Figure 5.

Perinatal and neonatal mono-colonization of either EcN-Chim or EcN-Ctrl activated both TLR2 and TLR4 expression and maintained ZO-1 levels in the lung. Mice were treated as indicated in Figure 2A. For immunohistochemistry analysis, lung sections were processed and stained as described in Material and Methods using antibodies against TLR2, TLR4, and ZO-1 followed by 3,3′-Diaminobenzidine (DAB) staining. Samples were analyzed under a light microscope with a 40x objective. Immunohistochemistry staining for TLR2 (A), TLR4 (B), and ZO-1 (E) is represented by a brown color (indicated by arrows). Quantification of TLR2 (C), TLR4 (D), and ZO-1 (F) expression was performed by optical density analysis (OD) of bronchial epithelial cell layer using ImageJ software. (C, D, F) represents mean ± SEM from two experiments (five mice per group were tested). Error bars show mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 by the One-way ANOVA followed by the Bonferroni’s Multiple Comparison Test.