Fig. 7: β-Glucan training induces robust changes in neutrophils phenotype and metabolism.

a, UMAP of 925 lung neutrophils isolated from nontreated mice and mice challenged with β-glucan for 4 and 7 d. b, Heatmap of scaled expression levels of select DEGs (FDR < 0.05) at day 4 post β-glucan treatment. c, Number of DEGs 4 and 7 d after β-glucan treatment. d, Enrichment plots of representative upregulated (FDR < 0.05) GO pathways 4 d after β-glucan treatment. e–h, Mice were treated with β-glucan 6 d before influenza infection. On day 9 post-β-glucan treatment, lung neutrophils from infected and noninfected mice were extracted and their surface phenotype was assessed by spectral flow cytometry. e, UMAP of CD11b⁺Ly6G⁺ neutrophils with and without influenza infection. Neutrophils separate in eight Flowsom Clusters. f, UMAP from b projected for the four experimental groups (PBS ± influenza, black and β-glucan ± influenza, dark blue). Clusters 5, 6 and 8 expand dramatically during influenza infection. g, Quantification of neutrophil present in influenza-driven clusters (clusters 5, 6 and 8). h, Mean fluorescence intensity (MFI) of selected markers projected on the UMAP from f. Influenza-driven clusters exhibit an activated phenotype (CD14high, CD24high CD11bhigh, CD62Llow, CD49dlow). UMAPs are based on 16 surface markers: CD45, Ly6G, CD101, CXCR2, CXCR4, CD62L, CD24, CD11b, CD49d, CD44, CCR2, Ly6C, CD80, MHC-II, CD16, CD14 (n = 7). i, Mice were treated with β-glucan. Neutrophils were purified from blood on day 4 post-βd-glucan treatment. j–m, Neutrophils’ cellular metabolism determined by Seahorse (j), basal respiration (k), maximal respiration (l) and ATP production (m). n,o, Representative histogram plot (n) and quantification (MFI) (o) for mitochondrial mass using Mitotracker Green dye in the neutrophils of β-glucan-treated mice (n = 4). p, Representative three-dimensional reconstructed lung intravital microscopy images from control or β-glucan-treated Ly6G-TdTom mice. Mitotracker Green dye was given i.v. before imaging. Scale bars, 50 μm. q, Mitochondria bright neutrophils quantified (n = 4). The horizontal lines represent the median, the bounds of the boxes indicate the 25th and 75th percentiles and the whiskers represent the minima and maxima. Each dot represents an individual sample (n = 4). Data are represented as mean ± s.e.m. Data were analyzed using two-way ANOVA followed by Šidák’s multiple-comparison tests (j) and two-tailed, unpaired Student’s t-test (k–m, o and q). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Norm., normalized. Illustration in i created using BioRender.com.

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