Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer

Abstract

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Discrimination of distinct chicken M cell subsets based on CSF1R expression

“…The analysis of the 2260 upregulated DEGs identified in 1-week-old M cells compared to IFE cells found that 13 of the 28 genes, originally identified by Nakato et al 21 , were represented in our dataset (Supplementary Table 1 ). However, of the missing genes, such as CLU and ANXA10, have been implicated in mammalian and chicken M cell biology 21 , 24 , 27 while genes not present in our DEG dataset, such as FLI1 , CXCR4 and VIM, implied that our enrichment approach excluded immune cells and fibroblasts 28 , 29 .…”

Disentangling the Innate Immune Responses of Intestinal Epithelial Cells and Lamina Propria Cells toSalmonellaTyphimurium Infection in Chickens

“…The aim of this study was to disentangle the contribution of avian intestinal epithelial cells and lamina propria cells to STm infection using unique enteroid culture models. In contrast to mammalian enteroids or intestinal organoids, the inner core of chicken 3D enteroids consists of cells present in a lamina propria including T and B cells, natural killer cells, macrophages, dendritic cells (DC) and heterophils (Nash et al, 2021;Nash et al, 2023), whereas few intraepithelial leukocytes are present in chicken 2D enteroids (Orr et al, 2021). Intestinal epithelial cells are equipped with pattern recognition receptors, such as TLRs, that initiate inflammatory responses in response to pathogen-associated molecular patterns if the epithelial barrier is breached.…”

“…For each independent culture, the intestines from five embryos were pooled. For the generation of 3D enteroids, the villi were released from the tissue as previously described (Nash et al, 2021) For 2D enteroid generation, freshly isolated intestinal villi were enzymatically digested with Accutase (TFS) as previously described (Orr et al, 2021). To remove the majority of the fibroblasts, cells were resuspended in FOM media supplemented with 1X N2 supplement (TFS), 100 ng/mL human (hu) epidermal growth factor (huEGF, TFS), 10 µM CHIR 99021 (Stratech Scientific), 10 µM Y27632 (Stem Cell Technologies) and 100 nM LDN193189 (Cambridge Bioscience).…”

“…For immuno-fluorescent staining, chicken 2D enteroids were grown on 2% Matrigel (Corning) coated transwell inserts (VWR, 0.33 cm 2 ) in 24 well plates (Orr et al, 2021) while 3D enteroids were grown as outlined above. Chicken 2D and 3D enteroids were treated with WT or ΔprgH STm, LPS or media alone as outlined above.…”

“…Avian floating 3D enteroids naturally develop in an advantageous apical-out conformation with apical microvilli facing the media and an inner core resembling the lamina propria, containing leukocytes, and mesenchymal and neuronal cells (Nash et al, 2021;Nash et al, 2023). In addition, a chicken 2D enteroid model that self-organises into an epithelial and mesenchymal sub-layer but lacks the underlying lamina propria cells has been developed (Orr et al, 2021). The aim of this study was to disentangle the innate immune response of epithelial cells and the immune response of the lamina propria cells to STm infection by comparing the gene expression profiles between uninfected and infected 2D and 3D enteroids.…”