Bacterial attachment and junctional transport function in induced apical-out polarized and differentiated canine intestinal organoids

Abstract

Dogs are increasingly recognized as valuable large animal models for understanding human intestinal diseases, as they naturally develop conditions similar to those in humans, such as Enterohemorrhagic E. coli, Clostridium difficile infection, inflammatory bowel disease, and ulcerative colitis. Given the similarity in gut flora between dogs and humans, canine in vitro intestinal models are ideal for translational research. However, conventional extracellular matrix-embedded organoids present challenges in accessing the lumen, which is critical for gut function. This study aimed to investigate the feasibility of inducing polarity reversal and differentiation in canine apical-out colonic organoids (colonoids), evaluate their barrier integrity, and visualize host-pathogen interactions. Our results demonstrated successful polarity reversal and differentiation induction while maintaining barrier integrity. Polarity reversal allowed for enhanced observation of host-pathogen interactions, facilitating visual assessments and membrane integrity evaluations using both pathogenic and nonpathogenic E. coli. This process led to the downregulation of stem cell marker LGR5 and upregulation of intestinal epithelial cell marker ALPI, indicating differentiation. Further differentiation was observed with the use of a differentiation culture medium, resulting in significant upregulation of ALPI and goblet cell marker MUC2. The findings suggest that apical-out canine colonoids can serve as physiologic and valuable models for studying the pathogenic mechanisms and clinical significance of intestinal diseases in dogs. This model has the potential to advance both canine and human gastrointestinal research, enhancing our understanding of gastrointestinal physiology and pathology and aiding in the development of novel therapeutics.

Keywords: apical-out; canine; differentiation; host-pathogen interaction; intestine; organoid; polarity reversal.

Figure 1

Development and characterization of the canine apical-out colonoids. (A) A schematic for polarity reversal of colonoids. (B) The percentage of apical-out colonoids (Apical, white box) and colonoids with mixed polarities (Mix, gray box) under confocal microscopy at 96 h (n = 3, each). Data is shown as mean ± standard error of the mean (SEM). 40–60 randomly selected organoids in each biological replicate were counted and classified as apical-out, basal-out, or mixed polarity. (C) Representative images from the phase-contrast microscope at 24 and 96 h. Scale bar = 25 μm. (D) Confocal microscopy images of an apical-out colonoid and Matrigel-embedded colonoid at 96 h with visualization of apical brush borders (F-actin, red) and basal nuclei (DAPI, blue). Scale bar = 10 μm.

Figure 2

Characterization of apical-out colonoids. (A) Comparison of relative gene expression levels of the following markers: LGR5 (stem cells), ALPI (Intestinal epithelial cells), MUC2 (goblet cells), and CHGA (enteroendocrine cells) of apical-out colonoids in EM (white box) versus DM (gray box). Data is shown as mean ± SEM. p < 0.05 was considered statistically significant using the Willcoxon rank-sum test, followed by Bonferroni correction. *p < 0.05; **p < 0.01, ***p < 0.001. (B) Confocal microscopy images of apical-out colonoids at 96 h in DM with visualization of apical brush borders (F-actin, red) and basal nuclei (DAPI, blue), chromogranin A (CHGA, yellow), and Sambucus nigra lectin (SNA, green). Scale bar = 10 μm.

Figure 3

The evaluation of barrier integrity using FITC-dextran assay at 96 h. (A) Representative image of untreated Apical-DM and EDTA-treated Apical-DM. Scale bar = 50 μm. (B) FITC permeability ratio of untreated (Control, white dots) and EDTA-treated Apical-DM (black dots). The median is shown as a horizontal line. For image acquisition, randomly selected 6–15 fields were used in each biological replicate. p < 0.05 was considered statistically significant using the Willcoxon rank-sum test. ***p < 0.001.

Figure 4

Host-pathogen interaction. (A) A representative confocal microscopy image of apical-DM infected with nonpathogenic E. coli (YFP-tagged, green) with visualization of apical brush borders (F-actin, red) and basal nuclei (DAPI, blue). Scale bar = 10 μm. (B) In barrier integrity assay, representative images of apical-out colonoids infected with nonpathogenic E. coli or EHEC. Scale bar = 50 μm. (C) FITC permeability ratio of untreated (Control, white dots), apical-out colonoids infected with nonpathogenic E. coli (gray dots), or EHEC (black dots). The median is shown as a horizontal line. For image acquisition, randomly selected 6–12 fields were used in each biological replicate. p < 0.05 was considered statistically significant using the Willcoxon rank-sum test. ***p < 0.001.