Summary of Study ST001996

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	ST001996
Study Title	Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells (Part 1)
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-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
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-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:	SU002077
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
SA186774	NecJ 4h 3+	4h engulfing
SA186775	NecJ 4h 1+	4h engulfing
SA186776	NecJ 4h 2+	4h engulfing
SA186777	NecJ 4h 1-	4h non-engulfing
SA186778	NecJ 4h 3-	4h non-engulfing
SA186779	NecJ 4h 2-	4h non-engulfing
SA186780	NecJ 4h 2	4h unexposed
SA186781	NecJ 4h 1	4h unexposed
SA186782	NecJ 4h 3	4h unexposed
SA186783	NecJ 8h 1+	8h engulfing
SA186784	NecJ 8h 3+	8h engulfing
SA186785	NecJ 8h 2+	8h engulfing
SA186786	NecJ 8h 3-	8h non-engulfing
SA186787	NecJ 8h 1-	8h non-engulfing
SA186788	NecJ 8h 2-	8h non-engulfing
SA186789	NecJ 8h 1	8h unexposed
SA186790	NecJ 8h 2	8h unexposed
SA186791	NecJ 8h 3	8h unexposed

Showing results 1 to 18 of 18

Showing results 1 to 18 of 18

Collection:

Collection ID:	CO002070
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. 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:	TR002089
Treatment Summary:	Apoptotic target cells Live Jurkat cells in RPMI-10 were exposed to 60,000 J of UV energy on a UV Stratalinker 2400 (Stratagene), then incubated at 37C for 4h to generate apoptotic Jurkat cells (ApoJ). Thymocytes, obtained by crushing WT murine thymi and filtering for a single cell suspension, were converted to apoptotic thymocytes in the same manner. For TAMRA-labeling, apoptotic targets were stained during the final hour by 15 minute incubation at 37C with 10M 5(6)-TAMRA,SE (Invitrogen) in PBS followed by two washes with RPMI-10 to remove excess TAMRA. Apoptotic targets were resuspended in fresh RPMI-10 and added to macrophages in culture at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 ApoJ were injected IP. Other cell targets Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior to opsonization or killing using the procedure described above. Labeled cells were incubated for 30 minutes on ice with 25g/mL purified anti-human CD3 (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 IgGJ were injected IP. Non-cell targets 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids Inc.) were used to generate PS-containing liposomes. Lipids suspended in chloroform were mixed at 70:30 POPC:POPS, then chloroform was evaporated under nitrogen. Dry lipids were resuspended in RPMI for a final concentration of 500M lipid and sonicated for 15 minutes until liposomes were formed and solution transitioned from opaque to clear. Liposomes were added to macrophages at a final concentration of 50M in RPMI-10. For other experiments, Flash Red 5m carboxylated polystyrene beads (PS/DVB-COOH·(660,690) carboxyl polystyrene beads, Bangs Laboratories Inc.) were added to macrophages at a final ratio of 1:2. Macrophages were co-cultured with non-cell targets for 1h and unengulfed targets were removed by washing macrophages three times with cold 1X PBS. Isotope-labeled metabolite use For some experiments, Jurkat T cells were cultured for 4 days in media containing [13C,15N]cell-free amino acid mixture (Sigma-Aldrich) (RPMI-1640 medium for SILAC, 10% v/v dyalized FBS, 1X Pen/Strep, 1mM HEPES, 4% v/v amino acid mixture). For some experiments, macrophages were cultured for 7 hours in media containing [13C,15N]Arginine (Cambridge Isotope Laboratories): RPMI-1640 medium for SILAC, 10% dyalized FBS v/v, 1X Pen/Strep, 1mM HEPES, 100M Sodium Pyruvate, 0.2 g/L [13C,15N]Arginine. FACS sorting engulfing and non-engulfing cells Single cell suspensions were incubated with unlabeled CD16/CD32 for 5 min to block non-specific FcR-mediated binding. Cells were stained with surface antibody panels for 20 min, and then washed. Cells were protected from light and incubations were performed on ice. HBSS containing 0.3% BSA and 0.3 mM EDTA was used as a buffer for all incubations. DAPI was added to cells immediately prior to sort to distinguish live and dead cells. For all in vitro target cell engulfment assays, live macrophages (murine peritoneal macrophages: CD45+F4/80+DAPI-; murine BMDM: CD45+CD88+DAPI-; HMDM: CD45+HLA-DR+DAPI-) were separated into engulfing (TAMRA+) and non-engulfing (TAMRA-) populations. For in vivo IP ApoJ or IgGJ engulfment assays, live macrophages were identified as CD45+Ly6G-CD88+CD11b+F4/80hiDAPI- and TAMRA+ or TAMRA-. For bead engulfment assays, live macrophages (CD45+F4/80+DAPI-) were sorted into engulfing (Flash Red+) and non-engulfing (Flash Red-) populations. Liposomes had no label; liposome-fed macrophages were only sorted for viability. Primary antibodies used (source/clone): unlabeled CD16/32 (eBioscience/93), Ly6G (BD/IA8), F4/80 (ebioscience/BM8), CD45 (BD/30-F11), CD11b (eBioscience/M1/70), CD88 (BioLegend/20/70), CD64 (Biolegend/X54-5/7.1). All primary antibodies were used at a 1:400 dilution. All macrophages were sorted into 0.06% BSA-coated tubes on a Synergy cell sorter (Sony Biotech).

Treatment ID:

TR002089

Treatment Summary:

Apoptotic target cells Live Jurkat cells in RPMI-10 were exposed to 60,000 J of UV energy on a UV Stratalinker 2400 (Stratagene), then incubated at 37C for 4h to generate apoptotic Jurkat cells (ApoJ). Thymocytes, obtained by crushing WT murine thymi and filtering for a single cell suspension, were converted to apoptotic thymocytes in the same manner. For TAMRA-labeling, apoptotic targets were stained during the final hour by 15 minute incubation at 37C with 10M 5(6)-TAMRA,SE (Invitrogen) in PBS followed by two washes with RPMI-10 to remove excess TAMRA. Apoptotic targets were resuspended in fresh RPMI-10 and added to macrophages in culture at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 ApoJ were injected IP. Other cell targets Jurkat cells destined to be IgGJ and NecJ were TAMRA-labeled prior to opsonization or killing using the procedure described above. Labeled cells were incubated for 30 minutes on ice with 25g/mL purified anti-human CD3 (Biolegend, Clone UCHT1) to generate IgGJ or incubated for 25 minutes at 55C to generate NecJ. All targets were resuspended in RPMI-10. IgG targets were added to macrophages at a ratio of 4:1 and co-cultured for 1h, NecJ targets were added to macrophages at a ratio of 5:1 and co-cultured for 1h. Unengulfed targets were removed by washing macrophages three times with cold 1X PBS. To assess in vivo uptake of exogenous targets, 2x106 IgGJ were injected IP. Non-cell targets 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids Inc.) were used to generate PS-containing liposomes. Lipids suspended in chloroform were mixed at 70:30 POPC:POPS, then chloroform was evaporated under nitrogen. Dry lipids were resuspended in RPMI for a final concentration of 500M lipid and sonicated for 15 minutes until liposomes were formed and solution transitioned from opaque to clear. Liposomes were added to macrophages at a final concentration of 50M in RPMI-10. For other experiments, Flash Red 5m carboxylated polystyrene beads (PS/DVB-COOH·(660,690) carboxyl polystyrene beads, Bangs Laboratories Inc.) were added to macrophages at a final ratio of 1:2. Macrophages were co-cultured with non-cell targets for 1h and unengulfed targets were removed by washing macrophages three times with cold 1X PBS. Isotope-labeled metabolite use For some experiments, Jurkat T cells were cultured for 4 days in media containing [13C,15N]cell-free amino acid mixture (Sigma-Aldrich) (RPMI-1640 medium for SILAC, 10% v/v dyalized FBS, 1X Pen/Strep, 1mM HEPES, 4% v/v amino acid mixture). For some experiments, macrophages were cultured for 7 hours in media containing [13C,15N]Arginine (Cambridge Isotope Laboratories): RPMI-1640 medium for SILAC, 10% dyalized FBS v/v, 1X Pen/Strep, 1mM HEPES, 100M Sodium Pyruvate, 0.2 g/L [13C,15N]Arginine. FACS sorting engulfing and non-engulfing cells Single cell suspensions were incubated with unlabeled CD16/CD32 for 5 min to block non-specific FcR-mediated binding. Cells were stained with surface antibody panels for 20 min, and then washed. Cells were protected from light and incubations were performed on ice. HBSS containing 0.3% BSA and 0.3 mM EDTA was used as a buffer for all incubations. DAPI was added to cells immediately prior to sort to distinguish live and dead cells. For all in vitro target cell engulfment assays, live macrophages (murine peritoneal macrophages: CD45+F4/80+DAPI-; murine BMDM: CD45+CD88+DAPI-; HMDM: CD45+HLA-DR+DAPI-) were separated into engulfing (TAMRA+) and non-engulfing (TAMRA-) populations. For in vivo IP ApoJ or IgGJ engulfment assays, live macrophages were identified as CD45+Ly6G-CD88+CD11b+F4/80hiDAPI- and TAMRA+ or TAMRA-. For bead engulfment assays, live macrophages (CD45+F4/80+DAPI-) were sorted into engulfing (Flash Red+) and non-engulfing (Flash Red-) populations. Liposomes had no label; liposome-fed macrophages were only sorted for viability. Primary antibodies used (source/clone): unlabeled CD16/32 (eBioscience/93), Ly6G (BD/IA8), F4/80 (ebioscience/BM8), CD45 (BD/30-F11), CD11b (eBioscience/M1/70), CD88 (BioLegend/20/70), CD64 (Biolegend/X54-5/7.1). All primary antibodies were used at a 1:400 dilution. All macrophages were sorted into 0.06% BSA-coated tubes on a Synergy cell sorter (Sony Biotech).

Sample Preparation:

Sampleprep ID:	SP002083
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. 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).

Sampleprep ID:

SP002083

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. 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).

Combined analysis:

Analysis ID	AN003255	AN003256
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:	CH002399
Chromatography Summary:	10 uL of each sample was injected. The LC was operated under isocratic conditions using 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 Rate:	250 ul/min
Solvent A:	5% acetonitrile/95%water; 0.1% formic acid
Solvent B:	isocratic
Chromatography Type:	Reversed phase

Chromatography ID:	CH002400
Chromatography Summary:	10 uL of each sample was injected. The LC was operated under isocratic conditions using 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:	5% acetonitrile/95%water; 0.1% formic acid
Solvent B:	isocratic
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003027
Analysis ID:	AN003255
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	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:	MS003028
Analysis ID:	AN003256
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	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