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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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.