Fig. 5

Inhibiting fungal colonization reduces hyperoxia-induced lung injury in mice. A Experimental schematic showing prenatal fluconazole exposure to inhibit neonatal fungal colonization. Hyperoxia (HO) or normoxia (NO) exposure was performed from day 3 to day 14 of life (P3–14). Tissue harvest and subsequent analyses were performed at P14. Schematic created using BioRender.com. Total sample size is 18–24 neonatal mice from three independent litters per experimental group. B Gut fungal microbiome analysis, showing alpha diversity as quantified by the Shannon diversity index and a PCoA plot showing Bray–Curtis dissimilarity. Significance testing by Mann–Whitney and PERMANOVA (n = 8 mice/group). C Gut bacterial microbiome analysis, showing alpha diversity as quantified by the Shannon diversity index and a principal coordinates analysis (PCoA) plot showing Bray–Curtis dissimilarity. Significance testing by Mann–Whitney and PERMANOVA (n = 8 mice/group). D Lung fungal microbiome analysis, showing alpha diversity as quantified by the Shannon diversity index and a PCoA plot showing Bray–Curtis dissimilarity. Significance testing by Mann–Whitney or PERMANOVA (n = 8 mice/group). E Lung bacterial microbiome analysis, showing alpha diversity as quantified by the Shannon diversity index and a PCoA plot showing Bray–Curtis dissimilarity. Significance testing by Mann–Whitney or PERMANOVA (n = 8 mice/group). F Representative hematoxylin and eosin confocal micrographs of the distal lung of 14-day-old neonatal mice born to mouse dams exposed to fluconazole or conventional specific pathogen-free (SPF), vehicle-exposed, control mice. Scale bars, 100 µm. Histomorphological analysis and forced oscillometry. Morphometry is quantified by mean linear intercept and radial alveolar count. Forced oscillometry is quantified by resistance and compliance. Significance testing by two-way ANOVA. G Representative immunofluorescence of alpha-smooth muscle actin. Quantification of vascular density of 20–50 µm vessels, and echocardiography quantification of right ventricular systolic pressure. Scale bars, 100 µm. Significance testing by two-way ANOVA. H Flow cytometric quantification of group 2 innate lymphoid cells (ILC2), expressed as a percentage of CD45⁺ cells. ILC2 were defined as live, lineage negative, CD45⁺, CD90.2⁺, CD127⁺, IL-33R (ST2)⁺, and GATA3⁺ cells. Significance testing by two-way ANOVA (n = 5–7 mice/group). I–K Hyperoxia exposure alters the lung transcriptome in the lungs in fluconazole-exposed or SPF mice by RNA-seq (n = 4 mice/group). I Volcano plot of the hyperoxia-induced transcriptomic alterations in fluconazole-exposed mice and SPF controls. J Venn diagram showing differential gene expression in fluconazole-exposed neonatal mice compared to SPF controls. K Heatmaps of the top 10 most differentially expressed genes either unique to SPF controls, shared, or unique to fluconazole-exposed mice. FiO₂, fraction of inspired oxygen. Values represent means ± SEM. See also Fig. S14–17, Table S4