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.
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