Summary of Study ST001998
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.
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 | ST001998 |
| Study Title | Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells (Part 3) |
| Study 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-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 Name | Haines |
| First Name | Julie |
| Address | 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA |
| julie.haines@cuanschutz.edu | |
| Phone | 3037243339 |
| Submit Date | 2021-11-22 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-12-08 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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-1or 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: | SU002079 |
| 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 |
|---|---|---|
| SA186810 | 1 + | engulfing |
| SA186811 | 2 + | engulfing |
| SA186812 | 3 + | engulfing |
| SA186813 | 3 - | non-engulfing |
| SA186814 | 2 - | non-engulfing |
| SA186815 | 1 - | non-engulfing |
| SA186816 | unexp2 | unexposed macrophages |
| SA186817 | unexp3 | unexposed macrophages |
| SA186818 | unexp1 | unexposed macrophages |
| Showing results 1 to 9 of 9 |
Collection:
| Collection ID: | CO002072 |
| 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 3 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: | TR002091 |
| 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 3 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: | SP002085 |
| 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 | AN003259 | AN003260 |
|---|---|---|
| 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: | CH002403 |
| Chromatography Summary: | Isocratic flow of 250 uL/min of 95% A (0.1% formic acid in water) and 5% 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 of 250 uL/min of 95% A (0.1% formic acid in water) and 5% 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: | CH002404 |
| Chromatography Summary: | Isocratic flow of 250 uL/min of 100% 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: | MS003031 |
| Analysis ID: | AN003259 |
| 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: | MS003032 |
| Analysis ID: | AN003260 |
| 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 |
