Summary of Study ST003610

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	ST003610
Study Title	13C-tracing metabolomics of peritoneal macrophages 1hour and 2hours after engulfment of 13C-labeled heat-killed E coli
Study Summary	13C-tracing analysis at 1h and 2h of peritoneal macrophages isolated from C57BL6/N mice injected with uniformly 13C-labelled heat-killed E. coli.
Institute	University of Colorado Anschutz Medical Campus
Last Name	Haines
First Name	Julie
Address	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email	julie.haines@cuanschutz.edu
Phone	3037243339
Submit Date	2024-11-26
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:	SU003739
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	Killed E coli labeling	Timepoint	Sample source
SA392771	AA83PM-5_r9-	13C-labelled KEC	1h	murine peritoneal macrophage cells
SA392772	AA83PM-2_r10-	13C-labelled KEC	1h	murine peritoneal macrophage cells
SA392773	AA83PM-1_r19-	13C-labelled KEC	1h	murine peritoneal macrophage cells
SA392774	AA83PM-3_r17-	13C-labelled KEC	1h	murine peritoneal macrophage cells
SA392775	AA83PM-4_r15-	13C-labelled KEC	1h	murine peritoneal macrophage cells
SA392776	AA83PM-10_r6-	13C-labelled KEC	2h	murine peritoneal macrophage cells
SA392777	AA83PM-12_r18-	13C-labelled KEC	2h	murine peritoneal macrophage cells
SA392778	AA83PM-13_r7-	13C-labelled KEC	2h	murine peritoneal macrophage cells
SA392779	AA83PM-14_r8-	13C-labelled KEC	2h	murine peritoneal macrophage cells
SA392780	AA83PM-11_r2-	13C-labelled KEC	2h	murine peritoneal macrophage cells
SA392781	AA83PM-6_r3-	Unlabeled KEC	1h	murine peritoneal macrophage cells
SA392782	AA83PM-7_r16-	Unlabeled KEC	1h	murine peritoneal macrophage cells
SA392783	AA83PM-8_r5-	Unlabeled KEC	1h	murine peritoneal macrophage cells
SA392784	AA83PM-9_r13-	Unlabeled KEC	1h	murine peritoneal macrophage cells
SA392785	AA83PM-15_r12-	Unlabeled KEC	2h	murine peritoneal macrophage cells
SA392786	AA83PM-16_r14-	Unlabeled KEC	2h	murine peritoneal macrophage cells
SA392787	AA83PM-17_r4-	Unlabeled KEC	2h	murine peritoneal macrophage cells
SA392788	AA83PM-18_r11-	Unlabeled KEC	2h	murine peritoneal macrophage cells
SA392789	AA83PM-19_r1-	Unlabeled KEC	2h	murine peritoneal macrophage cells

Showing results 1 to 19 of 19

Showing results 1 to 19 of 19

Collection:

Collection ID:	CO003732
Collection Summary:	Cells from the peritoneal cavity were harvested with cold PBS. Phagocytic cells (DAPI+ cells) were sorted by flow cytometry, frozen as dry cell pellet, and stored at -80˚C until processing.
Sample Type:	Macrophages

Treatment:

Treatment ID:	TR003748
Treatment Summary:	Preparation of macrophages: Mice were injected or not with 0.5 ml of thioglycolate medium to increase peritoneal macrophage abundance. Four days later, mice were injected with 1E9 killed U-[13C]Bacteria previously labeled with DAPI. One or two hours later, peritoneal cells were harvested, and sorted by flow cytometry based on DAPI. Preparation of viable and killed U-[13C]Bacteria: ThyA- E. coli were grown overnight with shaking in LB supplemented with thymidine (500 ug/ml) and trimethoprim (50 ug/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 ul 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 ug/ml thymidine, 50 ug/ml trimethoprim, and 0.5% U-[13C] 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. Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. For DAPI labelling, killed bacteria were incubated with 0.2 ug/ml of DAPI in PBS for 5min and washed 3 times with cold PBS.

Treatment ID:

TR003748

Treatment Summary:

Preparation of macrophages: Mice were injected or not with 0.5 ml of thioglycolate medium to increase peritoneal macrophage abundance. Four days later, mice were injected with 1E9 killed U-[13C]Bacteria previously labeled with DAPI. One or two hours later, peritoneal cells were harvested, and sorted by flow cytometry based on DAPI. Preparation of viable and killed U-[13C]Bacteria: ThyA- E. coli were grown overnight with shaking in LB supplemented with thymidine (500 ug/ml) and trimethoprim (50 ug/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 ul 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 ug/ml thymidine, 50 ug/ml trimethoprim, and 0.5% U-[13C] 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. Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. For DAPI labelling, killed bacteria were incubated with 0.2 ug/ml of DAPI in PBS for 5min and washed 3 times with cold PBS.

Sample Preparation:

Sampleprep ID:	SP003746
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	AN005933	AN005934
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 Orbitrap Exploris 120	Thermo Orbitrap Exploris 120
Ion Mode	NEGATIVE	POSITIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH004508
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:	10 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:	CH004509
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:	10 uL
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005650
Analysis ID:	AN005933
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 2.0 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (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:	MS005651
Analysis ID:	AN005934
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:	POSITIVE