Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf
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The intestinal epithelium is a major site of interaction with pathogens. In bovine intestinal epithelial cells (BIECs), Toll-like receptors (TLRs) play an important role in innate immune responses against enteric pathogens. This study is aimed at establishing a stable bovine intestinal epithelial cell line that can be maintained by a continuous passage so that studies on innate immune responses against various enteric pathogens can be performed. The main goal was to establish pure cultures of primary and immortalized bovine intestinal epithelial cells from the ileum and then characterize them biochemically and immunologically. Mixed epithelial and fibroblast bovine ileal intestinal cultures were first established from a 2-day old calf. Limiting dilution method was used to obtain a clone of epithelial cells which was characterized using immunocytochemistry (ICC). The selected clone BIEC-c4 was cytokeratin positive and expressed low levels of vimentin, confirming the epithelial cell phenotype. Early passage BIEC-c4 cells were transfected with either simian virus 40 (SV40) large T antigen or human telomerase reverse transcriptase (hTERT), or human papillomavirus (HPV) type 16E6/E7 genes to establish three immortalized BIEC cell lines. The expression of SV40, hTERT and HPV E6/E7 genes in immortalized BIECs was confirmed by a polymerase chain reaction (PCR). Immunocytochemistry and immunofluorescence assays also confirmed the expression of SV40, hTERT and HPV E6 proteins. The immortalized BIECs were cytokeratin positive and all except HPV-BIECs expressed low levels of vimentin. A growth kinetics study indicated that there were no significant differences in the doubling time of immortalized BIECs as compared to early passage BIEC-c4 cells. All four BIEC types expressed TLR 1-10 genes, with TLR 3 and 4 showing higher expression across all cell types. These newly established early passage and immortalized BIEC cell lines should serve as a good model for studying infectivity, pathogenesis and innate immune responses against enteric pathogens.
KeywordsIleum Bovine intestinal epithelial cells Immortalization SV40 hTERT HPV E6/E7 Toll-like-receptors
Radhey S. Kaushik, Pratik Katwal, Tirth Uprety, and Milton Thomas, and this research project were funded and supported by USDA, NIFA, SDSU Agricultural Experiment Station Hatch Grants # SD00H326-09, and SD00H547-15. This study was also funded by USDA/CSREES through project title JDIP: Johne’s Disease Integrated Program in Research, Education, and Extension, Grant Number # 2004-35605-14243; Sub-award No. Q6286224171. We acknowledge use of the SDSU-FGCF supported in part by NSF/EPSCoR Grant No. 0091948 and by the State of South Dakota.
RSK conceived and designed the study, received funding, and also conducted some of the experiments. RSK also reviewed the manuscript. PK, MT, and TU (graduate researchers) conducted the experiments, analyzed the results and wrote the paper. MH contributed to reagents/materials, analysis and helped in microscopy.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Bass DM, Mackow ER, Greenberg HB (1990) NS35 and not vp7 is the soluble rotavirus protein which binds to target cells. J Virol 64:322–330Google Scholar
- Birkner S, Weber S, Dohle A, Schmahl G, Follmann W (2004) Growth and characterisation of primary bovine colon epithelial cells in vitro. Altern Lab Anim ATLA 32:555–571Google Scholar
- Bols NC, Pham PH, Dayeh VR, Lee LEJ (2017) Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout. In Vitro Cell Dev Biol Anim 53:383–405. https://doi.org/10.1007/s11626-017-0142-5 CrossRefGoogle Scholar
- Booth C, Patel S, Bennion GR, Potten CS (1995) The isolation and culture of adult mouse colonic epithelium. Epithel Cell Biol 4:76–86Google Scholar
- Buckner D, Wilson S, Kurk S, Hardy M, Miessner N, Jutila MA (2006) Use of early passage fetal intestinal epithelial cells in semi-high-throughput screening assays: an approach to identify new innate immune system adjuvants. J Biomol Screen 11:664–671. https://doi.org/10.1177/1087057106289876 CrossRefGoogle Scholar
- Cencic A, Gradinik L, Vaukner M, Filipi B, Rannou O, Chingwaru W, Maragkoudakis P, Tsakalidou E (2007) Lefevre F intestinal cell models as alternative to experimental animals in research of food and water born viruses interactions with the host. Exp Pathol Health Sci 1:57Google Scholar
- Evans GS, Flint N, Potten CS (1994) Primary cultures for studies of cell regulation and physiology in intestinal epithelium. Annu Rev Physiol 56:399–417. https://doi.org/10.1146/annurev.ph.56.030194.002151 CrossRefGoogle Scholar
- Fiszer-Kierzkowska A, Vydra N, Wysocka-Wycisk A, Kronekova Z, Jarzab M, Lisowska KM, Krawczyk Z (2011) Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells. BMC Mol Biol 12:27. https://doi.org/10.1186/1471-2199-12-27 CrossRefGoogle Scholar
- Freitas M, Axelsson LG, Cayuela C, Midtvedt T, Trugnan G (2005) Indigenous microbes and their soluble factors differentially modulate intestinal glycosylation steps in vivo: use of a “lectin assay” to survey in vivo glycosylation changes. Histochem Cell Biol 124:423–433. https://doi.org/10.1007/s00418-005-0004-1 CrossRefGoogle Scholar
- Freshney RI (1994) Culture of animal cells: a manual of basic technique, 3rd edn. Wiley, New YorkGoogle Scholar
- He XY et al (2011) Recombinant adenovirus-mediated human telomerase reverse transcriptase gene can stimulate cell proliferation and maintain primitive characteristics in bovine mammary gland epithelial cells. Dev Growth Differ 53:312–322. https://doi.org/10.1111/j.1440-169X.2010.01236.x CrossRefGoogle Scholar
- Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Ann Rev Immunol 20:197–216. https://doi.org/10.1146/annurev.immunol.20.083001.084359 CrossRefGoogle Scholar
- Kaeffer B (2002) Mammalian intestinal epithelial cells in primary culture: a mini-review. In Vitro Cell Dev Biol Anim 38:123–134. https://doi.org/10.1290/1071-2690(2002)038%3c0123:MIECIP%3e2.0.CO;2 CrossRefGoogle Scholar
- Kaeffer B, Bottreau E, Velge P, Pardon P (1993) Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid. Eur J Cell Biol 62:152–162Google Scholar
- Koh SY, George S, Brozel V, Moxley R, Francis D, Kaushik RS (2008) Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli. Vet Microbiol 130:191–197. https://doi.org/10.1016/j.vetmic.2007.12.018 CrossRefGoogle Scholar
- Lee J, Yoo D, Redmond MJ, Attah-Poku SK, van den Hurk JV, Babiuk LA (1998) Characterization of the interaction between VP8 of bovine rotavirus C486 and cellular components on MA-104 cells and erythrocytes. Can J Vet Res 62:56–62Google Scholar
- Loret S, Rusu D, El Moualij B, Taminiau B, Heinen E, Dandrifosse G, Mainil J (2009) Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle. Res Vet Sci 87:123–132. https://doi.org/10.1016/j.rvsc.2008.12.002 CrossRefGoogle Scholar
- Mestecky J (2005) Mucosal immunology, 3rd edn. Elsevier Academic Press, BostonGoogle Scholar
- Moll R (1991) Molecular diversity of cytokeratins: significance for cell and tumor differentiation. Acta Histochem Suppl 41:117–127Google Scholar