Summary of Study ST003603

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 IDST003603
Study TitleComparison of metabolic responses in murine bone marrow derived macrophages upon LPS treatment versus following the engulfment of live or dead E coli.
Study SummaryGlobal metabolomics and 13C-tracing analysis of murine bone marrow derived macrophages exposed to live or heat-killed uniformly 13C-labelled E. coli in a trans-well system for 6h or 18h. Global metabolomics was performed in parallel on murine BMDMs treated with 500 ng/ml LPS for 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:SU003732
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 Sample source Timepoint (h) LPS treatment (ng/ml) E coli viability Multiplicity of infection well vs insert source
SA392565164murine macrophage cells 18 0 dead 25 NA
SA392566163murine macrophage cells 18 0 dead 25 NA
SA392567162murine macrophage cells 18 0 dead 25 NA
SA392568186murine macrophage cells 18 0 dead 50 well
SA392569188murine macrophage cells 18 0 dead 50 well
SA392570187murine macrophage cells 18 0 dead 50 well
SA392571159murine macrophage cells 18 0 live 25 NA
SA392572160murine macrophage cells 18 0 live 25 NA
SA392573161murine macrophage cells 18 0 live 25 NA
SA392559177murine macrophage cells 18 0 NA NA insert
SA392560179murine macrophage cells 18 0 NA NA insert
SA392561178murine macrophage cells 18 0 NA NA insert
SA392556157murine macrophage cells 18 0 NA NA NA
SA392557156murine macrophage cells 18 0 NA NA NA
SA392558158murine macrophage cells 18 0 NA NA NA
SA392562174murine macrophage cells 18 0 NA NA well
SA392563175murine macrophage cells 18 0 NA NA well
SA392564176murine macrophage cells 18 0 NA NA well
SA392574166murine macrophage cells 18 500 NA NA NA
SA392575165murine macrophage cells 18 500 NA NA NA
SA392576167murine macrophage cells 18 500 NA NA NA
SA392580182murine macrophage cells 6 0 dead 50 well
SA392581180murine macrophage cells 6 0 dead 50 well
SA392582181murine macrophage cells 6 0 dead 50 well
SA392577183murine macrophage cells 6 0 NA NA insert
SA392578184murine macrophage cells 6 0 NA NA insert
SA392579185murine macrophage cells 6 0 NA NA insert
Showing results 1 to 27 of 27

Collection:

Collection ID:CO003725
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:TR003741
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 to stimulation in 6-well plate and insert of a trans-well system (Falcon). Cells in wells were stimulated with live or killed labelled-E. coli at MOI 50 or with 500 ng/mL LPS. Cells in inserts were left untreated. Plates were 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:SP003739
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 AN005919 AN005920
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:CH004494
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:CH004495
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:MS005636
Analysis ID:AN005919
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:MS005637
Analysis ID:AN005920
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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