In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research
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.
Authors
- Sergio Rius‐Rocabert (ORCID: https://orcid.org/0000-0002-2193-5193)
- Jordi Ochando (ORCID: https://orcid.org/0000-0001-7037-1681)
- Alessia Zotta (ORCID: https://orcid.org/0000-0002-8274-7671)
- Maria González-Pérez
- Daniel Lozano‐Ojalvo (ORCID: https://orcid.org/0000-0002-3524-2929)
- David L. Williams (ORCID: https://orcid.org/0000-0002-4586-9290)
- Ana Dopazo (ORCID: https://orcid.org/0000-0002-4910-1684)
- Carlos de la Calle‐Fabregat (ORCID: https://orcid.org/0000-0002-3026-3069)
- Estanislao Nistal‐Villán (ORCID: https://orcid.org/0000-0003-2458-8833)
- Florent Ginhoux (ORCID: https://orcid.org/0000-0002-2857-7755)
- Leticia Pérez‐Rodríguez (ORCID: https://orcid.org/0000-0002-0311-9643)
- Xuedi Wang (ORCID: https://orcid.org/0009-0002-0709-8349)
- Raphaël Duivenvoorden (ORCID: https://orcid.org/0000-0003-1446-8336)
- Luke O'neill (ORCID: https://orcid.org/0000-0002-4333-2748)
- Jana Baranda (ORCID: https://orcid.org/0000-0002-9060-9228)
- Nikita Poddar (ORCID: https://orcid.org/0000-0003-3037-5519)
- Marcos J. Berges-Buxeda
- Joren C. Madsen (ORCID: https://orcid.org/0000-0003-2724-1333)
- Alexander G. Dimitrov (ORCID: https://orcid.org/0000-0002-1505-4322)
- Patricia Conde (ORCID: https://orcid.org/0000-0003-2022-5568)
- Dan Hasson (ORCID: https://orcid.org/0000-0001-7943-1636)
- Javier Arranz (ORCID: https://orcid.org/0000-0003-0891-1215)
Institutions
- Universidad San Pablo CEU (ES)
- East Tennessee State University (US)
- Radboud University Nijmegen (NL)
- Inserm (FR)
- Trinity College Dublin (IE)
- Instituto de Salud Carlos III (ES)
- Institut Gustave Roussy (FR)
- Radboud University Medical Center (NL)
- Massachusetts General Hospital (US)
- Tisch Hospital (US)
- Spanish National Centre for Cardiovascular Research (ES)
- Icahn School of Medicine at Mount Sinai (US)
Publication Details
- Journal
- Cell Reports
- Published
- 2025-09-27
- DOI
- https://doi.org/10.1016/j.celrep.2025.116202
- Citations
- 4
- Primary Topic
- Immune responses and vaccinations
- Type
- article
- Field-Weighted Citation Impact
- 3.19
Funders
- Georgia Clinical and Translational Science Alliance
- European Commission
- National Institutes of Health
- HORIZON EUROPE Framework Programme
- National Center for Advancing Translational Sciences