Summary of Study ST004061

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 PR002549. The data can be accessed directly via it's Project DOI: 10.21228/M8583B This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study IDST004061
Study TitleIn Vitro Method Demonstrating Trained Immunity as a Distinctive Functional Program: Implications for Biomarker Discovery, Adaptive Immune Responses, Sample Cryopreservation, and Mouse Genetics
Study SummaryTrained immunity is a distinct program of macrophages, enabling them to secrete varying levels of inflammatory cytokines according to their functional state during sequential stimulation. Experimental models that accurately replicate the defining steps of trained immunity are urgently needed. Here, we developed an in vitro methodology to study trained immunity using murine bone marrow-derived macrophages stimulated with β-glucan and lipopolysaccharide (LPS). Longitudinal analysis of interleukin 6 (IL6) and tumor necrosis factor (TNF) production demonstrated that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucancompared to unstimulated macrophages (step 1). After a resting period, trained macrophages return to basal levels of cytokine production (step 2) but rapidly produce enhanced levels of IL6 and TNF after secondary stimulation with LPS, compared to macrophages individually stimulated with either β-glucan (step 3) or LPS (step 4) alone. The combined cytokine production of macrophages after single stimulation with βglucan (stimulus 1) and LPS (stimulus 2) was significantly lower than the cytokine levels produced by trained macrophages sequentially stimulated with both β-glucan and LPS (stimulus 1+2) (step 5). These results experimentally reproduce, for the first time, the distinctive functional stages that macrophages undergo during the training process. Using this methodology, we identified serum amyloid A3 protein (SAA3) as a novel biomarker of trained immunity and revealed that trained macrophages induce T-cell proliferation via a contact dependent mechanisms. Additionally, our results uncovered adverse effects of cell cryopreservation, strain-specific differences, and the impact of mTOR genetic ablation in the induction of trained immunity. Standardization of methodologies to study trained immunity is critical for advancing our understanding of the fundamental mechanisms that regulate innate immune memory responses and develop therapies that specifically target trained immunity in multiple immune-mediated pathological conditions.
Institute
Instituto de Salud Carlos III
Last NameArranz Herrero
First NameJavier
AddressDalia 52, Mostoles, Madrid, 28933, Spain
Emailj.arranz3@usp.ceu.es
Phone655653445
Submit Date2025-07-07
Raw Data AvailableYes
Raw Data File Type(s)mzML, d
Analysis Type DetailLC-MS
Release Date2025-08-14
Release Version1
Javier Arranz Herrero Javier Arranz Herrero
https://dx.doi.org/10.21228/M8583B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002549
Project DOI:doi: 10.21228/M8583B
Project Title:In Vitro Method Demonstrating Trained Immunity as a Distinctive Functional Program: Implications for Biomarker Discovery, Adaptive Immune Responses, Sample Cryopreservation, and Mouse Genetics
Project Summary:Trained immunity is a distinct program of macrophages, enabling them to secrete varying levels of inflammatory cytokines according to their functional state during sequential stimulation. Experimental models that accurately replicate the defining steps of trained immunity are urgently needed. Here, we developed an in vitro methodology to study trained immunity using murine bone marrow-derived macrophages stimulated with β-glucan and lipopolysaccharide (LPS). Longitudinal analysis of interleukin 6 (IL6) and tumor necrosis factor (TNF) production demonstrated that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucancompared to unstimulated macrophages (step 1). After a resting period, trained macrophages return to basal levels of cytokine production (step 2) but rapidly produce enhanced levels of IL6 and TNF after secondary stimulation with LPS, compared to macrophages individually stimulated with either β-glucan (step 3) or LPS (step 4) alone. The combined cytokine production of macrophages after single stimulation with βglucan (stimulus 1) and LPS (stimulus 2) was significantly lower than the cytokine levels produced by trained macrophages sequentially stimulated with both β-glucan and LPS (stimulus 1+2) (step 5). These results experimentally reproduce, for the first time, the distinctive functional stages that macrophages undergo during the training process. Using this methodology, we identified serum amyloid A3 protein (SAA3) as a novel biomarker of trained immunity and revealed that trained macrophages induce T-cell proliferation via a contact dependent mechanisms. Additionally, our results uncovered adverse effects of cell cryopreservation, strain-specific differences, and the impact of mTOR genetic ablation in the induction of trained immunity. Standardization of methodologies to study trained immunity is critical for advancing our understanding of the fundamental mechanisms that regulate innate immune memory responses and develop therapies that specifically target trained immunity in multiple immune-mediated pathological conditions.
Institute:Instituto de Salud Carlos III
Last Name:Arranz Herrero
First Name:Javier
Address:Dalia 52, Mostoles, Madrid, 28933, Spain
Email:j.arranz3@usp.ceu.es
Phone:655653445

Subject:

Subject ID:SU004207
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 Sample source Condition
SA469518LPS_02macrophages LPS
SA469519LPS_08macrophages LPS
SA469520LPS_07macrophages LPS
SA469521LPS_06macrophages LPS
SA469522LPS_04macrophages LPS
SA469523LPS_03macrophages LPS
SA469524LPS_05macrophages LPS
SA469525LPS_01macrophages LPS
SA469526Naive_04macrophages Naive
SA469527Naive_08macrophages Naive
SA469528Naive_03macrophages Naive
SA469529Naive_01macrophages Naive
SA469530Naive_05macrophages Naive
SA469531Naive_02macrophages Naive
SA469532Naive_06macrophages Naive
SA469533Naive_07macrophages Naive
SA469534Trained_02macrophages Trained
SA469535Trained_03macrophages Trained
SA469536Trained_04macrophages Trained
SA469537Trained_05macrophages Trained
SA469538Trained_06macrophages Trained
SA469539Trained_07macrophages Trained
SA469540Trained_08macrophages Trained
SA469541Trained_01macrophages Trained
Showing results 1 to 24 of 24

Collection:

Collection ID:CO004200
Collection Summary:Bone marrow-derived macrophages (BMDMs) were obtained from C57BL/6 mice and cultured under standard conditions. Naive macrophages were either left untreated, activated with LPS, or trained with β-glucan before metabolite extraction. After stimulation, cells were washed, collected, and lysed in methanol for metabolomic analysis.
Sample Type:Macrophages

Treatment:

Treatment ID:TR004216
Treatment Summary:Murine bone marrow monocytes were isolated by negative selection and cultured in DMEM supplemented with 10% FBS, antibiotics, amino acids, and 30 ng/mL M-CSF. Naive macrophages were cultured for 6 days without additional stimulation. Activated macrophages were stimulated with 10 ng/mL LPS for 6 hours on day 6. Trained macrophages were primed with 10 µg/mL β-glucan on day 0, followed by a media change on day 3 and a 3-day resting period. On day 6, trained macrophages were re-stimulated with LPS (10 ng/mL) for 6 hours before metabolite extraction.

Sample Preparation:

Sampleprep ID:SP004213
Sampleprep Summary:After culture and treatment, macrophages were resuspended in 60 μL of methanol and ultrasonicated using a UP200S device with an S2 probe (Hielscher Ultrasonics). Organic and aqueous fractions were separated by centrifugation. Supernatants were collected and stored at -80°C until further analysis. Samples were then subjected to LC-MS/MS for metabolomic profiling.

Combined analysis:

Analysis ID AN006720
Chromatography ID CH005107
MS ID MS006419
Analysis type MS
Chromatography type HILIC
Chromatography system Agilent 6470
Column Agilent InfinityLab Poroshell 120 HILIC-Z (150 x 2.1mm, 2.7um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name Agilent 6470 QQQ
Ion Mode NEGATIVE
Units Relative intensity

Chromatography:

Chromatography ID:CH005107
Instrument Name:Agilent 6470
Column Name:Agilent InfinityLab Poroshell 120 HILIC-Z (150 x 2.1mm, 2.7um)
Column Temperature:50℃
Flow Gradient:Gradient elution from 96% to 65% B, followed by re-equilibration to 96% B: Linear gradient steps: 0.0–2.0 min at 96% B (isocratic), 2.0–5.5 min: linear decrease from 96% to 88% B, 5.5–8.5 min at 88% B, 8.5–9.0 min: linear decrease to 86% B, 9.0–14.0 min at 86% B, 14.0–17.0 min: linear decrease to 82% B, 17.0–22.0 min at 82% B, 22.0–23.0 min: linear decrease to 65% B, 23.0–24.0 min at 65% B, 24.5–39.0 min: re-equilibration at 96% B.
Flow Rate:0.250 mL/min
Solvent A:100% water; 10 mM ammonium acetate; 2.5 μM InfinityLab Deactivator Additive (pH 9)
Solvent B:15% water/85% acetonitrile; 10 mM ammonium acetate; 2.5 μM InfinityLab Deactivator Additive (pH 9)
Chromatography Type:HILIC

MS:

MS ID:MS006419
Analysis ID:AN006720
Instrument Name:Agilent 6470 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:The Agilent 6470 triple quadrupole system was operated in negative ion mode, with gas temperature at 225°C, gas flow 13 L/min, nebulizer pressure 60 psi, sheath gas temperature 250°C, sheath gas flow 12 L/min, capillary voltage –3500 V, and nozzle voltage 0 V. MS/MS acquisition was performed in dynamic Multiple Reaction Monitoring (MRM) mode, using optimized transitions for each compound. Data processing Comments: Data were processed using transition-specific MRM settings previously validated at the CEMBIO laboratory to ensure accurate quantification and compound identification. Chromatographic peaks were manually inspected and integrated. Software/procedures used for feature assignments: Feature assignment and quantification were performed using Agilent MassHunter Workstation software, with compound transitions and parameters optimized and validated internally at the CEMBIO laboratory.
Ion Mode:NEGATIVE
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