Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line

Abstract

The mucosal surface of intestine is continuously exposed to both potential pathogens and beneficial commensal microorganisms. Recent findings suggest that intestinal epithelial cells, which once considered as a simple physical barrier, are a crucial cell lineage necessary for maintaining intestinal immune homeostasis. Therefore, establishing a stable and reliable intestinal epithelial cell line for future research on the mucosal immune system is necessary. In the present study, we established a porcine intestinal epithelial cell line (ZYM-SIEC02) by introducing the human telomerase reverse transcriptase (hTERT) gene into small intestinal epithelial cells derived from a neonatal, unsuckled piglet. Morphological analysis revealed a homogeneous cobblestone-like morphology of the epithelial cell sheets. Ultrastructural indicated the presence of microvilli, tight junctions, and a glandular configuration typical of the small intestine. Furthermore, ZYM-SIEC02 cells expressed epithelial cell-specific markers including cytokeratin 18, pan-cytokeratin, sucrase-isomaltase, E-cadherin and ZO-1. Immortalized ZYM-SIEC02 cells remained diploid and were not transformed. In addition, we also examined the host cell response to Salmonella and LPS and verified the enhanced expression of mRNAs encoding IL-8 and TNF-α by infection with Salmonella enterica serovars Typhimurium (S. Typhimurium). Results showed that IL-8 protein expression were upregulated following Salmonella invasion. TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion. TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S. Typhimurium neither nor LPS. Taken together, these findings demonstrate that ZYM-SIEC02 cells retained the morphological and functional characteristics typical of primary swine intestinal epithelial cells and thus provide a relevant in vitro model system for future studies on porcine small intestinal pathogen-host cell interactions.

Figure 1. Morphological features of pSIECs and ZYM-SIEC02 cells.

A. Cellular morphology of pSIECs at 7 days in culture (100×) and at passage 5 (100×); a drug-resistant cell clone (100×) and ZYM-SIEC02 cells at passage 90 (100×).No obvious morphological differences were observed between ZYM-SIEC02 cells and primary SIECs. B. Electron micrographs of monolayer cultures of ZYM-SIEC02 cells indicate microvilli (MV) in 3 dimensions andin monolayer culture; tight junctions (TJ) and a small intestine glandular configuration (SIGC) (Red arrow).

Figure 2. Immunostaining and western blot analysis of ZYM-SIEC02 cell cultures.

A. pSIECs and ZYM-SIEC02 cells were stained with cytokeratin 18 antibodies (green) and propidium iodide (red). B. pSIECs and ZYM-SIEC02 cells were stained with pan-cytokeratin antibodies (green) and propidium iodide (red). C. expression of cytokeratin 18, E-cadherin, SI, villin, ZO-1 and occludin were determined by western blot analysis. Cytokeratin 18, pan-cytokeratin, E-cadherin, SI, villin, ZO-1 and occludin were expressed in both pSIECs (at passage 3) and ZYM-SIEC02 cells (at passages 15, 55, and 90).

Figure 3. Growth curves, apoptosis and cell cycle analysis of SIECs before and after hTERT transfection.

A. Growth of pSIECs and ZYM-SIEC02 cells. Data are represented as the mean ± SD of 3 independent experiments. B. Apoptosis analysis of control pSIECs and ZYM-SIEC02 cells, The percentage of apoptosis in pSIECs and ZYM-SIEC02 cells under basal growth conditions was 26.9% and 11.4% (Q2+Q4), respectively. C. Cell cycle distributions of control SIECs and ZYM-SIEC02 cells, the percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.66% and 31.65%, respectively.

Figure 4. Detection of Telomerase activity.

A. Expression of hTERT protein was detected by western blot analysis. Expression of hTERT was readily detected in ZYM-SIEC02 cells at passage 50 and 90, but was barely detectable in pSIECs at passage 3. The A549 cell line was used as a positive control. B. Silver staining method was used to detect telomerase activity. The laddering patterns indicated that ZYM-SIEC02 cells display higher telomerase activity as compared to pSIECs and A549 cell line.

Figure 5. Tumorigenicity assay of ZYM-SIEC02 cells.

A. Representative images of pSIECs and ZYM-SIEC02 cells growing in soft agar. Neither cell line formed colonies after 2 weeks in culture, indicating that the cells did not exhibit anchorage-independent growth (100×). B. Karyotype analysis of ZYM-SIEC02 cells at passage 55, indicated that the cells contained 38 chromosomes, consistent with a diploid karyotype. C. Tumor formation assay. pSIECs and ZYM-SIEC02 cells were injected subcutaneously into nude mice. After one month, neither group of mice had developed tumors, in contrast, mice injected with EMF6 cells rapidly developed tumors within 8 days. Histological examination indicated morphologically normal tissue below the injection site of pSIECs (passage 3) and ZYM-SIEC02 cells (passage 55), but a dense cellular mass below the EMF6 cell injection site.

Figure 6. Optimization of culture medium for ZYM-SIEC02 cells.

A. ZYM-SIEC02 cells were cultured for 24 h with 2%, 5%, 10%, 15%, or 20% FBS. 5% FBS was found to be the optimal concentration. B. Addition of 5% FBS or ITS, and/or EGF to the cell medium individually, compared with DMEM/F12 alone; Addition of ITS or ITS/EGF enhanced cell growthsimilar to the addition of 5% FBS, while addition of EGF alone did not have a significant effect. C. ITS/EGF or ITS significantly enhanced cell growth with the use of cell culture medium containing 5% FBS. D. Addition of ITS, EGF, or EGF/ITS to DMEM/F12 had little effect on the proliferation rate compared to the addition to 5% FBS. * P□0.05 versus basal conditions.

Figure 7. Invasion efficiency of Samonlla Thpyimurium in the ZYM-SIEC02 cell line.

Confluent monolayers of ZYM-SIEC02 cells and pSIECs were infected with a MOI of 10∶1 (S. Typhimurium to host cells) for 1 h, and incubated in media containing gentamicin for an additional hour. Cells used were pSIECs (open bars) and ZYM-SIEC02 cells (filled bars). Data shown are normalized to the efficiency (relative percent of input bacteria) and representative of 5 independent experiments performed in triplicate.

Figure 8. Cytokine expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS.

Confluent ZYM-SIEC02 cell monolayers were infected for 4 h with S. Typhimurium or LPS. A. Relative mRNA expression of IL-8 in ZYM-SIEC02 cells were upregulated approximately 2.5-fold within 4 h of S. Typhimurium challenge relative to control, while the cells that stimulated with LPS demonstrated a significant reduction; B. TNF-α mRNA expression was a modest increased by S. Typhimurium stimulation, while the cells that stimulated with LPS demonstrated a significant reduction; C. TLR4 and TLR6 mRNA expression were upregulated following stimulation with LPS. D. IL-8 protein expression in ZYM-SIEC02 cells infected with Salmonella typhimurium and LPS. ZYM-SIEC02 cells were incubated with S. Typhimurium at a MOI of 100∶1 followed by an incubation in media containing gentamicin, and then cell culture supernatants were harvested after 4 h. ZYM-SIEC02 cells were stimulated with LPS, and cells were harvested after 4 h. IL-8 concentrations were determined by ELISA. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001; ns, not significant.

Figure 9. Inhibition of LPS-mediated response.

ZYM-SIEC02 cells were stimulated with the indicated dose of E.coli 055:B55 LPS in the presence or absence of polymyxin B sulfate(Sigma) for 3 hours. A. Polymyxin B increased TNF-α mRNA expression when the concentration of LPS was lower than 250 ng/ml. B. Polymyxin B prevented the increase in IL-6 mRNA expression. The data shown are means±SEM of 3 independent experiments. * P<0.05;** P<0.01; *** P<0.001.