Figure 2

BCG generates catastrophic effects in the epithelial, but not endothelial component of the model. (a) Blue Dextran 2000 kDa (DB2000) permeability through endothelial (BPAEC), epithelial (BATII), co-cultures and co-cultures infected with BCG, compared to membrane only. BPAEC monocultures contribute considerably less than BATII cells to the barrier integrity of the co-culture (One-way ANOVA, Tukey’s Multiple Comparisons Test; ****P ≤ 0.0001, *P ≤ 0.05). Barrier properties of the co-culture are significantly reduced upon infection with BCG at MOI 10 (One-way ANOVA; ****P ≤ 0.0001). Data represent 3 experiments performed on different days, presented as Mean ± SD; n = 4. (b) TEER, measured from the point at which cultures were raised to air–liquid interface (ALI) until the day of experimentation (day 14; black arrow denotes BCG infection), with a final measurement being performed 48 h.p.i. A significant drop was observed in infected wells, compared to untreated controls (day 16) (Unpaired t-test; P ≤ 0.0001). Data represent 3 experiments performed on different days, presented as Mean ± SD; n = 3. (c) Regions of BATII epithelial (Ep) cells in (i) show a healthy and in this instance multi-layered structure, overlaid onto the endothelial (En) cell line BPAEC on the apical side of a permeable membrane (M). H&E staining reveals cuboidal alveolar type II cell morphology, apical microvillae and densely stained regions indicative of lamellar bodies. The underlying endothelial monolayer is distinguished by its elongated and flattened appearance. Following BCG infection (48 h.p.i, (ii)), the epithelial layer is stripped from the underlying endothelial cells almost completely. (d) Ziehl–Neelsen (carbol-fuchsin) staining, used in conjunction with the counterstain methylene blue indicates presence of BCG. Methylene blue highlights nuclei as more intensely stained structures, with other cell structures staining less intensely (i). At 48 h.p.i, BCG are observed as multiple red/pink bacilli (pink arrow) in a single surviving epithelial cell (Ep), whilst the methylene blue counterstain reveals a relatively intact endothelial layer (ii). All images taken on the Nikon Eclipse Ci upright microscope using 60 × objective.