Summary of Study ST003606

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 IDST003606
Study TitleGlobal metabolomics and tracing of E coli-derived metabolites following engulfment of live or heat-killed bacteria by bone marrow derived macrophages expressing constitutively active mutants of Kras or Hras
Study SummaryWe sought to identify possible mechanisms governing the recycling of microbially derived nutrients produced by the degradation of killed E coli in macrophages. Recent studies have demonstrated that extracellular protein utilization supports the cellular metabolism of cancer cells expressing activated Ras pathways. Here we tested the involvement of Kras and Hras pathway in metabolic recycling of microbial nutrients upon phagocytosis by macrophages using global and 13C-tracing metabolomics in bone marrow derived macrophages exposed to 13C-labeled E coli.
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

Select appropriate tab below to view additional metadata details:

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:SU003735
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) E coli treatment genotype Sample source
SA3926631-89-65-18 dead Cre KRAS +/+ murine bone marrow derived macrophages
SA3926641-89-64-18 dead Cre KRAS +/+ murine bone marrow derived macrophages
SA3926651-89-63-18 dead Cre KRAS +/+ murine bone marrow derived macrophages
SA3926661-89-80-18 dead Cre murine bone marrow derived macrophages
SA3926671-89-79-18 dead Cre murine bone marrow derived macrophages
SA3926681-89-78-18 dead Cre murine bone marrow derived macrophages
SA3926691-89-50-18 dead HRAS +/+ murine bone marrow derived macrophages
SA3926701-89-49-18 dead HRAS +/+ murine bone marrow derived macrophages
SA3926711-89-48-18 dead HRAS +/+ murine bone marrow derived macrophages
SA3926721-89-19-18 dead WT murine bone marrow derived macrophages
SA3926731-89-35-18 dead WT murine bone marrow derived macrophages
SA3926741-89-20-18 dead WT murine bone marrow derived macrophages
SA3926751-89-18-18 dead WT murine bone marrow derived macrophages
SA3926761-89-33-18 dead WT murine bone marrow derived macrophages
SA3926771-89-17-18 dead WT murine bone marrow derived macrophages
SA3926781-89-34-18 dead WT murine bone marrow derived macrophages
SA3926791-89-60-18 live Cre KRAS +/+ murine bone marrow derived macrophages
SA3926801-89-61-18 live Cre KRAS +/+ murine bone marrow derived macrophages
SA3926811-89-62-18 live Cre KRAS +/+ murine bone marrow derived macrophages
SA3926821-89-75-18 live Cre murine bone marrow derived macrophages
SA3926831-89-76-18 live Cre murine bone marrow derived macrophages
SA3926841-89-77-18 live Cre murine bone marrow derived macrophages
SA3926851-89-46-18 live HRAS +/+ murine bone marrow derived macrophages
SA3926861-89-45-18 live HRAS +/+ murine bone marrow derived macrophages
SA3926871-89-47-18 live HRAS +/+ murine bone marrow derived macrophages
SA3926881-89-30-18 live WT murine bone marrow derived macrophages
SA3926891-89-32-18 live WT murine bone marrow derived macrophages
SA3926901-89-16-18 live WT murine bone marrow derived macrophages
SA3926911-89-13-18 live WT murine bone marrow derived macrophages
SA3926921-89-31-18 live WT murine bone marrow derived macrophages
SA3926931-89-14-18 live WT murine bone marrow derived macrophages
SA3926941-89-15-18 live WT murine bone marrow derived macrophages
SA3926951-89-53-18 none Cre KRAS +/+ murine bone marrow derived macrophages
SA3926961-89-52-18 none Cre KRAS +/+ murine bone marrow derived macrophages
SA3926971-89-51-18 none Cre KRAS +/+ murine bone marrow derived macrophages
SA3926981-89-66-18 none Cre murine bone marrow derived macrophages
SA3926991-89-67-18 none Cre murine bone marrow derived macrophages
SA3927001-89-68-18 none Cre murine bone marrow derived macrophages
SA3927011-89-36-18 none HRAS +/+ murine bone marrow derived macrophages
SA3927021-89-37-18 none HRAS +/+ murine bone marrow derived macrophages
SA3927031-89-38-18 none HRAS +/+ murine bone marrow derived macrophages
SA3927041-89-23-18 none WT murine bone marrow derived macrophages
SA3927051-89-21-18 none WT murine bone marrow derived macrophages
SA3927061-89-22-18 none WT murine bone marrow derived macrophages
SA3927071-89-57-6 dead Cre KRAS +/+ murine bone marrow derived macrophages
SA3927081-89-59-6 dead Cre KRAS +/+ murine bone marrow derived macrophages
SA3927091-89-58-6 dead Cre KRAS +/+ murine bone marrow derived macrophages
SA3927101-89-74-6 dead Cre murine bone marrow derived macrophages
SA3927111-89-73-6 dead Cre murine bone marrow derived macrophages
SA3927121-89-72-6 dead Cre murine bone marrow derived macrophages
SA3927131-89-43-6 dead HRAS +/+ murine bone marrow derived macrophages
SA3927141-89-44-6 dead HRAS +/+ murine bone marrow derived macrophages
SA3927151-89-42-6 dead HRAS +/+ murine bone marrow derived macrophages
SA3927161-89-9-6 dead WT murine bone marrow derived macrophages
SA3927171-89-10-6 dead WT murine bone marrow derived macrophages
SA3927181-89-11-6 dead WT murine bone marrow derived macrophages
SA3927191-89-12-6 dead WT murine bone marrow derived macrophages
SA3927201-89-29-6 dead WT murine bone marrow derived macrophages
SA3927211-89-28-6 dead WT murine bone marrow derived macrophages
SA3927221-89-27-6 dead WT murine bone marrow derived macrophages
SA3927231-89-56-6 live Cre KRAS +/+ murine bone marrow derived macrophages
SA3927241-89-54-6 live Cre KRAS +/+ murine bone marrow derived macrophages
SA3927251-89-55-6 live Cre KRAS +/+ murine bone marrow derived macrophages
SA3927261-89-71-6 live Cre murine bone marrow derived macrophages
SA3927271-89-69-6 live Cre murine bone marrow derived macrophages
SA3927281-89-70-6 live Cre murine bone marrow derived macrophages
SA3927291-89-39-6 live HRAS +/+ murine bone marrow derived macrophages
SA3927301-89-40-6 live HRAS +/+ murine bone marrow derived macrophages
SA3927311-89-41-6 live HRAS +/+ murine bone marrow derived macrophages
SA3927321-89-26-6 live WT murine bone marrow derived macrophages
SA3927331-89-25-6 live WT murine bone marrow derived macrophages
SA3927341-89-24-6 live WT murine bone marrow derived macrophages
SA3927351-89-6-6 live WT murine bone marrow derived macrophages
SA3927361-89-8-6 live WT murine bone marrow derived macrophages
SA3927371-89-7-6 live WT murine bone marrow derived macrophages
SA3927381-89-5-6 live WT murine bone marrow derived macrophages
Showing results 1 to 76 of 76

Collection:

Collection ID:CO003728
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:TR003744
Treatment Summary:Tg.hUbC-cre-8 ERT2+/T::Kras+/G12V mice were originally generated in the laboratory of M. Barbacid and provided by D. Santamaria. HrasG12S mice were originally generated at the Institut Clinique de la Souris (ICS) and provided by R. Rossignol. All transgenic mice were on a C57BL/6N background. We used 8- to 10-week-old male or female animals for all experiments and did not observe any gender bias. 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). For Tg.hUbC-cre-ERT2+/T::Rragaf/f and Tg.hUbC-cre-ERT2+/T::Kras+/G12V mice, tamoxifen was added to the culture on day 2 or 3 to a final concentration of 1 µg/ml in order to induce CRE expression. 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 live or 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.

Sample Preparation:

Sampleprep ID:SP003742
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 AN005925 AN005926
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:CH004500
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:CH004501
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:MS005642
Analysis ID:AN005925
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:MS005643
Analysis ID:AN005926
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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