Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

Abstract

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.

Mucosal IFNγ production and potential role in protection in Escherichia coli O157:H7 vaccinated and challenged cattle

“…While Hep-2 cells are often used as a proxy for O157 attachment to epithelial cells, the O157 proteins involved in attachment to HEp-2 versus bovine cells may be different 30 . To examine the ability of IFNγ to limit O157 attachment to bovine cells, a bovine intestinal epithelial cell line (BIEC-c4) 31 was used in attachment assays. The addition of recombinant bovine IFNγ to BIEC-c4 cells limited subsequent attachment of O157 to the BIEC cells by about 40% (Fig.…”

“…After incubation at 37 °C for 24 h colonies that grew on these plates were counted. For determining O157 adherence to BIECs (kindly provided by Dr. Radhey Kaushik, South Dakota State University), confluent BIEC cells, grown as described previously 31 , 54 , were treated with media alone or rbIFNγ for approximately 16 h at 39 °C. Approximately 10 7 O157 cells were added to wells for 2 h, and non-adhered O157 bacteria were washed away with PBS.…”

Mucosal Immune System of Cattle

“…ME have important immune regulatory functions that affect the mucous barrier, tight junctions, and the innate and adaptive immune cells in the LP. 10 Like other immune cells, they use pattern-recognition receptors, 11 including the toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors, to monitor Contribution of commensals to ME cells and immunity. ME produce serum amyloid A protein after exposure to filamentous bacteria (related to Clostridium), which activates DCs to produce IL-6 and IL-23, resulting in the generation of Th17 cells that are important for T-cell development.…”

“…However, ME express TLR not on their surface but rather inside the cell, and are only upregulated when the cell is infected. 11 They also respond to microbial metabolites including short-chain fatty acids (ie, butyrate) and many normal commensal microbial components, such as Bacteroides fragilis capsular polysaccharide A (see Fig. 6.…”

Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells

“…The traditional immortalization strategies for bovine cells generally introduce human telomerase reverse transcriptase (hTERT) genes, oncogenes, or viral genomes into primary cells via transient transfection or a virus-mediated delivery system and construct long-term cultured cell lines in vitro [7]. However, there are some limitations and risks in the subsequent use of cell lines generated via these methods, such as the inefficiency of the immortalized cell lines caused by the short expression time of the immortalization factor via transient transfection, as well as the instability of the cell genome because of the integration of random exogenous immortalization factors through virus-mediated delivery [8][9][10][11][12][13]. Additionally, there are unknown obstacles for the immortalization of bovine cells.…”