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.