Figure 4. Bacterial infection of macrophage-enteroid monolayer induces morphological and physiological cell changes.
(a) MΦ projections extended through Transwell filters into DF enteroid monolayers following overnight EPEC infection. Actin, white; nuclei, blue; CD14 (MΦ), red; filter, dashed lines. (b) Adherent MΦ (left panel; *indicates p = 4.42 × 10−5) and number of MΦ projections (right panel; *indicates p = 2.33 × 10−9) increased upon apical overnight EPEC infection of co-cultures. UN, uninfected. (c) High resolution immunofluorescence confocal microscopy showed MΦ projections (arrowheads) going through permeable support and along lateral epithelial membranes upon overnight apical EPEC infection. Actin, white; CD14 (MΦ), red; filter, dashed lines. (d) ETEC adhered to DF enteroid monolayers alone (upper right panel) and co-cultured with MΦ (bottom right panel) following 3 h of infection. Uninfected cultures are represented in left panels. Actin, white; nuclei, blue; ETEC, green; CD14 (MΦ), red; filter, dashed lines. (e) Relative number of viable ETEC collected from the enteroid monolayer following 16 h of infection decreased in the presence of MΦ. *Indicates p = 0.0332. (f) DF enteroid monolayers infected with ETEC for 16 h displayed reduced TER values that were partially restored by the presence of MΦ in the co-culture. (g) Confocal Z-stack image (upper panel) and derivative 3D representation (lower panel) of MΦ extending projections through the membrane to reach the apical surface of DF epithelium infected 3 h with ETEC (arrowheads). Actin, white; CD14 (MΦ), red; nuclei, blue; filter, dashed lines. (h) Overnight ETEC infection did not significantly change the levels of IL-8, TGF-β1, IFN-γ, and IL-6 secreted by the cultures. IL8: *indicates p = 0.0447; **indicates p = 0.0455. Data are presented as the Mean + SEM of three independent experiments involving enteroids generated from three different donors with the exception of IFN-γ and IL-6 production that represent Mean + SEM of two independent experiments with enteroids generated from two different donors.