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.
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.
Study ID | ST003604 |
Study Title | Investigation of global metabolites and E coli-derived metabolites following engulfment of dead bacteria by wild type or RagAGTP-expressing bone marrow derived macrophages. |
Study Summary | Global 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 Name | Haines |
First Name | Julie |
Address | 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA |
julie.haines@cuanschutz.edu | |
Phone | 3037243339 |
Submit Date | 2024-11-25 |
Raw Data Available | Yes |
Raw Data File Type(s) | raw(Thermo) |
Analysis Type Detail | LC-MS |
Release Date | 2024-12-27 |
Release Version | 1 |
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: | 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 |
---|---|---|---|---|---|---|
SA392583 | CT RagA GTP 1 | 0 | no | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392584 | CT RagA GTP 4 | 0 | no | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392585 | CT RagA GTP 3 | 0 | no | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392586 | CT RagA GTP 2 | 0 | no | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392587 | CT WT 4 + Conca | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392588 | CT WT 3 + Conca | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392589 | CT WT 2 + Conca | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392590 | CT WT 1 + Conca | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392591 | CT WT 2 | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392592 | CT WT 1 | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392593 | CT WT 3 | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392594 | CT WT 4 | 0 | no | none | murine bone marrow derived macrophages | WT |
SA392595 | WT 4 18h + Conca | 18 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392596 | WT3 18h + Conca | 18 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392597 | WT 2 18h + Conca | 18 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392598 | WT 1 18h + Conca | 18 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392599 | WT 4 18h + Bafilo | 18 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392600 | WT3 18h + Bafilo | 18 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392601 | WT 2 18h + Bafilo | 18 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392602 | WT 1 18h + Bafilo | 18 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392603 | RagA GTP 3 18h | 18 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392604 | RagA GTP 1 18h | 18 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392605 | RagA GTP 2 18h | 18 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392606 | RagA GTP 4 18h | 18 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392607 | WT3 18h | 18 | yes | none | murine bone marrow derived macrophages | WT |
SA392608 | WT 4 18h | 18 | yes | none | murine bone marrow derived macrophages | WT |
SA392609 | WT 1 18h | 18 | yes | none | murine bone marrow derived macrophages | WT |
SA392610 | WT 2 18h | 18 | yes | none | murine bone marrow derived macrophages | WT |
SA392611 | WT 1 6h + Conca | 6 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392612 | WT 2 6h + Conca | 6 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392613 | WT 3 6h + Conca | 6 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392614 | WT 4 6h + Conca | 6 | yes | 150 nM concanamycin A | murine bone marrow derived macrophages | WT |
SA392615 | WT 2 6h + Bafilo | 6 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392616 | WT 4 6h + Bafilo | 6 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392617 | WT 3 6h + Bafilo | 6 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392618 | WT 1 6h + Bafilo | 6 | yes | 50 nM bafilomycin A1 | murine bone marrow derived macrophages | WT |
SA392619 | RagA GTP 2 6h | 6 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392620 | RagA GTP 3 6h | 6 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392621 | RagA GTP 1 6h | 6 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392622 | RagA GTP 4 6h | 6 | yes | none | murine bone marrow derived macrophages | RagA GTP mutant |
SA392623 | WT 2 6h | 6 | yes | none | murine bone marrow derived macrophages | WT |
SA392624 | WT 4 6h | 6 | yes | none | murine bone marrow derived macrophages | WT |
SA392625 | WT 3 6h | 6 | yes | none | murine bone marrow derived macrophages | WT |
SA392626 | WT 1 6h | 6 | 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 |