Fig. 1: LPS exposure induces trained immunity in alveolar macrophages.

a Experimental setup for i.n. LPS (1 ng/mouse) or saline exposure, followed by FACS analysis (BALF, lung) or AM cytokine analysis upon ex vivo bacterial challenge. b, c  Absolute numbers of BALF AMs and neutrophils, measured by flow cytometry 24 h (b) and six days (c) after in vivo treatment. d LEGENDplex analysis of LPS-exposed and control AMs upon ex vivo HISP-challenge (16 h). Absolute cytokine levels (top heat map) and log₂ fold change (log₂fc) of LPS-exposed AMs versus means of control AMs (bottom heat map) are shown. The right panel compares absolute levels and log₂fc of trained AMs. e IL-6 levels of LPS-exposed and control AMs upon ex vivo bacterial challenge two or six weeks after treatment. f Phagocytosis index (AU: arbitrary units) of trained and control AMs, isolated on day six after in vivo training, followed by ex vivo stimulation with FITC-labeled HISP and FACS analysis. g Experimental setup for PKH26 labeling eight days prior to in vivo training. h, i Representative histograms of PKH26 MFI (gated on CD11c⁺ Siglec F⁺ AMs) and percentage of PKH26⁺ AMs 24 h (h) and six days (i) after training. Graphs show means + SD of 7 (b) or 11–12 (c, h, i) biological replicates, means of 7–8 biological replicates (d) or means + SD of 3–5 technical replicates (e, f). Data (b, c, e, f) are representative of two independent experiments. Statistical analysis: student’s t-test. ns, not significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.