Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut

Abstract

Small intestinal organoids, or enteroids, represent a valuable model to study host-pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4⁺ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host-pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.

Keywords: ETEC; Enteroids; Intestinal stem cells; Pig; Swelling assay.

Figure 1

Development of porcine enteroids from small intestinal crypts. A Enteroids cultured using IESC or (B) L-WRN-conditioned IESC medium, respectively. Images were taken from day 1 until day 14 and are representative for enteroid development from jejunal crypts of 9 piglets. C Enteroids cultured using Intesticult organoid growth medium were followed from day 0 until day 4 and are representative for enteroid development from jejunal crypts of 4 piglets. D Comparison of enteroid cultures originating from duodenal, jejunal and ileal crypts. Images represent enteroids 6 days after passaging and are representative of enteroid cultures from crypts obtained from duodenum (n = 4), jejunum (n = 10) and ileum (n = 8). Scale bar equals 100 µm.

Figure 2

Porcine enteroids mimic the response of the small intestine to ETEC-derived enterotoxins. Spheroids derived from duodenum, jejunum and ileum 6 days after passaging were stimulated with enterotoxins or guanylin and imaged using live-cell microscopy. The surface area of the spheroids was measured using ImageJ. A Representative images displaying ileal spheroid swelling induced by guanylin (10 µM) at T0, T50 and T110 upon administration. B, C The average relative area increase of the spheroids was plotted in function of the time after enterotoxin administration. (n = 3 for all tissues). D Spheroid bursting upon guanylin (10 µM) stimulation. Images are representative for other tissues and swelling inducers. Scale bar = 100 µm. Relative IL8 secretion in medium supernatant (E) and Matrigel dome (F) of jejunal enteroids stimulated for 24 h with bacterial supernatant with (WT) or without enterotoxins (toxin negative) compared to non-immunogenic guanylin (n = 3; Kruskal–Wallis test).

Figure 3

Porcine enteroid monolayer development and interconvertibility with 3D culture. A Growth of jejunal crypts, plated on collagen-coated wells, using L-WRN-conditioned medium followed from days 1 to 7. B Comparison of 2D-monolayers from duodenum, jejunum and ileum at day 3 after passaging. C Interchange between 2 and 3D cultures and back of jejunal enteroids representative for 3 piglets. D 3D and (E) 2D-jejunal enteroid cultures were stained with anti-SOX9 or isotype control antibodies at days 7 and 3 respectively after passage. Images are representative for 2 piglets. Scale bar equals 100 µm.

Figure 4

F4-mediated bacterial adhesion on 2D-enteroid monolayers. A Jejunal and (B) ileal monolayers were grown until 100% confluent and infected with ETEC bacteria with (Gis26 WT) or without F4-fimbrae (GIS26∆faeG) at a MOI of 10 in duplicate (n = 3 for jejunum; n = 3 for ileum; Kruskal–Wallis test; * p < 0.05).