Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity

Abstract

Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery.

Keywords: GM-CSF; cholesterol biosynthesis; glycolysis; inflammation; innate immune memory; interleukin-1β; myelopoiesis; myelosuppression; trained innate immunity; β-glucan.

Graphical abstract

Figure 1

Administration of β-Glucan Drives Expansion of HSPC Subpopulations WT mice were injected with β-glucan or PBS, and BM analysis was performed after 24 hr. (A) Representative fluorescence-activated cell sorting (FACS) plots for the identification of hematopoietic progenitor cells. After gating for Lin⁻ cells, LSK cells were characterized as cKit⁺Sca1⁺ cells. LSK cells subpopulations were further characterized as MPP (CD48⁺CD150⁻LSK), ST-HSC (CD48⁻CD150⁻LSK), and LT-HSC (CD48⁻CD150⁺LSK). (B and C) Cell numbers of LSKs, MPPs, ST-HSCs, and LT-HSCs (B) and cell percentages of the same populations in total BM cells (C) of mice at 24 hr after the administration of PBS or β-glucan (ns = 11 and 12 mice). (D) Representative FACS plots for the identification of MPP subpopulations. After gating for LSK cells, MPP4 cells are characterized as CD48⁺Flt3⁺CD150⁻LSK, MPP3 cells are characterized as CD48⁺Flt3⁻CD150⁻LSK, and MPP2 cells are characterized as CD48⁺Flt3⁻CD150⁺LSK. (E) Frequency of MPP subpopulations in LSK cells in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group). (F and G) Representative FACS plots for the identification of CD41⁺ LT-HSCs (F) and frequency of CD41⁺ LT-HSCs (in total LT-HSCs) (G) in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group). Data are presented as mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S1.

Figure S1

Administration of β-Glucan Promotes Cell Proliferation of LT-HSCs, Related to Figure 1 (A and B) Cell cycle analysis was performed in LT-HSC at 24h after the administration of PBS or β-glucan by staining for Ki67 and DAPI. (A) Representative flow cytometry plots and (B) frequency of LT-HSC at different phases of the cell cycle (n = 5 mice per group). Data presented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 2

Sustained Increase in Myelopoiesis upon β-Glucan Administration (A–F) WT mice were injected with β-glucan or PBS, and BM analysis was performed after 7 days. (A) LSK, MPP and LT-HSC cell numbers in the BM of mice on day 7 after administration of PBS or β-glucan (n = 6 mice per group). (B) Frequency of MPP subpopulations in the LSK cells 7 days after β-glucan or PBS administration (n = 5 mice per group). (C) Frequency of CD41⁺ LT-HSCs (in total LT-HSCs) on day 7 after the administration of PBS or β-glucan (n = 5 mice per group). (D) Representative FACS plots for the identification of MyP subpopulations. (E and F) GMP cell numbers (E) and frequency within the MyP pool of GMPs (Lin⁻c-Kit⁺Sca1⁻CD16/32⁺CD34⁺) and CMPs (Lin⁻c-Kit⁺Sca1⁻CD16/32⁻CD34⁺) (F) in the BM of mice on day 7 after the administration of PBS or β-glucan (n = 6 mice per group). (G–I) WT mice were injected with β-glucan or PBS, and BM analysis was performed after 28 days. (G) LSK and LT-HSC cell numbers (n = 5 mice per group). (H and I) Frequency of MPP4 cells in total BM cells (H) and GMP cell numbers in the BM (I) of mice on day 28 after the administration of PBS or β-glucan (n = 5 mice per group). (J and K) Transplantation. (J) LT-HSCs (CD45.2⁺) were sorted 28 days after β-glucan or PBS administration and transplanted to lethally irradiated SJL/BL6 (CD45.1⁺) mice. CD45.1⁺ BM cells were co-transplanted in order to ensure the survival of recipients. (K) Lineage output of donor LT-HSCs (CD45.2⁺) in peripheral blood of recipients at week 12 post-transplant (n = 10 recipient mice per group). Data are presented as mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S2.

Figure S2

Peripheral Blood Chimerism in Recipients of LT-HSCs Isolated from β-Glucan-Administered or PBS-Injected Mice, Related to Figure 2 Briefly, LT-HSCs (CD45.2⁺) were sorted 28 days after β-glucan or PBS administration and transplanted to lethally irradiated SJL/BL6 (CD45.1⁺) mice. The percentage of donor-derived (CD45.2⁺) cells in peripheral blood of recipient mice at 12 weeks after transplantation is shown (n = 10 recipient mice per group). Data presented as mean ± SEM.

Figure 3

Single-Cell Transcriptional Analysis in LT-HSCs upon β-Glucan Administration (A–C) Single-cell qPCR in LT-HSCs isolated from mice at 24 hr after administration of PBS or β-glucan (n = 42 cells per condition). (A and B) Hierarchical clustering analysis (A) and distribution of LT-HSCs in the three identified clusters (B) at 24 hr after the administration of PBS or β-glucan. (C) Violin plots indicating genes with significantly altered expression between clusters 1 and 2. The y axis represents gene expression. The horizontal width of the plot shows the density of the data along the y axis. Color key represents the percentage of cells that express the specific gene. (D and E) Single-cell qPCR was performed in CD41⁻ and CD41⁺ LT-HSCs isolated from mice at 24 hr after the administration of PBS or β-glucan. Hierarchical clustering analysis (D) and violin plots indicating genes with significantly altered expression between CD41⁺ LT-HSCs from PBS and β-glucan-treated mice (E).

Figure 4

Training with β-Glucan Promotes a Beneficial Response to a Secondary Challenge (A) WT mice were injected with β-glucan or PBS, and after 28 days, they received a secondary challenge with LPS. (B and C) LSK, MPP, and LT-HSC cell numbers (B) and frequency of the same cells in total BM cells (C) at 24 hr after LPS injection (n = 10 mice per group). (D) Representative FACS plots and frequency of γ-H2AX-positive LT-HSCs at 24 hr after LPS injection (n = 10 mice per group, right plots and gray background). The frequency of γ-H2AX-positive LT-HSCs at 28 days after β-glucan administration in mice not injected with LPS (—) is also shown; ns = 4 and 5, left plots and white background. (E) Experimental protocol for the effect of β-glucan on the recovery of granulopoiesis after cyclophosphamide administration (4 rounds). (F and G) Total white blood cell (WBC) (F) and granulocyte (Gr1⁺CD11b⁺) (G) counts in the peripheral blood (n = 10 mice per group). (H) Experimental protocol for 5-FU administration. (I) Survival curves of 5-FU-treated mice treated with β-glucan or PBS control (n = 16 mice per group). Comparison of survival curves was performed by log-rank (Mantel-Cox) test, and p value is shown. (J and K) Mice were injected with β-glucan or PBS, and 7 days later, a single dose of 5-FU was administered. (J) Neutrophil numbers in peripheral blood at different time points after the administration of 5-FU (n = 5 mice per group). (K) Frequency of γ-H2AX-positive LT-HSCs 14 days after 5-FU administration (n = 10 mice per group). Data are presented as mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 5

β-Glucan-Induced Alterations in LT-HSC Metabolic Pathways Revealed by Transcriptomic Analysis (A–F) Transcriptome analysis in LT-HSCs sorted from mice on day 7 after β-glucan or PBS administration (n = 4 mice, PBS group; n = 3 mice, β-glucan group). (A) Differential gene expression in LT-HSCs from β-glucan-treated mice as compared to PBS-treated mice. Volcano plot showing the distribution of the adjusted p values (−log(P-adj.)) and the fold changes (log₂ fold change). Significant changes are indicated in red (FDR = 0.05). (B) Top overrepresented canonical pathways showing upregulated (red) or downregulated (blue) genes in LT-HSCs from β-glucan-treated mice, as compared to PBS-treated mice. (C) Heatmap of myeloid- and lymphoid-lineage-related genes. (D) Heatmap depicting the differential gene expression of transcription regulators. Log₂ fold change in cells derived from β-glucan-treated mice, as compared to PBS-treated mice, is indicated. (E) GSEA for glycolytic genes and genes related to cholesterol homeostasis. NES, normalized enrichment score. (F) Heatmap of genes involved in glycolysis and pentose phosphate pathway (PPP) and cholesterol homeostasis in LT-HSCs from β-glucan-treated mice compared to PBS-treated mice. (G) Bioenergetic extracellular flux analysis (Seahorse) in Lin⁻cKit⁺ BM progenitors sorted from mice 24 hr after β-glucan or PBS administration (n = 5 mice per group). Basal and maximal ECARs (after oligomycin) (left) and glycolytic reserve (right), calculated as the difference between basal and maximal ECARs, are indicated. (H) Glycolytic gene expression in LSKs from mice 24 hr after β-glucan or PBS administration using qPCR (ns = 4 and 5 mice). (I) Bioenergetic extracellular flux analysis of Lin⁻cKit⁺ cells sorted from mice at 7 days after β-glucan administration; glycolytic reserve is shown (n = 5 mice per group). Data are presented as mean ± SEM in (G)–(I). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. See also Figures S3 and S5.

Figure S3

Transcriptomic Analysis of LT-HSCs on Day 7 after β-Glucan Administration, Related to Figure 5 (A and B) Upstream regulator analysis in transcriptomic data using IPA. Genes regulated by the TFs (A) Cebpe and (B) Pax5. Genes with increased expression in LT-HSC from mice injected with β-glucan (as compared to cells from PBS-treated mice) are depicted in red, whereas genes with decreased expression are shown in blue.

Figure S4

Transcriptional Alterations in Hematopoietic Progenitors 28 Days after β-Glucan Administration, Related to Figure 5 LT-HSCs and MPPs were sorted from mice on day 28 after β-glucan or PBS administration and RNA sequencing was performed (n = 4 mice per group). (A and C) Differential gene expression in (A) LT-HSCs and (C) MPPs; volcano plots depicting the distribution of the adjusted p values (-log(P-adj.)) and the fold changes (log2 Fold Change). Significant changes are colored red (FDR = 0.05). (B and D) Overrepresented canonical pathways showing genes that were upregulated (red) or downregulated (blue) in (B) LT-HSCs and (D) MPPs from β-glucan–injected mice, as compared to PBS-treated mice. (E) Heatmap depicting the expression of lymphoid lineage-related genes in LT-HSCs and MPPs from β-glucan injected mice, as compared to PBS treated mice. Transcripts that are not significantly altered are shown in gray. (F) Differentially expressed genes in both LT-HSCs and MPPs. The number of upregulated (red) and downregulated (blue) genes is shown.

Figure S5

Mevalonate Pathway, Related to Figure 5 Schematic depiction of the mevalonate pathway with genes significantly upregulated in LT-HSC from β-glucan–injected mice (as compared to PBS-treated mice) shown in red.

Figure S6

GSEA in LT-HSC after 5-FU Administration, Related to Figure 5 Mice were injected with β-glucan or PBS and 7 days later a single dose of 5-FU was administered. LT-HSCs were sorted from mice on day 14 after 5-FU administration and transcriptomic analysis was performed (n = 4 mice per group). GSEA for genes related to glycolysis, OXPHOS and cholesterol biosynthesis.

Figure 6

Alterations in Lipid Metabolism in BM Progenitor Cells upon β-Glucan Administration Lin⁻cKit⁺ cells were sorted from mice 24 hr after β-glucan or PBS administration, and non-targeted metabolomic and lipidomic analyses were performed (n = 4 mice per group). (A and B) Not-targeted metabolomics. (A) Volcano plots depict the comparison of metabolite abundances between cells from β-glucan- and PBS-treated mice. Altered metabolites (q value < 0.1) involved in linoleate and arachidonic acid pathways (Kyoto Encyclopedia of Genes and Genomes [KEGG] pathways database) are indicated. (B) Heatmap depicts the abundance of differentially regulated metabolites (q value < 0.1). (C–H) Lipidomic analysis. (C) Principal-components analysis shows that the two conditions segregate along the first dimension, PC1 (Mann-Whitney U test, p value = 0.028). (D) Mol % abundance of lipid classes (CE, cholesterol esters; Cer, ceramides; CL, cardiolipin; DAG, diacylglycerols; HC, hexosyl ceramide; LPA, lysophosphatidic acid; LPC, lysophopshatidylcholines; LPE, lysophosphatidylethanolamine; LP^∗, LPC and LPE plasmalogens; PC, phosphatidylcholines; PCO, PC plasmalogens; PE, phosphatidylethanolamines; PEO, PE plasmalogens; PG, phosphatidylglycerols; PI, phosphatidylinositols; PS, phosphatidylserines; SM, sphingomyelins). Data are presented as mean ± SEM. ^∗p < 0.05. (E) Difference between the mean abundance of lipid species (excluding CL and cholesterol esters) grouped by the number of carbon atoms: blue bars indicate lipids enriched in cells from β-glucan-treated mice, while orange bars indicate lipids enriched in cells from PBS-treated mice. Lysolipids containing a single fatty-acyl chain are highlighted by a gray background. (F) Difference between the mean abundance of lipid species grouped by number of double bonds: blue bars indicate lipids enriched in cells from β-glucan-treated mice, while orange bars indicate lipids enriched in cells from PBS-treated mice. (G) Relative abundance of lipid species belonging to PCO−, PEO−, SM, PI, LPA, PS, and cholesterol esters. Relative difference of lipid species is shown; negative bars represent species that are more abundant in the PBS group, while positive bars represent species that are more abundant in the β-glucan group. (H) Lipid species containing arachidonic acid (fatty acid [FA] 20:4) are indicated; negative bars indicate species that are more abundant in the PBS group, while positive bars indicate species that are more abundant in the β-glucan group.

Figure 7

IL-1β, Glycolysis, Cholesterol Metabolism, and the GM-CSF/CD131 Axis Are Involved in β-Glucan-Dependent Training in the BM (A) Cytokine and G-CSF concentrations in the BM extracellular fluid of mice at 24 hr after the administration of PBS or β-glucan (n = 10 mice per group). (B and C) Mice were injected with β-glucan in the absence (vehicle control, Ctrl) or presence of IL1RA, and BM analysis was performed 24 hr later. (B) Cell-cycle analysis in LT-HSCs. (C) The frequency of MPP subpopulations in LSK cells in the BM is indicated (n = 5 mice per group). (D) Bioenergetic extracellular flux analysis in Lin⁻cKit⁺ cells sorted from mice 24 hr after β-glucan administration in the absence (vehicle control, Ctrl) or presence of IL1RA (n = 5 mice per group). Basal and maximal ECAR are indicated. (E) LSK cells were treated in vitro with IL-1β or PBS for 24 hr, and Seahorse analysis was performed (n = 5 cultures per group). Basal and maximal ECARs (left) and glycolytic reserve (right) are indicated. (F–I) Mice were injected with β-glucan on day 0. (F) Glycolysis and IL-1 were blocked by the administration of 2-DG and IL1RA, respectively, on days 0 and 1; PBS served as the vehicle control (Ctrl). (G) LSK, MPP, and LT-HSC numbers in the BM at day 7 after β-glucan administration (n = 5 mice per group). (H and I) GMP numbers in the BM (H) and frequency of GMPs within the MyP pool (I) at day 7 after β-glucan administration (n = 5 mice per group). (J and K) Mice were injected with PBS or β-glucan, and BM analysis was performed 24 hr later; representative FACS plots (J) and frequency of CD131⁺ LSKs, CD131⁺ MPPs, and CD131⁺ LT-HSCs (K) (n = 5 mice per group) are indicated. (L) Staining for pSTAT5 in LSK cells 24 hr after PBS or β-glucan administration. Representative FACS plots and median fluorescence intensity (MFI) are shown (n = 5 mice per group). (M–P), As indicated in (M), mice were injected with β-glucan on day 0. Cholesterol metabolism was blocked by the administration of atorvastatin on days 0 and 1; Ctrl represents the vehicle control. (N) LSK, LT-HSC, and MPP cell numbers; (O) frequency of the same cells in total BM cells; and (P) frequency of GMPs within the MyP pool at day 7 after β-glucan administration (n = 5 mice per group). (Q) Mice were injected with β-glucan on day 0. GM-CSF was blocked by specific anti-GM-CSF antibody on days 0 and 1. Immunoglobulin G (IgG) isotype served as the control. (R and S) LSK, LT-HSC, and MPP cell numbers (R) and frequency of the same cells in total BM cells (S) at day 7 after β-glucan administration are indicated (n = 5 mice per group). Data are presented as mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.