Summary of Study ST001835

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 PR001159. The data can be accessed directly via it's Project DOI: 10.21228/M8WH6D This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST001835
Study Title	Use of Integrated Metabolomics, Transcriptomics, and Signal Protein Profile to Characterize the Effector Function and Associated Metabotype of Polarized Macrophage Phenotypes
Study Type	Ex vivo macrophage polarization metabotyping
Study Summary	Macrophages (MΦs) display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 MΦs and a broad set of M2 MΦs, comprehensive characterization of functional phenotype and associated metabotype driving this diverse MΦ activation remains. Herein, we utilized an ex vivo model to produce six MΦ functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 MΦs, and then polarized into M1 (IFN-γ/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) MΦs. The MΦs were profiled using a bioanalyte matrix of four cell surface markers, ~50 secreted proteins, ~800 expressed myeloid genes, and ~450 identified metabolites relative to M0 MΦs. Signal protein and expressed gene profiles grouped the MΦs into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b MΦs shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO, and OXPHOS. Additionally, M2a, M2c, and M2d MΦs all profiled as tissue repair MΦs; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a MΦs, and angiogenesis and ECM assembly capabilities for M2b, M2c, and M2d MΦs. By integrating metabolomics into a systems analysis of MΦ phenotypes, we provide the most comprehensive map of MΦ diversity to date, along with the global metabolic shifts that correlate to MΦ functional plasticity in these phenotypes.
Institute	Idaho Veterans Research and Education Foundation
Department	Research
Laboratory	Ammons
Last Name	Ammons
First Name	Mary Cloud
Address	Mail Stop 151, Bldg 117, 500 W. Fort St. Boise, Idaho 83702
Email	MaryCloud.AmmonsAnderson@va.gov
Phone	208-422-1219
Submit Date	2021-06-18
Analysis Type Detail	LC-MS
Release Date	2021-09-17
Release Version	1

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Project:

Project ID:	PR001159
Project DOI:	doi: 10.21228/M8WH6D
Project Title:	Use of Integrated Metabolomics, Transcriptomics, and Signal Protein Profile to Characterize the Effector Function and Associated Metabotype of Polarized Macrophage Phenotypes
Project Type:	Systems biology profiling of ex vivo human macrophage polarization
Project Summary:	Macrophages (MΦs) display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 MΦs and a broad set of M2 MΦs, comprehensive characterization of functional phenotype and associated metabotype driving this diverse MΦ activation remains. Herein, we utilized an ex vivo model to produce six MΦ functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 MΦs, and then polarized into M1 (IFN-γ/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) MΦs. The MΦs were profiled using a bioanalyte matrix of four cell surface markers, ~50 secreted proteins, ~800 expressed myeloid genes, and ~450 identified metabolites relative to M0 MΦs. Signal protein and expressed gene profiles grouped the MΦs into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b MΦs shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO, and OXPHOS. Additionally, M2a, M2c, and M2d MΦs all profiled as tissue repair MΦs; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a MΦs, and angiogenesis and ECM assembly capabilities for M2b, M2c, and M2d MΦs. By integrating metabolomics into a systems analysis of MΦ phenotypes, we provide the most comprehensive map of MΦ diversity to date, along with the global metabolic shifts that correlate to MΦ functional plasticity in these phenotypes.
Institute:	Boise VA Medical Center
Department:	Research
Laboratory:	Ammons
Last Name:	Ammons
First Name:	Mary Cloud
Address:	Mail Stop 151, Bldg 117, 500 W. Fort St. Boise, Idaho 83702
Email:	MaryCloud.AmmonsAnderson@va.gov
Phone:	208-422-1219
Funding Source:	NIGMS

Subject:

Subject ID:	SU001912
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	hours	treatment
SA170650	M2c_037_081418	24	IL10
SA170651	M2c_016_092418	24	IL10
SA170652	M2c_038_081418	24	IL10
SA170653	M2a_016_092418	24	IL4_IL13
SA170654	M2a_037_081418	24	IL4_IL13
SA170655	M2a_038_081418	24	IL4_IL13
SA170656	M2d_037_081418	24	IL6_LIF
SA170657	M2d_016_092418	24	IL6_LIF
SA170658	M2d_038_081418	24	IL6_LIF
SA170659	M2b_038_081418	24	LPS_IC
SA170660	M2b_016_092418	24	LPS_IC
SA170661	M2b_037_081418	24	LPS_IC
SA170662	M1_016_092418	24	LPS_IFNG
SA170663	M1_037_081418	24	LPS_IFNG
SA170664	M1_038_081418	24	LPS_IFNG
SA170665	M0_016_092418	24	NT
SA170666	M0_038_081418	24	NT
SA170667	M0_037_081418	24	NT
SA170668	M2c_016_071218	72	IL10
SA170669	M2c_017_062818	72	IL10
SA170670	M2c_028_070518	72	IL10
SA170671	M2a_028_070518	72	IL4_IL13
SA170672	M2a_016_071218	72	IL4_IL13
SA170673	M2a_017_062818	72	IL4_IL13
SA170674	M2d_017_062818	72	IL6_LIF
SA170675	M2d_028_070518	72	IL6_LIF
SA170676	M2d_016_071218	72	IL6_LIF
SA170677	M2b_028_070518	72	LPS_IC
SA170678	M2b_016_071218	72	LPS_IC
SA170679	M2b_017_062818	72	LPS_IC
SA170680	M1_017_062818	72	LPS_IFNG
SA170681	M1_016_071218	72	LPS_IFNG
SA170682	M1_028_070518	72	LPS_IFNG
SA170683	M0_016_071218	72	NT
SA170684	M0_017_062818	72	NT
SA170685	M0_028_070518	72	NT

Showing results 1 to 36 of 36

Showing results 1 to 36 of 36

Collection:

Collection ID:	CO001905
Collection Summary:	CD14+ PBMCs were isolated from whole human blood, differentiated into macrophages with M-CSF, followed by polarization stimuli for 24-72hrs. Cells were counted, washed with PBS, and immediately frozen in liquid nitrogen.
Sample Type:	Blood (whole)

Treatment:

Treatment ID:	TR001925
Treatment Summary:	M0 macrophages were treated only with M-CSF; M1 macrophages were treated with LPS & IFN-gamma; M2a macrophages were treated with IL-4 & IL-13; M2b macrophages were treated with LPS & ICs; M2c macrophages were treated with IL-10; M2d macrophages were treated with IL-6 & LIF. Treatments course was 24hr or 72hr.

Sample Preparation:

Sampleprep ID:	SP001918
Sampleprep Summary:	With the goal of creating a comprehensive snapshot of cellular function, multiple experimental sample types were collected from each MΦ phenotype during cell harvest to obtain transcriptomic, proteomic, and metabolomic data from each experimental trial. At the desired time point, the cellular supernatant, composed of cell culture media and any non-adherent cells, was removed from the cell culture well and placed into falcon tubes on ice. One mL of ice-cold phosphate buffered saline (PBS) was added to the adherent cell layers and then removed to the tubes containing the cellular supernatant and the tubes centrifuged at 2000 rpm for 8 minutes. The collected supernatant was stored at -80° C until further analysis. Pelleted cells were flash-frozen in liquid nitrogen and stored on ice, while the remaining adherent cells were quenched with 350 µL of ice-cold 100% methanol (Honeywell; Muskegon, MI, USA) and 350 µL of ice-cold ultrapure distilled water (Invitrogen; Grand Island NY, USA) and removed through gentle cell scraping and added to the cell pellet fraction. Following the addition of 700 µL of ice-cold chloroform (Acros Organics; Thermo Fisher Scientific; Waltham, MA, USA), the collected cells were vortexed for 30 seconds and were transferred to FastPrep® lysing matrix D tubes (MP Biomedicals; Auckland, New Zealand). To achieve cell lysis, the tubes were homogenized during two cycles of 40 s each at 4.0 m/s with a 90 s delay between cycles utilizing the FastPrep-24™ 5G Homogenizer (MP Biomedicals; Auckland, New Zealand). The homogenized samples were centrifuged at 16,000 x g for 5 minutes at 4° C, and then placed immediately on ice. The polar (methanol/water) layer and non-polar (chloroform) layers were subsequently transferred to 1.5 mL protein low binding microcentrifuge tubes. These metabolite suspensions were lyophilized overnight without heat on a Thermo Scientific™ Savant™ ISS110 SpeedVac™ (Waltham, MA, USA) and stored at -80°C until the samples were shipped to Metabolon for further analysis. The remaining interphase layer was flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Sampleprep ID:

SP001918

Sampleprep Summary:

With the goal of creating a comprehensive snapshot of cellular function, multiple experimental sample types were collected from each MΦ phenotype during cell harvest to obtain transcriptomic, proteomic, and metabolomic data from each experimental trial. At the desired time point, the cellular supernatant, composed of cell culture media and any non-adherent cells, was removed from the cell culture well and placed into falcon tubes on ice. One mL of ice-cold phosphate buffered saline (PBS) was added to the adherent cell layers and then removed to the tubes containing the cellular supernatant and the tubes centrifuged at 2000 rpm for 8 minutes. The collected supernatant was stored at -80° C until further analysis. Pelleted cells were flash-frozen in liquid nitrogen and stored on ice, while the remaining adherent cells were quenched with 350 µL of ice-cold 100% methanol (Honeywell; Muskegon, MI, USA) and 350 µL of ice-cold ultrapure distilled water (Invitrogen; Grand Island NY, USA) and removed through gentle cell scraping and added to the cell pellet fraction. Following the addition of 700 µL of ice-cold chloroform (Acros Organics; Thermo Fisher Scientific; Waltham, MA, USA), the collected cells were vortexed for 30 seconds and were transferred to FastPrep® lysing matrix D tubes (MP Biomedicals; Auckland, New Zealand). To achieve cell lysis, the tubes were homogenized during two cycles of 40 s each at 4.0 m/s with a 90 s delay between cycles utilizing the FastPrep-24™ 5G Homogenizer (MP Biomedicals; Auckland, New Zealand). The homogenized samples were centrifuged at 16,000 x g for 5 minutes at 4° C, and then placed immediately on ice. The polar (methanol/water) layer and non-polar (chloroform) layers were subsequently transferred to 1.5 mL protein low binding microcentrifuge tubes. These metabolite suspensions were lyophilized overnight without heat on a Thermo Scientific™ Savant™ ISS110 SpeedVac™ (Waltham, MA, USA) and stored at -80°C until the samples were shipped to Metabolon for further analysis. The remaining interphase layer was flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Combined analysis:

Analysis ID	AN002977
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	raw area count

Chromatography:

Chromatography ID:	CH002206
Chromatography Summary:	Low pH polar
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002767
Analysis ID:	AN002977
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Proprietary analytical software for integration and peak picking
Ion Mode:	POSITIVE