In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research

Maria González-Pérez; Jana Baranda; Leticia Pérez-Rodríguez; Patricia Conde; Carlos de la Calle-Fabregat; Marcos J Berges-Buxeda; Alexander Dimitrov; Javier Arranz; Sergio Rius-Rocabert; Alessia Zotta; Ana Dopazo; Nikita Poddar; Xuedi Wang; Estanislao Nistal-Villán; Raphaël Duivenvoorden; Joren C Madsen; David L Williams; Dan Hasson; Daniel Lozano-Ojalvo; Florent Ginhoux; Luke A J O'Neill; Jordi Ochando

doi:10.1016/j.celrep.2025.116202

In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research

Maria González-Pérez
, Jana Baranda
, Leticia Pérez-Rodríguez
, Patricia Conde
, Carlos de la Calle-Fabregat
, Marcos J Berges-Buxeda
, Alexander Dimitrov
, Javier Arranz
, Sergio Rius-Rocabert
, Alessia Zotta
, Ana Dopazo
, Nikita Poddar
, Xuedi Wang
, Estanislao Nistal-Villán
, Raphaël Duivenvoorden
, Joren C Madsen
, David L Williams
, Dan Hasson
, Daniel Lozano-Ojalvo
, Florent Ginhoux

Luke A J O'Neill, Jordi Ochando

Department of Surgery

CEU-San Pablo University
Department of Oncological Sciences and Tisch Cancer Institute
National Institutes of Health Carlos III
Gustave Roussy Cancer Campus
Trinity College Dublin
National Cardiovascular Center (CNIC)
Tisch Cancer Institute Bioinformatics for Next Generation Sequencing
Radboud University Medical Center
Center for Transplantation Sciences and Division of Cardiac Surgery
Nutritional Immunology and Molecular Medicine Institute

Research output: Contribution to journal › Article › peer-review

Abstract

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 (IL)-6 and tumor necrosis factor (TNF) production demonstrates that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucan compared 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 IL-6 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 β-glucan (stimulus 1) and LPS (stimulus 2) is 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 the distinctive functional stages that macrophages undergo during the training process.

Original language	English
Pages (from-to)	116202
Journal	Cell Reports
Volume	44
Issue number	10
DOIs	https://doi.org/10.1016/j.celrep.2025.116202
State	E-pub ahead of print - Sep 25 2025

Access to Document

10.1016/j.celrep.2025.116202

Cite this

González-Pérez, M., Baranda, J., Pérez-Rodríguez, L., Conde, P., Calle-Fabregat, C. D. L., Berges-Buxeda, M. J., Dimitrov, A., Arranz, J., Rius-Rocabert, S., Zotta, A., Dopazo, A., Poddar, N., Wang, X., Nistal-Villán, E., Duivenvoorden, R., Madsen, J. C., Williams, D. L., Hasson, D., Lozano-Ojalvo, D., ... Ochando, J. (2025). In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research. Cell Reports, 44(10), 116202. Advance online publication. https://doi.org/10.1016/j.celrep.2025.116202

González-Pérez, M, Baranda, J, Pérez-Rodríguez, L, Conde, P, Calle-Fabregat, CDL, Berges-Buxeda, MJ, Dimitrov, A, Arranz, J, Rius-Rocabert, S, Zotta, A, Dopazo, A, Poddar, N, Wang, X, Nistal-Villán, E, Duivenvoorden, R, Madsen, JC, Williams, DL, Hasson, D, Lozano-Ojalvo, D, Ginhoux, F, O'Neill, LAJ & Ochando, J 2025, 'In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research', Cell Reports, vol. 44, no. 10, pp. 116202. https://doi.org/10.1016/j.celrep.2025.116202

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title = "In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research",

abstract = "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 (IL)-6 and tumor necrosis factor (TNF) production demonstrates that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucan compared 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 IL-6 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 β-glucan (stimulus 1) and LPS (stimulus 2) is 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 the distinctive functional stages that macrophages undergo during the training process.",

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T2 - Implications on biomarker discovery and translational research

AU - González-Pérez, Maria

AU - Baranda, Jana

AU - Pérez-Rodríguez, Leticia

AU - Conde, Patricia

AU - Calle-Fabregat, Carlos de la

AU - Berges-Buxeda, Marcos J

AU - Dimitrov, Alexander

AU - Arranz, Javier

AU - Rius-Rocabert, Sergio

AU - Zotta, Alessia

AU - Dopazo, Ana

AU - Poddar, Nikita

AU - Wang, Xuedi

AU - Nistal-Villán, Estanislao

AU - Duivenvoorden, Raphaël

AU - Madsen, Joren C

AU - Williams, David L

AU - Hasson, Dan

AU - Lozano-Ojalvo, Daniel

AU - Ginhoux, Florent

AU - O'Neill, Luke A J

AU - Ochando, Jordi

PY - 2025/9/25

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AB - 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 (IL)-6 and tumor necrosis factor (TNF) production demonstrates that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucan compared 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 IL-6 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 β-glucan (stimulus 1) and LPS (stimulus 2) is 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 the distinctive functional stages that macrophages undergo during the training process.

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