Summary of Study ST003605

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.

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 IDST003605
Study TitleGlobal metabolomics and tracing of E coli-derived metabolites following engulfment of heat-killed bacteria by wild type or RagAGTP-deficient bone marrow derived macrophages.
Study SummaryGlobal metabolomics and 13C-tracing analysis of wild type or RagA-deficient 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

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:SU003734
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 genotype treatment time (h) Sample source
SA392627Dead 18h 479 n°3Cre RagA KO dead bacteria 18 murine bone marrow derived macrophages
SA392628Dead 18h 479 n°2Cre RagA KO dead bacteria 18 murine bone marrow derived macrophages
SA392629Dead 18h 479 n°1Cre RagA KO dead bacteria 18 murine bone marrow derived macrophages
SA392630Dead 18h 477 n°3Cre RagA KO dead bacteria 18 murine bone marrow derived macrophages
SA392631Dead 18h 477 n°2Cre RagA KO dead bacteria 18 murine bone marrow derived macrophages
SA392632Dead 18h 477 n°1Cre RagA KO dead bacteria 18 murine bone marrow derived macrophages
SA392633Dead 6h 477 n°3Cre RagA KO dead bacteria 6 murine bone marrow derived macrophages
SA392634Dead 6h 477 n°1Cre RagA KO dead bacteria 6 murine bone marrow derived macrophages
SA392635Dead 6h 477 n°2Cre RagA KO dead bacteria 6 murine bone marrow derived macrophages
SA392636Dead 6h 479 n°2Cre RagA KO dead bacteria 6 murine bone marrow derived macrophages
SA392637Dead 6h 479 n°1Cre RagA KO dead bacteria 6 murine bone marrow derived macrophages
SA392638Dead 6h 479 n°3Cre RagA KO dead bacteria 6 murine bone marrow derived macrophages
SA392639CT 477 n°2Cre RagA KO none 18 murine bone marrow derived macrophages
SA392640CT 479 n°3Cre RagA KO none 18 murine bone marrow derived macrophages
SA392641CT 479 n°2Cre RagA KO none 18 murine bone marrow derived macrophages
SA392642CT 479 n°1Cre RagA KO none 18 murine bone marrow derived macrophages
SA392643CT 477 n°3Cre RagA KO none 18 murine bone marrow derived macrophages
SA392644CT 477 n°1Cre RagA KO none 18 murine bone marrow derived macrophages
SA392645Dead 18h 390 n°2WT dead bacteria 18 murine bone marrow derived macrophages
SA392646Dead 18h 390 n°1WT dead bacteria 18 murine bone marrow derived macrophages
SA392647Dead 18h 389 n°3WT dead bacteria 18 murine bone marrow derived macrophages
SA392648Dead 18h 389 n°2WT dead bacteria 18 murine bone marrow derived macrophages
SA392649Dead 18h 389 n°1WT dead bacteria 18 murine bone marrow derived macrophages
SA392650Dead 18h 390 n°3WT dead bacteria 18 murine bone marrow derived macrophages
SA392651Dead 6h 390 n°1WT dead bacteria 6 murine bone marrow derived macrophages
SA392652Dead 6h 390 n°3WT dead bacteria 6 murine bone marrow derived macrophages
SA392653Dead 6h 390 n°2WT dead bacteria 6 murine bone marrow derived macrophages
SA392654Dead 6h 389 n°3WT dead bacteria 6 murine bone marrow derived macrophages
SA392655Dead 6h 389 n°2WT dead bacteria 6 murine bone marrow derived macrophages
SA392656Dead 6h 389 n°1WT dead bacteria 6 murine bone marrow derived macrophages
SA392657CT 390 n°1WT none 18 murine bone marrow derived macrophages
SA392658CT 389 n°3WT none 18 murine bone marrow derived macrophages
SA392659CT 389 n°2WT none 18 murine bone marrow derived macrophages
SA392660CT 390 n°2WT none 18 murine bone marrow derived macrophages
SA392661CT 390 n°3WT none 18 murine bone marrow derived macrophages
SA392662CT 389 n°1WT none 18 murine bone marrow derived macrophages
Showing results 1 to 36 of 36

Collection:

Collection ID:CO003727
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:TR003743
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). For Tg.hUbC-cre-ERT2+/T::Rragaf/f and RagAGTP/delta 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:SP003741
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 AN005923 AN005924
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:CH004498
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:CH004499
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:MS005640
Analysis ID:AN005923
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:MS005641
Analysis ID:AN005924
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
  logo