Fig. 4: Environmental adaptation induced by β-glucan significantly improves pulmonary bacterial clearance and outcome of bleomycin-induced fibrosis.
a,b, Extracellular acidification rate (ECAR; a) and glycolysis (b) in BALF cells 7 days after PBS or β-glucan stimulation of WT mice measured by Seahorse (n = 6 mice, one independent experiment). c,d, AM cells from day 7 PBS- or β-glucan-experienced WT mice were selected by adherence and subsequently treated with 2.5 µg pHrodo S. aureus bioparticles (n = 3 mice pooled per condition, technical replicates: 6 control wells, 12–19 treated wells per group, one of two independent experiments shown). c, Representative curve of absolute phagocytosis+ AM numbers over the time course of 7 h (here shown as mean ± s.d. of all technical replicates). d, Absolute numbers of phagocytosis+ AMs 2 h after adding the pHrodo S. aureus bioparticles. e,f, C57BL/6J WT mice were intranasally stimulated with PBS or β-glucan followed by intratracheal infection with 5 × 106 colony-forming units (CFUs) L. pneumophilia at day 7 after primary stimulation and analysis at day nine (n = 9–10 mice, two independent experiments). Quantification of bacterial load in BALF (e) and absolute numbers of ApoE+CD11b+ AMs by flow cytometry 9 days after primary stimulation (f). g,h, Representative confocal images (g) and SMA area quantification (h) of BALOs co-cultured with PBS- or β-glucan-experienced AMs 48 h after induction of fibrosis via TGF-β. Seven days after stimulation, 2.5 × 104 AMs of PBS- or β-glucan-experienced WT mice were co-cultured with day 21 lung BALOs for 24 h. AM–organoid co-cultures were subsequently treated with 1.05 ng ml−1 TGF-β for 48 h before fixation and antibody staining. Myofibroblasts were stained for α-SMA (n = 6–8 organoids per condition from two replicate wells; one of two independent experiments shown). Scale bars, 50 µm (g). Data are depicted as the mean ± s.d. Significance was assessed using unpaired two-tailed student’s t-test (b, e and f), ordinary one-way ANOVA with Tukey’s multiple comparisons (d) and two-tailed Mann–Whitney test (h).
