HSPC-derived macrophages phenotype in other models of trained immunity, and modulation of inflammatory tolerization. (A) C57BL/6 mice were injected IV with PBS, 1.5 million PCA2 yeasts or 300 μg per mouse of depleted zymosan and 24 hours later LKS⁻ CD34⁺ FcγR⁺ or LKS⁺ cells were isolated from the bone marrow, and differentiated into macrophages with M-CSF (50 ng/mL) for 6 or 8 days, respectively; TNF-α and IL-6 production was measured by ELISA in supernatants upon LPS stimulation (B) and TNF-α by intracellular flow cytometry upon LPS stimulation (C). (D) LKS⁺ cells isolated from the bone marrow of mice were cultured in vitro with M-CSF (50 ng/mL) + SCF (20 ng/mL) and stimulated with or without GM-CSF (50 ng/mL) for 24 hours, washed, cultured with M-CSF for 2 more days, restimulated with or without GM-CSF (50 ng/mL) for 24 hours, washed, and differentiated into macrophages with M-CSF for 5 more days. (E) Purified LKS⁺ cells or day 3 LKS⁺-derived cells, treated or not with GM-CSF for the first 24 hours, were analyzed for FcγR and CD116 expression by flow cytometry. (F) CD116 MFI from LKS⁺–derived FcγR⁺ cells are expressed as means + SD. (G) TNF-α and IL-6 production was measured by intracellular flow cytometry in macrophages obtained as in panel D upon 4 hours of LPS stimulation and brefeldin A for the final 1 hour. (H) Human CD34⁺ CD38⁻ (selected as CD11b⁻ CD10⁻ CD34⁺ CD38⁻ cells) and CD34⁺ CD38⁺ (selected as CD11b⁻ CD10⁻ CD34⁺ CD38⁺ CD45RA⁺ cells), were isolated from human peripheral blood mononuclear cells of human bone marrow, cultured in vitro with hM-CSF (50 ng/mL) + hSCF (50 ng/mL) and stimulated with or without hGM-CSF (50 ng/mL) for 24 hours, washed, differentiated into macrophages with hM-CSF for 14 or 7 days, respectively, and TNF-α production was measured by intracellular flow cytometry upon LPS stimulation. For the ELISA measurements triplicate samples were analyzed and expressed as means + SD and for intracellular cytokine measurements, percentage of cytokine-producing cells and MFI are indicated in the histograms or heatmaps, and bar graphs for MFIs and box plots for cell percentages are expressed as means + SD. Data presented are 1 representative experiment of at least 3 independent experiments. Statistical significance was assessed by the Student t test (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). depl. zym., depleted zymosan; mφ, macrophage.