Figure 3.

Modeling of atopic dermatitis (AD) by S. aureus colonization and infection of ALI-skin organoids

(A) Schematic outline of a S. aureus and ALI-skin organoid co-culture method.

(B) S. aureus immunostaining to visualize bacteria and the KRT5⁺ basal epithelial layer maker (red) in 0 CFU (control; PBS only), 10⁵ CFU, 10⁶ CFU, or 10⁷ CFU S. aureus-infected ALI-skin organoids (left) and S. aureus infection in the dermal layer as mean fluorescence intensity (MFI) was analyzed in each group (right; independent replicates = 5). Scale bars, 50 μm.

(C) Epidermal barrier markers were analyzed in ALI-skin organoids infected with 0 CFU, 10⁵ CFU, 10⁶ CFU, or 10⁷ CFU SA. Immunostaining images for KRT5⁺ basal and KRT10⁺ spinous epithelial layer markers (left) and quantification of KRT10 expression level (MFI; right) on the top panel, immunostaining images for LOR⁺ granular layer marker (left) and its quantification analysis (MFI; right) on the middle panel, and immunostaining images for FLG⁺ cornified epidermal layer marker (left) and its quantification analysis (MFI; right) on the bottom panel are shown. One-way ANOVA with Bonferroni post-hoc test; ∗∗∗p < 0.001, compared to 0 CFU; independent replicates = 5. Scale bars, 50 μm.

(D) Immunostaining images for TSLP in green and KRT14 in red in ALI-skin organoids infected with 0 CFU, 10⁵ CFU, 10⁶ CFU, or 10⁷ CFU S aureus (left) and quantification of TSLP expression on the epidermal layer (MFI; right; independent replicates = 4). Scale bars, 50 μm.

Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. The results are presented as the means ± SEM.