Work

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

Year: 2025

Type: article

Abstract: 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-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 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 β-glucan (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.

Source: Cell Reports

DOI: 10.1016/j.celrep.2025.116202