Figure 2.
Trained Immunity Shapes the Transcriptional Profile of TANs and Neutrophils from β-Glucan-Trained Mice Suppress Tumor Growth via ROS Production
(A–E) WT mice were treated with β-glucan or PBS and after 7 days were inoculated with B16-F10 melanoma cells. TANs (CD45+CD11c−CD11b+Ly6c−Ly6g+) were sorted and RNA sequencing analysis was performed (n = 5 mice in the PBS group; n = 4 mice in the β-glucan group) 14 days after the tumor injection. In (A), (C) and (E), false discovery rate (FDR) ≤ 0.05.
(A) Differential gene expression in TANs from mice pre-treated with β-glucan compared with TANs from PBS-treated mice. Volcano plot showing the distribution of the adjusted p values (−log10(padj)) and fold changes (log2 fold change).
(B) GSEA for genes related to a gene set previously implicated in the TAN1 phenotype (Shaul et al., 2016) was used to analyze the transcriptomic effect induced in TANs by β-glucan administration in mice. Abbreviation is as follows: NES, normalized enrichment score.
(C) Top 10 enriched canonical pathways identified by IPA in TANs from β-glucan-treated mice compared to PBS-treated mice.
(D and E ) Shown in (D) is a GSEA for genes related to ROS pathway, and in (E) is a heatmap of genes involved in ROS metabolic process in TANs from β-glucan-treated mice as compared with TANs from PBS-treated mice.
(F) WT mice were treated with β-glucan or PBS and after 7 days were inoculated with B16-F10 melanoma cells. Staining for ROS in TANs (CD45+CD11b+Ly6g+) was performed by flow cytometry 14 days after the tumor cell injection. Relative mean fluorescence intensity (MFI) is shown. MFI of ROS was measured and expressed in relation to the PBS group, set as 1. Data are presented as mean ± SEM (n = 15 per group).
(G) Splenic neutrophils were isolated from mice 7 days after injection with β-glucan or PBS. Neutrophils were co-cultured with luciferase expressing B16-F10 cells for 24 h. Tumor cell survival was assessed by measuring luminescence (n = 5 per group).
(H) Experimental scheme.
(I) As indicated in (H), WT or NCF1-deficient mice were injected with β-glucan or PBS, and after 7 days splenic neutrophils were isolated and were adoptively transferred together with B16-F10 cells into WT recipients. Tumor volume is shown. Data are presented as mean ± SEM (n = 7 mice in the PBS WT group; n = 12 mice in the β-glucan WT group; n = 11 mice in the PBS NCF1-deficient group; n = 14 mice in the β-glucan NCF1-deficient group).
∗p < 0.05, ∗∗∗∗p < 0.0001.
See also Figure S2.