Interleukin-1β induces trained innate immunity in human hematopoietic progenitor cells in vitro

Abstract

Innate immune cells can develop a long-lasting hyperresponsive phenotype, termed trained immunity, mediated by epigenetic and metabolic reprogramming. In mice, exposure to Bacille Calmette-Guérin (BCG), β-glucan, or Western diet induces trained immunity by reprogramming hematopoietic progenitor cells (HPCs), through interleukin-1β (IL-1β) signaling in the bone marrow (BM). We investigated whether IL-1β induces trained immunity in primary human BM-derived HPCs in vitro. We exposed human BM-derived HPCs to IL-1β for 4 h. HPCs were expanded and differentiated into monocytes followed by functional and transcriptomic characterization. IL-1β-exposed HPCs showed higher granulocyte-macrophage colony-forming units. The monocyte offspring produced more tumor necrosis factor (TNF) and IL-1β after restimulation with lipopolysaccharide (LPS) and Pam3Cys and is metabolically more active. Transcriptomic analysis showed upregulation of key atherogenic and inflammatory pathways. In conclusion, brief exposure of human BM-derived HPCs to IL-1β in vitro induces a trained immunity phenotype.

Keywords: IL-1β; bone marrow; hematopoietic progenitor cells; macrophages; monocytes; trained immunity.

Graphical abstract

Figure 1

Schematic overview, proliferation and differentiation during expansion and differentiation of HPCs (A) Schematic overview of the experimental design. (B) Proliferation and differentiation capacity of human bone marrow hematopoietic progenitor cells (HPCs). Number and percentages of colonies counted after 14 days of incubation of control (IMDM) and IL-1β -exposed HPCs (IL-1β 10 and 100 ng/mL). 500 HPCs were initially seeded per condition in duplicate (n = 4 independent HPCs donors, Wilcoxon matched-pairs signed-rank test, ^∗p < 0,05 compared to IMDM control). (C) Schematic overview of the flow cytometry panels to identify stem cell progenitor populations during expansion and mature cells during differentiation. (D) Progenitor populations in controls and IL-1β-exposed cells (10 and 100 ng/mL) at day 0, day 6, and day 10 of expansion (n = 4 independent HPC donors, Wilcoxon matched-pairs signed-rank test, differences were not significant). (E) Monocyte subsets identified at the beginning and end of differentiation of HPCs with M-CSF in control and cells exposed to IL-1β (10 and 100 ng/mL). IL-1β induces an increase of classical monocytes at the beginning of the differentiation (n = 5 independent HPC donors, Wilcoxon matched-pairs signed-rank test, differences were not significant). (F) CD11b and CCR2 activation markers expression and median fluorescence intensity (MFI) in the different bone-marrow-derived monocyte subsets after 7 days of differentiation. (n = 5 independent HPC donors, Wilcoxon matched-pairs signed-rank test). See also Figures S1–S3.

Figure 2

Short exposure of HPCs to IL-1β augments cytokine production capacity and affects cellular metabolism of bone-marrow-derived monocytes HPCs were exposed to IL-1β (10 and 100 ng/mL) for 4 h. After 10 days of expansion and 7 days of differentiation. (A) Cells were restimulated with LPS and Pam3Cys for 24 h and IL-6, TNF, IL-1β, IL-10, and IL-1RA production were measured (n = 5 independent HPC donors, ^∗p < 0.05, Wilcoxon matched-pairs signed-rank test). (B) Extracellular acidification rate (ECAR) over time during subsequent injection of glucose, oligomycin A, and 2DG. Oxygen consumption rate over time during subsequent injection of oligomycin A, FCCP, and antimycin A/rotenone (n = 5 independent HPC donors for glycostress test and n = 4 independent HPC donors for mito stress test, Wilcoxon matched-pairs signed-rank test). (C) Bar graphs with individual points of basal glycolysis, glucose metabolism, and glycolytic capacity of IL-1β-trained cells compared to control (n = 5 independent HPC donors, Wilcoxon matched-pairs signed-rank test). (D) Bar graphs with individual points of basal respiration, proton leak, ATP-linked respiration, maximum respiration, and spare mitochondrial capacity of IL-1β-trained cells compared to control (n = 4 independent HPC donors, Wilcoxon matched-pairs signed-rank test).

Figure 3

Short exposure to IL-1β 10 ng/mL and restimulation with LPS induce transcriptional changes in HPC-derived monocytes (A) Schematic overview of the protocol used to collect RNA samples of IL-1β-trained cells. Magnetically sorted monocytes were stimulated with 10 ng/mL LPS for 4 h. Samples were collected before and after LPS restimulation for RNA-seq. (B) Volcano plot showing up and down-regulated genes between IMDM and IL-1β-exposed monocytes before LPS exposure. p values were adjusted for multiple comparisons. (C) Top biological pathways enriched in the differentially expressed gene (DEG) list, according to p value and fraction of DEG present. (D) Transcriptional response to LPS restimulation. Genes were clustered in 3 groups: IL-1β trained (119 genes), IL-1β attenuated (911 genes), and unaffected (38 genes). (E) KEGG pathway analysis showing the influence of IL-1β upon LPS restimulation in the trained, IL-1β-attenuated, and unaffected clusters according to p value and fraction of DEG present. For all the analysis, n = 3 independent HPC donors was used. (F) Biological Process (BP) enrichment and (G) KEGG pathway enrichment analysis on the 371 dynamic genes (p < 0.05, FC > 1.2), in response to at rest or after LPS exposure, relative to IMDM macrophages.

Figure 4

IL-1β induces increased phagocytosis and a suggestively higher endothelial cell interaction with HPC-derived monocytes (A) After 4 h of HPC exposure to IL-1β 100 ng/mL or untrained control, HPC-derived monocytes were differentiated into macrophages. After that, we added fluorescent Latex beads for 3 h and after washing made images with the EVOS microscope to measure phagocytosis capacity. Macrophages are visualized with 10× magnification, scale bar, 200 μm. (B) Phagocytosis rate quantification of IL-1β-trained macrophages compared to untrained control (n = 3 independent HPC donors, Wilcoxon matched-pairs signed-rank test). (C) General overview of the IBIDI system to measure endothelial cell (EC)-monocyte interaction. (D) Visualization of HPC-derived monocytes attached to EC of IL-1β-trained cells compared to the control (10× magnification, scale bar, 400 μm). (E) Relative adherence of IL-1β 100 ng/mL trained monocytes to EC relative to baseline (n = 2 independent HPC donors). (F) Relative mRNA expression of cell adhesion molecules and endothelial activation markers indicating various stages of leukocyte extravasation (n = 2 independent HPC donors).