β-glucan-mediated trained immunity increases LPS-induced ALI.

A, Schematic of the β-glucan-induced training seven days before lipopolysaccharide (LPS)-induced acute lung injury (ALI) model. Experiments were performed in sex- and age-matched 10-12 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) WT mice. B, Lung micro-CT scan, percentage of poorly- or non-aerated lung and average lung Hounsfield unit. C, Alveolar capillary membrane permeability assessed by lung Evans blue dye concentration. D, Lung histology after staining with haematoxylin and eosin. E, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec-F-, CD11b+, Ly6G+). F, G, H BAL chemokine and pro-inflammatory cytokines concentrations (left to right) (CXCL1: chemokine C-X-C motif ligand 1, IL-6: interleukin-6 and TNF-α: tumor necrosis factor α). I, Quantification of BAL alveolar macrophages frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c+, Siglec-F+). Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. * p<0.05, *** p<0.001.

Systemic administration of β-glucan enhances ALI via AMs.

A, Schematic of the clodronate-mediated alveolar macrophages (AM) depletion experiments, performed in sex- and age-matched 10-12 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) WT mice. B, Alveolar capillary membrane permeability assessed by lung Evans blue dye concentration. C, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec-F-, CD11b+, Ly6G+). D, BAL chemokine C-X-C motif ligand 1 (CXCL1) and pro-inflammatory cytokines (IL-6: interleukin-6 and TNF-α: tumor necrosis factor α) concentrations. E, Schematic of the β-glucan-induced training and lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in sex- and age-matched 6 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) Csf2rb-/- mice. F, BAL total protein concentration. G, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec-F-, CD11b+, Ly6G+). H, BAL CXCL1, IL-6 and TNF-α concentrations. I, Schematic of the adoptive transfer of control (i.p. PBS, white bars) or β-glucan-trained (i. p. β-glucan, green bars) AMs collected from adult WT mice to 2 days old Csf2rb-/- mice. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) was performed 6 weeks after adoptive transfer. J, BAL total protein concentration. K, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c, Siglec-F-, CD11b+, Ly6G+). L, BAL CXCL1, IL-6 and TNF-α concentrations. Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001.

β-glucan reprograms AMs.

A, Schematic of control (i.p. PBS) or β-glucan-trained (i. p. β-glucan) AMs collected from adult WT mice ex vivo stimulation with LPS (LPS-: unstimulated, LPS+: stimulated in RNAseq analysis). B, Discovery plot. C, AM differential expression of genes in response to β-glucan training. D, Gene ontology in response to β-glucan training. E, GSEA in response to β-glucan training. F, AM differential expression of genes in response to LPS stimulation. G, Gene ontology in response to LPS stimulation. H, GSEA in response to LPS stimulation. I, AM gene expression in response to LPS in β-glucan-trained AMs. J, Examples of genes expression in response to LPS in control vs β-glucan-trained AMs. K, Chemokine C-X-C motif ligand 1 (CXCL1) and tumor necrosis factor α (TNF-α) concentrations after ex vivo LPS stimulation. L, GSEA of oxidative phosphorylation (left) and glycolysis (right) pathways according to β-glucan-training in unstimulated (LPS-) and LPS stimulated (LPS+) AMs. M, Evaluation of AM metabolism: basal respiration (upper left), ATP production (upper right), extracellular acidification rate (ECAR, lower left), oxygen consumption rate (OCR, lower right). Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. ** p<0.01, *** p<0.001.

IFNγ and neutrophils are required in β-glucan-mediated AM reprogramming.

A, Schematic of the β-glucan-induced training and lipopolysaccharide (LPS)-induced acute lung injury (ALI) model. Experiments were performed in sex- and age-matched 10-12 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) IfngR-/-mice. B, Alveolar capillary membrane permeability assessed by lung Evans blue dye concentration. C, Quantification of BAL neutrophils proportion (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec-F-, CD11b+, Ly6G+). D, BAL chemokine C-X-C motif ligand 1 (CXCL1) concentration (left) and pro-inflammatory cytokines (IL-6: interleukin-6 – middle- and TNF-α: tumor necrosis factor α –right) concentrations. E, Schematic of control (i.p. PBS, white bars) or β-glucan-trained (i. p. β-glucan, green bars) AMs collected from adult IfngR-/- mice ex vivo stimulation with LPS. F, Chemokine C-X-C motif ligand 1 (CXCL1) and tumor necrosis factor α (TNF-α) concentrations after ex vivo LPS stimulation. G, Schematic of the analysis of the effect of i.p. β-glucan injection on interferon-γ (IFNγ) production and neutrophils expansion before (white bars) and Day 1, Day 3, Day 5 and Day 7 post-injection (green bars). H, BAL IFNγ concentrations. I, Quantification of BAL neutrophils proportion (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec-F-, CD11b+, Ly6G+). J, Quantification of lung neutrophils proportion (left) and absolute count (right). K, Schematic of control (i.p. PBS, white bars) or β-glucan-trained (i. p. β-glucan, green bars) AMs ex vivo stimulation with 50ng/mL LPS. AMs were collected from control (i.p. injection of isotypes), neutrophils depleted (i.p. injection of anti-Ly6G antibodies) or IFNγ antibody-depleted (i.p. injection of anti-IFNγ antibodies) adult WT mice. L, CXCL1 and TNF-α concentrations after ex vivo LPS stimulation. Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. * p<0.05, *** p<0.001.

Long-term effects of β-glucan-mediated trained immunity on LPS-induced ALI.

A, Schematic of the β-glucan-induced training twenty-eight days before lipopolysaccharide (LPS)-induced acute lung injury (ALI) model. Experiments were performed in sex- and age-matched 10-12 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) WT mice. B, Alveolar capillary membrane permeability assessed by lung Evans blue dye concentration. C, Lung histology after staining with haematoxylin and eosin. D, BAL chemokine C-X-C motif ligand 1 (CXCL1) concentration (left) and pro-inflammatory cytokines (IL-6: interleukin-6 (middle) and TNF-α: tumor necrosis factor α (right)). E, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec-F-, CD11b+, Ly6G+). F, quantification of BAL alveolar macrophages frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c+, Siglec-F+). G, Schematic of control (i.p. PBS, white bars) or 28 days β-glucan-trained (i. p. β-glucan, green bars) AMs collected from adult WT mice ex vivo stimulation with LPS. H, Chemokine C-X-C motif ligand 1 (CXCL1) and tumor necrosis factor α (TNF-α) concentrations after ex vivo LPS stimulation. Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001.

β-glucan-mediated trained immunity increases poly(I:C)- induced ALI.

A, Schematic of the β-glucan-induced training seven days before poly(I:C)- induced acute lung injury (ALI) model. Experiments were performed in sex- and age-matched 10-12 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) WT mice. B, Alveolar capillary membrane permeability assessed by lung Evans blue dye concentration. C, Lung histology after staining with haematoxylin and eosin. D, BAL chemokine C-X-C motif ligand 1 (CXCL1) concentration (left) and pro-inflammatory cytokines (IL-6: interleukin-6 (middle) and TNF-α: tumor necrosis factor α (right). E, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec- F-, CD11b+, Ly6G+). F, Quantification of BAL alveolar macrophages frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c+, Siglec-F+). G, Schematic of control (i.p. PBS, white bars) β-glucan-trained (i. p. β-glucan, green bars) AMs collected from adult WT mice ex vivo stimulation with LPS. Differential expression of viral defense genes in response to LPS in β-glucan-trained AMs. Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001.

β-glucan-mediated AM reprogramming is independent of Dectin-1 and type I interferon signaling.

A, Schematic of the β-glucan-induced training seven days before lipopolysaccharide (LPS)-induced acute lung injury (ALI) model. Experiments were performed in sex- and age-matched 10-12 weeks old control (i.p. PBS, white bars) and trained (i. p. β-glucan, green bars) Dectin1-/- mice. B, BAL chemokine C-X-C motif ligand 1 (CXCL1) concentration (left) and pro-inflammatory cytokines (IL-6: interleukin-6 (middle) and TNF-α: tumor necrosis factor α (right)). C, Quantification of BAL neutrophils frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c-, Siglec- F-, CD11b+, Ly6G+). D, Quantification of BAL alveolar macrophages frequency (left) and absolute count (right) (gated on single live cells, CD45.2+, CD11c+, Siglec-F+). E, Schematic of control (i.p. PBS, white bars) β-glucan-trained (i. p. β-glucan, green bars) AMs collected from adult Dectin1-/-mice ex vivo stimulation with LPS. F, Chemokine C-X-C motif ligand 1 (CXCL1) and tumor necrosis factor α (TNF-α) concentrations after ex vivo LPS stimulation. G, Schematic of control (i.p. PBS, white bars) β-glucan-trained (i. p. β-glucan, green bars) AMs collected from adult IfnaR-/- mice ex vivo stimulation with LPS. H, Chemokine C-X-C motif ligand 1 (CXCL1) and tumor necrosis factor α (TNF-α) concentrations after ex vivo LPS stimulation. Data were analysed using one-way ANOVA followed by Dunn’s multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001.