Pathogen-epithelium interactions and inflammatory responses in Salmonella Dublin infections using ileal monolayer models derived from adult bovine organoids

Abstract

Salmonella enterica serovar Dublin (S. Dublin) is an important enteric pathogen affecting cattle and poses increasing public health risks. Understanding the pathophysiology and host-pathogen interactions of S. Dublin infection are critical for developing effective control strategies, yet studies are hindered by the lack of physiologically relevant in vitro models. This study aimed to generate a robust ileal monolayer derived from adult bovine organoids, validate its feasibility as an in vitro infection model with S. Dublin, and evaluate the epithelial response to infection. A stable, confluent monolayer with a functional epithelial barrier was established under optimized culture conditions. The model's applicability for studying S. Dublin infection was confirmed by documenting intracellular bacterial invasion and replication, impacts on epithelial integrity, and a specific inflammatory response, providing insights into the pathogen-epithelium interactions. The study underscores the utility of organoid-derived monolayers in advancing our understanding of enteric infections in livestock and highlights implications for therapeutic strategy development and preventive measures, with potential applications extending to both veterinary and human medicine. The established bovine ileal monolayer offers a novel and physiologically relevant in vitro platform for investigating enteric pathogen-host interactions, particularly for pathogens like S. Dublin.

Keywords: S. Dublin; Salmonella; Adult stem cells; Bovine ileum; In vitro infection model; Organoid-derived monolayer.

Figure 1

Characterization of adult bovine ileal organoid-derived monolayers. (a) A representative phase-contrast image of the monolayer at Day 6 of culture, exhibiting characteristic cobblestone morphology. Bar: 20 μm. (b,c) Representative scanning electron microscopy images of the monolayers at Day 6 of culture in low (b) and high (c) magnifications. The entire surface of the monolayer was uniformly covered by densely packed microvilli. Bars: 2 μm. (d) A representative transmission electron microscopy image of the monolayer at Day 5 of culture, demonstrating the formation of apical microvilli (MV) covered with glycocalyx (GLX), inter-cellular tight junctions (TJ) and desmosome (D). Bar: 500 nm. (e) The transepithelial electrical resistance (TEER) measured daily from Day 1 to 7 and apparent permeability (P_app) of fluorescein isothiocyanate-dextran across the monolayer measured at Day 1, 3, and 5 of culture. Results are expressed as mean ± s.e.m. obtained from three independent experiments with at least two technical replicates per experiment using three biological replicates. **p < 0.01, ***p < 0.001 compared with the previous day. (f–i) Immunofluorescence staining of the monolayers against F-actin (f, red), E-cadherin (g, green), Sambucus nigra agglutinin (SNA) (h, green), and EdU (i, cyan) at Day 6 of culture. Nuclei were stained with DAPI (blue). Top-down view (top) and cross-sectional images (z-stack, bottom) are shown. Bars: 20 μm. (j) RT-qPCR of the cells within the monolayers at Day 4 of culture. Relative expression levels of stem and lineage cell marker genes were evaluated using GAPDH, RPL0 and ACTB as internal controls. Results are expressed as mean ± s.e.m. obtained from two technical replicates from three biological replicates. LGR5 leucine rich repeat containing G protein-coupled receptor 5, CHGA chromogranin A, LYZC lysozyme C, MUC2 mucin 2, FABP2 fatty acid-binding protein 2, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, RPL0 ribosomal protein L0 and ACTB β-actin.

Figure 2

A schematic of experimental design for the infection of bovine ileal organoid-derived monolayers with S. Dublin. An experimental timeline from generation of ileal monolayers to infection with S. Dublin and subsequent analyses is shown. Single cells derived from adult bovine ileal organoids are seeded onto 24-well cell culture inserts utilizing the optimized monolayer culture media without antibiotics. Once the formation of confluent monolayers is confirmed by stable TEER measurements (3–4 days after seeding), the epithelial cells are exposed apically with 1 × 10⁶ CFU/well of S. Dubin for 1 h in antibiotic-free culture medium, facilitating bacterial invasion of the epithelial cells. The control cells are treated with the same volume of the monolayer culture medium without bacteria. After the invasion period, the monolayers undergo a treatment phase to eliminate non-invaded bacteria. Initially, this involves an hour-long incubation with a medium containing 50 μg/mL of gentamicin, aimed at killing extracellular bacteria. Following this, the monolayers are maintained in a medium supplemented with 10 μg/mL of gentamicin for the remainder of the experiment (24 h post infection). This step ensures the suppression of any residual bacteria, preventing their overgrowth in the culture medium. Created with BioRender.com.

Figure 3

Invasion and replication of S. Dublin in bovine ileal organoid-derived monolayers. (a) Representative scanning (SEM, left) and transmission (TEM, center and right) electron microscopy images of uninfected monolayers. Apical surface of the monolayer is covered with uniform microvilli. (b) Representative SEM (top left and bottom) and TEM (top center and right) images of S. Dublin-infected monolayers at 24 h post infection. SEM revealed adhering bacteria on the apical surface of the monolayer (top left) and mucus (bottom left) as well as invading bacteria into the epithelial cell through disruption of the apical membrane (bottom center and right). TEM revealed intracellular bacteria within vesicles in the cytoplasm of the epithelial cell (arrowheads) and effacement of microvilli on the apical surface of the monolayer (arrow). The right column images show magnification of the white dashed area in both (a) and (b). N: nuclei, MV: microvilli. Bars: 2 μm. (c) Enumeration of intracellular bacteria at 2- and 24 h post infection relative to the inoculum. Results are expressed as mean ± s.e.m. obtained from three independent experiments with two technical replicates per experiment using three biological replicates. *p < 0.05.

Figure 4

Impacts of S. Dublin infection on epithelial barrier integrity of the bovine ileal organoid-derived monolayers. (a) Representative phase-contrast (left) and immunofluorescent images (center: F-actin, red and right: E-cadherin, green) of uninfected (top) and infected (bottom) monolayers at 24 h post infection. Nuclei were stained with DAPI (blue). Top-down view and cross-sectional images are shown. Arrowheads indicate contractile actin rings. Bars: 20 μm. (b) Changes in the TEER relative to the pre-infection value over the course of the experiment. TEER was measured at 0-, 4-, 8-, 12- and 24 h post infection with S. Dublin, normalized by the pre-infection value, and compared between the control and infected monolayers at each time point. (c) Normalized TEER (left) and P_app (right) values at 0- and 24 h post infection. Significant differences were noted between control and S. Dublin-infected monolayers at 24 h in both TEER and P_app. For both (b) and (c), results are expressed as mean ± s.e.m. obtained from four independent experiments with at least two technical replicates per experiment using three biological replicates. **p < 0.01.

Figure 5

Immune response of bovine ileal organoid-derived monolayers to S. Dublin infection. (a) RT-qPCR of the cells within the monolayers at 24-h post infection. Relative gene expressions for cytokines (TNFA, IL6, and IL8) were evaluated using GAPDH, RPL0 and ACTB as internal controls. (b) IL8 concentrations in the apical and basolateral culture media were determined with ELISA. For both evaluations, results are expressed as mean ± s.e.m obtained from three independent experiments with two technical replicates per experiment using three biological replicates and compared between the control and infected monolayers. TNFA tumor necrosis factor alpha, IL6 interleukin 6, IL8 interleukin 8. *p < 0.05, **p < 0.01, ***p < 0.001.