Summary of Study ST001997

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 IDST001997
Study TitlePolyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells (Part 2)
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-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 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:SU002078
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
SA1867924h 3+4h engulfing
SA1867934h 1+4h engulfing
SA1867944h 2+4h engulfing
SA1867954h 1-4h non-engulfing
SA1867964h 3-4h non-engulfing
SA1867974h 2-4h non-engulfing
SA1867984h 24h unexposed
SA1867994h 14h unexposed
SA1868004h 34h unexposed
SA1868018h 1+8h engulfing
SA1868028h 3+8h engulfing
SA1868038h 2+8h engulfing
SA1868048h 3-8h non-engulfing
SA1868058h 1-8h non-engulfing
SA1868068h 2-8h non-engulfing
SA1868078h 18h unexposed
SA1868088h 28h unexposed
SA1868098h 38h unexposed
Showing results 1 to 18 of 18

Collection:

Collection ID:CO002071
Collection Summary:Experimental animals This study was approved and performed in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee at National Jewish Health. C57BL/6J (000664), B6-CD45.1 (002014) and CCR2KO (004999) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). DsRed.T3 mice (available as 006051 at Jackson Labs) were generously shared by the Nagy laboratory (Vintersten et al., 2004). CCR2KO mice were crossed with DsRed mice until double homozygous. Mice used in experiments were between 8-16 weeks of age. Both male and female sex were used in experiments except bone marrow chimera experiments, which used only male mice. Human tissue This study used deidentified donor peripheral blood mononuclear cells (PBMC) collected and isolated (Young et al., 2011) by the National Jewish Blood Prep Core (IRB No. HS-1285). Age and sex of deidentified blood donors in this study is not known. Cell Lines Jurkat (human T lymphocytes, clone E6-1) were obtained from ATCC and cultured in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (v/v) (GeminiBio), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium Pyruvate. Cells were cultured in a humidified CO2 incubator at 37C. The Jurkat cell line was established from peripheral blood of a 14-year-old male diagnosed with T cell leukemia. Murine primary cell cultures Peritoneal cells were isolated from naïve mice by lavage with 10mL of PBS containing 0.5mM EDTA. Lavage was estimated to contain 50% macrophages. Peritoneal macrophages were isolated by adhesion purification of lavage cells in RPMI-10 (RPMI-1640 containing 10% heat-inactivated fetal bovine serum (v/v), 1X Pen/Strep/Glut, 1mM HEPES, and 100M Sodium Pyruvate) plated 1.3x106 macrophages/well in 6-well plates for FACS sorting or 4x106 macrophage/well in 24-well plates for flow cytometry. Non-adherent cells were removed by washing after 3-5 hours, and macrophages were cultured in fresh RPMI-10 overnight. 400ng/mL purified E. coli O111:B4 LPS (List Biological Laboratories Inc.) was added to generate inflammatory macrophages 12h before the addition of target cells. Bone marrow-derived macrophages were generated by 8-day culture of murine bone marrow cells grown in media containing M-CSF (High glucose DMEM containing 10% v/v FBS, 20% v/v L929-conditioned media, 1X Pen/Strep/Glut, 100M Sodium Pyruvate, 55M beta-Mercaptoethanol). All macrophages were removed from culture dishes by 3-minute incubation with 1X Trypsin-EDTA (Sigma) plus gentle scraping.
Sample Type:Macrophages

Treatment:

Treatment ID:TR002090
Treatment Summary:LPS-primed murine peritoneal macrophages were fed fluorescently-tagged IgG-opsonized live Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were left to degrade targets for a further 3 or 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:SP002084
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.

Combined analysis:

Analysis ID AN003257 AN003258
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:CH002401
Chromatography Summary:Isocratic flow of 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% formic acid in acetonitrile) at 250 uL/min.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:25
Flow Gradient:Isocratic flow of 95% phase A (0.1% formic acid in water) and 5% phase B (0.1% formic acid in acetonitrile) at 250 uL/min.
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:CH002402
Chromatography Summary:Isocratic flow of 100% phase A (95% water, 5% acetonitrile, 10 mM ammonium acetate) at 250 uL/min.
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:MS003029
Analysis ID:AN003257
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:MS003030
Analysis ID:AN003258
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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