Fig. 4
Bifidobacterium bifidum strains W23 and W28 enhance intestinal barrier properties. (A) Immunofluorescence confocal images of HRT18 cells grown in glass slides until full confluency and infected with B. bifidum strains W23, W28, and 20456 at MOI 10 for 20 h. Cells were stained for occludin (green), ZO-1 (red), and DAPI (white). The maximum intensity projection is depicted. White scale bars represent 20 µM. (B) Immunofluorescence images of HRT18 cultures grown in Transwells incubated with W23, W28, 20456, W1, and W37 at MOI 100 for 28 h. Cultures were stained for occludin (green), ZO-1 (red), and DAPI (white). Z-stacks were captured from the apical surface until the appearance of nuclei and maximum intensity projections are depicted. White scale bars represent 20 µM. (C) Immunoblot analysis of MUC13 modification and expression of claudins-1, -3, and -4 in HRT18 Transwell cultures after incubation with bacterial strains W23, W28, 20456, W1, and W37 at MOI 100 for 28 h and during a challenge with 100 ng/mL IL-1β and 100 ng/mL TNF-α. The control sample only received the cytokine challenge without bacteria. All immunoblots used for quantification are available in Figure S7. (D) Quantification of protein expression of MUC13 and claudins-1, -3, and -4 (relative to actin) as shown in (C). All graphs depict the SEM of three independent experiments. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (E) TEER measurements of HRT18 Transwell cultures over time during incubation with W23, W28, 20456, W1, and W37 strains at MOI 100 and cytokine challenge at baseline as described for C. (F) TEER values at the 28 h timepoint depicted in E. Graphs depict SEM of three independent biological replicates. Statistical test: two-way ANOVA with Dunnett’s post hoc test. **** p < 0.0001.
