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MTBLS23:  Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation

 Authors: Thomas Metz , Charles Ansong , Joshua Adkins

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  Submitted: 24-Aug-2012 , Release date: 05-Sep-2012 , Update date: 17-Dec-2015

 Submitted by:  Tom Metz  |   Study status: Public

Study Description

Macrophages are central players in immune response, manifesting divergent phenotypes to control inflammation and innate immunity through release of cytokines and other signaling factors. Recently, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features that are critical for macrophage activation. A genome-scale metabolic network for the RAW 264.7 cell line was constructed to determine metabolic modulators of activation. Metabolites well-known to be associated with immunoactivation (glucose and arginine) and immunosuppression (tryptophan and vitamin D3) were among the most critical effectors. Intracellular metabolic mechanisms were assessed, identifying a suppressive role for de-novo nucleotide synthesis. Finally, underlying metabolic mechanisms of macrophage activation were identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. This study demonstrates that the role of metabolism in regulating activation may be greater than previously anticipated and elucidates underlying connections between activation and metabolic effectors. This submission corresponds to the metabolomics data from this study.

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  Organism(s)

Mus musculus

  Study Design Description

untargeted metabolites

CHMO:gas chromatography-mass spectrometry

immunometabolism

endometabolome

genome-scale modeling

metabolic network reconstruction

  Experimental Factors

Treatment

Protocol Description
Sample collection Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, then grown for 2 days and stimulated for 24 hours with 100 ng/ml of lipopolysaccharide diluted in fresh medium. A control culture was run in parallel by incubating for the same period of time with fresh medium only. Three biological replicates were performed per condition, and two dishes were used for each replicate. After stimulation, cells were washed twice with Dulbecco’s PBS, scraped out and harvested into 15-ml centrifuge tubes.
Extraction Cell suspensions were softly centrifuged (230 × g for 5 min) and as much buffer as possible was removed. Then, 170 µl of 150 mM ammonium bicarbonate was added to the cell pellet and the cell suspensions were transferred to 2 ml micro-centrifuge tubes for extraction. Subsequently, the water soluble metabolites were extracted with four volumes of chilled (-20 °C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator.

Metabolite extracts were evaporated to dryness using a vacuum concentrator. For chemical derivatization, 30 µl of methoxyamine in pyridine (30 mg/ml) were added to each sample, followed by incubation at 37 ºC with shaking for 90 min to protect carbonyl groups. Next, 120 µl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37 ºC with shaking for 30 min to derivatize hydroxyl and amine groups. The samples were then allowed to cool to room temperature prior to analysis by GC-MS.
Chromatography Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m × 0.25 mm × 0.25 μm film thickness).
Mass spectrometry Samples were analyzed in duplicate using an Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Santa Clara, CA) using a HP-5MS column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies). One microliter of each sample was injected in splitless mode and with the port temperature held at 250 °C throughout analysis. The GC oven was initially maintained at 60 °C for 1 min and then ramped to 325 °C by 10 °C/min and kept at this final temperature for 5 min to complete an analysis cycle.
Data transformation After converting raw data to netCDF format, the data were processed by the MetaboliteDetector software for peak deconvolution and chromatographic alignment. Retention indices (RI) were calculated based on the analysis of a mixture of fatty acid methyl esters (C8 - C30) (Agilent Technologies) as external retention time standards, then their retention index information was subsequently applied to all experimental chromatograms for retention time alignment. MetaboliteDetector parameters for peak detection and deconvolution are as follows: Peak threshold, 7; minimum peak height, 7; deconvolution width, 8.
Metabolite identification Deconvoluted features were identified by matching to the Agilent Fiehn Metabolomics Retention Time Locked Library, which contains mass spectral and retention indix information for approximately 700 metabolites. Each initial match to the library was manually inspected to confirm a confident identification. The data were also matched against the NIST 08 library to identify additional peaks not included in the Fiehn library. MetaboliteDetector software was used for database matching and batch identification/quantification parameters are as follows: required score, 0.6; ΔRI, 25; minimum S/N, 20; maximum peak discrepancy index, 100. Ions 73 and 143 were excluded from use as metabolite quantification ions, since these are due to fragmentation of the trimethylsilyl groups. Otherwise, three unique fragment ions were assigned to each metabolite for quantification and used for each individual GC-MS analysis when processing the data in batch mode. The summed areas of the three quantification ions were exported from MetaboliteDetector and used in further statistical anayses. All identifications were manually validated by inspection of retention index and spectrum matches.
Source Name Organism Organism part Protocol REF Sample Name Treatment
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_Control1_A Control
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_Control1_B Control
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_Control2_A Control
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_Control2_B Control
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_Control3_A Control
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_Control3_B Control
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_LPSactivated1_A Lipopolysaccharide stimulated
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_LPSactivated1_B Lipopolysaccharide stimulated
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_LPSactivated2_A Lipopolysaccharide stimulated
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_LPSactivated2_B Lipopolysaccharide stimulated
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_LPSactivated3_A Lipopolysaccharide stimulated
ATTC Mus musculus RAW-264.7 cell Sample collection SBEP_Metab_LPSactivated3_B Lipopolysaccharide stimulated
Validations marked with (*) have been allowed by the MetaboLights Curators.
Click here for the detailed description of Validations.
Condition Status Description Requirement Group Message
PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
PASSES Study text successfully parsed OPTIONAL STUDY OK
PASSES Study Contact(s) have listed email MANDATORY CONTACT OK
PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
PASSES Publication(s) associated with this Study MANDATORY PUBLICATION OK
PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Sample Collection protocol MANDATORY PROTOCOLS OK
PASSES Protocols text successfully parsed OPTIONAL PROTOCOLS OK
PASSES Organism name MANDATORY ORGANISM OK
PASSES Organism part MANDATORY ORGANISM OK
PASSES Study Factors MANDATORY FACTORS OK
PASSES Assay platform information OPTIONAL ASSAYS OK
PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
PASSES Raw files in the Assay(s) have the correct format MANDATORY FILES OK
PASSES Assay(s) MANDATORY ASSAYS OK
PASSES All Assays have Metabolite Assignment File (MAF) referenced OPTIONAL FILES OK
PASSES Metabolite Assignment File (MAF) is present in Study folder MANDATORY FILES OK
PASSES Metabolite Assignment File (MAF) has correct format MANDATORY FILES OK
PASSES Metabolite Identification File (MAF) content MANDATORY FILES OK
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Assay 

Assay file name: a_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry.txt
Measurement: metabolite profiling
Technology: mass spectrometry
Platform: 5975C Series GC/MSD (Agilent)

Data

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
SBEP_Metab_Control1_A Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_Control1_A Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_Control1_A.D SBEP_Metab_Control1_A.D.zip Data transformation SBEP_Metab_Control1_A.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_Control1_B Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_Control1_B Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_Control1_B.D SBEP_Metab_Control1_B.D.zip Data transformation SBEP_Metab_Control1_B.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_Control2_A Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_Control2_A Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_Control2_A.D SBEP_Metab_Control2_A.D.zip Data transformation SBEP_Metab_Control2_A.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_Control2_B Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_Control2_B Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_Control2_B.D SBEP_Metab_Control2_B.D.zip Data transformation SBEP_Metab_Control2_B.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_Control3_A Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_Control3_A Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_Control3_A.D SBEP_Metab_Control3_A.D.zip Data transformation SBEP_Metab_Control3_A.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_Control3_B Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_Control3_B Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_Control3_B.D SBEP_Metab_Control3_B.D.zip Data transformation SBEP_Metab_Control3_B.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_LPSactivated1_A Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_LPSactivated1_A Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_LPSactivated1_A.D SBEP_Metab_LPSactivated1_A.D.zip Data transformation SBEP_Metab_LPSactivated1_A.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_LPSactivated1_B Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_LPSactivated1_B Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_LPSactivated1_B.D SBEP_Metab_LPSactivated1_B.D.zip Data transformation SBEP_Metab_LPSactivated1_B.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_LPSactivated2_A Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_LPSactivated2_A Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_LPSactivated2_A.D SBEP_Metab_LPSactivated2_A.D.zip Data transformation SBEP_Metab_LPSactivated2_A.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_LPSactivated2_B Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_LPSactivated2_B Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_LPSactivated2_B.D SBEP_Metab_LPSactivated2_B.D.zip Data transformation SBEP_Metab_LPSactivated2_B.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_LPSactivated3_A Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_LPSactivated3_A Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_LPSactivated3_A.D SBEP_Metab_LPSactivated3_A.D.zip Data transformation SBEP_Metab_LPSactivated3_A.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
SBEP_Metab_LPSactivated3_B Extraction After extraction with cold (-20°C) chloroform/methanol (2:1, v/v) in a 4-fold excess over the sample volume, water- and lipid-soluble extracts were dried in vacuo. sylilation SBEP_Metab_LPSactivated3_B Chromatography Agilent 7890A GC HP-5ms GC (0.25 µm, 0.25 mm x 30 m; Agilent Technologies) low polarity Mass spectrometry positive 50-550 Agilent 5975C MSD MS:electron ionization quadrupole SBEP_Metab_LPSactivated3_B.D SBEP_Metab_LPSactivated3_B.D.zip Data transformation SBEP_Metab_LPSactivated3_B.CDF Metabolite identification m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv

Pathways - Assay 



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)
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Aspera Download Details:

List of study files   Subset

File
audit
metexplore_mapping.json
MacrophageMetabolicImmunomodulators_MetaboliteAssignment.xlsx
SBEP_Metab_Control1_A.CDF
SBEP_Metab_Control1_A.D.zip
SBEP_Metab_Control1_B.CDF
SBEP_Metab_Control1_B.D.zip
SBEP_Metab_Control2_A.CDF
SBEP_Metab_Control2_A.D.zip
SBEP_Metab_Control2_B.CDF
SBEP_Metab_Control2_B.D.zip
SBEP_Metab_Control3_A.CDF
SBEP_Metab_Control3_A.D.zip
SBEP_Metab_Control3_B.CDF
SBEP_Metab_Control3_B.D.zip
SBEP_Metab_LPSactivated1_A.CDF
SBEP_Metab_LPSactivated1_A.D.zip
SBEP_Metab_LPSactivated1_B.CDF
SBEP_Metab_LPSactivated1_B.D.zip
SBEP_Metab_LPSactivated2_A.CDF
SBEP_Metab_LPSactivated2_A.D.zip
SBEP_Metab_LPSactivated2_B.CDF
SBEP_Metab_LPSactivated2_B.D.zip
SBEP_Metab_LPSactivated3_A.CDF
SBEP_Metab_LPSactivated3_A.D.zip
SBEP_Metab_LPSactivated3_B.CDF
SBEP_Metab_LPSactivated3_B.D.zip
i_Investigation.txt
a_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry.txt
a_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_maf.csv
~$MacrophageMetabolicImmunomodulators_MetaboliteAssignment.xlsx
m_model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation_metabolite profiling_mass spectrometry_v2_maf.tsv
s_Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation.txt

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