water; 5% acetonitrile; 1mM ammonium acetate
Solvent B:95% acetonitrile; 5% water; 1 mM ammonium acetate
Chromatography Type:Reversed phase
  
Chromatography ID:CH004127
Chromatography Summary:After sample randomization, 10 μL of extracts were injected into a Thermo Vanquish UHPLC system (San Jose, CA, USA) and resolved on a Kinetex C18 column (150 × 2.1 mm, 1.7 μm, Phenomenex, Torrance, CA, USA) at 450 μL/min through a 5 min gradient from 5 to 95% organic solvent B (mobile phases: A = water, 0.1% formic acid; B = acetonitrile, 0.1% formic acid) in positive ion mode. Solvent gradient: 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B.
Flow Rate:0.45 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005164
Analysis ID:AN005438
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS Acquisition: The mass spectrometer scanned in Full MS mode at 70,000 resolution in the 65–975 m/z range, 4 kV spray voltage, 45 sheath gas and 15 auxiliary gas. Data Processing/Feature Assignment: Sample runs were converted to .mzxml files using MSConvert (ProteoWizard). Metabolite mass spectra peaks were identified (against an in-house standards library), annotated, and quantified (by peak area) using the El-Maven v0.12.0. For more details, see: Nemkov, T. et al. (2019) ‘High-Throughput Metabolomics: Isocratic and Gradient Mass Spectrometry-Based Methods’, Methods in molecular biology (Clifton, N.J.), 1978, pp. 13–26.
Ion Mode:NEGATIVE
  
MS ID:MS005165
Analysis ID:AN005439
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS Acquisition: The mass spectrometer scanned in Full MS mode at 70,000 resolution in the 65–975 m/z range, 4 kV spray voltage, 45 sheath gas and 15 auxiliary gas. Data Processing/Feature Assignment: Sample runs were converted to .mzxml files using MSConvert (ProteoWizard). Metabolite mass spectra peaks were identified (against an in-house standards library), annotated and quantified (by peak area) using the El-Maven v0.12.0. For more details, see: Nemkov, T. et al. (2019) ‘High-Throughput Metabolomics: Isocratic and Gradient Mass Spectrometry-Based Methods’, Methods in molecular biology (Clifton, N.J.), 1978, pp. 13–26.
Ion Mode:POSITIVE
  logo