Summary of Study ST003604

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 PR002231. The data can be accessed directly via it's Project DOI: 10.21228/M87R79 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST003604
Study TitleInvestigation of global metabolites and E coli-derived metabolites following engulfment of dead bacteria by wild type or RagAGTP-expressing bone marrow derived macrophages.
Study SummaryGlobal metabolomics and 13C-tracing analysis of wild type or RagAGTP-expressing bone marrow derived macrophages exposed to heat-killed uniformly 13C-labelled E. coli in a trans-well system for 6h or 18h.
Institute
University of Colorado Anschutz Medical Campus
Last NameHaines
First NameJulie
Address12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Emailjulie.haines@cuanschutz.edu
Phone3037243339
Submit Date2024-11-25
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-12-27
Release Version1
Julie Haines Julie Haines
https://dx.doi.org/10.21228/M87R79
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002231
Project DOI:doi: 10.21228/M87R79
Project Title:Macrophages recycle phagocytosed bacteria to fuel immunometabolic responses
Project Summary:Macrophages specialize in phagocytosis, a cellular process that eliminates extracellular matter, including microbes, through internalization and degradation. Despite the critical role of phagocytosis during bacterial infection, the fate of phagocytosed microbial cargo and its impact on host cell is poorly understood. Here, we reveal that ingested bacteria constitute an alternative nutrient source that skews immunometabolic host responses. Tracing stable isotope-labelled bacteria, we found that phagolysosomal degradation of bacteria provides carbon atoms and amino acids that are recycled into various metabolic pathways, including glutathione and itaconate biosynthesis, and satisfy macrophage bioenergetic needs. Metabolic recycling of microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and intricately tied to microbial viability. Dead bacteria, as opposed to live ones, are enriched in cyclic- adenosine monophosphate (AMP), sustain the cellular AMP pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1. Consequently, killed bacteria strongly fuel metabolic recycling and support macrophage survival, but elicit decreased reactive oxygen species (ROS) production and a reduced IL-1β secretion compared to viable bacteria. These results reveal a novel insight into the fate of engulfed microbes and highlights a microbial viability-associated metabolite that triggers host metabolic and immune responses. Our findings hold promise for shaping immunometabolic intervention in various immune-related pathologies.
Institute:University of Colorado Anschutz Medical Campus
Laboratory:Lab of Angelo D'Alessandro in collaboration with lab of Johan Garaude (INSERM, Fr)
Last Name:Haines
First Name:Julie
Address:12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email:julie.haines@cuanschutz.edu
Phone:3037243339

Subject:

Subject ID:SU003733
Subject Type:Cultured cells
Subject Species:Mus musculus
Gender:Not applicable

Factors:

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

mb_sample_id local_sample_id time (h) Dead E coli treatment inhibitor Sample source genotype
SA392583CT RagA GTP 10 no none murine bone marrow derived macrophages RagA GTP mutant
SA392584CT RagA GTP 40 no none murine bone marrow derived macrophages RagA GTP mutant
SA392585CT RagA GTP 30 no none murine bone marrow derived macrophages RagA GTP mutant
SA392586CT RagA GTP 20 no none murine bone marrow derived macrophages RagA GTP mutant
SA392587CT WT 4 + Conca0 no none murine bone marrow derived macrophages WT
SA392588CT WT 3 + Conca0 no none murine bone marrow derived macrophages WT
SA392589CT WT 2 + Conca0 no none murine bone marrow derived macrophages WT
SA392590CT WT 1 + Conca0 no none murine bone marrow derived macrophages WT
SA392591CT WT 20 no none murine bone marrow derived macrophages WT
SA392592CT WT 10 no none murine bone marrow derived macrophages WT
SA392593CT WT 30 no none murine bone marrow derived macrophages WT
SA392594CT WT 40 no none murine bone marrow derived macrophages WT
SA392595WT 4 18h + Conca18 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392596WT3 18h + Conca18 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392597WT 2 18h + Conca18 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392598WT 1 18h + Conca18 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392599WT 4 18h + Bafilo18 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392600WT3 18h + Bafilo18 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392601WT 2 18h + Bafilo18 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392602WT 1 18h + Bafilo18 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392603RagA GTP 3 18h18 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392604RagA GTP 1 18h18 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392605RagA GTP 2 18h18 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392606RagA GTP 4 18h18 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392607WT3 18h18 yes none murine bone marrow derived macrophages WT
SA392608WT 4 18h18 yes none murine bone marrow derived macrophages WT
SA392609WT 1 18h18 yes none murine bone marrow derived macrophages WT
SA392610WT 2 18h18 yes none murine bone marrow derived macrophages WT
SA392611WT 1 6h + Conca6 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392612WT 2 6h + Conca6 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392613WT 3 6h + Conca6 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392614WT 4 6h + Conca6 yes 150 nM concanamycin A murine bone marrow derived macrophages WT
SA392615WT 2 6h + Bafilo6 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392616WT 4 6h + Bafilo6 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392617WT 3 6h + Bafilo6 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392618WT 1 6h + Bafilo6 yes 50 nM bafilomycin A1 murine bone marrow derived macrophages WT
SA392619RagA GTP 2 6h6 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392620RagA GTP 3 6h6 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392621RagA GTP 1 6h6 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392622RagA GTP 4 6h6 yes none murine bone marrow derived macrophages RagA GTP mutant
SA392623WT 2 6h6 yes none murine bone marrow derived macrophages WT
SA392624WT 4 6h6 yes none murine bone marrow derived macrophages WT
SA392625WT 3 6h6 yes none murine bone marrow derived macrophages WT
SA392626WT 1 6h6 yes none murine bone marrow derived macrophages WT
Showing results 1 to 44 of 44

Collection:

Collection ID:CO003726
Collection Summary:BMDMs were seeded at 2.5E6 cell/well in a non-tissue treated 6-well plate (2 wells per condition) the day before the experiment. To harvest, BMDMs were washed with cold PBS and harvested with 5 mM EDTA in PBS, frozen as dry cell pellet, and stored at -80˚C until processing.
Sample Type:Macrophages

Treatment:

Treatment ID:TR003742
Treatment Summary:Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were generated as described previously, in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Gibco). BMDMs were used on day 5 to 7 after seeding. Period of differentiation of the cells, concentration of cells when replating and time-lapse between replating and stimulation with bacteria were important parameters to maintain metabolic backgrounds homogenous between experiments. Preparation of viable and killed U-[13C]Bacteria: ThyA- E. coli were grown overnight with shaking in LB supplemented with thymidine (500 µg/ml) and trimethoprim (50 µg/ml), diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts before addition to cells. For labeling of bacteria, 10 µl of an overnight cultured of thyA- E. coli was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, 500 µg/ml thymidine, 50 µg/ml trimethoprim, and 0.5% U-[13C6] glucose (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For antibiotic killing, bacteria were incubated for 6h to 12h with Streptomycin or Gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. Treatment of macrophages: 2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated 6-well plate (BD Falcons). Cells were then stimulated with killed labelled-E. coli at MOI 50, centrifuged at 2000 rpm for 5min. BMDMs were incubated for 5 min and washed with PBS to remove non-ingested bacteria and further incubated for 6h or 18h. To inhibit lysosomal degradation, cells were treated with 50 nM Bafilomycin A1. To inhibit v-ATPase, cells were treated with 150 nM concanamycin A. All the above inhibitors were added to the cultured medium at least 2h before stimulation of the BMDMs.

Sample Preparation:

Sampleprep ID:SP003740
Sampleprep Summary:Metabolites from frozen pellets were extracted at 4e6 cells per mL using ice cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 degrees C followed by centrifugation as described for 10 min at 18,000 g. Supernatants were maintained at 4°C until analysis that same day.
Processing Storage Conditions:4℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN005921 AN005922
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 peak area peak area

Chromatography:

Chromatography ID:CH004496
Chromatography Summary:Negative C18
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
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:450 uL/min
Sample Injection:6 uL
Solvent A:95% water/5% acetonitrile; 1 mM ammonium acetate
Solvent B:95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:Reversed phase
  
Chromatography ID:CH004497
Chromatography Summary:Positive C18
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
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:450 uL/min
Sample Injection:6 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005638
Analysis ID:AN005921
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:NEGATIVE
  
MS ID:MS005639
Analysis ID:AN005922
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:POSITIVE
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