Human Caco-2 enterocytes were polarized on standard plates (a) or Transwell inserts (b to h) for 14–21 days. (b to h) Human Raji B lymphocytes were added in the lower compartment of the culture chamber in order to obtain a cellular layer composed of enterocytes and M cells and the cell culture medium was supplemented with E2-P4 concentrations equivalent to those found at birth (E2: 10−8M, P4: 10−6M; E2-P4 C0 condition) and 7 days later (E2: 10−9M, P4: 10−7M; E2-P4 C7 condition) for 6 days before infection. Control cells were maintained in regular cell culture medium without E2 and P4 supplementation. Epithelial cells were infected by their apical side with bacteria at a multiplicity of infection 10 (a) and 100 (c to h) for 2 hr. (a) Bacterial adherence and invasion of a cellular monolayer composed of enterocytes. (b) Representative confocal microscopy image of the epithelial layer obtained following Caco-2 and Raji co-culture showing enterocytes and M cells. (c) Transcytosis of CC17 GBS across a monolayer strictly composed of enterocytes and a monolayer composed of both enterocytes and M cells. (d) Representative confocal microscopy images showing CC17 and CC23 GBS adherence to the enterocytes + M cells epithelial layer. (e) Bacterial adherence of CC17 and CC23 GBS to the enterocytes + M cells epithelial layer. (f) Quantitative imaging analysis of bacterial association with M cells expressed as the proportion of bacteria associated with GP2 positive cells relative to total adherent bacteria. (g) Representative confocal microscopy image showing the presence of several CC17 GBS cocci at the surface and inside an M cell. (g1 to g3) Orthogonal views of (g). (d to g) Experiments were carried out in E2-P4 C7 condition. (h) Quantitative imaging analysis of CC17 GBS association with M cells in E2-P4 C0, E2-P4 C7, and control condition. (a, c, e, f, h) Results are expressed as mean ± SEM.≥3 experiments in duplicate. (b, d, g) Tight junctions are labeled with anti-ZO1 antibody, M cells with anti-GP2 antibody, GBS with anti-GBS antibody, and actin with phalloidin. Multiple-group comparisons were performed by Kruskal-Wallis test (c and h) and single-group comparisons by Mann-Whitney test (e and f). *p<0.05; **p<0.01, ***p<0.001.