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. 2024 Jan 19;17(1):25.
doi: 10.1186/s13071-023-06090-8.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species

Huifang Chen #  1   2 , Juan Li #  2 , Xiaoting Pan  1   2 , Zhichao Hu  1 , Jianfeng Cai  1 , Zijie Xia  3 , Nanshan Qi  2 , Shenquan Liao  2 , Zachary Spritzer  3 , Yinshan Bai  4 , Mingfei Sun  5
Affiliations

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species

Huifang Chen et al. Parasit Vectors. .

Abstract

Background: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes.

Methods: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed.

Results: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines.

Conclusions: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.

Keywords: Avian embryo; Avian intestinal epithelial cell line (AIEC); Culture model; E. tenella.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Development of avian embryonic intestinal tissue visualized by hematoxylin and eosin (H&E) histochemistry and immunostaining for marker LGR5. A Avian embryos of several ages were obtained, and intestinal tissues were isolated to observe their morphology. B The development of the small intestine at E9, E11, E13, E15, and E18 were observed by H&E staining. C LGR5 expression by immunofluorescence (IF) detection. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, blue), and LGR5 was stained with anti-LGR5 (green). Scale bars: 100 μm
Fig. 2
Fig. 2
Morphology of primary epithelial cells isolated from E9. A Cellular morphology in 12, 48, and 120 h of culture after isolation. B SV40T antigen was successfully transfected into embryonic kidney epithelial cells (293FT) and integrated into the small avian intestinal epithelial cell line (AIEC) of E9 embryos. C F10 and F30 AIEC marker genes (KRT-18, CDH1, CLDN1, TJP1, OCLN, and VILL) expression detection. D Morphology of F5, F10, F20, and F30 AIEC (light/green). E Representative images of immunofluorescence staining of F30 AIEC for TJP1 showing a single cell layer, including nuclei of cells stained in blue (HoChest33342) and TJP1 in red. The images were captured with a fluorescence microscope. Scale bars: 50 μm
Fig. 3
Fig. 3
Comparison of gene expression. A Heatmap of differentially expressed genes (DEGs). B F10 and F20 volcano plots of DEGs. C F10 and F30 volcano plots of DEGs. D F20 and F30 volcano plots of DEGs. E Venn diagram showing co-expression of differential genes (a, AIECs F10-F20; b, AIECs F10-F30; c, AIECs F20-F30). F Phylogenetic tree of F10, F20 and F30; log2(FC) < 1, adjusted P-value < 0.05)
Fig. 4
Fig. 4
Enrichment analysis of avian intestinal epithelial cell line (AIEC) in F10 and F30. A Gene Ontology (GO) analysis. B Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of AIECs
Fig. 5
Fig. 5
The expression of cell cycle genes in the F30 of the avian intestinal epithelial cell line (AIEC) was detected and measured by quantitative polymerase chain reaction (qPCR). A–D Upregulated gene expression in F30 AIECs. E, F Downregulated gene expression in F30 AIECs
Fig. 6
Fig. 6
Infection and development of E. tenella in AIECs. Cells were infected with E. tenella sporozoites. A After 4 h of infection, most sporozoites could find and invade AIECs. B After 6 h of infection, the invasion was successful and the sporophyte formed. C After 24 h of infection, the formation of mononuclear trophozoites were observed. D After 48 h of infection, the formation of first-generation immature schizonts was observed. E After 72 h of infection, the formation of first-generation mature schizonts with budding merozoites was observed. F After 96 h of infection, the second-generation schizonts and merozoites were observed (H&E staining). G At different time points (6, 24, 48, 72, 96, 120, 144, and 168 h), the effect of E. tenella activity on the endogenous developmental stage was evaluated by measuring gene expression of EtGAPDH. H Infection efficiency of E. tenella sporozoites inoculated with DF-1 and AIECs at 6 h, 24 h and 48 h. I Inoculation of E. tenella sporozoites on F10 and F30 AIECs at 24 h and 48 h. Scale bars: 20 μm
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