The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research. Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64.

Keywords: 2D cell culture; 3D cell model; Mycobacterium paratuberculosis (MAP); cattle; in vitro model; organoid.

Figure 1

3D enteroids cultivated from bovine intestinal tissue. Representative images showing crypts isolated from bovine ileal tissue and maintained in a Matrigel dome with IntestiCult medium, from 5 independent animals. (A–C) Brightfield images of 3D enteroids cultured for 24 h (A), 3 days (B) and 7 days (C) demonstrating that they bud and proliferate over time. By 7 days the enteroid lumen fills with debris from cells sloughed off and are ready to be passaged. (D–H) Confocal images of 3D bovine enteroids stained for epithelial cell fate markers. (D) Nuclei (DAPI, blue), tight junctions (ZO-1, green) and proliferative cells (Ki-67, red). (E) Nuclei (DAPI, blue), actin (Phalloidin, green) and Paneth cells (lysozyme, red). (F) Nuclei (DAPI, blue), F-actin (Phalloidin, green) and enteroendocrine cells (chromogranin A, red). (G) Split panel of enteroids stained for nuclei (DAPI, blue), F-actin (Phalloidin, green), and tight junctions between cells (ZO-1, red). (H) Split panel of enteroids stained for nuclei (DAPI, blue), glycolipids (UEA-1, green) and tight junctions (ZO-1, red). Scale bar = 50 μm.

Figure 2

2D epithelial monolayers cultured on collagen matrix. Single cells were seeded onto collagen coated wells. The 2D monolayers were cultured with IntestiCult containing the relevant inhibitors and maintained for up to 10 days. (A–F) Representative confocal microscopy images of 2D monolayers cultured on collagen coated wells from 3 separate amimals, demonstrating presence of specific cell marker proteins. (A) F-actin (phalloidin, green), nuclei (DAPI, blue) and proliferative cells (Ki67, red). (B) Nuclei (DAPI, blue), F-actin (Phalloidin, green) and Paneth cells (lysozyme, red). (C) Nuclei (DAPI, blue) and glycolipids (UEA-1, green). (D) 2D monolayer imaged by brightfield microscopy stained for Periodic Acid Schiff to show mucins produced by goblet cells. (E) Split panel of monolayer stained for nuclei (DAPI, blue), tight junctions between cells (ZO-1, green), and proliferative cells (Ki-67, red). Scale bar = 20 μm. (F) Split panel of monolayer stained for nuclei (DAPI, blue), F-actin (Phalloidin, green) and enteroendocrine cells (chromogranin A, red). Scale bar = 50 μm.

Figure 3

Apical-out bovine enteroids show epithelial barrier integrity when established from freshly harvested intestinal crypts. Apical-out enteroids cultivated from freshly isolated intestinal crypts and cultured in suspension in Intesticult at 1 day (A) and 7 days (B). Images are representative of enteroids derived from 3 independent animals. (C,D) Confocal images of bovine inside out enteroids (7 days of culture) immersed in FITC-dextran 4kDa showing epithelial barrier integrity in untreated (C) and EDTA-treated conditions (D). Immunofluorescence staining of bovine inside out intestinal organoids shown in split panel demonstrates epithelial differentiation (E–J). Apical-out enteroids show reverse polarisation compared to basal-out 3D enteroids from the brush border facing the external medium, represented by F-actin staining (Phalloidin, green); and a dense internal core of cells (nuclei stained with DAPI, blue) (E). Nuclei (DAPI, blue), proliferative cells (Ki67, red), and tight junctions between cells (ZO-1, green) (F). Cross section of an apical-out enteroid from z-stack images of apical-out enteroids stained for nuclei (DAPI, blue), F-actin (Phalloidin, green) and Paneth cells (Lysozyme 1, red) (G). Split panel of apical-out enteroids stained for nuclei (DAPI, blue), actin (Phalloidin, green) and tight junctions between cells (ZO-1, red) (H). Split panel of apical-out enteroids stained for nuclei (DAPI, blue), F- actin (Phalloidin, green) and enteroendocrine cells (Chromogranin A, red) (I). Split panel of apical-out enteroids stained for nuclei (DAPI, blue), glycoproteins/ glycolipids (UEA-1, green) and tight junctions (ZO-1, red) (J). Scale bar = 20 μm.

Figure 4

RT-qPCR of mRNA expression indicative of specific cell types in enteroid-derived models. The gene expression was determined by RT-qPCR and calculated as fold change relative to the expression of ACTB and REPS1 as endogenous reference genes. Total RNA was isolated from the samples and confirmed to be free of genomic DNA. The results are from samples derived from 2 independent animals, with 3 technical repeats and presented as mean values ± standard deviation (SD). Statistical analysis was performed with a one-way ANOVA; * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Figure 5

Infection of basal-out 3D enteroids with 2 different strains of MAP. Enteroids were infected with an MOI of 100 in 3 biological replicates using enteroids generated from the same calf at passage 15, 17 and 20. The bovine (A) and bacterial (B) cell number was quantified using qPCR for the spastin gene and F57 sequence element respectively via qPCR (data presented as mean ± SD). Statistical analysis performed using 2-way ANOVA followed by a post hoc Tukey's test. P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***; P < 0.0001 = ****. (C–D) Immunofluorescence microscopy images of infected 3D enteroids 24 h post infection. Enteroids were stained for nuclei (DAPI, blue), F-actin (Phalloidin, green) and MAP (anti-MAP, red). 3D enteroids are shown as a cross section from Z-stack images infected with MAP K10 (C) and MAP C49 (D). Scale bar = 10μm.

Figure 6

Infection of 2D monolayers with 2 different strains of MAP. Monolayers were infected with an MOI of 10 in 3 independent experiments. The bovine (A) and bacterial (B) cell number was quantified using qPCR for the spastin gene and F57 sequence element respectively via qPCR (data presented as mean ± SD). Statistical analysis performed using 2-way ANOVA followed by a post hoc Tukey's test. P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***; P < 0.0001 = ****. (C,D) Immunofluorescence staining of monolayers imaged 24 hours post infection. Infected monolayers were fixed and stained for nuclei (DAPI, blue), F-actin (Phalloidin, green) and MAP (anti-MAP, red). Cells were infected with MAP K10 (C) and as a cross section from Z-stack images infected with MAP C49 (D). Scale bar = 10μm.

Figure 7

Infection of 3D Apical-out enteroids with 2 different strains of MAP. Apical-out enteroids were infected with an MOI of 100 using apical-out enteroids generated from 3 separate calves. The bovine (A) and bacterial (B) cell number was quantified using qPCR for the spastin gene and F57 sequence element respectively via qPCR (data presented as mean ± SD). Statistical analysis performed using 2-way ANOVA followed by a post hoc Tukey's test. P < 0.05 = ^*; P < 0.01 = ^**; P < 0.001 = ^***; P < 0.0001 = ^****. (C,D) Immunofluorescence staining of 3D apical-out enteroids imaged 24 h post infection. Slides with infected apical-out enteroids were fixed and stained for nuclei (DAPI, blue), F-actin (Phalloidin, green) and MAP (anti-MAP, red). Apical-out enteroids were infected with MAP K10 (C) and as a cross section from Z-stack images infected with MAP C49 (D). Scale bar = 10μm.