Transcytosis of Bacillus subtilis extracellular vesicles through an in vitro intestinal epithelial cell model

Abstract

Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.

Figure 1

Representative plot of EVs concentration isolated from B. subtilis 168 versus particle size obtained by nanoparticle tracking analysis (NTA). Mean ± SD, n = 3.

Figure 2

B. subtilis EVs are taken up efficiently by Caco-2 cells in a time-dependent active manner. Caco-2 cells differentiated monolayer were incubated with CFSE-labeled EVs for different times (0, 15, 30, 45, 60, 120 and 240 min). After incubation, cells nuclear DNA was stained with To-Pro3 and actin filaments were stained with Rhodamine Phalloidin, and images visualized by CLSM. (a) Arrows point out EVs. (b) Number of EVs internalized per cell at the different times of incubation. (c) CFSE fluorescence per cell at the different times of incubation. In b and c data represents Mean ± SD. One-way ANOVA with Tukey’s post hoc test were carried out (n = 3). Means with a common letter are not significantly different (p > 0.05).

Figure 3

EVs derived from B. subtilis 168 did not affect cellular proliferation, viability or cytotoxicity of Caco-2 cells. Data were obtained from MTT Assay. Mean ± SD. One-way ANOVA (n = 8). Means with a common letter are not significantly different (p > 0.05).

Figure 4

Intact B. subtilis 168 EVs are transported across Caco-2 cells in a time-dependent active manner. (a) TEER was measured every 2–3 days to follow the Caco-2 cells monolayer formation. On day 14 after cell seeding, TEER measurement stabilized and Caco-2 cells monolayers were used for transcytosis assays. (b) CFSE-labeled EVs were incubated with Caco-2 cells in the upper chamber of transwell system. CFSE fluorescence and the number of EVs (NTA) were measured of the medium collected from the lower chamber at different times. Mean ± SD (n = 3). (c) After 120 min incubation CFSE-labeled EVs were visualized using Epi-fluorescence Microscopy. Arrows point out EVs.

Figure 5

B. subtilis 168 EVs move through Z axis in Caco-2 cell monolayers. Caco-2 cell differentiated monolayers were incubated with CFSE-labeled EVs and series of time-lapse scans were taken at intervals of 10 min during 120 min. One color was assigned to three selected times 0 min (red), 60 min (green) and 120 min (blue) and then images were overlapped to infer EVs movement (FIJI software). (a) Panoramic view of a representative image. (b) Zoom of the selected area. (c) 3D Reconstruction of EVs within selected area. (d) Z-slices projection of the selected area. (e,f) are reference for time and spatial coordinates (XYZ) of c and d respectively.