Summary of Study ST001999

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 PR001268. The data can be accessed directly via it's Project DOI: 10.21228/M8T39S 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 IDST001999
Study TitlePolyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells (Part 4)
Study SummaryPhagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naïve and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to unique increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis, but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage IL-1beta or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.
Institute
University of Colorado Denver
Last NameHaines
First NameJulie
Address12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Emailjulie.haines@cuanschutz.edu
Phone3037243339
Submit Date2021-11-22
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2021-12-08
Release Version1
Julie Haines Julie Haines
https://dx.doi.org/10.21228/M8T39S
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001268
Project DOI:doi: 10.21228/M8T39S
Project Title:Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells
Project Summary:Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naïve and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to unique increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis, but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage IL-1or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.
Institute:University of Colorado Denver
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:SU002080
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Category
SA1868191 +AC 8hengulfing
SA1868202 +AC 8hengulfing
SA1868213 +AC 8hengulfing
SA1868223 -AC 8hnon-engulfing
SA1868232 -AC 8hnon-engulfing
SA1868241 -AC 8hnon-engulfing
SA186825ctrl 2unexposed macrophages
SA186826ctrl 3unexposed macrophages
SA186827ctrl 1unexposed macrophages
Showing results 1 to 9 of 9

Collection:

Collection ID:CO002073
Collection Summary:LPS-primed murine peritoneal macrophages were fed fluorescently-tagged apoptotic Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were left to degrade targets for a further 7 hours. Macrophages were then collected and sorted into non-engulfing or engulfing populations, alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were snap frozen and stored at -80C until metabolite extraction.
Sample Type:Macrophages

Treatment:

Treatment ID:TR002092
Treatment Summary:LPS-primed murine peritoneal macrophages were fed fluorescently-tagged apoptotic Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were left to degrade targets for a further 7 hours. Macrophages were then collected and sorted into non-engulfing or engulfing populations, alongside control unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were snap frozen and stored at -80C until metabolite extraction.

Sample Preparation:

Sampleprep ID:SP002086
Sampleprep Summary:To process cells for assessment of intracellular metabolites, cells were pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and discarded; residual liquid was carefully wicked away from the pellet with a kimwipe. Dry pellets were immediately snap frozen and stored at -80C until processing. To process culture supernatants for assessment of metabolites, supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant was then transferred to a fresh tube, snap frozen, and stored at -80C until processing. Ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) was performed by the University of Colorado School of Medicine Metabolomics Core. Metabolites from frozen cell pellets were extracted at 2e6 cells/mL in ice cold 5:3:2 MeOH:acetonitrile:water (v/v/v). Media was thawed on ice and a 10 L aliquot treated with 240 L of the same extraction solution. Extractions were carried out using vigorous vortexing for 30 min at 4C. Supernatants were clarified by centrifugation (10 min, 18,000 g, 4C) and analyzed using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer.

Combined analysis:

Analysis ID AN003261 AN003262
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002405
Chromatography Summary:Isocratic flow at 250 uL/min using 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% formic acid in acetonitrile).
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:25
Flow Gradient:Isocratic flow at 250 uL/min using 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% formic acid in acetonitrile).
Flow Rate:250 ul/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH002406
Chromatography Summary:Isocratic flow at 250 uL/min using 100% phase A (95% water, 5% acetonitrile, 10 mM ammonium acetate).
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:25
Flow Gradient:isocratic
Flow Rate:250 ul/min
Solvent A:95% water/5% acetonitrile; 10 mM ammonium acetate
Solvent B:N/A
Chromatography Type:Reversed phase

MS:

MS ID:MS003033
Analysis ID:AN003261
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Global metabolomics analyses were performed using a 3 min isocratic run in positive and negative ion modes (separate runs) as described previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in conjunction with the KEGG database), integrated, and quality control performed using Maven (Princeton University) as described. Stable isotope tracing results were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017).
Ion Mode:POSITIVE
  
MS ID:MS003034
Analysis ID:AN003262
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Global metabolomics analyses were performed using a 3 min isocratic run in positive and negative ion modes (separate runs) as described previously (Nemkov et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) as described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in conjunction with the KEGG database), integrated, and quality control performed using Maven (Princeton University) as described. Stable isotope tracing results were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and 15N2 (Nemkov et al., 2017).
Ion Mode:NEGATIVE
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